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Sample records for cell-penetrating peptides decreases

  1. Intracellular transduction using cell-penetrating peptides.

    PubMed

    Sawant, Rupa; Torchilin, Vladimir

    2010-04-01

    Cell penetrating peptides (CPPs), TATp, in particular, has been used widely for intracellular delivery of various agents ranging from small molecules to proteins, peptides, range of pharmaceutical nanocarriers and imaging agents. This review highlights the mechanisms of CPP-mediated delivery and summarizes numerous examples illustrating the potential of CPPs in the fields of biology and medicine. PMID:20237640

  2. Membranotropic Cell Penetrating Peptides: The Outstanding Journey

    PubMed Central

    Falanga, Annarita; Galdiero, Massimiliano; Galdiero, Stefania

    2015-01-01

    The membrane bilayer delimits the interior of individual cells and provides them with the ability to survive and function properly. However, the crossing of cellular membranes constitutes the principal impediment to gaining entry into cells, and the potential therapeutic application of many drugs is predominantly dependent on the development of delivery tools that should take the drug to target cells selectively and efficiently with only minimal toxicity. Cell-penetrating peptides are short and basic peptides are widely used due to their ability to deliver a cargo across the membrane both in vitro and in vivo. It is widely accepted that their uptake mechanism involves mainly the endocytic pathway, the drug is catched inside endosomes and lysosomes, and only a small quantity is able to reach the intracellular target. In this wide-ranging scenario, a fascinating novel hypothesis is that membranotropic peptides that efficiently cross biological membranes, promote lipid-membrane reorganizing processes and cause a local and temporary destabilization and reorganization of the membrane bilayer, may also be able to enter cells circumventing the endosomal entrapment; in particular, by either favoring the escape from the endosome or by direct translocation. This review summarizes current data on membranotropic peptides for drug delivery. PMID:26512649

  3. Cell-penetrating peptides: breaking through to the other side.

    PubMed

    Koren, Erez; Torchilin, Vladimir P

    2012-07-01

    Cell-penetrating peptides (CPPs) have been previously shown to be powerful transport vector tools for the intracellular delivery of a large variety of cargoes through the cell membrane. Intracellular delivery of plasmid DNA (pDNA), oligonucleotides, small interfering RNAs (siRNAs), proteins and peptides, contrast agents, drugs, as well as various nanoparticulate pharmaceutical carriers (e.g., liposomes, micelles) has been demonstrated both in vitro and in vivo. This review focuses on the peptide-based strategy for intracellular delivery of CPP-modified nanocarriers to deliver small molecule drugs or DNA. In addition, we discuss the rationales for the design of 'smart' pharmaceutical nanocarriers in which the cell-penetrating properties are hidden until triggered by exposure to appropriate environmental conditions (e.g., a particular pH, temperature, or enzyme level), applied local microwave, ultrasound, or radiofrequency radiation. PMID:22682515

  4. Cell-penetrating peptides: Possible transduction mechanisms and therapeutic applications

    PubMed Central

    GUO, ZHENGRONG; PENG, HUANYAN; KANG, JIWEN; SUN, DIANXING

    2016-01-01

    Cell-penetrating peptides (CPPs), also known as protein transduction domains, are a class of diverse peptides with 5–30 amino acids. CPPs are divided into cationic, amphipathic and hydrophobic CPPs. They are able to carry small molecules, plasmid DNA, small interfering RNA, proteins, viruses, imaging agents and other various nanoparticles across the cellular membrane, resulting in internalization of the intact cargos. However, the mechanisms of CPP internalization remain to be elucidated. Recently, CPPs have received considerable attention due to their high transduction efficiency and low cytotoxicity. These peptides have a significant potential for diagnostic and therapeutic applications, such as delivery of fluorescent or radioactive compounds for imaging, delivery of peptides and proteins for therapeutic application, and delivery of molecules into induced pluripotent stem cells for directing differentiation. The present study reviews the classifications and transduction mechanisms of CPPs, as well as their potential applications. PMID:27123243

  5. Cell Penetrating Elastin-like Polypeptides for Therapeutic Peptide Delivery

    PubMed Central

    Bidwell, Gene L.; Raucher, Drazen

    2010-01-01

    Current treatment of solid tumors is limited by side effects that result from the nonspecific delivery of drugs to the tumor site. Alternative targeted therapeutic approaches for localized tumors would significantly reduce systemic toxicity. Peptide therapeutics are a promising new strategy for targeted cancer therapy because of the ease of peptide design and the specificity of peptides for their intracellular molecular targets. However, the utility of peptides is limited by their poor pharmacokinetic parameters and poor tissue and cellular membrane permeability in vivo. This review article summarizes the development of elastin-like polypeptide (ELP) as a potential carrier for thermally targeted delivery of therapeutic peptides (TP), and the use of cell penetrating peptides (CPP) to enhance the intracellular delivery of the ELP-fused TPs. CPP-fused ELPs have been used to deliver a peptide inhibitor of c-Myc function and a peptide mimetic of p21 in several cancer models in vitro, and both polypeptides are currently yielding promising results in in vivo models of breast and brain cancer. PMID:20478348

  6. Translocation and Endocytosis for Cell-penetrating Peptide Internalization

    PubMed Central

    Jiao, Chen-Yu; Delaroche, Diane; Burlina, Fabienne; Alves, Isabel D.; Chassaing, Gérard; Sagan, Sandrine

    2009-01-01

    Cell-penetrating peptides (CPPs) share the property of cellular internalization. The question of how these peptides reach the cytoplasm of cells is still widely debated. Herein, we have used a mass spectrometry-based method that enables quantification of internalized and membrane-bound peptides. Internalization of the most used CPP was studied at 37 °C (endocytosis and translocation) and 4 °C (translocation) in wild type and proteoglycan-deficient Chinese hamster ovary cells. Both translocation and endocytosis are internalization pathways used by CPP. The choice of one pathway versus the other depends on the peptide sequence (not the number of positive changes), the extracellular peptide concentration, and the membrane components. There is no relationship between the high affinity of these peptides for the cell membrane and their internalization efficacy. Translocation occurs at low extracellular peptide concentration, whereas endocytosis, a saturable and cooperative phenomenon, is activated at higher concentrations. Translocation operates in a narrow time window, which implies a specific lipid/peptide co-import in cells. PMID:19833724

  7. Intracellular Delivery of Nanoparticles with Cell Penetrating Peptides.

    PubMed

    Salzano, Giuseppina; Torchilin, Vladimir P

    2015-01-01

    The functionalization of nanoparticles (NPs) with cell penetrating peptides (CPPs) constitutes a breakthrough for the intracellular delivery of therapeutic and diagnostic payloads. In late 1998, a significant cellular uptake of a small protein from the HIV-1 virus, namely TAT peptide (TATp), was observed. Thereafter, research began on design of similarly acting peptides, and the coupling of NPs with these novel CPPs. Here, we describe recent methods used to modify the surface of NPs with CPPs and the in vitro and in vivo effects of such functionalization on the intracellular delivery of various cargos. In particular, we highlight recent advances aimed at reducing the non-selectivity of CPPs and the prevention of their enzymatic cleavage en route to target tissues. PMID:26202282

  8. Identification and characterization of a new family of cell-penetrating peptides: cyclic cell-penetrating peptides.

    PubMed

    Cascales, Laura; Henriques, Sónia T; Kerr, Markus C; Huang, Yen-Hua; Sweet, Matthew J; Daly, Norelle L; Craik, David J

    2011-10-21

    Cell-penetrating peptides can translocate across the plasma membrane of living cells and thus are potentially useful agents in drug delivery applications. Disulfide-rich cyclic peptides also have promise in drug design because of their exceptional stability, but to date only one cyclic peptide has been reported to penetrate cells, the Momordica cochinchinensis trypsin inhibitor II (MCoTI-II). MCoTI-II belongs to the cyclotide family of plant-derived cyclic peptides that are characterized by a cyclic cystine knot motif. Previous studies in fixed cells showed that MCoTI-II could penetrate cells but kalata B1, a prototypic cyclotide from a separate subfamily of cyclotides, was bound to the plasma membrane and did not translocate into cells. Here, we show by live cell imaging that both MCoTI-II and kalata B1 can enter cells. Kalata B1 has the same cyclic cystine knot structural motif as MCoTI-II but differs significantly in sequence, and the mechanism by which these two peptides enter cells also differs. MCoTI-II appears to enter via macropinocytosis, presumably mediated by interaction of positively charged residues with phosphoinositides in the cell membrane, whereas kalata B1 interacts directly with the membrane by targeting phosphatidylethanolamine phospholipids, probably leading to membrane bending and vesicle formation. We also show that another plant-derived cyclic peptide, SFTI-1, can penetrate cells. SFTI-1 includes just 14 amino acids and, with the exception of its cyclic backbone, is structurally very different from the cyclotides, which are twice the size. Intriguingly, SFTI-1 does not interact with any of the phospholipids tested, and its mechanism of penetration appears to be distinct from MCoTI-II and kalata B1. The ability of diverse disulfide-rich cyclic peptides to penetrate cells enhances their potential in drug design, and we propose a new classification for them, i.e. cyclic cell-penetrating peptides. PMID:21873420

  9. Identification and characterization of a new family of cell-penetrating peptides: cyclic cell-penetrating peptides.

    PubMed

    Cascales, Laura; Henriques, Sónia T; Kerr, Markus C; Huang, Yen-Hua; Sweet, Matthew J; Daly, Norelle L; Craik, David J

    2011-10-21

    Cell-penetrating peptides can translocate across the plasma membrane of living cells and thus are potentially useful agents in drug delivery applications. Disulfide-rich cyclic peptides also have promise in drug design because of their exceptional stability, but to date only one cyclic peptide has been reported to penetrate cells, the Momordica cochinchinensis trypsin inhibitor II (MCoTI-II). MCoTI-II belongs to the cyclotide family of plant-derived cyclic peptides that are characterized by a cyclic cystine knot motif. Previous studies in fixed cells showed that MCoTI-II could penetrate cells but kalata B1, a prototypic cyclotide from a separate subfamily of cyclotides, was bound to the plasma membrane and did not translocate into cells. Here, we show by live cell imaging that both MCoTI-II and kalata B1 can enter cells. Kalata B1 has the same cyclic cystine knot structural motif as MCoTI-II but differs significantly in sequence, and the mechanism by which these two peptides enter cells also differs. MCoTI-II appears to enter via macropinocytosis, presumably mediated by interaction of positively charged residues with phosphoinositides in the cell membrane, whereas kalata B1 interacts directly with the membrane by targeting phosphatidylethanolamine phospholipids, probably leading to membrane bending and vesicle formation. We also show that another plant-derived cyclic peptide, SFTI-1, can penetrate cells. SFTI-1 includes just 14 amino acids and, with the exception of its cyclic backbone, is structurally very different from the cyclotides, which are twice the size. Intriguingly, SFTI-1 does not interact with any of the phospholipids tested, and its mechanism of penetration appears to be distinct from MCoTI-II and kalata B1. The ability of diverse disulfide-rich cyclic peptides to penetrate cells enhances their potential in drug design, and we propose a new classification for them, i.e. cyclic cell-penetrating peptides.

  10. Reinventing cell penetrating peptides using glycosylated methionine sulfonium ion sequences

    SciTech Connect

    Kramer, Jessica R.; Schmidt, Nathan W.; Mayle, Kristine M.; Kamei, Daniel T.; Wong, Gerard C.L.; Deming, Timothy J.

    2015-04-15

    Cell penetrating peptides (CPPs) are intriguing molecules that have received much attention, both in terms of mechanistic analysis and as transporters for intracellular therapeutic delivery. Most CPPs contain an abundance of cationic charged residues, typically arginine, where the amino acid compositions, rather than specific sequences, tend to determine their ability to enter cells. Hydrophobic residues are often added to cationic sequences to create efficient CPPs, but typically at the penalty of increased cytotoxicity. Here, we examined polypeptides containing glycosylated, cationic derivatives of methionine, where we found these hydrophilic polypeptides to be surprisingly effective as CPPs and to also possess low cytotoxicity. X-ray analysis of how these new polypeptides interact with lipid membranes revealed that the incorporation of sterically demanding hydrophilic cationic groups in polypeptides is an unprecedented new concept for design of potent CPPs.

  11. Reinventing cell penetrating peptides using glycosylated methionine sulfonium ion sequences

    DOE PAGES

    Kramer, Jessica R.; Schmidt, Nathan W.; Mayle, Kristine M.; Kamei, Daniel T.; Wong, Gerard C.L.; Deming, Timothy J.

    2015-04-15

    Cell penetrating peptides (CPPs) are intriguing molecules that have received much attention, both in terms of mechanistic analysis and as transporters for intracellular therapeutic delivery. Most CPPs contain an abundance of cationic charged residues, typically arginine, where the amino acid compositions, rather than specific sequences, tend to determine their ability to enter cells. Hydrophobic residues are often added to cationic sequences to create efficient CPPs, but typically at the penalty of increased cytotoxicity. Here, we examined polypeptides containing glycosylated, cationic derivatives of methionine, where we found these hydrophilic polypeptides to be surprisingly effective as CPPs and to also possess lowmore » cytotoxicity. X-ray analysis of how these new polypeptides interact with lipid membranes revealed that the incorporation of sterically demanding hydrophilic cationic groups in polypeptides is an unprecedented new concept for design of potent CPPs.« less

  12. Anionic activators for differential sensing with cell-penetrating peptides.

    PubMed

    Montenegro, Javier; Matile, Stefan

    2011-02-01

    The design, synthesis, and evaluation of small peptides with one to three negative charges and one to three hydrazides as key components of membrane-based synthetic sensing systems are reported. Their spontaneous reaction with hydrophobic aldehydes or ketones gives rapid access to small collections of amphiphilic anions. These anionic amphiphiles can activate polycations as anion transporters in lipid-bilayer membranes. Odorants are used as representative hydrophobic aldehydes and ketones, and cell-penetrating peptides (CPPs) as polycationic transporters in fluorogenic vesicles. Different activities obtained with different counterion activators are used to generate multidimensional patterns that can be recognized by principal component and hierarchical cluster analysis to extract unique "fingerprints" for individual analytes (including enantiomers, cis-trans isomers or perfumes as illustrative analyte mixtures). Comparison of the peptide activators reveals that carboxylates perform better than phosphonates. Gemini-like activators containing two carboxylates and two hydrophobic hydrazone tails are best, whereas excessive charges and tails give weaker activities. This result differs from cationic activators of polyanionic transporters such as DNA, which worked best with octopus amphiphiles with one cationic head and four hydrophobic tentacles.

  13. Inhibition of regulated cell death by cell-penetrating peptides.

    PubMed

    Krautwald, Stefan; Dewitz, Christin; Fändrich, Fred; Kunzendorf, Ulrich

    2016-06-01

    Development of the means to efficiently and continuously renew missing and non-functional proteins in diseased cells remains a major goal in modern molecular medicine. While gene therapy has the potential to achieve this, substantial obstacles must be overcome before clinical application can be considered. A promising alternative approach is the direct delivery of non-permeant active biomolecules, such as oligonucleotides, peptides and proteins, to the affected cells with the purpose of ameliorating an advanced disease process. In addition to receptor-mediated endocytosis, cell-penetrating peptides are widely used as vectors for rapid translocation of conjugated molecules across cell membranes into intracellular compartments and the delivery of these therapeutic molecules is generally referred to as novel prospective protein therapy. As a broad coverage of the enormous amount of published data in this field is unrewarding, this review will provide a brief, focused overview of the technology and a summary of recent studies of the most commonly used protein transduction domains and their potential as therapeutic agents for the treatment of cellular damage and the prevention of regulated cell death. PMID:27048815

  14. Effects of the properties of short peptides conjugated with cell-penetrating peptides on their internalization into cells.

    PubMed

    Matsumoto, Ryo; Okochi, Mina; Shimizu, Kazunori; Kanie, Kei; Kato, Ryuji; Honda, Hiroyuki

    2015-01-01

    Peptides, especially intracellular functional peptides that can play a particular role inside a cell, have attracted attention as promising materials to control cell fate. However, hydrophilic materials like peptides are difficult for cells to internalize. Therefore, the screening and design of intracellular functional peptides are more difficult than that of extracellular ones. An effective high-throughput screening system for intracellular functional peptides has not been reported. Here, we demonstrate a novel peptide array system for screening intracellular functional peptides, in which both cell-penetrating peptide (CPP) domain and photo-cleavable linkers are used. By using this screening system, we determined how the cellular uptake properties of CPP-conjugated peptides varied depending on the properties of the conjugated peptides. We found that the internalization ability of CPP-conjugated peptides varied greatly depending on the property of the conjugated peptides, and anionic peptides drastically decreased the uptake ability. We summarized our data in a scatter diagram that plots hydrophobicity versus isoelectric point (pI) of conjugated peptides. These results define a peptide library suitable for screening of intracellular functional peptides. Thus, our system, including the diagram, is a promising tool for searching biological active molecules such as peptide-based drugs. PMID:26256261

  15. Poly(NIPAm-AMPS) nanoparticles for targeted delivery of anti-inflammatory cell penetrating peptides

    NASA Astrophysics Data System (ADS)

    Bartlett, Rush Lloyd, II

    Inflammatory diseases such as osteoarthritis and rheumatoid arthritis cause $127.8 billion in US healthcare expenditures each year and are the cause of disability for 27% of disabled persons in the United States. Current treatment options rarely halt disease progression and often result in significant unwanted and debilitating side effects. Our laboratory has previously developed a family of cell penetrating peptides (CPPs) which inhibit the activity of mitogen activated protein kinase activate protein kinase 2 (MK2). MK2 mediates the inflammatory response by activating Tristetraprline (TTP). Once activated, TTP rapidly stabilizes AU rich regions of pro-inflammatory cytokine mRNA which allows translation of pro-inflammatory cytokines to occur. Blocking MK2 with our labs CPPs yields a decrease in inflammatory activity but CPPs by are highly non specific and prone to rapid enzymatic degradation in vivo.. In order to increase the potency of MK2 inhibiting CPPs we have developed a novel nanoparticle drug carrier composed of poly(N-isopropylacrylamide-co-2-acrylamido-2-methyl-1-propanesulfonic acid). This drug carrier has been shown to have preliminary efficacy in vitro and ex vivo for suppressing pro-inflammatory cytokine production when releasing CPPs. This thesis will present progress made on three aims: Specific Aim 1) Create and validate a NIPAm based drug delivery system that mimics the binding and release previously observed between cell penetrating peptides and glycosaminoglycans. Specific Aim 2) Engineer degradability into poly(NIPAm-AMPS) nanoparticles to enable more drug to be released and qualify that system in vitro. Specific Aim 3) Validate poly(NIPAm-AMPS) nanoparticles for targeted drug delivery in an ex vivo inflammatory model. Overall we have developed a novel anionic nanoparticle system that is biocompatible and efficient at loading and releasing cell penetrating peptides to inflamed tissue. Once loaded with a CPP the nanoparticle drug complex is

  16. Mitochondrial transit peptide exhibits cell penetration ability and efficiently delivers macromolecules to mitochondria.

    PubMed

    Jain, Aastha; Chugh, Archana

    2016-09-01

    Mitochondrial malfunction under various circumstances can lead to a variety of disorders. Effective targeting of macromolecules (drugs) is important for restoration of mitochondrial function and treatment of related disorders. We have designed a novel cell-penetrating mitochondrial transit peptide (CpMTP) for delivery of macromolecules to mitochondria. Comparison between properties of cell-penetrating peptides (CPPs) and mitochondrial signal sequences enabled prediction of peptides with dual ability for cellular translocation and mitochondrial localization. Among the predicted peptides, CpMTP translocates across HeLa cells and shows successful delivery of noncovalently conjugated cargo molecules to mitochondria. CpMTP may have applications in transduction and transfection of mitochondria for therapeutics.

  17. Cell-penetrating recombinant peptides for potential use in agricultural pest control applications

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Several important areas of interest intersect in a class of peptides characterized by their highly cationic and partly hydrophobic structure. These molecules have been called cell-penetrating peptides (CPPs) because they possess the ability to translocate across cell membranes. This ability makes ...

  18. A Cell-Penetrating Peptide with a Guanidinylethyl Amine Structure Directed to Gene Delivery

    NASA Astrophysics Data System (ADS)

    Oba, Makoto; Kato, Takuma; Furukawa, Kaori; Tanaka, Masakazu

    2016-01-01

    A peptide composed of lysine with a guanidinylethyl (GEt) amine structure in the side chain [Lys(GEt)] was developed as a cell-penetrating peptide directed to plasmid DNA (pDNA) delivery. The GEt amine adopted a diprotonated form at neutral pH, which may have led to the more efficient cellular uptake of a Lys(GEt)-peptide than an arginine-peptide at a low concentration. Lys(GEt)-peptide/pDNA complexes showed the highest transfection efficiency due to efficient endosomal escape without any cytotoxicity. Lys(GEt)-peptide may be a promising candidate as a gene delivery carrier.

  19. Cell-penetrating peptides and their analogues as novel nanocarriers for drug delivery

    PubMed Central

    Jafari, Samira; Maleki Dizaj, Solmaz; Adibkia, Khosro

    2015-01-01

    Introduction: The impermeability of biological membranes is a major obstacle in drug delivery; however, some peptides have transition capabilities of biomembranes. In recent decades, cell-penetrating peptides (CPPs) have been introduced as novel biocarriers that are able to translocate into the cells. CPPs are biologically potent tools for non-invasive cellular internalization of cargo molecules. Nevertheless, the non-specificity of these peptides presents a restriction for targeting drug delivery; therefore, a peptidic nanocarrier sensitive to matrix metalloproteinase (MMP) has been prepared, called activatable cell-penetrating peptide (ACPP). In addition to the cell-penetrating peptide dendrimer (DCPP), other analogues of CPPs have been synthesized. Methods: In this study, the most recent literature in the field of biomedical application of CPPs and their analogues, ACPP and DCCP, were reviewed. Results: This review focuses on CPP and its analogues, ACPP and DCPP, as novel nanocarriers for drug delivery. In addition, nanoconjugates and bioconjugates of these peptide sequences are discussed. Conclusion: DCCP, branched CPPs, compared to linear peptides have advantages such as resistance to rapid biodegradation, high loading capacities and large-scale production capability. PMID:26191505

  20. In vivo characterization of activatable cell penetrating peptides for targeting protease activity in cancer

    PubMed Central

    Olson, Emilia S.; Aguilera, Todd A.; Jiang, Tao; Ellies, Lesley G.; Nguyen, Quyen T.; Wong, Edmund; Gross, Larry; Tsien, Roger Y.

    2009-01-01

    Activatable cell penetrating peptides (ACPPs) are novel in vivo targeting agents comprised of a polycationic cell penetrating peptide (CPP) connected via a cleavable linker to a neutralizing polyanion (Fig. 1A). Adsorption and uptake into cells are inhibited until the linker is proteolyzed1–3. An ACPP cleavable by matrix metalloproteinase-2 (MMP-2) in vitro was the first one demonstrated to work in a tumor model in vivo, but only HT-1080 xenografts and resected human squamous cell carcinomas were tested. Generality to other cancer types, in vivo selectivity of ACPPs for MMPs, and spatial resolution require further characterization. We now show that ACPPs can target many xenograft tumor models from different cancer sites, as well as a well thoroughly studied transgenic model of spontaneous breast cancer (mouse mammary tumor virus promoter driving polyoma middle T antigen, MMTV-PyMT). Pharmacological inhibitors and genetic knockouts indicate that current ACPPs are selective for MMP-2 and MMP-9 in the above in vivo models. In accord with the known local distribution of MMP activity, accumulation is strongest at the tumor-stromal interface in primary tumors and associated metastases, indicating better spatial resolution (<50 µm) than other currently available MMP-cleavable probes4. We also find that background uptake of ACPPs into normal tissues such as liver and kidney can be decreased by appending inert macromolecules of 30–50 KDa to the polyanionic inhibitory domain. Our results validate an approach that should generally deliver imaging agents and chemotherapeutics to sites of invasion, tumor-promoting inflammation, and metastasis. PMID:20023745

  1. Membrane-active peptides from marine organisms--antimicrobials, cell-penetrating peptides and peptide toxins: applications and prospects.

    PubMed

    Ponnappan, Nisha; Budagavi, Deepthi Poornima; Yadav, Bhoopesh Kumar; Chugh, Archana

    2015-03-01

    Marine organisms are known to be a rich and unique source of bioactive compounds as they are exposed to extreme conditions in the oceans. The present study is an attempt to briefly describe some of the important membrane-active peptides (MAPs) such as antimicrobial peptides (AMPs), cell-penetrating peptides (CPPs) and peptide toxins from marine organisms. Since both AMPs and CPPs play a role in membrane perturbation and exhibit interchangeable role, they can speculatively fall under the broad umbrella of MAPs. The study focuses on the structural and functional characteristics of different classes of marine MAPs. Further, AMPs are considered as a potential remedy to antibiotic resistance acquired by several pathogens. Peptides from marine organisms show novel post-translational modifications such as cysteine knots, halogenation and histidino-alanine bridge that enable these peptides to withstand harsh marine environmental conditions. These unusual modifications of AMPs from marine organisms are expected to increase their half-life in living systems, contributing to their increased bioavailability and stability when administered as drug in in vivo systems. Apart from AMPs, marine toxins with membrane-perturbing properties could be essentially investigated for their cytotoxic effect on various pathogens and their cell-penetrating activity across various mammalian cells. The current review will help in identifying the MAPs from marine organisms with crucial post-translational modifications that can be used as template for designing novel therapeutic agents and drug-delivery vehicles for treatment of human diseases.

  2. Conjugation of cell-penetrating peptides to parathyroid hormone affects its structure, potency, and transepithelial permeation.

    PubMed

    Kristensen, Mie; de Groot, Anne Marit; Berthelsen, Jens; Franzyk, Henrik; Sijts, Alice; Nielsen, Hanne Mørck

    2015-03-18

    Delivery of therapeutic peptides and proteins by the use of cell-penetrating peptides (CPPs) as carriers has been suggested as a feasible strategy. The aim of the present study was to investigate the effect of conjugating a series of well-known CPPs to the biologically active part of parathyroid hormone, i.e., PTH(1-34), and to evaluate the effect with regard to secondary structure, potency in Saos-2 cells, immunogenicity, safety, as well as the transepithelial permeation across monolayers by using the Caco-2 cell culture model. Further, co-administration of CPP and PTH(1-34) as an alternative to covalent conjugation was compared with regard to the transepithelial permeation. CPP-conjugated PTH(1-34) fusion peptides were successfully expressed in Escherichia coli and purified from inclusion bodies. No clear correlation between the degree of secondary structure of the CPP-conjugated PTH(1-34) fusion peptides and their potency was found, albeit a general decrease in permeation was observed for both N- and C-terminally CPP-conjugated PTH(1-34) as compared to native PTH(1-34). However, attachment of CPP to the N-terminus significantly increased permeation across Caco-2 cell monolayers as compared to the corresponding C-terminally CPP-conjugated PTH(1-34). In addition, the nonaarginine sequence proved to be the only CPP capable of increasing permeation when conjugated to PTH(1-34) as compared to co-administration of CPP and PTH(1-34). This enhancement effect was, however, associated with an unacceptably low level of cell viability. In conclusion, covalent conjugation of CPPs to PTH(1-34) influenced the secondary structure, potency, and transepithelial permeation efficiency of the resulting conjugate, and hence this approach appears not to be favorable as compared to co-administration when optimizing CPP-mediated permeation of PTH(1-34) across an intestinal epithelium.

  3. Nucleolar localization signals of LIM kinase 2 function as a cell-penetrating peptide.

    PubMed

    Kobayashi, Nahoko; Niwa, Mikio; Hao, Yang; Yoshida, Tetsuhiko

    2010-12-01

    LIM Kinase 2 (LIMK2) is a LIM domain-containing protein kinase which regulates actin polymerization thorough phosphorylation of the actin depolymerizing factor cofilin. It is also known to function as a shuttle between the cytoplasm and nucleus in endothelial cells. A basic amino acid-rich motif in LIMK2 was previously identified to be responsible for this shuttling function, as a nucleolar localization signal (NoLS). Here it is shown that this nucleolar localization signal sequence also has the characteristic function of a cell-penetrating peptide (CPP). We synthesized LIMK2 NoLS-conjugated peptides and a protein and analyzed their cell-penetrating abilities in various types of cells. The BC-box motif of the Von Hippel-Lindau (VHL) protein was used for the peptide. This motif previously has been reported to be involved in the neural differentiation of bone marrow stromal cells and skin-derived precursor cells. Green fluorescence protein (GFP) was used as a large biologically active biomolecule for the protein. The LIMK2 NoLS-conjugated peptides and protein translocated across the cell membranes of fibroblast cells, neural stem cells, and even iPS cells. These results suggest that LIMK2 NoLS acts as a cell-penetrating peptide and its cell-penetrating ability is not restricted by cell type. Moreover, from an in vivo assay using a mouse brain, it was confirmed that NoLS has potential for transporting biomolecules across the blood-brain barrier. PMID:20937035

  4. Cell-Penetrating Ability of Peptide Hormones: Key Role of Glycosaminoglycans Clustering.

    PubMed

    Neree, Armelle Tchoumi; Nguyen, Phuong Trang; Bourgault, Steve

    2015-11-16

    Over the last two decades, the potential usage of cell-penetrating peptides (CPPs) for the intracellular delivery of various molecules has prompted the identification of novel peptidic identities. However, cytotoxic effects and unpredicted immunological responses have often limited the use of various CPP sequences in the clinic. To overcome these issues, the usage of endogenous peptides appears as an appropriate alternative approach. The hormone pituitary adenylate-cyclase-activating polypeptide (PACAP38) has been recently identified as a novel and very efficient CPP. This 38-residue polycationic peptide is a member of the secretin/glucagon/growth hormone-releasing hormone (GHRH) superfamily, with which PACAP38 shares high structural and conformational homologies. In this study, we evaluated the cell-penetrating ability of cationic peptide hormones in the context of the expression of cell surface glycosaminoglycans (GAGs). Our results indicated that among all peptides evaluated, PACAP38 was unique for its potent efficiency of cellular uptake. Interestingly, the abilities of the peptides to reach the intracellular space did not correlate with their binding affinities to sulfated GAGs, but rather to their capacity to clustered heparin in vitro. This study demonstrates that the uptake efficiency of a given cationic CPP does not necessarily correlate with its affinity to sulfated GAGs and that its ability to cluster GAGs should be considered for the identification of novel peptidic sequences with potent cellular penetrating properties.

  5. Effect of poly-glutamate on uptake efficiency and cytotoxicity of cell penetrating peptides.

    PubMed

    Farkhani, Samad Mussa; Shirani, Ali; Mohammadi, Samaneh; Zakeri-Milani, Parvin; Shahbazi Mojarrad, Javid; Valizadeh, Hadi

    2016-04-01

    Cell penetrating peptides (CPPs) were developed as vehicles for efficient delivery of various molecules. An ideal CPP-peptide should not display any toxicity against cancer cells as well as healthy cells and efficiently enter into the cell. Because of the cationic nature and the intrinsic vector capabilities, these peptides can cause cytotoxicity. One of the possible reasons for toxicity of CPPs is direct translocation and consequently, pore formation on the plasma membrane. In this study it was demonstrated that interaction of poly-glutamate with CPP considerably reduced their cytotoxicity in A549 cell. This strategy could be useful for efficient drug delivery mediated by CPP. PMID:27074859

  6. Conjugation with Cationic Cell-Penetrating Peptide Increases Pulmonary Absorption of Insulin

    PubMed Central

    Patel, Leena N.; Wang, Jeffrey; Kim, Kwang-Jin; Borok, Zea; Crandall, Edward; Shen, Wei-Chiang

    2009-01-01

    In this study, we determined if cell-penetrating peptides (CPPs) can be used to enhance the absorption rate of insulin (INS) across the alveolar epithelial barrier. Using a heterobifuctional crosslinker, INS was conjugated to a series of cationic CPPs, including Tat peptide, oligoarginine (r9) or oligolysine (k9), via disulfide bridge to a D-isoform cysteine (c) present at the N-terminal of the peptide sequence, yielding INS-cTat, INS-cr9, and INS-ck9, respectively. SDS-PAGE and MALDI-TOF mass spectroscopy confirmed homogenous conjugates with a 1:1 ratio of INS and various CPPs. Transport of INS and INS-CPPs across primary cultured rat alveolar epithelial cell monolayers was in the order INS-cr9 > INS-cTat > INS-ck9 > INS, with 27-, 19- and 4-fold increase compared to native INS, respectively. Transport of INS-cr9 was temperature- and time-dependent. Covalent conjugation between r9 and INS, as opposed to adding unconjugated INS and r9 together into donor fluid, was necessary to enhance transport of INS. Absorption of INS-cr9 across the alveolar epithelial barrier appeared to be in part transcellular, since INS-cr9 transport in the presence of heparin and protamine was decreased by ~20%. Adsorptive transcytosis appeared to be in part responsible for INS-cr9 absorption, as INS-cr9 did not compete with free INS in binding assays for INS receptors. Finally, intratracheal instillation of INS-cr9 in diabetic rats resulted in a steady decrease in blood glucose level that was more sustained over time when compared with INS. These results suggest that oligoarginine can be used to increase the alveolar absorption rate of insulin (and potentially other macromolecules as well). PMID:19228019

  7. Topical Delivery of Protein and Peptide Using Novel Cell Penetrating Peptide IMT-P8.

    PubMed

    Gautam, Ankur; Nanda, Jagpreet Singh; Samuel, Jesse S; Kumari, Manisha; Priyanka, Priyanka; Bedi, Gursimran; Nath, Samir K; Mittal, Garima; Khatri, Neeraj; Raghava, Gajendra Pal Singh

    2016-01-01

    Skin, being the largest organ of the body, is an important site for drug administration. However, most of the drugs have poor permeability and thus drug delivery through the skin is very challenging. In this study, we examined the transdermal delivery capability of IMT-P8, a novel cell-penetrating peptide. We generated IMT-P8-GFP and IMT-P8-KLA fusion constructs and evaluated their internalization into mouse skin after topical application. Our results demonstrate that IMT-P8 is capable of transporting green fluorescent protein (GFP) and proapoptotic peptide, KLA into the skin and also in different cell lines. Interestingly, uptake of IMT-P8-GFP was considerably higher than TAT-GFP in HeLa cells. After internalization, IMT-P8-KLA got localized to the mitochondria and caused significant cell death in HeLa cells signifying an intact biological activity. Further in vivo skin penetration experiments revealed that after topical application, IMT-P8 penetrated the stratum corneum, entered into the viable epidermis and accumulated inside the hair follicles. In addition, both IMT-P8-KLA and IMT-P8-GFP internalized into the hair follicles and dermal tissue of the skin following topical application. These results suggested that IMT-P8 could be a potential candidate to be used as a topical delivery vehicle for various cosmetic and skin disease applications. PMID:27189051

  8. Topical Delivery of Protein and Peptide Using Novel Cell Penetrating Peptide IMT-P8

    PubMed Central

    Gautam, Ankur; Nanda, Jagpreet Singh; Samuel, Jesse S.; Kumari, Manisha; Priyanka, Priyanka; Bedi, Gursimran; Nath, Samir K.; Mittal, Garima; Khatri, Neeraj; Raghava, Gajendra Pal Singh

    2016-01-01

    Skin, being the largest organ of the body, is an important site for drug administration. However, most of the drugs have poor permeability and thus drug delivery through the skin is very challenging. In this study, we examined the transdermal delivery capability of IMT-P8, a novel cell-penetrating peptide. We generated IMT-P8-GFP and IMT-P8-KLA fusion constructs and evaluated their internalization into mouse skin after topical application. Our results demonstrate that IMT-P8 is capable of transporting green fluorescent protein (GFP) and proapoptotic peptide, KLA into the skin and also in different cell lines. Interestingly, uptake of IMT-P8-GFP was considerably higher than TAT-GFP in HeLa cells. After internalization, IMT-P8-KLA got localized to the mitochondria and caused significant cell death in HeLa cells signifying an intact biological activity. Further in vivo skin penetration experiments revealed that after topical application, IMT-P8 penetrated the stratum corneum, entered into the viable epidermis and accumulated inside the hair follicles. In addition, both IMT-P8-KLA and IMT-P8-GFP internalized into the hair follicles and dermal tissue of the skin following topical application. These results suggested that IMT-P8 could be a potential candidate to be used as a topical delivery vehicle for various cosmetic and skin disease applications. PMID:27189051

  9. Coexistence of a Two-States Organization for a Cell-Penetrating Peptide in Lipid Bilayer

    PubMed Central

    Plénat, Thomas; Boichot, Sylvie; Dosset, Patrice; Milhiet, Pierre-Emmanuel; Le Grimellec, Christian

    2005-01-01

    Primary amphipathic cell-penetrating peptides transport cargoes across cell membranes with high efficiency and low lytic activity. These primary amphipathic peptides were previously shown to form aggregates or supramolecular structures in mixed lipid-peptide monolayers, but their behavior in lipid bilayers remains to be characterized. Using atomic force microscopy, we have examined the interactions of P(α), a primary amphipathic cell-penetrating peptide which remains α-helical whatever the environment, with dipalmitoylphosphatidylcholine (DPPC) bilayers. Addition of P(α) at concentrations up to 5 mol % markedly modified the supported bilayers topography. Long and thin filaments lying flat at the membrane surface coexisted with deeply embedded peptides which induced a local thinning of the bilayer. On the other hand, addition of P(α) only exerted very limited effects on the corresponding liposome's bilayer physical state, as estimated from differential scanning calorimetry and diphenylhexatriene fluorescence anisotropy experiments. The use of a gel-fluid phase separated supported bilayers made of a dioleoylphosphatidylcholine/dipalmitoylphosphatidylcholine mixture confirmed both the existence of long filaments, which at low peptide concentration were preferentially localized in the fluid phase domains and the membrane disorganizing effects of 5 mol % P(α). The simultaneous two-states organization of P(α), at the membrane surface and deeply embedded in the bilayer, may be involved in the transmembrane carrier function of this primary amphipathic peptide. PMID:16199494

  10. Cell-penetrating TAT peptide in drug delivery systems: Proteolytic stability requirements

    PubMed Central

    Koren, Erez; Apte, Anjali; Sawant, Rupa R.; Grunwald, Jacob; Torchilin, Vladimir P.

    2012-01-01

    The stability and activity of the HIV cell-penetrating TAT peptide (TATp) on the surface of TATp-modified micelles and liposomes in relation to its proteolytic cleavage was investigated. TATp moieties were attached to the surface of these nanocarriers using TATp modified with a conjugate of phosphatidyl ethanolamine with a ‘short’ PEG (PEG-PE). Following pre-incubation with trypsin, elastase, or collagenase, the proteolytic stability of TATp on the surface of these modified carriers was studied by HPLC with fluorescence detection using fluorenylmethyl chloroformate (FMOC) labeling. All tested enzymes produced a dose-dependent cleavage of TATp as shown by the presence of TATp Arg-Arg fragments. Inhibition of TATp cleavage occurred when these TATp-micelles were modified by the addition of longer PEG-PE blocks, indicating an effective shielding of TATp from proteolysis by these blocks. TATp-modified carriers were also tested for their ability to accumulate in EL-4, HeLa, and B16-F10 cells. Trypsin treatment of TATp-modified liposomes and micelles resulted in decreased uptake and cell interaction, as measured by fluorescence microscopy and fluorescence-activated cell sorting techniques. Furthermore, a decrease in the cytotoxicity of TATp-modified liposomes loaded with doxorubicin (Doxil) was observed following trypsin treatment. In conclusion, steric shielding of TATp is essential to ensure its in vivo therapeutic function. PMID:21438724

  11. Cell-penetrating TAT peptide in drug delivery systems: proteolytic stability requirements.

    PubMed

    Koren, Erez; Apte, Anjali; Sawant, Rupa R; Grunwald, Jacob; Torchilin, Vladimir P

    2011-07-01

    The stability and activity of the HIV cell-penetrating TAT peptide (TATp) on the surface of TATp-modified micelles and liposomes in relation to its proteolytic cleavage was investigated. TATp moieties were attached to the surface of these nanocarriers using TATp modified with a conjugate of phosphatidyl ethanolamine with a 'short' PEG (PEG-PE). Following pre-incubation with trypsin, elastase, or collagenase, the proteolytic stability of TATp on the surface of these modified carriers was studied by HPLC with fluorescence detection using fluorenylmethyl chloroformate (FMOC) labeling. All tested enzymes produced a dose-dependent cleavage of TATp as shown by the presence of TATp Arg-Arg fragments. Inhibition of TATp cleavage occurred when these TATp-micelles were modified by the addition of longer PEG-PE blocks, indicating an effective shielding of TATp from proteolysis by these blocks. TATp-modified carriers were also tested for their ability to accumulate in EL-4, HeLa, and B16-F10 cells. Trypsin treatment of TATp-modified liposomes and micelles resulted in decreased uptake and cell interaction, as measured by fluorescence microscopy and fluorescence-activated cell sorting techniques. Furthermore, a decrease in the cytotoxicity of TATp-modified liposomes loaded with doxorubicin (Doxil) was observed following trypsin treatment. In conclusion, steric shielding of TATp is essential to ensure its in vivo therapeutic function. PMID:21438724

  12. Biodistribution, Stability, and Blood Distribution of the Cell Penetrating Peptide Maurocalcine in Mice

    PubMed Central

    Perret, Pascale; Ahmadi, Mitra; Riou, Laurent; Bacot, Sandrine; Pecher, Julien; Poillot, Cathy; Broisat, Alexis; Ghezzi, Catherine; De Waard, Michel

    2015-01-01

    Maurocalcine (MCa) is the first natural cell penetrating peptide to be discovered in animal venom. In addition to the fact that it represents a potent vector for the cell penetration of structurally diverse therapeutic compounds, MCa also displays several distinguishing features that make it a potential peptide of choice for clinical and biotechnological applications. The aim of the present study was to gain new information about the properties of MCa in vivo in order to delineate the future potential applications of this vector. For this purpose, two analogues of this peptide with (Tyr-MCa) and without (Lin-Tyr-MCa) disulfide bridges were synthesized, radiolabeled with 125I, and their in vitro stabilities were first evaluated in mouse blood. The results indicated that 125I-Tyr-MCa was stable in vitro and that the disulfide bridges conferred a competitive advantage for the stability of peptide. Following in vivo injection in mice, 125I-Tyr-MCa targeted peripheral organs with interesting quantitative differences and the main route of peptide elimination was renal. PMID:26610471

  13. Neuroprotective peptides fused to arginine-rich cell penetrating peptides: Neuroprotective mechanism likely mediated by peptide endocytic properties.

    PubMed

    Meloni, Bruno P; Milani, Diego; Edwards, Adam B; Anderton, Ryan S; O'Hare Doig, Ryan L; Fitzgerald, Melinda; Palmer, T Norman; Knuckey, Neville W

    2015-09-01

    Several recent studies have demonstrated that TAT and other arginine-rich cell penetrating peptides (CPPs) have intrinsic neuroprotective properties in their own right. Examples, we have demonstrated that in addition to TAT, poly-arginine peptides (R8 to R18; containing 8-18 arginine residues) as well as some other arginine-rich peptides are neuroprotective in vitro (in neurons exposed to glutamic acid excitotoxicity and oxygen glucose deprivation) and in the case of R9 in vivo (after permanent middle cerebral artery occlusion in the rat). Based on several lines of evidence, we propose that this neuroprotection is related to the peptide's endocytosis-inducing properties, with peptide charge and arginine residues being critical factors. Specifically, we propose that during peptide endocytosis neuronal cell surface structures such as ion channels and transporters are internalised, thereby reducing calcium influx associated with excitotoxicity and other receptor-mediated neurodamaging signalling pathways. We also hypothesise that a peptide cargo can act synergistically with TAT and other arginine-rich CPPs due to potentiation of the CPPs endocytic traits rather than by the cargo-peptide acting directly on its supposedly intended intracellular target. In this review, we systematically consider a number of studies that have used CPPs to deliver neuroprotective peptides to the central nervous system (CNS) following stroke and other neurological disorders. Consequently, we critically review evidence that supports our hypothesis that neuroprotection is mediated by carrier peptide endocytosis. In conclusion, we believe that there are strong grounds to regard arginine-rich peptides as a new class of neuroprotective molecules for the treatment of a range of neurological disorders.

  14. Cellular uptake of antisense oligonucleotides after complexing or conjugation with cell-penetrating model peptides.

    PubMed

    Oehlke, J; Birth, P; Klauschenz, E; Wiesner, B; Beyermann, M; Oksche, A; Bienert, M

    2002-08-01

    The uptake by mammalian cells of phosphorothioate oligonucleotides was compared with that of their respective complexes or conjugates with cationic, cell-penetrating model peptides of varying helix-forming propensity and amphipathicity. An HPLC-based protocol for the synthesis and purification of disulfide bridged conjugates in the 10-100 nmol range was developed. Confocal laser scanning microscopy (CLSM) in combination with gel-capillary electrophoresis and laser induced fluorescence detection (GCE-LIF) revealed cytoplasmic and nuclear accumulationin all cases. The uptake differences between naked oligonucleotides and their respective peptide complexes or conjugates were generally confined to one order of magnitude. No significant influence of the structural properties of the peptide components upon cellular uptake was found. Our results question the common belief that the increased biological activity of oligonucleotides after derivatization with membrane permeable peptides may be primarily due to improved membrane translocation.

  15. Electroporation-based delivery of cell-penetrating peptide conjugates of peptide nucleic acids for antisense inhibition of intracellular bacteria.

    PubMed

    Ma, Sai; Schroeder, Betsy; Sun, Chen; Loufakis, Despina Nelie; Cao, Zhenning; Sriranganathan, Nammalwar; Lu, Chang

    2014-10-01

    Cell penetrating peptides (CPPs) have been used for a myriad of cellular delivery applications and were recently explored for delivery of antisense agents such as peptide nucleic acids (PNAs) for bacterial inhibition. Although these molecular systems (i.e. CPP-PNAs) have shown ability to inhibit growth of bacterial cultures in vitro, they show limited effectiveness in killing encapsulated intracellular bacteria in mammalian cells such as macrophages, presumably due to difficulty involved in the endosomal escape of the reagents. In this report, we show that electroporation delivery dramatically increases the bioavailability of CPP-PNAs to kill Salmonella enterica serovar Typhimurium LT2 inside macrophages. Electroporation delivers the molecules without involving endocytosis and greatly increases the antisense effect. The decrease in the average number of Salmonella per macrophage under a 1200 V cm(-1) and 5 ms pulse was a factor of 9 higher than that without electroporation (in an experiment with a multiplicity of infection of 2 : 1). Our results suggest that electroporation is an effective approach for a wide range of applications involving CPP-based delivery. The microfluidic format will allow convenient functional screening and testing of PNA-based reagents for antisense applications.

  16. A Cell-penetrating Peptide Derived from Human Lactoferrin with Conformation-dependent Uptake Efficiency

    PubMed Central

    Duchardt, Falk; Ruttekolk, Ivo R.; Verdurmen, Wouter P. R.; Lortat-Jacob, Hugues; Bürck, Jochen; Hufnagel, Hansjörg; Fischer, Rainer; van den Heuvel, Maaike; Löwik, Dennis W. P. M.; Vuister, Geerten W.; Ulrich, Anne; de Waard, Michel; Brock, Roland

    2009-01-01

    The molecular events that contribute to the cellular uptake of cell-penetrating peptides (CPP) are still a matter of intense research. Here, we report on the identification and characterization of a 22-amino acid CPP derived from the human milk protein, lactoferrin. The peptide exhibits a conformation-dependent uptake efficiency that is correlated with efficient binding to heparan sulfate and lipid-induced conformational changes. The peptide contains a disulfide bridge formed by terminal cysteine residues. At concentrations exceeding 10 μm, this peptide undergoes the same rapid entry into the cytoplasm that was described previously for the arginine-rich CPPs nona-arginine and Tat. Cytoplasmic entry strictly depends on the presence of the disulfide bridge. To better understand this conformation dependence, NMR spectroscopy was performed for the free peptide, and CD measurements were performed for free and lipid-bound peptide. In solution, the peptides showed only slight differences in secondary structure, with a predominantly disordered structure both in the presence and absence of the disulfide bridge. In contrast, in complex with large unilamellar vesicles, the conformation of the oxidized and reduced forms of the peptide clearly differed. Moreover, surface plasmon resonance experiments showed that the oxidized form binds to heparan sulfate with a considerably higher affinity than the reduced form. Consistently, membrane binding and cellular uptake of the peptide were reduced when heparan sulfate chains were removed. PMID:19858187

  17. Nonhemolytic Cell-Penetrating Peptides: Site Specific Introduction of Glutamine and Lysine Residues into the α-Helical Peptide Causes Deletion of Its Direct Membrane Disrupting Ability but Retention of Its Cell Penetrating Ability.

    PubMed

    Kim, Seoyeon; Hyun, Soonsil; Lee, Yuri; Lee, Yan; Yu, Jaehoon

    2016-09-12

    Cell-penetrating peptides (CPPs) often have cationic and amphipathic characteristics that are commonly associated with α-helical peptides. These features give CPPs both membrane demolishing and penetrating abilities. To make CPPs safe for biomedical applications, their toxicities resulting from their membrane demolishing abilities must be removed while their cell penetrating abilities must be retained. In this study, we systematically constructed mutants of the amphipathic α-helical model peptide (LKKLLKLLKKLLKLAG, LK peptide). The hydrophobic amino acid leucine in the LK peptide was replaced with hydrophilic amino acids to reduce hemolytic or cell toxicity. Most of the mutants were found to have weakened membrane disrupting abilities, but their cell penetrating abilities were also weakened. However, the L8Q and L8K mutants were found to have low micromolar range cell penetrating ability and almost no membrane disrupting ability. These selected mutants utilize energy-dependent endocytosis mechanisms instead of an energy-independent direct cell penetrating mechanism to enter cells. In addition, the mutants can be used to deliver the anticancer drug methotrexate (MTX) to cells, thereby overcoming resistance to this drug. To determine if the effect of these mutations on the membrane disrupting and cell penetrating abilities is general, Q and K mutations of the natural amphipathic α-helical antimicrobial peptide (AMP), LL37, were introduced. Specific positional Q and K mutants of LL37 were found to have lower hemolytic toxicities and preserved the ability to penetrate eukaryotic cells such as MDA-MB-231 cells. Taken together, observations made in this work suggest that interrupting the global hydrophobicity of amphipathic α-helical CPPs and AMPs, by replacing hydrophobic residues with mildly hydrophilic amino acids such as Q and K, might be an ideal strategy for constructing peptides that have strong cell penetrating abilities and weak cell membrane disrupting

  18. Nonhemolytic Cell-Penetrating Peptides: Site Specific Introduction of Glutamine and Lysine Residues into the α-Helical Peptide Causes Deletion of Its Direct Membrane Disrupting Ability but Retention of Its Cell Penetrating Ability.

    PubMed

    Kim, Seoyeon; Hyun, Soonsil; Lee, Yuri; Lee, Yan; Yu, Jaehoon

    2016-09-12

    Cell-penetrating peptides (CPPs) often have cationic and amphipathic characteristics that are commonly associated with α-helical peptides. These features give CPPs both membrane demolishing and penetrating abilities. To make CPPs safe for biomedical applications, their toxicities resulting from their membrane demolishing abilities must be removed while their cell penetrating abilities must be retained. In this study, we systematically constructed mutants of the amphipathic α-helical model peptide (LKKLLKLLKKLLKLAG, LK peptide). The hydrophobic amino acid leucine in the LK peptide was replaced with hydrophilic amino acids to reduce hemolytic or cell toxicity. Most of the mutants were found to have weakened membrane disrupting abilities, but their cell penetrating abilities were also weakened. However, the L8Q and L8K mutants were found to have low micromolar range cell penetrating ability and almost no membrane disrupting ability. These selected mutants utilize energy-dependent endocytosis mechanisms instead of an energy-independent direct cell penetrating mechanism to enter cells. In addition, the mutants can be used to deliver the anticancer drug methotrexate (MTX) to cells, thereby overcoming resistance to this drug. To determine if the effect of these mutations on the membrane disrupting and cell penetrating abilities is general, Q and K mutations of the natural amphipathic α-helical antimicrobial peptide (AMP), LL37, were introduced. Specific positional Q and K mutants of LL37 were found to have lower hemolytic toxicities and preserved the ability to penetrate eukaryotic cells such as MDA-MB-231 cells. Taken together, observations made in this work suggest that interrupting the global hydrophobicity of amphipathic α-helical CPPs and AMPs, by replacing hydrophobic residues with mildly hydrophilic amino acids such as Q and K, might be an ideal strategy for constructing peptides that have strong cell penetrating abilities and weak cell membrane disrupting

  19. Effects of cargo molecules on membrane perturbation caused by transportan10 based cell-penetrating peptides.

    PubMed

    Vasconcelos, Luís; Madani, Fatemeh; Arukuusk, Piret; Pärnaste, Ly; Gräslund, Astrid; Langel, Ulo

    2014-12-01

    Cell-penetrating peptides with the ability to escape endosomes and reach the target are of great value as delivery vectors for different bioactive cargoes and future treatment of human diseases. We have studied two such peptides, NickFect1 and NickFect51, both originated from stearylated transportan10 (PF3). To obtain more insight into the mechanism(s) of peptide delivery and the biophysical properties of an efficient vector system, we investigated the effect of different bioactive oligonucleotide cargoes on peptide-membrane perturbation and peptide structural induction. We studied the membrane interactions of the peptides with large unilamellar vesicles and compared their effects with parent peptides transportan10 and PF3. In addition, cellular uptake and peptide-mediated oligonucleotide delivery were analyzed. Calcein leakage experiments showed that similar to transportan10, NickFect51 caused a significant degree of membrane leakage, whereas NickFect1, similar to PF3, was less membrane perturbing. The results are in agreement with previously published results indicating that NickFect51 is a more efficient endosomal escaper. However, the presence of a large cargo like plasmid DNA inhibited NickFect's membrane perturbation and cellular uptake efficiency of the peptide was reduced. We conclude that the pathway for cellular uptake of peptide complexes is cargo dependent, whereas the endosomal escape efficacy depends on peptide hydrophobicity and chemical structure. For small interfering RNA delivery, NickFect51 appears to be optimal. The biophysical signature shows that the peptide alone causes membrane perturbation, but the cargo complex does not. These two biophysical characteristics of the peptide and its cargo complex may be the signature of an efficient delivery vector system.

  20. Nose-to-brain delivery of macromolecules mediated by cell-penetrating peptides.

    PubMed

    Lin, Tingting; Liu, Ergang; He, Huining; Shin, Meong Cheol; Moon, Cheol; Yang, Victor C; Huang, Yongzhuo

    2016-07-01

    Brain delivery of macromolecular therapeutics (e.g., proteins) remains an unsolved problem because of the formidable blood-brain barrier (BBB). Although a direct pathway of nose-to-brain transfer provides an answer to circumventing the BBB and has already been intensively investigated for brain delivery of small drugs, new challenges arise for intranasal delivery of proteins because of their larger size and hydrophilicity. In order to overcome the barriers and take advantage of available pathways (e.g., epithelial tight junctions, uptake by olfactory neurons, transport into brain tissues, and intra-brain diffusion), a low molecular weight protamine (LMWP) cell-penetrating peptide was utilized to facilitate nose-to-brain transport. Cell-penetrating peptides (CPP) have been widely used to mediate macromolecular delivery through many kinds of biobarriers. Our results show that conjugates of LMWP-proteins are able to effectively penetrate into the brain after intranasal administration. The CPP-based intranasal method highlights a promising solution for protein therapy of brain diseases.

  1. Cell-Penetrating Peptide Penetratin as a Potential Tool for Developing Effective Nasal Vaccination Systems.

    PubMed

    Muto, Keiya; Kamei, Noriyasu; Yoshida, Mia; Takayama, Kozo; Takeda-Morishita, Mariko

    2016-06-01

    Nasal vaccination is considered an attractive strategy to prevent the infection and spread of viruses. However, the vaccine formulations available on the market remain imperfect on account of their limited effectiveness. In the present study, we hypothesized that the nasal coadministration of antigens with cell-penetrating peptides promotes antigen delivery immune response in the nasal mucosa, thereby enhancing the production of mucosal IgA and systemic IgG. The levels of ovalbumin (OVA)-specific IgG and IgA in plasma and nasal perfusate, respectively, increased after 2 or 4 weeks on nasal coadministration of OVA with l- or d-penetratin, suggesting that OVA antigen was effectively delivered by penetratin to the nasal epithelium. An additional study demonstrated that the production of systemic IgG and nasal mucosal IgA against influenza A virus was specifically promoted by nasal coadministration of influenza A virus with d-penetratin. The results of this study suggested that cell-penetrating peptides are a promising tool for the delivery of vaccines to the nasal mucosa and for the subsequent dual stimulation of systemic and mucosal immune responses. PMID:27155764

  2. Cell penetrating peptide-mediated transport enables the regulated secretion of accumulated cargoes from mast cells.

    PubMed

    Howl, John; Jones, Sarah

    2015-03-28

    The in vivo utility of technologies employing cell penetrating peptides and bioportides may be compromised by the general capacity of polycationic peptides to activate mast cell secretion. Moreover, the same technologies could be exploited in a clinical setting either to directly modulate intrinsic exocytotic mechanisms or to load mast cells with bioactive cargoes. Comparative investigations identified two cell penetrating vectors, Tat and C105Y, which readily translocate into mast cells without inducing receptor-independent exocytosis. Efficient Tat transduction also enabled the intracellular delivery and accumulation of cargoes within discrete intracellular compartments. A tetramethylrhodamine-Tat conjugate is effectively translocated into the secretory lysosomes of RBL-2H3 cells. In contract, the intracellular delivery of avidin, as a non-covalent complex with a biotinylated Tat vector, is also efficient but the protein is predominantly accumulated outside of secretory lysosomes. Significantly, both cargoes can be subsequently released following mast cell stimulation either by mastoparan, a wasp venom secretagogue, or by the physiological mechanism of antigen-induced aggregation of high affinity IgE receptors. These studies indicate that mast cells could be exploited to direct the delivery of bioactive agents to disease sites as an innovative cell-mediated therapy. PMID:25660072

  3. Cationic cell-penetrating peptides as vehicles for siRNA delivery.

    PubMed

    Beloor, Jagadish; Zeller, Skye; Choi, Chang Seon; Lee, Sang-Kyung; Kumar, Priti

    2015-01-01

    RNA interference mediated gene silencing has tremendous applicability in fields ranging from basic biological research to clinical therapy. However, delivery of siRNA across the cell membrane into the cytoplasm, where the RNA silencing machinery is located, is a significant hurdle in most primary cells. Cell-penetrating peptides (CPPs), peptides that possess an intrinsic ability to translocate across cell membranes, have been explored as a means to achieve cellular delivery of siRNA. Approaches using CPPs by themselves or through incorporation into other siRNA delivery platforms have been investigated with the intent of improving cytoplasmic delivery. Here, we review the utilization of CPPs for siRNA delivery with a focus on strategies developed to enhance cellular uptake, endosomal escape and cytoplasmic localization of CPP/siRNA complexes.

  4. Intracellular Delivery of Proteins with Cell-Penetrating Peptides for Therapeutic Uses in Human Disease.

    PubMed

    Dinca, Ana; Chien, Wei-Ming; Chin, Michael T

    2016-02-22

    Protein therapy exhibits several advantages over small molecule drugs and is increasingly being developed for the treatment of disorders ranging from single enzyme deficiencies to cancer. Cell-penetrating peptides (CPPs), a group of small peptides capable of promoting transport of molecular cargo across the plasma membrane, have become important tools in promoting the cellular uptake of exogenously delivered proteins. Although the molecular mechanisms of uptake are not firmly established, CPPs have been empirically shown to promote uptake of various molecules, including large proteins over 100 kiloDaltons (kDa). Recombinant proteins that include a CPP tag to promote intracellular delivery show promise as therapeutic agents with encouraging success rates in both animal and human trials. This review highlights recent advances in protein-CPP therapy and discusses optimization strategies and potential detrimental effects.

  5. Intracellular Delivery of Proteins with Cell-Penetrating Peptides for Therapeutic Uses in Human Disease

    PubMed Central

    Dinca, Ana; Chien, Wei-Ming; Chin, Michael T.

    2016-01-01

    Protein therapy exhibits several advantages over small molecule drugs and is increasingly being developed for the treatment of disorders ranging from single enzyme deficiencies to cancer. Cell-penetrating peptides (CPPs), a group of small peptides capable of promoting transport of molecular cargo across the plasma membrane, have become important tools in promoting the cellular uptake of exogenously delivered proteins. Although the molecular mechanisms of uptake are not firmly established, CPPs have been empirically shown to promote uptake of various molecules, including large proteins over 100 kiloDaltons (kDa). Recombinant proteins that include a CPP tag to promote intracellular delivery show promise as therapeutic agents with encouraging success rates in both animal and human trials. This review highlights recent advances in protein-CPP therapy and discusses optimization strategies and potential detrimental effects. PMID:26907261

  6. Cell-Penetrating Peptides Selectively Cross the Blood-Brain Barrier In Vivo

    PubMed Central

    Stalmans, Sofie; Bracke, Nathalie; Wynendaele, Evelien; Gevaert, Bert; Peremans, Kathelijne; Burvenich, Christian; Polis, Ingeborgh; De Spiegeleer, Bart

    2015-01-01

    Cell-penetrating peptides (CPPs) are a group of peptides, which have the ability to cross cell membrane bilayers. CPPs themselves can exert biological activity and can be formed endogenously. Fragmentary studies demonstrate their ability to enhance transport of different cargoes across the blood-brain barrier (BBB). However, comparative, quantitative data on the BBB permeability of different CPPs are currently lacking. Therefore, the in vivo BBB transport characteristics of five chemically diverse CPPs, i.e. pVEC, SynB3, Tat 47–57, transportan 10 (TP10) and TP10-2, were determined. The results of the multiple time regression (MTR) analysis revealed that CPPs show divergent BBB influx properties: Tat 47–57, SynB3, and especially pVEC showed very high unidirectional influx rates of 4.73 μl/(g × min), 5.63 μl/(g × min) and 6.02 μl/(g × min), respectively, while the transportan analogs showed a negligible to low brain influx. Using capillary depletion, it was found that 80% of the influxed peptides effectively reached the brain parenchyma. Except for pVEC, all peptides showed a significant efflux out of the brain. Co-injection of pVEC with radioiodinated bovine serum albumin (BSA) did not enhance the brain influx of radiodionated BSA, indicating that pVEC does not itself significantly alter the BBB properties. A saturable mechanism could not be demonstrated by co-injecting an excess dose of non-radiolabeled CPP. No significant regional differences in brain influx were observed, with the exception for pVEC, for which the regional variations were only marginal. The observed BBB influx transport properties cannot be correlated with their cell-penetrating ability, and therefore, good CPP properties do not imply efficient brain influx. PMID:26465925

  7. Cell penetrating peptide-based polyplexes shelled with polysaccharide to improve stability and gene transfection

    NASA Astrophysics Data System (ADS)

    Li, Wenyu; Liu, Yajie; Du, Jianwei; Ren, Kefeng; Wang, Youxiang

    2015-04-01

    Cell-penetrating peptides (CPP) have been widely developed as a strategy to enhance cell penetrating ability and transfection. In this work, octa-arginine modified dextran gene vector with pH-sensitivity was developed via host-guest interactions. α-Cyclodextrin was modified with octa-arginine (CDR), which had excellent cell penetrating ability. Dextran was selected as a backbone and modified with azobenzene as guest units by acid-labile imine bonds (Az-I-Dex). The supramolecular polymer CDR/Az-I-Dex with high a C/A molar ratio (molar ratio of CD on CDR to Az on Az-I-Dex) was unfavorable for DNA condensation. The dextran shell of CDR/Az-I-Dex/DNA polyplexes improved the stability under physiological conditions. However, once treated with acetate buffer (pH 5.4) for 3 h, large aggregates formed rapidly due to the cleavage of the dextran shell. As expected, the vector had cell viability of 80% even when the CDR concentration increased to 100 μg mL-1. Moreover, due to the effective cellular uptake efficiency, CDR/Az-I-Dex/DNA polyplexes had 6-300 times higher transfection efficiency than CDR/DNA polyplexes. It was even higher than high molecular weight PLL-based polyplexes of HEK293 T cells. Importantly, chloroquine as an endosomal escape agent could not improve the transfection of CDR/Az-I-Dex/DNA polyplexes, which indicated that the CDR/Az-I-Dex supramolecular polymer had its own ability for endosomal escape. These results suggested that the CPP-based polyplexes shelled with polysaccharide can be promising non-viral gene delivery carriers.Cell-penetrating peptides (CPP) have been widely developed as a strategy to enhance cell penetrating ability and transfection. In this work, octa-arginine modified dextran gene vector with pH-sensitivity was developed via host-guest interactions. α-Cyclodextrin was modified with octa-arginine (CDR), which had excellent cell penetrating ability. Dextran was selected as a backbone and modified with azobenzene as guest units by acid

  8. Cell penetrating peptide-based polyplexes shelled with polysaccharide to improve stability and gene transfection.

    PubMed

    Li, Wenyu; Liu, Yajie; Du, Jianwei; Ren, Kefeng; Wang, Youxiang

    2015-05-14

    Cell-penetrating peptides (CPP) have been widely developed as a strategy to enhance cell penetrating ability and transfection. In this work, octa-arginine modified dextran gene vector with pH-sensitivity was developed via host-guest interactions. α-Cyclodextrin was modified with octa-arginine (CDR), which had excellent cell penetrating ability. Dextran was selected as a backbone and modified with azobenzene as guest units by acid-labile imine bonds (Az-I-Dex). The supramolecular polymer CDR/Az-I-Dex with high a C/A molar ratio (molar ratio of CD on CDR to Az on Az-I-Dex) was unfavorable for DNA condensation. The dextran shell of CDR/Az-I-Dex/DNA polyplexes improved the stability under physiological conditions. However, once treated with acetate buffer (pH 5.4) for 3 h, large aggregates formed rapidly due to the cleavage of the dextran shell. As expected, the vector had cell viability of 80% even when the CDR concentration increased to 100 μg mL(-1). Moreover, due to the effective cellular uptake efficiency, CDR/Az-I-Dex/DNA polyplexes had 6-300 times higher transfection efficiency than CDR/DNA polyplexes. It was even higher than high molecular weight PLL-based polyplexes of HEK293 T cells. Importantly, chloroquine as an endosomal escape agent could not improve the transfection of CDR/Az-I-Dex/DNA polyplexes, which indicated that the CDR/Az-I-Dex supramolecular polymer had its own ability for endosomal escape. These results suggested that the CPP-based polyplexes shelled with polysaccharide can be promising non-viral gene delivery carriers. PMID:25893559

  9. Cell-Penetrating Recombinant Peptides for Potential Use in Agricultural Pest Control Applications

    PubMed Central

    Hughes, Stephen R.; Dowd, Patrick F.; Johnson, Eric T.

    2012-01-01

    Several important areas of interest intersect in a class of peptides characterized by their highly cationic and partly hydrophobic structure. These molecules have been called cell-penetrating peptides (CPPs) because they possess the ability to translocate across cell membranes. This ability makes these peptides attractive candidates for delivery of therapeutic compounds, especially to the interior of cells. Compounds with characteristics similar to CPPs and that, in addition, have antimicrobial properties are being investigated as antibiotics with a reduced risk of causing resistance. These CPP-like membrane-acting antimicrobial peptides (MAMPs) are α-helical amphipathic peptides that interact with and perturb cell membranes to produce their antimicrobial effects. One source of MAMPs is spider venom. Because these compounds are toxic to insects, they also show promise for development as biological agents for control of insecticide-resistant agricultural pests. Spider venom is a potential source of novel insect-specific peptide toxins. One example is the small amphipathic α-helical peptide lycotoxin-1 (Lyt-1 or LCTX) from the wolf spider (Lycosa carolinensis). One side of the α-helix has mostly hydrophilic and the other mainly hydrophobic amino acid residues. The positive charge of the hydrophilic side interacts with negatively charged prokaryotic membranes and the hydrophobic side associates with the membrane lipid bilayer to permeabilize it. Because the surface of the exoskeleton, or cuticle, of an insect is highly hydrophobic, to repel water and dirt, it would be expected that amphipathic compounds could permeabilize it. Mutagenized lycotoxin 1 peptides were produced and expressed in yeast cultures that were fed to fall armyworm (Spodoptera frugiperda) larvae to identify the most lethal mutants. Transgenic expression of spider venom toxins such as lycotoxin-1 in plants could provide durable insect resistance. PMID:24281256

  10. Cell-penetrating recombinant peptides for potential use in agricultural pest control applications.

    PubMed

    Hughes, Stephen R; Dowd, Patrick F; Johnson, Eric T

    2012-09-28

    Several important areas of interest intersect in a class of peptides characterized by their highly cationic and partly hydrophobic structure. These molecules have been called cell-penetrating peptides (CPPs) because they possess the ability to translocate across cell membranes. This ability makes these peptides attractive candidates for delivery of therapeutic compounds, especially to the interior of cells. Compounds with characteristics similar to CPPs and that, in addition, have antimicrobial properties are being investigated as antibiotics with a reduced risk of causing resistance. These CPP-like membrane-acting antimicrobial peptides (MAMPs) are α-helical amphipathic peptides that interact with and perturb cell membranes to produce their antimicrobial effects. One source of MAMPs is spider venom. Because these compounds are toxic to insects, they also show promise for development as biological agents for control of insecticide-resistant agricultural pests. Spider venom is a potential source of novel insect-specific peptide toxins. One example is the small amphipathic α-helical peptide lycotoxin-1 (Lyt-1 or LCTX) from the wolf spider (Lycosa carolinensis). One side of the α-helix has mostly hydrophilic and the other mainly hydrophobic amino acid residues. The positive charge of the hydrophilic side interacts with negatively charged prokaryotic membranes and the hydrophobic side associates with the membrane lipid bilayer to permeabilize it. Because the surface of the exoskeleton, or cuticle, of an insect is highly hydrophobic, to repel water and dirt, it would be expected that amphipathic compounds could permeabilize it. Mutagenized lycotoxin 1 peptides were produced and expressed in yeast cultures that were fed to fall armyworm (Spodoptera frugiperda) larvae to identify the most lethal mutants. Transgenic expression of spider venom toxins such as lycotoxin-1 in plants could provide durable insect resistance.

  11. Peptide based DNA nanocarriers incorporating a cell-penetrating peptide derived from neurturin protein and poly-L-lysine dendrons.

    PubMed

    Rosli, Nurlina; Christie, Michelle P; Moyle, Peter M; Toth, Istvan

    2015-05-15

    Multicomponent gene delivery systems incorporating cell-penetrating peptides (CPP) from the human neurturin protein (NRTN-30, NRTN(132-161); NRTN-17, NRTN(145-161)) and a poly-l-lysine (PLL) dendron, were synthesized and characterized for plasmid DNA (pDNA) delivery. Acetylated NRTN peptides (Ac-CPP) and peptides conjugated to a PLL dendron (DEN-CPP) efficiently condensed and stabilized pDNA. Complexes between pDNA and DEN-CPP formed smaller and more stable nanoparticles. Flow cytometry experiments showed that pDNA-DEN-CPPs were taken up more efficiently into HeLa cells. There was also no significant difference between NRTN-30 and NRTN-17 for pDNA uptake, indicating that the truncated peptide alone is sufficient as a CPP for pDNA delivery.

  12. Tumor homing cell penetrating peptide decorated nanoparticles used for enhancing tumor targeting delivery and therapy.

    PubMed

    Gao, Huile; Zhang, Qianyu; Yang, Yuting; Jiang, Xinguo; He, Qin

    2015-01-15

    Specific targeting ability and good tissue penetration are two critical requirements for tumor targeted delivery systems. Systematical selected peptides from a library may meet these two requirements. RLW was such a cell penetrating peptide that could specifically target to non-small cell lung cancer cells (A549). In this study, RLW was linked onto nanoparticles (RNPs) and then the RNPs were used for lung cancer targeting delivery. A traditional cell penetrating peptide, R8 (RRRRRRRR), was used as control. In vitro cellular uptake study demonstrated that modification with RLW specifically enhanced the uptake by A549 cells rather than human umbilical vein endothelial cells, while modification with R8 increased the uptake by both cells. Furthermore, the modification with RLW specifically elevated the penetration into A549 tumor spheroids rather than glioma cell (U87, used as in vivo control) spheroids. And the in vivo imaging further demonstrated RNPs could target to A549 xenografts rather than U87 xenografts. Importantly, the distribution of RNPs in normal organs was approximately the same as that of unmodified nanoparticles. However, R8 modified nanoparticles elevated the distribution in almost all the tissues. These results demonstrated that RLW was superior in A549 tumor targeted delivery. After loaded with docetaxel, an anti-microtube agent, different formulations could effectively induce the A549 cell apoptosis, and inhibit the growth of A549 spheroids in vitro. While in vivo, RNPs displayed the best antitumor effect. The tumor volume was significantly lower than other groups, which was only 33.3% as that of saline group. In conclusion, in vitro RLW could specifically target to A549 cells and enhance the cytotoxicity of docetaxel. In vivo, RLW could significantly enhance the A549 xenografts targeting delivery and led to improved antitumor effect.

  13. CPPsite 2.0: a repository of experimentally validated cell-penetrating peptides

    PubMed Central

    Agrawal, Piyush; Bhalla, Sherry; Usmani, Salman Sadullah; Singh, Sandeep; Chaudhary, Kumardeep; Raghava, Gajendra P. S.; Gautam, Ankur

    2016-01-01

    CPPsite 2.0 (http://crdd.osdd.net/raghava/cppsite/) is an updated version of manually curated database (CPPsite) of cell-penetrating peptides (CPPs). The current version holds around 1850 peptide entries, which is nearly two times than the entries in the previous version. The updated data were curated from research papers and patents published in last three years. It was observed that most of the CPPs discovered/ tested, in last three years, have diverse chemical modifications (e.g. non-natural residues, linkers, lipid moieties, etc.). We have compiled this information on chemical modifications systematically in the updated version of the database. In order to understand the structure-function relationship of these peptides, we predicted tertiary structure of CPPs, possessing both modified and natural residues, using state-of-the-art techniques. CPPsite 2.0 also maintains information about model systems (in vitro/in vivo) used for CPP evaluation and different type of cargoes (e.g. nucleic acid, protein, nanoparticles, etc.) delivered by these peptides. In order to assist a wide range of users, we developed a user-friendly responsive website, with various tools, suitable for smartphone, tablet and desktop users. In conclusion, CPPsite 2.0 provides significant improvements over the previous version in terms of data content. PMID:26586798

  14. CPPsite 2.0: a repository of experimentally validated cell-penetrating peptides.

    PubMed

    Agrawal, Piyush; Bhalla, Sherry; Usmani, Salman Sadullah; Singh, Sandeep; Chaudhary, Kumardeep; Raghava, Gajendra P S; Gautam, Ankur

    2016-01-01

    CPPsite 2.0 (http://crdd.osdd.net/raghava/cppsite/) is an updated version of manually curated database (CPPsite) of cell-penetrating peptides (CPPs). The current version holds around 1850 peptide entries, which is nearly two times than the entries in the previous version. The updated data were curated from research papers and patents published in last three years. It was observed that most of the CPPs discovered/ tested, in last three years, have diverse chemical modifications (e.g. non-natural residues, linkers, lipid moieties, etc.). We have compiled this information on chemical modifications systematically in the updated version of the database. In order to understand the structure-function relationship of these peptides, we predicted tertiary structure of CPPs, possessing both modified and natural residues, using state-of-the-art techniques. CPPsite 2.0 also maintains information about model systems (in vitro/in vivo) used for CPP evaluation and different type of cargoes (e.g. nucleic acid, protein, nanoparticles, etc.) delivered by these peptides. In order to assist a wide range of users, we developed a user-friendly responsive website, with various tools, suitable for smartphone, tablet and desktop users. In conclusion, CPPsite 2.0 provides significant improvements over the previous version in terms of data content. PMID:26586798

  15. Cell-Penetrating Peptide as a Means of Directing the Differentiation of Induced Pluripotent Stem Cells

    PubMed Central

    Kaitsuka, Taku; Tomizawa, Kazuhito

    2015-01-01

    Protein transduction using cell-penetrating peptides (CPPs) is useful for the delivery of large protein molecules, including some transcription factors. This method is safer than gene transfection methods with a viral vector because there is no risk of genomic integration of the exogenous DNA. Recently, this method was reported as a means for the induction of induced pluripotent stem (iPS) cells, directing the differentiation into specific cell types and supporting gene editing/correction. Furthermore, we developed a direct differentiation method to obtain a pancreatic lineage from mouse and human pluripotent stem cells via the protein transduction of three transcription factors, Pdx1, NeuroD, and MafA. Here, we discuss the possibility of using CPPs as a means of directing the differentiation of iPS cells and other stem cell technologies. PMID:26561805

  16. Interaction of nanoparticles and cell-penetrating peptides with skin for transdermal drug delivery

    PubMed Central

    Desai, Pinaki; Patlolla, Ram R.; Singh, Mandip

    2011-01-01

    Topical or transdermal drug delivery is challenging because the skin acts as a natural and protective barrier. Therefore, several methods have been examined to increase the permeation of therapeutic molecules into and through the skin. One approach is to use the nanoparticulate delivery system. Starting with liposomes and other vesicular systems, several other types of nanosized drug carriers have been developed such as solid lipid nanoparticles, nanostructured lipid carriers, polymer-based nanoparticles and magnetic nanoparticles for dermatological applications. This review article discusses how different particulate systems can interact and penetrate into the skin barrier. In this review, the effectiveness of nanoparticles, as well as possible mode of actions of nanoparticles, is presented. In addition to nanoparticles, cell-penetrating peptide (CPP)-mediated drug delivery into the skin and the possible mechanism of CPP-derived delivery into the skin is discussed. Lastly, the effectiveness and possible mechanism of CPP-modified nanocarriers into the skin are addressed. PMID:21028936

  17. Visualization of the Nucleolus in Living Cells with Cell-Penetrating Fluorescent Peptides.

    PubMed

    Martin, Robert M; Herce, Henry D; Ludwig, Anne K; Cardoso, M Cristina

    2016-01-01

    The nucleolus is the hallmark of nuclear compartmentalization and has been shown to exert multiple roles in cellular metabolism besides its main function as the place of ribosomal RNA synthesis and assembly of ribosomes. The nucleolus plays also a major role in nuclear organization as the largest compartment within the nucleus. The prominent structure of the nucleolus can be detected using contrast light microscopy providing an approximate localization of the nucleolus, but this approach does not allow to determine accurately the three-dimensional structure of the nucleolus in cells and tissues. Immunofluorescence staining with antibodies specific to nucleolar proteins albeit very useful is time consuming, normally antibodies recognize their epitopes only within a small range of species and is applicable only in fixed cells. Here, we present a simple method to selectively and accurately label this ubiquitous subnuclear compartment in living cells of a large range of species using a fluorescently labeled cell-penetrating peptide.

  18. Visualization of the Nucleolus in Living Cells with Cell-Penetrating Fluorescent Peptides.

    PubMed

    Martin, Robert M; Herce, Henry D; Ludwig, Anne K; Cardoso, M Cristina

    2016-01-01

    The nucleolus is the hallmark of nuclear compartmentalization and has been shown to exert multiple roles in cellular metabolism besides its main function as the place of ribosomal RNA synthesis and assembly of ribosomes. The nucleolus plays also a major role in nuclear organization as the largest compartment within the nucleus. The prominent structure of the nucleolus can be detected using contrast light microscopy providing an approximate localization of the nucleolus, but this approach does not allow to determine accurately the three-dimensional structure of the nucleolus in cells and tissues. Immunofluorescence staining with antibodies specific to nucleolar proteins albeit very useful is time consuming, normally antibodies recognize their epitopes only within a small range of species and is applicable only in fixed cells. Here, we present a simple method to selectively and accurately label this ubiquitous subnuclear compartment in living cells of a large range of species using a fluorescently labeled cell-penetrating peptide. PMID:27576711

  19. Interaction of nanoparticles and cell-penetrating peptides with skin for transdermal drug delivery.

    PubMed

    Desai, Pinaki; Patlolla, Ram R; Singh, Mandip

    2010-10-01

    Topical or transdermal drug delivery is challenging because the skin acts as a natural and protective barrier. Therefore, several methods have been examined to increase the permeation of therapeutic molecules into and through the skin. One approach is to use the nanoparticulate delivery system. Starting with liposomes and other vesicular systems, several other types of nanosized drug carriers have been developed such as solid lipid nanoparticles, nanostructured lipid carriers, polymer-based nanoparticles and magnetic nanoparticles for dermatological applications. This review article discusses how different particulate systems can interact and penetrate into the skin barrier. In this review, the effectiveness of nanoparticles, as well as possible mode of actions of nanoparticles, is presented. In addition to nanoparticles, cell-penetrating peptide (CPP)-mediated drug delivery into the skin and the possible mechanism of CPP-derived delivery into the skin is discussed. Lastly, the effectiveness and possible mechanism of CPP-modified nanocarriers into the skin are addressed. PMID:21028936

  20. Methods to Study the Role of the Glycocalyx in the Uptake of Cell-Penetrating Peptides.

    PubMed

    Schmidt, Samuel; Wallbrecher, Rike; van Kuppevelt, Toin H; Brock, Roland

    2015-01-01

    Cells are covered by a layer of negatively charged oligo- and polysaccharides, the glycocalyx. Cell-penetrating peptides and other drug delivery vehicles first encounter these polyanions before contacting the lipid bilayer of the plasma membrane. While a large body of data supports the notion that interactions with the glycocalyx promote or even trigger uptake, in some cases, the glycocalyx compromises delivery. As a consequence there is a need to address the role of the glycocalyx in delivery for each specific delivery vehicle and for each particular type of cell. Here, we describe protocols to obtain information on the composition and dynamics of the glycocalyx, and the role of individual glycocalyx components in the uptake of drug delivery vehicles.

  1. Hemocompatible poly(NIPAm-MBA-AMPS) colloidal nanoparticles as carriers of anti-inflammatory cell penetrating peptides.

    PubMed

    Bartlett, Rush L; Medow, Matthew R; Panitch, Alyssa; Seal, Brandon

    2012-04-01

    Anionic copolymer systems containing sulfated monomers have great potential for delivery of cationic therapeutics, but N-isopropylacrylamide (NIPAm) 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) copolymer nanoparticles have seen limited characterization to date with regard to physical properties relevant to loading and release of therapeutics. Characterization of polymeric nanoparticles incorporating AMPS showed an increased size and decreased thermodynamic swelling ratios of AMPS containing particles as compared to NIPAm nanoparticles lacking AMPS. Particles with increasing AMPS addition showed an increased propensity for uniformity, intraparticle colloidal stability, and drug loading capacity. Peptide encapsulated in particles was shielded from peptide degradation in serum. Particles were shown not impede blood coagulation or to cause hemolysis. This study has demonstrated that AMPS incorporation into traditional NIPAm nanoparticles presents a tunable parameter for changing particle LCST, size, swelling ratio, ζ potential, and cationic peptide loading potential. This one-pot synthesis results in a thermosensitive anionic nanoparticle system that is a potentially useful platform to deliver cationic cell penetrating peptides.

  2. Hemocompatible poly(NIPAm-MBA-AMPS) colloidal nanoparticles as carriers of anti-inflammatory cell penetrating peptides.

    PubMed

    Bartlett, Rush L; Medow, Matthew R; Panitch, Alyssa; Seal, Brandon

    2012-04-01

    Anionic copolymer systems containing sulfated monomers have great potential for delivery of cationic therapeutics, but N-isopropylacrylamide (NIPAm) 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) copolymer nanoparticles have seen limited characterization to date with regard to physical properties relevant to loading and release of therapeutics. Characterization of polymeric nanoparticles incorporating AMPS showed an increased size and decreased thermodynamic swelling ratios of AMPS containing particles as compared to NIPAm nanoparticles lacking AMPS. Particles with increasing AMPS addition showed an increased propensity for uniformity, intraparticle colloidal stability, and drug loading capacity. Peptide encapsulated in particles was shielded from peptide degradation in serum. Particles were shown not impede blood coagulation or to cause hemolysis. This study has demonstrated that AMPS incorporation into traditional NIPAm nanoparticles presents a tunable parameter for changing particle LCST, size, swelling ratio, ζ potential, and cationic peptide loading potential. This one-pot synthesis results in a thermosensitive anionic nanoparticle system that is a potentially useful platform to deliver cationic cell penetrating peptides. PMID:22452800

  3. A Heparan Sulfate-Binding Cell Penetrating Peptide for Tumor Targeting and Migration Inhibition

    PubMed Central

    Kuo, Ping-Hsueh; Chang, Pei-Lin; Wang, Wen-Ching; Chuang, Yung-Jen; Chang, Margaret Dah-Tsyr

    2015-01-01

    As heparan sulfate proteoglycans (HSPGs) are known as co-receptors to interact with numerous growth factors and then modulate downstream biological activities, overexpression of HS/HSPG on cell surface acts as an increasingly reliable prognostic factor in tumor progression. Cell penetrating peptides (CPPs) are short-chain peptides developed as functionalized vectors for delivery approaches of impermeable agents. On cell surface negatively charged HS provides the initial attachment of basic CPPs by electrostatic interaction, leading to multiple cellular effects. Here a functional peptide (CPPecp) has been identified from critical HS binding region in hRNase3, a unique RNase family member with in vitro antitumor activity. In this study we analyze a set of HS-binding CPPs derived from natural proteins including CPPecp. In addition to cellular binding and internalization, CPPecp demonstrated multiple functions including strong binding activity to tumor cell surface with higher HS expression, significant inhibitory effects on cancer cell migration, and suppression of angiogenesis in vitro and in vivo. Moreover, different from conventional highly basic CPPs, CPPecp facilitated magnetic nanoparticle to selectively target tumor site in vivo. Therefore, CPPecp could engage its capacity to be developed as biomaterials for diagnostic imaging agent, therapeutic supplement, or functionalized vector for drug delivery. PMID:26064887

  4. Applications and Challenges for Use of Cell-Penetrating Peptides as Delivery Vectors for Peptide and Protein Cargos

    PubMed Central

    Kristensen, Mie; Birch, Ditlev; Mørck Nielsen, Hanne

    2016-01-01

    The hydrophilic nature of peptides and proteins renders them impermeable to cell membranes. Thus, in order to successfully deliver peptide and protein-based therapeutics across the plasma membrane or epithelial and endothelial barriers, a permeation enhancing strategy must be employed. Cell-penetrating peptides (CPPs) constitute a promising tool and have shown applications for peptide and protein delivery into cells as well as across various epithelia and the blood-brain barrier (BBB). CPP-mediated delivery of peptides and proteins may be pursued via covalent conjugation of the CPP to the cargo peptide or protein or via physical complexation obtained by simple bulk-mixing of the CPP with its cargo. Both approaches have their pros and cons, and which is the better choice likely relates to the physicochemical properties of the CPP and its cargo as well as the route of administration, the specific barrier and the target cell. Besides the physical barrier, a metabolic barrier must be taken into consideration when applying peptide-based delivery vectors, such as the CPPs, and stability-enhancing strategies are commonly employed to prolong the CPP half-life. The mechanisms by which CPPs translocate cell membranes are believed to involve both endocytosis and direct translocation, but are still widely investigated and discussed. The fact that multiple factors influence the mechanisms responsible for cellular CPP internalization and the lack of sensitive methods for detection of the CPP, and in some cases the cargo, further complicates the design and conduction of conclusive mechanistic studies. PMID:26840305

  5. Angiopep-2 and activatable cell penetrating peptide dual modified nanoparticles for enhanced tumor targeting and penetrating.

    PubMed

    Mei, Ling; Zhang, Qianyu; Yang, Yuting; He, Qin; Gao, Huile

    2014-10-20

    Delivering chemotherapeutics by nanoparticles into tumor was influenced by at least two factors: specific targeting and highly efficient penetrating of the nanoparticles. In this study, two targeting ligands, angiopep-2 and activatable cell penetrating peptide (ACP), were functionalized onto nanoparticles for tumor targeting delivery. In this system, angiopep-2 is a ligand of low-density lipoprotein receptor-related protein-1 (LRP1) which was highly expressed on tumor cells, and the ACP was constructed by the conjugation of RRRRRRRR (R8) with EEEEEEEE through a matrix metalloproteinase-2 (MMP-2) sensitive linker, enabling the ACP with tumor microenvironment-responsive cell penetrating property. 4h incubation of ACP with MMP-2 leads to over 80% cleavage of ACP, demonstrating ACP indeed possessed MMP-2 responsive property. The constructed dual targeting nanoparticles (AnACNPs) were approximately 110 nm with a polydispersity index of 0.231. In vitro, ACP modification and angiopep-2 modification could both enhance the U-87 MG cell uptake because of the high expression of MMP-2 and LRP-1 on C6 cells. AnACNPs showed higher uptake level than the single ligand modified nanoparticles. The uptake of all particles was time- and concentration-dependent and endosomes were involved. In vivo, AnACNPs showed best tumor targeting efficiency. The distribution of AnACNPs in tumor was higher than all the other particles. After microvessel staining with anti-CD31 antibody, the fluorescent distribution demonstrated AnACNPs could distribute in the whole tumor with the highest intensity. In conclusion, a novel drug delivery system was developed for enhanced tumor dual targeting and elevated cell internalization.

  6. Paramagnetic particles carried by cell-penetrating peptide tracking of bone marrow mesenchymal stem cells, a research in vitro

    SciTech Connect

    Liu Min; Guo Youmin . E-mail: mikie0763@126.com; Wu Qifei; Yang Junle; Wang Peng; Wang Sicen; Guo Xiaojuan; Qiang Yongqian; Duan Xiaoyi

    2006-08-18

    The ability to track the distribution and differentiation of stem cells by high-resolution imaging techniques would have significant clinical and research implications. In this study, a model cell-penetrating peptide was used to carry gadolinium particles for magnetic resonance imaging of the mesenchymal stem cells. The mesenchymal stem cells were isolated from rat bone marrow by Percoll and identified by osteogenic differentiation in vitro. The cell-penetrating peptides labeled with fluorescein-5-isothiocyanate and gadolinium were synthesized by a solid-phase peptide synthesis method and the relaxivity of cell-penetrating peptide-gadolinium paramagnetic conjugate on 400 MHz nuclear magnetic resonance was 5.7311 {+-} 0.0122 mmol{sup -1} s{sup -1}, higher than that of diethylenetriamine pentaacetic acid gadolinium (p < 0.05). Fluorescein imaging confirmed that this new peptide could internalize into the cytoplasm and nucleus. Gadolinium was efficiently internalized into mesenchymal stem cells by the peptide in a time- or concentration-dependent fashion, resulting in intercellular T1 relaxation enhancement, which was obviously detected by 1.5 T magnetic resonance imaging. Cytotoxicity assay and flow cytometric analysis showed the intercellular contrast medium incorporation did not affect cell viability and membrane potential gradient. The research in vitro suggests that the newly constructed peptides could be a vector for tracking mesenchymal stem cells.

  7. Generation of GFP Native Protein for Detection of Its Intracellular Uptake by Cell-Penetrating Peptides.

    PubMed

    Kadkhodayan, S; Sadat, S M; Irani, S; Fotouhi, F; Bolhassani, A

    2016-01-01

    Different types of lipid- and polymer-based vectors have been developed to deliver proteins into cells, but these methods showed relatively poor efficiency. Recently, a group of short, highly basic peptides known as cell-penetrating peptides (CPPs) were used to carry polypeptides and proteins into cells. In this study, expression and purification of GFP protein was performed using the prokaryotic pET expression system. We used two amphipathic CPPs (Pep-1 and CADY-2) as a novel delivery system to transfer the GFP protein into cells. The morphological features of the CPP/GFP complexes were studied by scanning electron microscopy (SEM), Zetasizer, and SDS-PAGE. The efficiency of GFP transfection using Pep-1 and CADY-2 peptides and TurboFect reagent was compared with FITC-antibody protein control delivered by these transfection vehicles in the HEK-293T cell line. SEM data confirmed formation of discrete nanoparticles with a diameter of below 300 nm. Moreover, formation of the complexes was detected using SDS-PAGE as two individual bands, indicating non-covalent interaction. The size and homogeneity of Pep-1/GFP and CADY-2/GFP complexes were dependent on the ratio of peptide/cargo formulations, and responsible for their biological efficiency. The cells transfected by Pep-1/GFP and CADY-2/GFP complexes at a molar ratio of 20 : 1 demonstrated spreading green regions using fluorescent microscopy. Flow cytometry results showed that the transfection efficiency of Pep-based nanoparticles was similar to CADY-based nanoparticles and comparable with TurboFect-protein complexes. These data open an efficient way for future therapeutic purposes. PMID:27516189

  8. Comparative mechanisms of protein transduction mediated by cell-penetrating peptides in prokaryotes.

    PubMed

    Liu, Betty Revon; Huang, Yue-Wern; Aronstam, Robert S; Lee, Han-Jung

    2015-04-01

    Bacterial and archaeal cell envelopes are complex multilayered barriers that serve to protect these microorganisms from their extremely harsh and often hostile environments. Import of exogenous proteins and nanoparticles into cells is important for biotechnological applications in prokaryotes. In this report, we demonstrate that cell-penetrating peptides (CPPs), both bacteria-expressed nona-arginine peptide (R9) and synthetic R9 (SR9), are able to deliver noncovalently associated proteins or quantum dots into four representative species of prokaryotes: cyanobacteria (Synechocystis sp. PCC 6803), bacteria (Escherichia coli DH5α and Arthrobacter ilicis D-50), and archaea (Thermus aquaticus). Although energy-dependent endocytosis is generally accepted as a hallmark that distinguishes eukaryotes from prokaryotes, cellular uptake of uncomplexed green fluorescent protein (GFP) by cyanobacteria was mediated by classical endocytosis. Mechanistic studies revealed that macropinocytosis plays a critical and major role in CPP-mediated protein transduction in all four prokaryotes. Membrane damage was not observed when cyanobacterial cells were treated with R9/GFP complexes, nor was cytotoxicity detected when bacteria or archaea were treated with SR9/QD complexes in the presence of macropinocytic inhibitors. These results indicate that the uptake of protein is not due to a compromise of membrane integrity in cyanobacteria, and that CPP can be an effective and safe carrier for membrane trafficking in prokaryotic cells. Our investigation provides important new insights into the transport of exogenous proteins and nanoparticles across the complex membrane systems of prokaryotes.

  9. Cell-penetrating Peptides Split into Two Groups Based on Modulation of Intracellular Calcium Concentration*

    PubMed Central

    Lorents, Annely; Kodavali, Praveen Kumar; Oskolkov, Nikita; Langel, Ülo; Hällbrink, Mattias; Pooga, Margus

    2012-01-01

    Cell-penetrating peptides (CPPs) promote the uptake of different cargo molecules, e.g. therapeutic compounds, making the harnessing of CPPs a promising strategy for drug design and delivery. However, the internalization mechanisms of CPPs are still under discussion, and it is not clear how cells compensate the disturbances induced by peptides in the plasma membrane. In this study, we demonstrate that the uptake of various CPPs enhances the intracellular Ca2+ levels in Jurkat and HeLa cells. The elevated Ca2+ concentration in turn triggers plasma membrane blebbing, lysosomal exocytosis, and membrane repair response. Our results indicate that CPPs split into two major classes: (i) amphipathic CPPs that modulate the plasma membrane integrity inducing influx of Ca2+ and activating downstream responses starting from low concentrations; (ii) non-amphipathic CPPs that do not evoke changes at relevant concentrations. Triggering of the membrane repair response may help cells to replace distorted plasma membrane regions and cells can recover from the influx of Ca2+ if its level is not drastically elevated. PMID:22437827

  10. In cellulo phosphorylation induces pharmacological reprogramming of maurocalcin, a cell-penetrating venom peptide

    PubMed Central

    Ronjat, Michel; Feng, Wei; Dardevet, Lucie; Dong, Yao; Al Khoury, Sawsan; Chatelain, Franck C.; Vialla, Virginie; Chahboun, Samir; Lesage, Florian; Darbon, Hervé; Pessah, Isaac N.; De Waard, Michel

    2016-01-01

    The venom peptide maurocalcin (MCa) is atypical among toxins because of its ability to rapidly translocate into cells and potently activate the intracellular calcium channel type 1 ryanodine receptor (RyR1). Therefore, MCa is potentially subjected to posttranslational modifications within recipient cells. Here, we report that MCa Thr26 belongs to a consensus PKA phosphorylation site and can be phosphorylated by PKA both in vitro and after cell penetration in cellulo. Unexpectedly, phosphorylation converts MCa from positive to negative RyR1 allosteric modulator. Thr26 phosphorylation leads to charge neutralization of Arg24, a residue crucial for MCa agonist activity. The functional effect of Thr26 phosphorylation is partially mimicked by aspartyl mutation. This represents the first case, to our knowledge, of both ex situ posttranslational modification and pharmacological reprogramming of a small natural cystine-rich peptide by target cells. So far, phosphorylated MCa is the first specific negative allosteric modulator of RyR1, to our knowledge, and represents a lead compound for further development of phosphatase-resistant analogs. PMID:27071086

  11. Therapeutic Potential of Cell Penetrating Peptides (CPPs) and Cationic Polymers for Chronic Hepatitis B

    PubMed Central

    Ndeboko, Bénédicte; Lemamy, Guy Joseph; Nielsen, Peter. E; Cova, Lucyna

    2015-01-01

    Chronic hepatitis B virus (HBV) infection remains a major health problem worldwide. Because current anti-HBV treatments are only virostatic, there is an urgent need for development of alternative antiviral approaches. In this context, cell-penetrating peptides (CPPs) and cationic polymers, such as chitosan (CS), appear of particular interest as nonviral vectors due to their capacity to facilitate cellular delivery of bioactive cargoes including peptide nucleic acids (PNAs) or DNA vaccines. We have investigated the ability of a PNA conjugated to different CPPs to inhibit the replication of duck hepatitis B virus (DHBV), a reference model for human HBV infection. The in vivo administration of PNA-CPP conjugates to neonatal ducklings showed that they reached the liver and inhibited DHBV replication. Interestingly, our results indicated also that a modified CPP (CatLip) alone, in the absence of its PNA cargo, was able to drastically inhibit late stages of DHBV replication. In the mouse model, conjugation of HBV DNA vaccine to modified CS (Man-CS-Phe) improved cellular and humoral responses to plasmid-encoded antigen. Moreover, other systems for gene delivery were investigated including CPP-modified CS and cationic nanoparticles. The results showed that these nonviral vectors considerably increased plasmid DNA uptake and expression. Collectively promising results obtained in preclinical studies suggest the usefulness of these safe delivery systems for the development of novel therapeutics against chronic hepatitis B. PMID:26633356

  12. Nanocarriers Conjugated with Cell Penetrating Peptides: New Trojan Horses by Modern Ulysses.

    PubMed

    Zappavigna, Silvia; Misso, Gabriella; Falanga, Annarita; Perillo, Emiliana; Novellino, Ettore; Galdiero, Massimiliano; Grieco, Paolo; Caraglia, Michele; Galdiero, Stefania

    2016-01-01

    Nanomedicine has opened the way to the design of more efficient diagnostics and therapeutics. Moreover, recent literature has illustrated the use of short cationic and/or amphipathic peptides, known as cell-penetrating peptides (CPPs), for mediating advanced drug delivery. CPPs exploit their ability to enter cells and enhance the uptake of many cargoes ranging from small molecules to proteins. The distinctive properties of nanocarriers (NC) based systems provide unforeseen benefits over pure drugs for biomedical applications and constitute a challenging research field particularly focused on imaging and delivery; nonetheless, several problems have to be overcome to make them a viable option in clinic. The use of CPPs improves significantly their delivery to specific intracellular targets and thus readily contributes to their use both for effective tumor therapy and gene therapy. A key issue is related to their mechanism of uptake, because although classical CPPs enhance NCs' uptake, the entry mechanism involves the endocytic pathway, which means that the delivered material is sequestered within vesicles and only a small amount will escape from this environment and reach the desired target. In this review, we will summarize recent advances in the use of CPP for enhanced delivery of nanocarriers, nucleic acids, and drugs, we will discuss their uptake mechanisms and we will describe novel approaches to improve endosomal escape of internalized nanosystems. PMID:27087493

  13. Impact of different cell penetrating peptides on the efficacy of antisense therapeutics for targeting intracellular pathogens

    PubMed Central

    Abushahba, Mostafa F. N.; Mohammad, Haroon; Thangamani, Shankar; Hussein, Asmaa A. A.; Seleem, Mohamed N.

    2016-01-01

    There is a pressing need for novel and innovative therapeutic strategies to address infections caused by intracellular pathogens. Peptide nucleic acids (PNAs) present a novel method to target intracellular pathogens due to their unique mechanism of action and their ability to be conjugated to cell penetrating peptides (CPP) to overcome challenging delivery barriers. In this study, we targeted the RNA polymerase α subunit (rpoA) using a PNA that was covalently conjugated to five different CPPs. Changing the conjugated CPP resulted in a pronounced improvement in the antibacterial activity observed against Listeria monocytogenes in vitro, in cell culture, and in a Caenorhabditis elegans (C. elegans) infection model. Additionally, a time-kill assay revealed three conjugated CPPs rapidly kill Listeria within 20 minutes without disrupting the bacterial cell membrane. Moreover, rpoA gene silencing resulted in suppression of its message as well as reduced expression of other critical virulence genes (Listeriolysin O, and two phospholipases plcA and plcB) in a concentration-dependent manner. Furthermore, PNA-inhibition of bacterial protein synthesis was selective and did not adversely affect mitochondrial protein synthesis. This study provides a foundation for improving and developing PNAs conjugated to CPPs to better target intracellular pathogens. PMID:26860980

  14. Use of Cell-Penetrating Peptides in Dendritic Cell-Based Vaccination

    PubMed Central

    Lim, Sangho; Koo, Ja-Hyun

    2016-01-01

    Cell-penetrating peptides (CPPs) are short amino acids that have been widely used to deliver macromolecules such as proteins, peptides, DNA, or RNA, to control cellular behavior for therapeutic purposes. CPPs have been used to treat immunological diseases through the delivery of immune modulatory molecules in vivo. Their intracellular delivery efficiency is highly synergistic with the cellular characteristics of the dendritic cells (DCs), which actively uptake foreign antigens. DC-based vaccines are primarily generated by pulsing DCs ex vivo with various immunomodulatory antigens. CPP conjugation to antigens would increase DC uptake as well as antigen processing and presentation on both MHC class II and MHC class I molecules, leading to antigen specific CD4+ and CD8+ T cell responses. CPP-antigen based DC vaccination is considered a promising tool for cancer immunotherapy due to the enhanced CTL response. In this review, we discuss the various applications of CPPs in immune modulation and DC vaccination, and highlight the advantages and limitations of the current CPP-based DC vaccination. PMID:26937230

  15. Cell penetrating peptide conjugated liposomes as transdermal delivery system of Polygonum aviculare L. extract.

    PubMed

    Kwon, Soon Sik; Kim, Sun Young; Kong, Bong Ju; Kim, Kyeong Jin; Noh, Geun Young; Im, Na Ri; Lim, Ji Won; Ha, Ji Hoon; Kim, Junoh; Park, Soo Nam

    2015-04-10

    In this study, Polygonum aviculare L. extract, which has superior antioxidative and cellular membrane protective activity, was loaded onto cell penetrating peptide (CPP) conjugated liposomes to enhance transdermal delivery. The physical characteristics of typical liposomes and CPP-conjugated liposomes containing P. aviculare extract were evaluated. The particle sizes of both liposomes were approximately 150 nm. Whereas the zeta potential of typical liposomes was -45 mV, that of CPP-conjugated liposomes was +42 mV. The loading efficiency of P. aviculare extract in both liposomes was calculated to be about 83%. Fluorescent-labeled liposomes were prepared to evaluate cellular uptake and skin permeation efficiency. Using flow cytometry, we found that CPP-conjugated liposomes improved cellular uptake of the fluorescent dye as compared with the typical liposomes. In addition, the skin permeation of CPP-conjugated liposomes was proved higher than that of typical liposomes by confocal laser scanning microscopy studies and Franz diffusion cell experiments. The improved cellular uptake and skin permeation of the CPP-conjugated liposomes were due to the cationic arginine-rich peptide. In vivo studies also determined that the CPP-conjugated liposomes were more effective in depigmentation and anti-wrinkle studies than typical liposomes. These results indicate that the CPP-conjugated liposomes could be effective for transdermal drug delivery of antioxidant and anti-aging therapeutics.

  16. Applicability and Limitations of Cell-Penetrating Peptides in Noncovalent Mucosal Drug or Carrier Delivery Systems.

    PubMed

    Kamei, Noriyasu; Bech Nielsen, Ebbe Juel; Nakakubo, Takayuki; Aoyama, Yukina; Rahbek, Ulrik Lytt; Pedersen, Betty Lomstein; Takeda-Morishita, Mariko

    2016-02-01

    Our recent studies show that cell-penetrating peptides (CPPs) have potential to improve the intestinal absorption of peptide and protein drugs safely and effectively when used in the noncovalent drug--CPP approach. To clarify the applicability and limitations of this strategy, the present study examined the effects of cargo size on the absorption-stimulatory effect of CPPs. Different sizes of hydrophilic macromolecular dextran (4.4, 10, and 70 kDa) and polystyrene-based nanoparticles (20, 100, and 200 nm) were chosen as the model cargos in this study. In an in situ rat intestinal absorption study, CPPs (octaarginine, Tat, penetratin, and PenetraMax) increased the intestinal absorption of dextran, and the efficiency varied according to the molecular size of dextran. Among the CPPs, D-penetratin showed an enhancing effect even when coadministered with the largest dextran (70 kDa). By contrast, an in vitro study of Caco-2 cell uptake showed that the ability of CPPs to deliver nanoparticles into epithelial cells was dependent on their particle size and that the relatively poor internalization of 200-nm nanoparticles could be facilitated by coincubation with CPPs. These findings suggest that the intrinsic uptake properties of macromolecules and particulate cargos determine the effectiveness of their intestinal mucosal delivery using the noncovalent CPP method. PMID:26869427

  17. Cell-penetrating peptides: achievements and challenges in application for cancer treatment

    PubMed Central

    Shin, Meong Cheol; Zhang, Jian; Min, Kyoung Ah; Lee, Kyuri; Byun, Youngro; David, Allan E.; He, Huining; Yang, Victor C.

    2014-01-01

    One of the major hurdles to cure cancer lies in the low potency of currently available drugs, which could eventually be solved by using more potent therapeutic macromolecules, such as proteins or genes. However, although these macromolecules possess greater potency inside the cancer cells, the barely permeable cell membrane remains a formidable barrier to exert their efficacy. A widely used strategy is to use cell penetrating peptides (CPPs) to improve their intracellular uptake. Since the discovery of the first CPP, numerous CPPs have been derived from natural or synthesized products. Both in vitro and in vivo studies have demonstrated that those CPPs are highly efficient in transducing cargoes into almost all cell types. Therefore, to date, CPPs have been widely used for intracellular delivery of various cargoes, including peptides, proteins, genes, and even nanoparticles. In addition, recently, based on the successes of CPPs in cellular studies, their applications in vivo have been actively pursued. This review will focus on the advanced applications of CPP-based in vivo delivery of therapeutics (e.g., small molecule drugs, proteins, and genes). In addition, we will highlight certain updated applications of CPPs for intracellular delivery of nanoparticulate drug carriers, as well as several ‘smart’ strategies for tumor targeted delivery of CPP-cargoes. PMID:23852939

  18. Targeting a c-Myc inhibitory polypeptide to specific intracellular compartments using cell penetrating peptides.

    PubMed

    Bidwell, Gene L; Davis, Aisha N; Raucher, Drazen

    2009-04-01

    The therapeutic index of current anti-cancer chemotherapeutics can be improved by two major mechanisms: 1) developing drugs which are specifically toxic to the cancer cells and 2) developing methods to deliver drugs to the tumor site. In an attempt to combine these approaches, we developed a thermally responsive polypeptide inhibitor of c-Myc. This polypeptide is based on the thermally responsive Elastin-like polypeptide (ELP). When injected systemically, ELP-fused drugs will aggregate and accumulate at the tumor site where local hyperthermia is applied. ELP was fused to a peptide which blocks c-Myc/Max dimerization (H1), thereby inhibiting transcription activation by c-Myc (ELP-H1). In this study, the cellular uptake, intracellular distribution, and potency of the Pen, Tat and Bac cell penetrating peptides fused to ELP-H1 were evaluated. While Pen-ELP-H1 and Tat-ELP-H1 were localized in the cytoplasm, Bac-ELP-H1 localized to the nucleus in a subset of the cells and was the most potent inhibitor of MCF-7 cell proliferation. This data demonstrates that ELP can be targeted to the desired cellular compartment simply by choice of the CPP used, resulting in a more potent nuclear targeted c-Myc inhibitory polypeptide which may be beneficial in cancer therapy.

  19. Impact of different cell penetrating peptides on the efficacy of antisense therapeutics for targeting intracellular pathogens.

    PubMed

    Abushahba, Mostafa F N; Mohammad, Haroon; Thangamani, Shankar; Hussein, Asmaa A A; Seleem, Mohamed N

    2016-02-10

    There is a pressing need for novel and innovative therapeutic strategies to address infections caused by intracellular pathogens. Peptide nucleic acids (PNAs) present a novel method to target intracellular pathogens due to their unique mechanism of action and their ability to be conjugated to cell penetrating peptides (CPP) to overcome challenging delivery barriers. In this study, we targeted the RNA polymerase α subunit (rpoA) using a PNA that was covalently conjugated to five different CPPs. Changing the conjugated CPP resulted in a pronounced improvement in the antibacterial activity observed against Listeria monocytogenes in vitro, in cell culture, and in a Caenorhabditis elegans (C. elegans) infection model. Additionally, a time-kill assay revealed three conjugated CPPs rapidly kill Listeria within 20 minutes without disrupting the bacterial cell membrane. Moreover, rpoA gene silencing resulted in suppression of its message as well as reduced expression of other critical virulence genes (Listeriolysin O, and two phospholipases plcA and plcB) in a concentration-dependent manner. Furthermore, PNA-inhibition of bacterial protein synthesis was selective and did not adversely affect mitochondrial protein synthesis. This study provides a foundation for improving and developing PNAs conjugated to CPPs to better target intracellular pathogens.

  20. Cell Penetrating Peptide POD Mediates Delivery of Recombinant Proteins to Retina, Cornea and Skin

    PubMed Central

    Johnson, Leslie N.; Cashman, Siobhan M.; Read, Sarah Parker; Kumar-Singh, Rajendra

    2009-01-01

    Recently we described a novel cell penetrating peptide, POD (peptide for ocular delivery) that could deliver small molecules including fluorescent dyes into retinal cells. The objective of the current study was to examine whether biologically relevant macromolecules such as proteins, genetically fused with POD could also be delivered into retinal tissues in vivo. We generated a POD-GFP fusion protein and examined its cell and tissue penetrating properties. We found that endogenously expressed POD-GFP fusion protein localized to the nucleus, suggesting that POD acts as a nuclear localization signal. Adenovirus (Ad) vectors expressing POD-GFP fusion protein were constructed and the recombinant protein was purified from Ad-infected human embryonic retinoblasts (HER). Exogenously supplied POD-GFP fusion protein rapidly transduced A549 and HER cells and colocalized in part with markers of late endosomes, from which it could escape. Following subretinal delivery, POD-GFP localized to the retinal pigment epithelium and the photoreceptor cell bodies. When injected into the vitreous, POD-GFP localized to the ganglion cells and the inner nuclear layer of the retina as well as the lens capsule. Topical application of POD-GFP to ocular surfaces resulted in uptake by the corneal epithelium. POD-GFP also transduced non-ocular tissues, including the epidermis of the skin following topical application. PMID:19733192

  1. Functionalization of gold nanoparticles and CdS quantum dots with cell penetrating peptides

    NASA Astrophysics Data System (ADS)

    Berry, Catherine C.; de la Fuente, Jesus M.

    2009-02-01

    During the last decade, there has been great deal of interest in the self-assembly fabrication of hybrid materials from inorganic nanoparticles and biomolecules. Nanoparticles are similar in size range to many common biomolecules, thus, nanoparticles appear to be natural companions in hybrid systems. At present, it is straightforward to control and modify properties of nanostructures to better suit their integration with biological systems; for example, controlling their size, modifying their surface layer for enhanced aqueous solubility, biocompatibility, or biorecognition. A particularly desirable target for therapeutic uses is the cell nucleus, because the genetic information is there. We review in this article the synthesis developed by our research group of water-soluble gold nanoparticles and CdS nanocrystals functionalized with a Tat protein-derived peptide sequence by straightforward and economical methodologies. The particles were subsequently tested in vitro with a human fibroblast cell line using optical and transmission electron microscopy to determine the biocompatibility of these nanoparticles and whether the functionalization with the cell penetrating peptide allowed particles to transfer across the cell membrane and locate into the nucleus.

  2. Nanocarriers Conjugated with Cell Penetrating Peptides: New Trojan Horses by Modern Ulysses.

    PubMed

    Zappavigna, Silvia; Misso, Gabriella; Falanga, Annarita; Perillo, Emiliana; Novellino, Ettore; Galdiero, Massimiliano; Grieco, Paolo; Caraglia, Michele; Galdiero, Stefania

    2016-01-01

    Nanomedicine has opened the way to the design of more efficient diagnostics and therapeutics. Moreover, recent literature has illustrated the use of short cationic and/or amphipathic peptides, known as cell-penetrating peptides (CPPs), for mediating advanced drug delivery. CPPs exploit their ability to enter cells and enhance the uptake of many cargoes ranging from small molecules to proteins. The distinctive properties of nanocarriers (NC) based systems provide unforeseen benefits over pure drugs for biomedical applications and constitute a challenging research field particularly focused on imaging and delivery; nonetheless, several problems have to be overcome to make them a viable option in clinic. The use of CPPs improves significantly their delivery to specific intracellular targets and thus readily contributes to their use both for effective tumor therapy and gene therapy. A key issue is related to their mechanism of uptake, because although classical CPPs enhance NCs' uptake, the entry mechanism involves the endocytic pathway, which means that the delivered material is sequestered within vesicles and only a small amount will escape from this environment and reach the desired target. In this review, we will summarize recent advances in the use of CPP for enhanced delivery of nanocarriers, nucleic acids, and drugs, we will discuss their uptake mechanisms and we will describe novel approaches to improve endosomal escape of internalized nanosystems.

  3. In vivo fluorescence imaging of atherosclerotic plaques with activatable cell-penetrating peptides targeting thrombin activity†

    PubMed Central

    Olson, Emilia S.; Whitney, Michael A.; Friedman, Beth; Aguilera, Todd A.; Crisp, Jessica L.; Baik, Fred M.; Jiang, Tao; Baird, Stephen M.; Tsimikas, Sotirios; Tsien, Roger Y.

    2012-01-01

    Thrombin and other coagulation enzymes have been shown to be important during atherosclerotic disease development. Study of these proteases is currently limited because of lack of robust molecular imaging agents for imaging protease activity in vivo. Activatable cell penetrating peptides (ACPPs) have been used to monitor MMP activity in tumors and, in principle, can be modified to detect other proteases. We have developed a probe that incorporates the peptide sequence DPRSFL from the proteinase activated receptor 1 (PAR-1) into an ACPP and shown that it is preferentially cleaved by purified thrombin. Active thrombin in serum cleaves DPRSFL–ACPP with >90% inhibition by lepirudin or argatroban. The DPRSFL–ACPP cleavage product accumulated in advanced atherosclerotic lesions in living mice, with 85% reduction in retention upon pre-injection of mice with hirudin. Uptake of the ACPP cleavage product was highest in plaques with histological features associated with more severe disease. Freshly resected human atheromas bathed in DPRSFL–ACPP retained 63% greater cleavage product compared to control ACPP. In conclusion, DPRSFL–ACPP can be used to study thrombin activity in coagulation and atherosclerosis with good spatial and temporal resolution. Thrombin-sensitive ACPPs may be developed into probes for early detection and intraoperative imaging of high risk atherosclerotic plaques. PMID:22534729

  4. Development of a Cell-penetrating Peptide that Exhibits Responsive Changes in its Secondary Structure in the Cellular Environment.

    PubMed

    Yamashita, Hiroko; Kato, Takuma; Oba, Makoto; Misawa, Takashi; Hattori, Takayuki; Ohoka, Nobumichi; Tanaka, Masakazu; Naito, Mikihiko; Kurihara, Masaaki; Demizu, Yosuke

    2016-01-01

    Cell-penetrating peptides (CPP) are received a lot of attention as an intracellular delivery tool for hydrophilic molecules such as drugs, proteins, and DNAs. We designed and synthesized nona-arginine analogues 1-5 [FAM-β-Ala-(l-Arg-l-Arg-l-Pro)3-(Gly)3-NH2 (1), FAM-β-Ala-(l-Arg-l-Arg-l-Pro(NH2))3-(Gly)3-NH2 (2), FAM-β-Ala-(l-Arg-l-Arg-l-Pro(Gu))3-(Gly)3-NH2 (3), FAM-β-Ala-(l-Arg)2-(l-Pro(Gu))2-(l-Arg)4-l-Pro(Gu)-(Gly)3-NH2 (4), and FAM-β-Ala-(l-Arg)6-(l-Pro(Gu))3-(Gly)3-NH2 (5)] containing l-proline (l-Pro) or cationic proline derivatives (l-Pro(NH2) and l-Pro(Gu)), and investigated their cell-penetrating abilities. Interestingly, only peptide 3 having the side-chain guanidinyl l-Pro(Gu) exhibited a secondary structural change in cellular environment. Specifically, peptide 3 formed a random structure in hydrophilic conditions, whereas it formed a helical structure under amphipathic conditions. Furthermore, during cellular permeability tests, peptide 3 demonstrated greater cell-penetrating activity than other peptides and effectively transported plasmid DNA into HeLa cells. Thus, l-Pro(Gu)-containing peptide 3 may be a useful candidate as a gene delivery carrier. PMID:27609319

  5. Development of a Cell-penetrating Peptide that Exhibits Responsive Changes in its Secondary Structure in the Cellular Environment

    PubMed Central

    Yamashita, Hiroko; Kato, Takuma; Oba, Makoto; Misawa, Takashi; Hattori, Takayuki; Ohoka, Nobumichi; Tanaka, Masakazu; Naito, Mikihiko; Kurihara, Masaaki; Demizu, Yosuke

    2016-01-01

    Cell-penetrating peptides (CPP) are received a lot of attention as an intracellular delivery tool for hydrophilic molecules such as drugs, proteins, and DNAs. We designed and synthesized nona-arginine analogues 1–5 [FAM-β-Ala-(l-Arg-l-Arg-l-Pro)3-(Gly)3-NH2 (1), FAM-β-Ala-(l-Arg-l-Arg-l-ProNH2)3-(Gly)3-NH2 (2), FAM-β-Ala-(l-Arg-l-Arg-l-ProGu)3-(Gly)3-NH2 (3), FAM-β-Ala-(l-Arg)2-(l-ProGu)2-(l-Arg)4-l-ProGu-(Gly)3-NH2 (4), and FAM-β-Ala-(l-Arg)6-(l-ProGu)3-(Gly)3-NH2 (5)] containing l-proline (l-Pro) or cationic proline derivatives (l-ProNH2 and l-ProGu), and investigated their cell-penetrating abilities. Interestingly, only peptide 3 having the side-chain guanidinyl l-ProGu exhibited a secondary structural change in cellular environment. Specifically, peptide 3 formed a random structure in hydrophilic conditions, whereas it formed a helical structure under amphipathic conditions. Furthermore, during cellular permeability tests, peptide 3 demonstrated greater cell-penetrating activity than other peptides and effectively transported plasmid DNA into HeLa cells. Thus, l-ProGu-containing peptide 3 may be a useful candidate as a gene delivery carrier. PMID:27609319

  6. Development of a Cell-penetrating Peptide that Exhibits Responsive Changes in its Secondary Structure in the Cellular Environment.

    PubMed

    Yamashita, Hiroko; Kato, Takuma; Oba, Makoto; Misawa, Takashi; Hattori, Takayuki; Ohoka, Nobumichi; Tanaka, Masakazu; Naito, Mikihiko; Kurihara, Masaaki; Demizu, Yosuke

    2016-01-01

    Cell-penetrating peptides (CPP) are received a lot of attention as an intracellular delivery tool for hydrophilic molecules such as drugs, proteins, and DNAs. We designed and synthesized nona-arginine analogues 1-5 [FAM-β-Ala-(l-Arg-l-Arg-l-Pro)3-(Gly)3-NH2 (1), FAM-β-Ala-(l-Arg-l-Arg-l-Pro(NH2))3-(Gly)3-NH2 (2), FAM-β-Ala-(l-Arg-l-Arg-l-Pro(Gu))3-(Gly)3-NH2 (3), FAM-β-Ala-(l-Arg)2-(l-Pro(Gu))2-(l-Arg)4-l-Pro(Gu)-(Gly)3-NH2 (4), and FAM-β-Ala-(l-Arg)6-(l-Pro(Gu))3-(Gly)3-NH2 (5)] containing l-proline (l-Pro) or cationic proline derivatives (l-Pro(NH2) and l-Pro(Gu)), and investigated their cell-penetrating abilities. Interestingly, only peptide 3 having the side-chain guanidinyl l-Pro(Gu) exhibited a secondary structural change in cellular environment. Specifically, peptide 3 formed a random structure in hydrophilic conditions, whereas it formed a helical structure under amphipathic conditions. Furthermore, during cellular permeability tests, peptide 3 demonstrated greater cell-penetrating activity than other peptides and effectively transported plasmid DNA into HeLa cells. Thus, l-Pro(Gu)-containing peptide 3 may be a useful candidate as a gene delivery carrier.

  7. Design of aromatic-containing cell-penetrating peptide mimics with structurally modified π electronics.

    PubMed

    deRonde, Brittany M; Birke, Alexander; Tew, Gregory N

    2015-02-01

    Cell-penetrating peptides (CPPs) and their synthetic mimics (CPPMs) represent a class of molecules that facilitate the intracellular delivery of various cargo. Previous studies indicated that the presence of aromatic functionalities improved CPPM activity. Given that aromatic functionalities play prominent roles in membrane biology and participate in various π interactions, we explored whether these interactions could be optimized for improved CPPM activity. CPPMs were synthesized by ring-opening metathesis polymerization by using monomers that contained aromatic rings substituted with electron-donating and electron-withdrawing groups and covered an electrostatic potential range from -29.69 to +15.57 kcal mol(-1) . These groups altered the quadrupole moments of the aromatic systems and were used to test if such structural modifications changed CPPM activity. CPPMs were added to dye-loaded vesicles and the release of carboxyfluorescein was monitored as a function of polymer concentration. Changes in the effective polymer concentration to release 50% of the dye (effective concentration, EC50 ) were monitored. Results from this assay showed that the strength of the electron-donating and electron-withdrawing groups incorporated in the CPPMs did not alter polymer EC50 values or activity. This suggests that other design parameters may have a stronger impact on CPPM activity. In addition, these results indicate that a wide range of aromatic groups can be incorporated without negatively impacting polymer activity.

  8. Cholesterol prevents interaction of the cell-penetrating peptide transportan with model lipid membranes.

    PubMed

    Arsov, Zoran; Nemec, Marjana; Schara, Milan; Johansson, Henrik; Langel, Ulo; Zorko, Matjaz

    2008-12-01

    Interaction of the cell-penetrating peptide (CPP) cysteine-transportan (Cys-TP) with model lipid membranes was examined by spin-label electron paramagnetic resonance (EPR). Membranes were labeled with lipophilic spin probes and the influence of Cys-TP on membrane structure was studied. The influence of Cys-TP on membrane permeability was monitored by the reduction of a liposome-trapped water-soluble spin probe. Cys-TP caused lipid ordering in membranes prepared from pure dimyristoylphosphatidylcholine (DMPC) and in DMPC membranes with moderate cholesterol concentration. In addition, Cys-TP caused a large increase in permeation of DMPC membranes. In contrast, with high cholesterol content, at which model lipid membranes are in the so-called liquid-ordered phase, no effect of Cys-TP was observed, either on the membrane structure or on the membrane permeability. The interaction between Cys-TP and the lipid membrane therefore depends on the lipid phase. This could be of great importance for understanding of the CPP-lipid interaction in laterally heterogeneous membranes, while it implies that the CPP-lipid interaction can be different at different points along the membrane. PMID:18683276

  9. When cationic cell-penetrating peptides meet hydrocarbons to enhance in-cell cargo delivery.

    PubMed

    Di Pisa, Margherita; Chassaing, Gérard; Swiecicki, Jean-Marie

    2015-05-01

    Cell-penetrating peptides (CPPs) are short sequences often rich in cationic residues with the remarkable ability to cross cell membranes. In the past 20 years, CPPs have gained wide interest and have found numerous applications in the delivery of bioactive cargoes to the cytosol and even the nucleus of living cells. The covalent or non-covalent addition of hydrocarbon moieties to cationic CPPs alters the hydrophobicity/hydrophilicity balance in their sequence. Such perturbation dramatically influences their interaction with the cell membrane, might induce self-assembling properties and modifies their intracellular trafficking. In particular, the introduction of lipophilic moieties changes the subcellular distribution of CPPs and might result in a dramatically increase of the internalization yield of the co-transported cargoes. Herein, we offer an overview of different aspects of the recent findings concerning the properties of CPPs covalently or non-covalently associated to hydrocarbons. We will focus on the impact of the hydrocarbon moieties on the delivery of various cargoes, either covalently or non-covalently bound to the modified CPPs. We will also provide some key elements to rationalize the influence of the hydrocarbons moieties on the cellular uptake. Furthermore, the recent in vitro and in vivo successful applications of acylated CPPs will be summarized to provide a broad view of the versatility of these modified CPPs as small-molecules and oligonucleotides vectors.

  10. The Role of Cell-Penetrating Peptide and Transferrin on Enhanced Delivery of Drug to Brain

    PubMed Central

    Sharma, Gitanjali; Lakkadwala, Sushant; Modgil, Amit; Singh, Jagdish

    2016-01-01

    The challenge of effectively delivering therapeutic agents to brain has led to an entire field of active research devoted to overcome the blood brain barrier (BBB) and efficiently deliver drugs to brain. This review focusses on exploring the facets of a novel platform designed for the delivery of drugs to brain. The platform was constructed based on the hypothesis that a combination of receptor-targeting agent, like transferrin protein, and a cell-penetrating peptide (CPP) will enhance the delivery of associated therapeutic cargo across the BBB. The combination of these two agents in a delivery vehicle has shown significantly improved (p < 0.05) translocation of small molecules and genes into brain as compared to the vehicle with only receptor-targeting agents. The comprehensive details of the uptake mechanisms and properties of various CPPs are illustrated here. The application of this technology, in conjunction with nanotechnology, can potentially open new horizons for the treatment of central nervous system disorders. PMID:27231900

  11. Cell Penetrating Peptide Conjugated Chitosan for Enhanced Delivery of Nucleic Acid

    PubMed Central

    Layek, Buddhadev; Lipp, Lindsey; Singh, Jagdish

    2015-01-01

    Gene therapy is an emerging therapeutic strategy for the cure or treatment of a spectrum of genetic disorders. Nevertheless, advances in gene therapy are immensely reliant upon design of an efficient gene carrier that can deliver genetic cargoes into the desired cell populations. Among various nonviral gene delivery systems, chitosan-based carriers have gained increasing attention because of their high cationic charge density, excellent biocompatibility, nearly nonexistent cytotoxicity, negligible immune response, and ideal ability to undergo chemical conjugation. However, a major shortcoming of chitosan-based carriers is their poor cellular uptake, leading to inadequate transfection efficiency. The intrinsic feature of cell penetrating peptides (CPPs) for transporting diverse cargoes into multiple cell and tissue types in a safe manner suggests that they can be conjugated to chitosan for improving its transfection efficiency. In this review, we briefly discuss CPPs and their classification, and also the major mechanisms contributing to the cellular uptake of CPPs and cargo conjugates. We also discuss immense improvements for the delivery of nucleic acids using CPP-conjugated chitosan-based carriers with special emphasis on plasmid DNA and small interfering RNA. PMID:26690119

  12. Porous silicon-cell penetrating peptide hybrid nanocarrier for intracellular delivery of oligonucleotides.

    PubMed

    Rytkönen, Jussi; Arukuusk, Piret; Xu, Wujun; Kurrikoff, Kaido; Langel, Ulo; Lehto, Vesa-Pekka; Närvänen, Ale

    2014-02-01

    The largest obstacle to the use of oligonucleotides as therapeutic agents is the delivery of these large and negatively charged biomolecules through cell membranes into intracellular space. Mesoporous silicon (PSi) is widely recognized as a potential material for drug delivery purposes due to its several beneficial features like large surface area and pore volume, high loading capacity, biocompatibility, and biodegradability. In the present study, PSi nanoparticles stabilized by thermal oxidation or thermal carbonization and subsequently modified by grafting aminosilanes on the surface are utilized as an oligonucleotide carrier. Splice correcting oligonucleotides (SCOs), a model oligonucleotide drug, were loaded into the positively charged PSi nanoparticles with a loading degree as high as 14.3% (w/w). Rapid loading was achieved by electrostatic interactions, with the loading efficiencies reaching 100% within 5 min. The nanoparticles were shown to deliver and release SCOs, in its biologically active form, inside cells when formulated together with cell penetrating peptides (CPP). The biological effect was monitored with splice correction assay and confocal microscopy utilizing HeLa pLuc 705 cells. Furthermore, the use of PSi carrier platform in oligonucleotide delivery did not reduce the cell viability. Additionally, the SCO-CPP complexes formed in the pores of the carrier were stabilized against proteolytic digestion. The advantageous properties of protecting and releasing the cargo and the possibility to further functionalize the carrier surface make the hybrid nanoparticles a potential system for oligonucleotide delivery.

  13. Cell Penetrating Peptide Conjugated Chitosan for Enhanced Delivery of Nucleic Acid.

    PubMed

    Layek, Buddhadev; Lipp, Lindsey; Singh, Jagdish

    2015-12-04

    Gene therapy is an emerging therapeutic strategy for the cure or treatment of a spectrum of genetic disorders. Nevertheless, advances in gene therapy are immensely reliant upon design of an efficient gene carrier that can deliver genetic cargoes into the desired cell populations. Among various nonviral gene delivery systems, chitosan-based carriers have gained increasing attention because of their high cationic charge density, excellent biocompatibility, nearly nonexistent cytotoxicity, negligible immune response, and ideal ability to undergo chemical conjugation. However, a major shortcoming of chitosan-based carriers is their poor cellular uptake, leading to inadequate transfection efficiency. The intrinsic feature of cell penetrating peptides (CPPs) for transporting diverse cargoes into multiple cell and tissue types in a safe manner suggests that they can be conjugated to chitosan for improving its transfection efficiency. In this review, we briefly discuss CPPs and their classification, and also the major mechanisms contributing to the cellular uptake of CPPs and cargo conjugates. We also discuss immense improvements for the delivery of nucleic acids using CPP-conjugated chitosan-based carriers with special emphasis on plasmid DNA and small interfering RNA.

  14. Transferrin and cell-penetrating peptide dual-functioned liposome for targeted drug delivery to glioma

    PubMed Central

    Zheng, Chuanyi; Ma, Chunyang; Bai, Enqi; Yang, Kun; Xu, Ruxiang

    2015-01-01

    A brain drug delivery system for glioma chemotherapy based on transferrin and cell-penetrating peptide dual-functioned liposome, Tf/TAT-lip, was made and evaluated with doxorubicin (DOX) as a model drug. TAT conjugated liposome (TAT-lip) loaded with doxorubicin (DOX) were prepared by the thin film hydration methods (lip-DOX) and then conjugated with transferrin (Tf) to yield Tf/TAT-lip-DOX which was characterized for their various physicochemical and pharmaceutical properties. Cellular uptakes were explored in both brain capillary endothelial cells (BCECs) of rats and U87 cells. The blood brain barrier model in vitro was established to evaluate the trans-endothelial ability crossing the BBB. The biodistribution of each formulation was further identified. The Tf/TAT-lip-DOX presents the best anti-proliferative activity against U87 cells. The orthotropic glioma model was established for the evaluation of anti-glioma effect. In conclusion, the experimental data in vitro and in vivo indicated that the Tf/TAT-lip was a promising brain drug delivery system due to its high delivery efficiency across the BBB. PMID:25932094

  15. Sodium diclofenac and cell-penetrating peptides embedded in H(II) mesophases: physical characterization and delivery.

    PubMed

    Cohen-Avrahami, Marganit; Libster, Dima; Aserin, Abraham; Garti, Nissim

    2011-09-01

    Glycerol monooleate (GMO)-based mesophases offer extensive prospects for incorporation of various bioactive molecules. This work deals with the solubilization of selected cell-penetrating peptides (CPPs) together with sodium diclofenac (Na-DFC) within the H(II) mesophase for transdermal applications. The effect of CPPs such as RALA (an amphipatic CPP), penetratin (PEN), and oligoarginine (NONA) on Na-DFC skin permeation kinetics to provide controlled release and tune the drug transdermal diffusion was studied. The location of the drug and the CPPs within the mesophase was probed by DSC and FTIR. Na-DFC was found to be located at the interfacial region between the surfactant chains, leading to denser H(II) mesophase. The hydrophilic NONA was intercalated into the aqueous cylinders and caused their swelling. It induced a significant decrease in the hydrogen binding between the GMO carbonyls and their surrounding. The amphiphilic PEN was entrapped within two different regions, depending on its concentration. PEN and NONA improved Na-DFC permeation by 100%, whereas RALA enhanced permeation by 50%. When estimating Na-DFC migration rate out of the mesophase toward surrounding aqueous media, it appeared to be slower with the CPPs. The peptides were not involved at this diffusion-controlled step. It seems that their effect on skin permeation is based on their specific interaction with the skin.

  16. [Cell-penetrating chimeric apoptotic peptide AVPI-LMWP/DNA co-delivery system for cancer therapy].

    PubMed

    Tan, Jiao; Wang, Ya-Ping; Wang, Hui-Xin; Liang, Jian-Ming; Zhang, Meng; Sun, Xun; Huang, Yong-Zhuo

    2014-12-01

    To develop a cell-penetrating chimeric apoptotic peptide AVPI-LMWP/DNA co-delivery system for cancer therapy, we prepared the AVPI-LMWP/pTRAIL self-assembled complexes containing a therapeutic combination of peptide drug AVPI and DNA drug TRAIL. The chimeric apoptotic peptide AVPI-LMWP was synthesized using the standard solid-phase synthesis. The cationic AVPI-LMWP could condense pTRAIL by electrostatic interaction. The physical-chemical properties of the AVPI-LMWP/pTRAIL complexes were characterized. The cellular uptake efficiency and the inhibitory activity of the AVPI-LMWP/pTRAIL complexes on tumor cell were also performed. The results showed that the AVPI-LMWP/pTRAIL complexes were successfully prepared by co-incubation. With the increase of mass ratio (AVPI-LMWP/DNA), the particle size was decreased and the zeta potential had few change. Agarose gel electrophoresis showed that AVPI-LMWP could fully bind and condense pTRAIL at a mass ratio above 15:1. Cellular uptake efficiency was improved along with the increased ratio of W(AVPI-LMWP)/WpTRAIL. The in vitro cytotoxicity experiments demonstrated that the AVPI-LMWP/pTRAIL (W:W = 20:1) complexes was significantly more effective than the pTRAIL, AVPI-LMWP alone or LMWP/pTRAIL complexes on inhibition of HeLa cell growth. Our studies indicated that the AVPI-LMWP/pTRAIL co-delivery system could deliver plasmid into HeLa cell and induce tumor cell apoptosis efficiently, which showed its potential in cancer therapy using combination of apoptoic peptide and gene drugs.

  17. Modeling the endosomal escape of cell-penetrating peptides using a transmembrane pH gradient.

    PubMed

    Madani, Fatemeh; Abdo, Rania; Lindberg, Staffan; Hirose, Hisaaki; Futaki, Shiroh; Langel, Ulo; Gräslund, Astrid

    2013-04-01

    Cell-penetrating peptides (CPPs) can internalize into cells with covalently or non-covalently bound biologically active cargo molecules, which by themselves are not able to pass the cell membrane. Direct penetration and endocytosis are two main pathways suggested for the cellular uptake of CPPs. Cargo molecules which have entered the cell via an endocytotic pathway must be released from the endosome before degradation by enzymatic processes and endosomal acidification. Endosomal entrapment seems to be a major limitation in delivery of these molecules into the cytoplasm. Bacteriorhodopsin (BR) asymmetrically introduced into large unilamellar vesicles (LUVs) was used to induce a pH gradient across the lipid bilayer. By measuring pH outside the LUVs, we observed light-induced proton pumping mediated by BR from the outside to the inside of the LUVs, creating an acidic pH inside the LUVs, similar to the late endosomes in vivo. Here we studied the background mechanism(s) of endosomal escape. 20% negatively charged LUVs were used as model endosomes with incorporated BR into the membrane and fluorescein-labeled CPPs entrapped inside the LUVs, together with a fluorescence quencher. The translocation of different CPPs in the presence of a pH gradient across the membrane was studied. The results show that the light-induced pH gradient induced by BR facilitates vesicle membrane translocation, particularly for the intermediately hydrophobic CPPs, and much less for hydrophilic CPPs. The presence of chloroquine inside the LUVs or addition of pyrenebutyrate outside the LUVs destabilizes the vesicle membrane, resulting in significant changes of the pH gradient across the membrane.

  18. Activation of Cell-Penetrating Peptides with Ionpair-π Interactions and Fluorophiles.

    PubMed

    Chuard, Nicolas; Fujisawa, Kaori; Morelli, Paola; Saarbach, Jacques; Winssinger, Nicolas; Metrangolo, Pierangelo; Resnati, Giuseppe; Sakai, Naomi; Matile, Stefan

    2016-09-01

    In this report, we elaborate on two new concepts to activate arginine-rich cell-penetrating peptides (CPPs). Early on, we have argued that repulsion-driven ion-pairing interactions with anionic lipids account for their ability to move across hydrophobic cell membranes and that hydrophobic anions such as pyrenebutyrate can accelerate this process to kinetically outcompete endosomal capture. The original explanation that the high activity of pyrenebutyrate might originate from ionpair-π interactions between CPP and activator implied that replacement of the π-basic pyrene with polarized push-pull aromatics should afford more powerful CPP activators. To elaborate on this hypothesis, we prepared a small collection of anionic amphiphiles that could recognize cations by ionpair-π interactions. Consistent with theoretical predictions, we find that parallel but not antiparallel ionpair-π interactions afford operational CPP activators in model membranes and cells. The alternative suggestion that the high activity of pyrenebutyrate might originate from self-assembly in membranes was explored with perfluorinated fatty acids. Their fluorophilicity was expected to promote self-assembly in membranes, while their high acidity should prevent charge neutralization in response to self-assembly, i.e., generate repulsion-driven ion-pairing interactions. Consistent with these expectations, we find that perfluorinated fatty acids are powerful CPP activators in HeLa cells but not in model membranes. These findings support parallel ionpair-π interactions and repulsion-driven ion pairing with self-assembled fluorophiles as innovative concepts to activate CPPs. These results also add much corroborative support for counterion-mediated uptake as the productive mode of action of arginine-rich CPPs. PMID:27568814

  19. The tetrapeptide core of the carrier peptide Xentry is cell-penetrating: novel activatable forms of Xentry

    PubMed Central

    Montrose, Kristopher; Yang, Yi; Krissansen, Geoffrey W.

    2014-01-01

    Here we describe a structure-function analysis of the cell-penetrating peptide Xentry derived from the X-protein of the hepatitis B virus. Remarkably, the tetrapeptide core LCLR retains the cell-penetrating ability of the parental peptide LCLRPVG, as either an L- or D-enantiomer. Substitution of the cysteine with leucine revealed that the cysteine is essential for activity. In contrast, the C-terminal arginine could be substituted in the L-isomer with lysine, histidine, glutamic acid, glutamine, and asparagine, though the resulting peptides displayed distinct cell-type-specific uptake. Substitution of the leucines in the D-isomer with other hydrophobic residues revealed that leucines are optimal for activity. Surprisingly, linear di- and tetra-peptide forms of Xentry are not cell-permeable. Protease-activatable forms of Xentry were created by fusing Xentry to itself via a protease-cleavable peptide, or by attaching a heparin mimic peptide to the N-terminus. These novel activatable forms of Xentry were only taken up by MCF-7 cells after cleavage by matrix metalloproteinase 9, and could be used to deliver drugs specifically to tumours. PMID:24811205

  20. Enhanced and selective permeability of gold nanoparticles functionalized with cell penetrating peptide derived from maurocalcine animal toxin.

    PubMed

    Khamehchian, Sedigheh; Nikkhah, Maryam; Madani, Rasool; Hosseinkhani, Saman

    2016-11-01

    Functionalization of gold nanoparticles (GNPs) is suitable for many applications such as biomedical imaging, clinical diagnosis, and targeted delivery by conjugating cell-penetrating peptides (CPPs). Here, we investigated intracellular uptake of GNP conjugated to MCaUF1-9(Ala) , a CPP derived from maurocalcine (MCa) animal toxin, and compared it with TAT functionalized GNP. Peptide conjugated GNP was characterized using UV-Visible spectroscopy, dynamic light scattering, zeta potential, and transmission electron microscopy. Uptake of MCaUF1-9(Ala) and TAT functionalized GNPs was evaluated in three cell lines, HeLa, MDA-MB-231, and A431, using dark field imaging and atomic absorption spectroscopy. According to peptide sequences and type of cells different cell penetrating activity was observed. Peptide functionalized GNP had little effect on cell viability and respect to net charge difference between peptide, showed interesting selectivity against three cell types. Peptide conjugated to GNPs displayed higher uptake than bare GNPs in the all cell lines except HeLa cell with lowest internalization. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2693-2700, 2016.

  1. Insights on the structural characteristics of Vim-TBS (58-81) peptide for future applications as a cell penetrating peptide.

    PubMed

    Saini, Avneet; Jaswal, Radhika R; Negi, Riteshwari; Nandel, Fateh S

    2013-10-01

    The plasma membrane presents a remarkable barrier for the delivery of peptide and nucleic acid based drugs to the inside of cells. This restraint in the path of their development as therapeutic agents can be offset by their conjugation to cell penetrating peptides (CPPs) that can lead to an improved pharmacological profile. In this context, conformational behavior of Vimentin Tubulin Binding Site (TBS) peptide, Vim-TBS (58-81), was investigated for its acknowledged cell penetrating properties along with Trans-activating Tat (48-60) peptide and a pro-apoptogenic peptide of p21/WAFI protein (p10). Also, the fusion peptides Vim- TBS (58-81)-p10 & Tat (48-60)-p10 were studied using molecular mechanics (MM) and molecular dynamics (MD) based strategies. MM results revealed formation of stable α-helix like secondary structures in Vim-TBS (58-81), Tat (48-60) and p10 peptides. In water, three peptides adopted either a helical structure or a random conformation; the stability of either of the two states being governed by the formation of polar contacts with the solvent. The fusion peptides formed helical structures after MD simulations but the structure obtained for the fusion peptide, Vim-TBS-p10 is relatively better characterized in terms of its amphipathic nature with a hydrophilic face formed by the positively charged residues facilitating a better interaction of this fusion peptide with the membrane as compared to that of Tat-p10 peptide. This is the first report on the conformational characteristics of the Vim-TBS (58-81) peptide and the fusion peptide, Vim-TBS (58-81)-p10. The results presented here are significant for their potential role in guiding and facilitating the future efforts of designing peptide based cell penetrating drugs.

  2. Effects of Lipid Composition on the Entry of Cell-Penetrating Peptide Oligoarginine into Single Vesicles.

    PubMed

    Sharmin, Sabrina; Islam, Md Zahidul; Karal, Mohammad Abu Sayem; Alam Shibly, Sayed Ul; Dohra, Hideo; Yamazaki, Masahito

    2016-08-01

    The cell-penetrating peptide R9, an oligoarginine comprising nine arginines, has been used to transport biological cargos into cells. However, the mechanisms underlying its translocation across membranes remain unclear. In this report, we investigated the entry of carboxyfluorescein (CF)-labeled R9 (CF-R9) into single giant unilamellar vesicles (GUVs) of various lipid compositions and the CF-R9-induced leakage of a fluorescent probe, Alexa Fluor 647 hydrazide (AF647), using a method developed recently by us. First, we investigated the interaction of CF-R9 with dioleoylphosphatidylglycerol (DOPG)/dioleoylphosphatidylcholine (DOPC) GUVs containing AF647 and small DOPG/DOPC vesicles. The fluorescence intensity of the GUV membrane due to CF-R9 (i.e., the rim intensity) increased with time to a steady-state value, and then the fluorescence intensity of the membranes of the small vesicles in the GUV lumen increased without leakage of AF647. This result indicates that CF-R9 entered the GUV lumen from the outside by translocating across the lipid membrane without forming pores through which AF647 could leak. The fraction of entry of CF-R9 at 6 min in the absence of pore formation, Pentry (6 min), increased with an increase in CF-R9 concentration, but the CF-R9 concentration in the lumen was low. We obtained similar results for dilauroyl-PG (DLPG)/ditridecanoyl-PC (DTPC) (2/8) GUVs. The values of Pentry (6 min) of CF-R9 for DLPG/DTPC (2/8) GUVs were larger than those obtained with DOPG/DOPC (2/8) GUVs at the same CF-R9 concentrations. In contrast, a high concentration of CF-R9 induced pores in DLPG/DTPC (4/6) GUVs through which CF-R9 entered the GUV lumen, so the CF-R9 concentration in the lumen was higher. However, CF-R9 could not enter DOPG/DOPC/cholesterol (2/6/4) GUVs. Analysis of the rim intensity showed that CF-R9 was located only in the outer monolayer of the DOPG/DOPC/cholesterol (2/6/4) GUVs. On the basis of analyses of these results, we discuss the elementary

  3. riDOM, a cell-penetrating peptide. Interaction with DNA and heparan sulfate.

    PubMed

    Québatte, Gabriela; Kitas, Eric; Seelig, Joachim

    2013-09-19

    DNA condensation in the presence of polycationic molecules is a well-known phenomenon exploited in gene delivery. riDOM (retro-inverso dioleoylmelittin) is a cell-penetrating peptide with excellent transporter properties for DNA. It is a chimeric molecule where ri-melittin is fused to dioleoylphosphoethanolamine. The physical-chemical properties of riDOM in solution and in the presence of DNA and heparan sulfate were investigated with spectroscopic and thermodynamic methods. Dynamic light scattering shows that riDOM in solution aggregates to well-defined nanoparticles with a diameter of ∼13 nm and a ζ-potential of 22 mV, composed of about 220-270 molecules. Binding of riDOM to DNA was studied with dynamic light scattering, ζ-potential measurements, and isothermal titration calorimetry and was compared with authentic melittin-DNA interaction. riDOM binds tightly to DNA with a microscopic binding constant of 5 × 10(7) M(-1) and a stoichiometry of 12 riDOM per 10 DNA base pairs. In the complex the DNA double strand is completely shielded by the more hydrophobic riDOM molecules. Authentic melittin binds to DNA with a much lower binding constant of 5 × 10(6) M(-1) and lower stoichiometry of 5 melittin per 10 DNA base pairs. The binding enthalpies for riDOM and melittin are small and the binding reactions are entropy-driven. Sulfated glycosaminoglycans such as heparan sulfate are also linear molecules with a negative charge. riDOM binding to heparan sulfate on cell surfaces can therefore interfere with DNA-riDOM binding. riDOM-heparan sulfate complex formation was characterized by isothermal titration calorimetry and spectroscopic methods. The binding constant of riDOM for heparan sulfate is K ≈ 2 × 10(6) M(-1). Authentic melittin has a similar binding constant but riDOM shows a 3-fold higher packing density on heparan sulfate than the distinctly smaller melittin.

  4. Cyclization of a cell-penetrating peptide via click-chemistry increases proteolytic resistance and improves drug delivery.

    PubMed

    Reichart, Florian; Horn, Mareike; Neundorf, Ines

    2016-06-01

    In this work we report synthesis and biological evaluation of a cell-penetrating peptide (CPP), that is partly cyclized via a triazole bridge. Recently, beneficious properties have been reported for cyclized peptides concerning their metabolic stability and intracellular uptake. A CPP based on human calcitonin was used in this study, and side chain cyclization was achieved via copper catalyzed alkyne-azide click reaction. Cell viability studies in several cell-lines revealed no cytotoxic effects. Furthermore, efficient uptake in breast cancer MCF-7 cells could be determined. Moreover, preliminary studies using this novel peptide as drug transporter for daunorubicin were performed. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd. PMID:27197760

  5. Cellular Internalization Mechanism and Intracellular Trafficking of Filamentous M13 Phages Displaying a Cell-Penetrating Transbody and TAT Peptide

    PubMed Central

    Shin, Seung-Min; Pham, Chuong D.; Choi, Dong-Ki; Kwon, Myung-Hee; Kim, Yong-Sung

    2012-01-01

    Cellular internalization of bacteriophage by surface-displayed cell penetrating peptides has been reported, though the underlying mechanism remains elusive. Here we describe in detail the internalization mechanism and intracellular trafficking and stability of filamentous M13 phages, the cellular entry of which is mediated by surface-displayed cell-penetrating light chain variable domain 3D8 VL transbody (3D8 VL-M13) or TAT peptide (TAT-M13). Recombinant 3D8 VL-M13 and TAT-M13 phages were efficiently internalized into living mammalian cells via physiologically relevant, energy-dependent endocytosis and were recovered from the cells in their infective form with the yield of 3D8 VL-M13 being higher (0.005∼0.01%) than that of TAT-M13 (0.001∼0.005%). Biochemical and genetic studies revealed that 3D8 VL-M13 was internalized principally by caveolae-mediated endocytosis via interaction with heparan sulfate proteoglycans as cell surface receptors, whereas TAT-M13 was internalized by clathrin- and caveolae-mediated endocytosis utilizing chondroitin sulfate proteoglycans as cell surface receptors, suggesting that phage internalization occurs by physiological endocytotic mechanism through specific cell surface receptors rather than non-specific transcytotic pathways. Internalized 3D8 VL-M13 phages routed to the cytosol and remained stable for more than 18 h without further trafficking to other subcellular compartments, whereas TAT-M13 phages routed to several subcellular compartments before being degraded in lysosomes even after 2 h of internalization. Our results suggest that the internalizing mechanism and intracellular trafficking of filamentous M13 bacteriophages largely follow the attributes of the displayed cell-penetrating moiety. Efficient internalization and cytosolic localization of 3D8 VL transbody-displayed phages will provide a useful tool for intracellular delivery of polar macromolecules such as proteins, peptides, and siRNAs. PMID:23251631

  6. Fluorescent sterols monitor cell penetrating peptide Pep-1 mediated uptake and intracellular targeting of cargo protein in living cells.

    PubMed

    Petrescu, Anca D; Vespa, Aude; Huang, Huan; McIntosh, Avery L; Schroeder, Friedhelm; Kier, Ann B

    2009-02-01

    Although cell-penetrating peptides (CPP) facilitate endocytic uptake of proteins, little is known regarding the extent to which CPPs facilitate protein cargo exit from endocytic vesicles for targeting to other intracellular sites. Since the plasma membrane and less so intracellular membranes contain cholesterol, the fluorescent sterol analogues dansyl-cholestanol (DChol) and dehydroergosterol (DHE) were used to monitor the uptake and intracellular distribution of fluorescent-tagged acyl coenzyme A binding protein (ACBP) into COS-7 cells and rat hepatoma cells. Confocal microscopy colocalized DChol and Texas Red-ACBP (TR-ACBP) with markers for the major endocytosis pathways, especially fluorescent-labeled cholera toxin (marker of ganglioside GM1 in plasma membrane lipid rafts) and dextran (macropinocytosis marker), but less so with transferrin (clathrin-mediated endocytosis marker). These findings were confirmed by multiphoton laser scanning microscopy colocalization of TR-ACBP with DHE (naturally-fluorescent sterol) and by double immunofluorescence labeling of native endogenous ACBP. Serum greatly and Pep-1 further 2.4-fold facilitated uptake of TR-ACBP, but neither altered the relative proportion of TR-ACBP colocalized with membranes/organelles (nearly 80%) vs cytoplasm and/or nucleoplasm (20%). Interestingly, Pep-1 selectively increased TR-ACBP associated with mitochondria while concomitantly decreasing that in endoplasmic reticulum. In summary, fluorescent sterols (DChol, DHE) were useful markers for comparing the distributions of both transported and endogenous proteins. Pep-1 modestly enhanced the translocation and altered the intracellular targeting of exogenous-delivered (TR-ACBP) in living cells.

  7. Full membrane spanning self-assembled monolayers as model systems for UHV-based studies of cell-penetrating peptides

    SciTech Connect

    Franz, Johannes; Graham, Daniel J.; Baio, Joe E.; Lelle, Marco; Peneva, Kalina; Müllen, Klaus; Castner, David G.; Weidner, Tobias

    2015-03-01

    Biophysical studies of the interaction of peptides with model membranes provide a simple yet effective approach to understand the transport of peptides and peptide based drug carriers across the cell membrane. Therein, the authors discuss the use of self-assembled monolayers fabricated from the full membrane-spanning thiol (FMST) 3-((14-((4'-((5-methyl-1-phenyl-35-(phytanyl)oxy-6,9,12,15,18,21,24,27,30,33,37-undecaoxa-2,3-dithiahenpentacontan-51-yl)oxy)-[1,1'-biphenyl]-4-yl)oxy)tetradecyl)oxy)-2-(phytanyl)oxy glycerol for ultrahigh vacuum (UHV) based experiments. UHV-based methods such as electron spectroscopy and mass spectrometry can provide important information about how peptides bind and interact with membranes, especially with the hydrophobic core of a lipid bilayer. Moreover, near-edge x-ray absorption fine structure spectra and x-ray photoelectron spectroscopy (XPS) data showed that FMST forms UHV-stable and ordered films on gold. XPS and time of flight secondary ion mass spectrometry depth profiles indicated that a proline-rich amphipathic cell-penetrating peptide, known as sweet arrow peptide is located at the outer perimeter of the model membrane.

  8. Modeling the endosomal escape of cell-penetrating peptides: transmembrane pH gradient driven translocation across phospholipid bilayers.

    PubMed

    Magzoub, Mazin; Pramanik, Aladdin; Gräslund, Astrid

    2005-11-15

    Cell-penetrating peptides (CPPs) are able to mediate the efficient cellular uptake of a wide range of cargoes. Internalization of a number of CPPs requires uptake by endocytosis, initiated by binding to anionic cell surface heparan sulfate (HS), followed by escape from endosomes. To elucidate the endosomal escape mechanism, we have modeled the process for two CPPs: penetratin (pAntp) and the N-terminal signal peptide of the unprocessed bovine prion protein (bPrPp). Large unilamellar phospholipid vesicles (LUVs) were produced encapsulating either peptide, and an ionophore, nigericin, was used to create a transmembrane pH gradient (DeltapH(mem), inside acidic) similar to the one arising in endosomes in vivo. In the absence of DeltapH(mem), no pAntp escape from the LUVs is observed, while a fraction of bPrPp escapes. In the presence of DeltapH(mem), a significant amount of pAntp escapes and an even higher degree of bPrPp escape takes place. These results, together with the differences in kinetics of escape, indicate different escape mechanisms for the two peptides. A minimum threshold peptide concentration exists for the escape of both peptides. Coupling of the peptides to a cargo reduces the fraction escaping, while complexation with HS significantly hinders the escape. Fluorescence correlation spectroscopy results show that during the escape process the LUVs are intact. Taken together, these results suggest a model for endosomal escape of CPPs: DeltapH(mem)-mediated mechanism, following dissociation from HS of the peptides, above a minimum threshold peptide concentration, in a process that does not involve lysis of the vesicles.

  9. The spacer arm length in cell-penetrating peptides influences chitosan/siRNA nanoparticle delivery for pulmonary inflammation treatment

    NASA Astrophysics Data System (ADS)

    Jeong, Eun Ju; Choi, Moonhwan; Lee, Jangwook; Rhim, Taiyoun; Lee, Kuen Yong

    2015-11-01

    Although chitosan and its derivatives have been frequently utilized as delivery vehicles for small interfering RNA (siRNA), it is challenging to improve the gene silencing efficiency of chitosan-based nanoparticles. In this study, we hypothesized that controlling the spacer arm length between a cell-penetrating peptide (CPP) and a nanoparticle could be critical to enhancing the cellular uptake as well as the gene silencing efficiency of conventional chitosan/siRNA nanoparticles. A peptide consisting of nine arginine units (R9) was used as a CPP, and the spacer arm length was controlled by varying the number of glycine units between the peptide (R9Gn) and the nanoparticle (n = 0, 4, and 10). Various physicochemical characteristics of R9Gn-chitosan/siRNA nanoparticles were investigated in vitro. Increasing the spacing arm length did not significantly affect the complex formation between R9Gn-chitosan and siRNA. However, R9G10-chitosan was much more effective in delivering genes both in vitro and in vivo compared with non-modified chitosan (without the peptide) and R9-chitosan (without the spacer arm). Chitosan derivatives modified with oligoarginine containing a spacer arm can be considered as potential delivery vehicles for various genes.Although chitosan and its derivatives have been frequently utilized as delivery vehicles for small interfering RNA (siRNA), it is challenging to improve the gene silencing efficiency of chitosan-based nanoparticles. In this study, we hypothesized that controlling the spacer arm length between a cell-penetrating peptide (CPP) and a nanoparticle could be critical to enhancing the cellular uptake as well as the gene silencing efficiency of conventional chitosan/siRNA nanoparticles. A peptide consisting of nine arginine units (R9) was used as a CPP, and the spacer arm length was controlled by varying the number of glycine units between the peptide (R9Gn) and the nanoparticle (n = 0, 4, and 10). Various physicochemical characteristics of

  10. Enhancement of intracellular concentration and biological activity of PNA after conjugation with a cell-penetrating synthetic model peptide.

    PubMed

    Oehlke, Johannes; Wallukat, Gerd; Wolf, Yvonne; Ehrlich, Angelika; Wiesner, Burkhard; Berger, Hartmut; Bienert, Michael

    2004-07-01

    In order to evaluate the ability of the cell-penetrating alpha-helical amphipathic model peptide KLALKLALKALKAALKLA-NH(2) (MAP) to deliver peptide nucleic acids (PNAs) into mammalian cells, MAP was covalently linked to the 12-mer PNA 5'-GGAGCAGGAAAG-3' directed against the mRNA of the nociceptin/orphanin FQ receptor. The cellular uptake of both the naked PNA and its MAP-conjugate was studied by means of capillary electrophoresis combined with laser-induced fluorescence detection, confocal laser scanning microscopy and fluorescence-activated cell sorting. Incubation with the fluorescein-labelled PNA-peptide conjugate led to three- and eightfold higher intracellular concentrations in neonatal rat cardiomyocytes and CHO cells, respectively, than found after exposure of the cells to the naked PNA. Correspondingly, pretreatment of spontaneously-beating neonatal rat cardiomyocytes with the PNA-peptide conjugate and the naked PNA slowed down the positive chronotropic effect elicited by the neuropeptide nociceptin by 10- and twofold, respectively. The main reasons for the higher bioavailability of the PNA-peptide conjugate were found to be a more rapid cellular uptake in combination with a lowered re-export and resistance against influences of serum.

  11. Insights into the uptake mechanism of NrTP, a cell-penetrating peptide preferentially targeting the nucleolus of tumour cells.

    PubMed

    Rádis-Baptista, Gandhi; de la Torre, Beatriz G; Andreu, David

    2012-06-01

    Nucleolar targeting peptides are 14-15 residue-long sequences designed by structural minimization of a snake toxin (J Med Chem 2008;50:7041). Peptides such as NrTP1 (YKQCHKKGGKK GSG) and analogues are capable of penetrating human cervix epithelial carcinoma cells and homing into their nucleoli. We now show that NrTP1 similarly penetrates and localizes in the nucleolus of tumour cells derived from human pancreatic (BxPC-3) and human ductal mammary gland (BT-474) carcinomas. Live cell confocal microscopy imaging, combined with flow cytometry analysis of cells arrested to defined phases of their cycle, confirms that NrTP1 uptake and nucleolar homing are independent of cell cycle phase. Peptide uptake is significantly reduced at low temperature. Also, drugs inhibiting chlatrin-mediated endocytosis severely decrease uptake, pointing to a clathrin-dependent route as the primary NrTP1 internalization mechanism. These results highlight nucleolar targeting peptides not only as a novel and efficient class of cell-penetrating peptides but also for their exceptional ability to target preferentially an essential and dynamic subnuclear structure such as the nucleolus. PMID:22405142

  12. Syndecan-4 Is a Receptor for Clathrin-Mediated Endocytosis of Arginine-Rich Cell-Penetrating Peptides.

    PubMed

    Kawaguchi, Yoshimasa; Takeuchi, Toshihide; Kuwata, Keiko; Chiba, Junya; Hatanaka, Yasumaru; Nakase, Ikuhiko; Futaki, Shiroh

    2016-04-20

    Arginine-rich cell-penetrating peptides (CPPs) such as Tat and oligoarginine peptides have been widely used as carriers for intracellular delivery of bioactive molecules. Despite accumulating evidence for involvement of endocytosis in the cellular uptake of arginine-rich CPPs, the primary cell-surface receptors for these peptide carriers that would initiate endocytic processes leading to intracellular delivery of bioactive cargoes have remained poorly understood. Our previous attempt to identify membrane receptors for octa-arginine (R8) peptide, one of the representative arginine-rich CPPs, using the photo-cross-linking probe bearing a photoreactive diazirine was not successful due to considerable amounts of cellular proteins nonspecifically bound to the affinity beads. To address this issue, here we developed a photo-cross-linking probe in which a cleavable linker of a diazobenzene moiety was employed to allow selective elution of cross-linked proteins by reducing agent-mediated cleavage. We demonstrated that introduction of the diazobenzene moiety into the photoaffinity probe enables efficient purification of cross-linked proteins with significant reduction of nonspecific binding proteins, leading to successful identification of 17 membrane-associated proteins that would interact with R8 peptide. RNAi-mediated knockdown experiments in combination with the pharmacological inhibitors revealed that, among the proteins identified, syndecan-4, one of the heparan sulfate proteoglycans, is an endogenous membrane-associated receptor for the cellular uptake of R8 peptide via clathrin-mediated endocytosis. This syndecan-4-dependent pathway was also involved in the intracellular delivery of bioactive proteins mediated by R8 peptide. These results reveal that syndecan-4 is a primary cell-surface target for R8 peptide that allows intracellular delivery of bioactive cargo molecules via clathrin-mediated endocytosis. PMID:27019270

  13. Functionalization with C-terminal cysteine enhances transfection efficiency of cell-penetrating peptides through dimer formation

    SciTech Connect

    Amand, Helene L.

    2012-02-17

    Highlights: Black-Right-Pointing-Pointer Reversible CPP dimerisation is a simple yet efficient strategy to improve delivery. Black-Right-Pointing-Pointer Dimer formation enhances peptiplex stability, resulting in increased transfection. Black-Right-Pointing-Pointer By dimerisation, the CPP EB1 even gain endosomal escape properties while lowering cytotoxicity. -- Abstract: Cell-penetrating peptides have the ability to stimulate uptake of macromolecular cargo in mammalian cells in a non-toxic manner and therefore hold promise as efficient and well tolerated gene delivery vectors. Non-covalent peptide-DNA complexes ('peptiplexes') enter cells via endocytosis, but poor peptiplex stability and endosomal entrapment are considered as main barriers to peptide-mediated delivery. We explore a simple, yet highly efficient, strategy to improve the function of peptide-based vectors, by adding one terminal cysteine residue. This allows the peptide to dimerize by disulfide bond formation, increasing its affinity for nucleic acids by the 'chelate effect' and, when the bond is reduced intracellularly, letting the complex dissociate to deliver the nucleic acid. By introducing a single C-terminal cysteine in the classical CPP penetratin and the penetratin analogs PenArg and EB1, we show that this minor modification greatly enhances the transfection capacity for plasmid DNA in HEK293T cells. We conclude that this effect is mainly due to enhanced thermodynamic stability of the peptiplexes as endosome-disruptive chloroquine is still required for transfection and the effect is more pronounced for peptides with lower inherent DNA condensation capacity. Interestingly, for EB1, addition of one cysteine makes the peptide able to mediate transfection in absence of chloroquine, indicating that dimerisation can also improve endosomal escape properties. Further, the cytotoxicity of EB1 peptiplexes is considerably reduced, possibly due to lower concentration of free peptide dimer resulting from

  14. State of the Art in the Studies on Crotamine, a Cell Penetrating Peptide from South American Rattlesnake

    PubMed Central

    Kerkis, Irina; Hayashi, Mirian A. F.; Prieto da Silva, Alvaro R. B.; Pereira, Alexandre; De Sá Júnior, Paulo Luiz; Zaharenko, Andre J.; Rádis-Baptista, Gandhi; Kerkis, Alexandre; Yamane, Tetsuo

    2014-01-01

    Animal venoms comprise a naturally selected cocktail of bioactive peptides/proteins and other molecules, each of which playing a defined role thanks to the highly specific interactions with diverse molecular targets found in the prey. Research focused on isolation, structural, and functional characterizations of novel natural biologics (bioactive peptides/proteins from natural sources) has a long way to go through from the basic science to clinical applications. Herein, we overview the structural and functional characteristics of the myoneurotoxin crotamine, firstly isolated from the South American rattlesnake venom. Crotamine is the first venom peptide classified as a natural cell penetrating and antimicrobial peptide (CPP and AMP) with a more pronounced antifungal activity. In contrast to other known natural CPPs and AMPs, crotamine demonstrates a wide spectrum of biological activities with potential biotechnological and therapeutic values. More recent studies have demonstrated the selective in vitro anticancer activity of crotamine. In vivo, using a murine melanoma model, it was shown that crotamine delays tumor implantation, inhibits tumor cells proliferation, and also increases the survival of mice engrafted with subcutaneous melanoma. The structural and functional properties and also the possible biotechnological applications of minimized molecules derived from crotamine are also discussed. PMID:24551848

  15. Transduction of proteins into leishmania tarentolae by formation of non-covalent complexes with cell-penetrating peptides.

    PubMed

    Keller, Andrea-Anneliese; Breitling, Reinhard; Hemmerich, Peter; Kappe, Katarina; Braun, Maria; Wittig, Berith; Schaefer, Buerk; Lorkowski, Stefan; Reissmann, Siegmund

    2014-02-01

    Cell-penetrating peptides (CPPs) are used to transport peptides, proteins, different types of ribonucleic acids (or mimics of these molecules), and DNA into live cells, both plant and mammalian. Leishmania belongs to the class of protozoa having, in comparison to mammalian cells, a different lipid composition of the membrane, proteoglycans on the surface, and signal pathways. We investigated the uptake of two different and easily detectable proteins into the non-pathogenic strain Leishmania tarentolae. From the large number of CPPs available, six and a histone were chosen specifically for their ability to form non-covalent complexes. For Leishmania we used the enzyme β-galactosidase and fluorescent labeled bovine serum albumin as cargoes. The results are compared to similar internalization studies using mammalian cells [Mussbach et al., ]. Leishmania cells can degrade CPPs by a secreted and membrane-bound chymotrypsin-like protease. Both cargo proteins were internalized with sufficient efficiency and achieved intramolecular concentrations similar to mammalian cells. The transport efficiencies of the CPPs differed from each other, and showed a different rank order for both cargoes. The intracellular distribution of fluorescent-labeled bovine serum albumin showed highest concentrations in the nucleus and kinetoplast. Leishmania are susceptible to high concentrations of some CPPs, although comparably dissimilar to mammalian cells. MPG-peptides are more cytotoxic in Leishmania than in mammalian cells, acting as antimicrobial peptides. Our results contribute to a better understanding of molecular interactions in Leishmania cells and possibly to new treatments of leishmaniasis.

  16. Secondary conformational conversion is involved in glycosaminoglycans-mediated cellular uptake of the cationic cell-penetrating peptide PACAP.

    PubMed

    Tchoumi Neree, Armelle; Nguyen, Phuong Trang; Chatenet, David; Fournier, Alain; Bourgault, Steve

    2014-12-20

    Glycosaminoglycans (GAGs) contribute to the cellular uptake of cationic cell-penetrating peptides (CPPs). However, molecular details about the contributions of GAGs in CPP internalization remain unclear. In this study, we examined the cellular uptake mechanism of the arginine-rich CPP pituitary adenylate-cyclase-activating polypeptide (PACAP). We observed that the uptake efficacy of PACAP is dependent on the expression of cell surface GAGs. As the binding of PACAP to sulfated GAGs induced a random coil-to-α-helix conformational conversion, we investigated the role of the helical formation in PACAP internalization. Whereas this secondary structure was not crucial for efficient internalization in GAGs-deficient cells, PACAP α-helix was essential for GAGs-dependent uptake.

  17. Cell-Penetrating Peptide as a Means of Directing the Differentiation of Induced-Pluripotent Stem Cells.

    PubMed

    Kaitsuka, Taku; Tomizawa, Kazuhito

    2015-01-01

    Protein transduction using cell-penetrating peptides (CPPs) is useful for the delivery of large protein molecules, including some transcription factors. This method is safer than gene transfection methods with a viral vector because there is no risk of genomic integration of the exogenous DNA. Recently, this method was reported as a means for the induction of induced pluripotent stem (iPS) cells, directing the differentiation into specific cell types and supporting gene editing/correction. Furthermore, we developed a direct differentiation method to obtain a pancreatic lineage from mouse and human pluripotent stem cells via the protein transduction of three transcription factors, Pdx1, NeuroD, and MafA. Here, we discuss the possibility of using CPPs as a means of directing the differentiation of iPS cells and other stem cell technologies. PMID:26561805

  18. Cell-Penetrating Peptide as a Means of Directing the Differentiation of Induced-Pluripotent Stem Cells.

    PubMed

    Kaitsuka, Taku; Tomizawa, Kazuhito

    2015-11-06

    Protein transduction using cell-penetrating peptides (CPPs) is useful for the delivery of large protein molecules, including some transcription factors. This method is safer than gene transfection methods with a viral vector because there is no risk of genomic integration of the exogenous DNA. Recently, this method was reported as a means for the induction of induced pluripotent stem (iPS) cells, directing the differentiation into specific cell types and supporting gene editing/correction. Furthermore, we developed a direct differentiation method to obtain a pancreatic lineage from mouse and human pluripotent stem cells via the protein transduction of three transcription factors, Pdx1, NeuroD, and MafA. Here, we discuss the possibility of using CPPs as a means of directing the differentiation of iPS cells and other stem cell technologies.

  19. The influence of cell penetrating peptide branching on cellular uptake of QDs

    NASA Astrophysics Data System (ADS)

    Breger, Joyce; Delehanty, James; Susumu, Kimihiro; Anderson, George; Muttenhaler, Markus; Dawson, Philip; Medintz, Igor

    2016-03-01

    Semiconductor quantum dots (QDs) serve as a valuable platform for understating the intricacies of nanoparticle cellular uptake and fate for the development of theranostics. Developing novel internalization peptides that maximize cellular uptake while minimizing the amount of peptide is important to allow space on the nanoparticle for other cargo (e.g. drugs). We have designed a range of branched, dendritic internalization peptides composed of polyarginine (Arg9) branches (1 to 16 repeats) attached a dendritic wedge based on the sequence WP9G2H6. By attaching these branched dendritic peptides to QD's, we can study the influence of branching on cellular uptake as a function of time, ratio, and degree of branching.

  20. Anti-HIV screening for cell-penetrating peptides using chloroquine and identification of anti-HIV peptides derived from matrix proteins.

    PubMed

    Mizuguchi, Takaaki; Ohashi, Nami; Nomura, Wataru; Komoriya, Mao; Hashimoto, Chie; Yamamoto, Naoki; Murakami, Tsutomu; Tamamura, Hirokazu

    2015-08-01

    Previously, compounds which inhibit the HIV-1 replication cycle were found in overlapping peptide libraries covering the whole sequence of an HIV-1 matrix (MA) protein constructed with the addition of an octa-arginyl group. The two top lead compounds are sequential fragments MA-8L and MA-9L. In the present study, the addition of chloroquine in cell-based anti-HIV assays was proven to be an efficient method with which to find anti-HIV compounds among several peptides conjugated by cell-penetrating signals such as an octa-arginyl group: the conjugation of an octa-arginyl group to individual peptides contained in whole proteins in combination with the addition of chloroquine in cells is a useful assay method to search active peptides. To find more potent fragment peptides, individual peptides between MA-8L and MA-9L, having the same peptide chain length but with sequences shifted by one amino acid residue, were synthesized in this paper and their anti-HIV activity was evaluated with an anti-HIV assay using chloroquine. As a result, the peptides in the C-terminal side of the series, which are relatively close to MA-9L, showed more potent inhibitory activity against both X4-HIV-1 and R5-HIV-1 than the peptides in the N-terminal side.

  1. H(II) mesophase and peptide cell-penetrating enhancers for improved transdermal delivery of sodium diclofenac.

    PubMed

    Cohen-Avrahami, Marganit; Aserin, Abraham; Garti, Nissim

    2010-06-01

    This study develops a novel transdermal delivery vehicle for the enhanced delivery of sodium diclofenac (Na-DFC). The system utilizes the advantages of reversed hexagonal lyotropic liquid crystals (H(II)LC), combined with a peptide cell penetration enhancer (CPE), creating together an adaptable system that provides versatile options in the field of transdermal delivery. This enhancer peptide is based on a family of amphipatic peptides that exhibit improved membrane permeability. Franz permeation cell experiments revealed that the peptide enhancer (RALA) improved Na-DFC skin penetration of the liquid crystal 2.2-fold. We studied the structural effects of RALA solubilization on the H(II) mesophase. RALA acts as a chaotropic agent, interfering in the structure of the water, and causes a measurable swelling of the aqueous cylinders by 5A. Small angle X-ray scattering (SAXS) and attenuated total reflectance-Fourier transform infrared (ATR-FTIR) measurements reveal enhanced hydration of the glycerol monooleate (GMO) headgroups and a 6.5% increase in the fraction of non-freezable water resulting from RALA incorporation. RALA caused a gradual increase in the GMO effective headgroup area due to the hydration, leading eventually to a transform of the hexagonal structure towards a lamellar one. Circular dichroism and ATR-FTIR measurements showed a conservation of the peptide structure when incorporated into the H(II) mesophase. The combined H(II)LC-CPE systems can serve as high potential vehicles for a variety of drugs, as they can easily be modified by varying the composition and temperature, according to the required dose and delivery features. PMID:20189781

  2. H(II) mesophase and peptide cell-penetrating enhancers for improved transdermal delivery of sodium diclofenac.

    PubMed

    Cohen-Avrahami, Marganit; Aserin, Abraham; Garti, Nissim

    2010-06-01

    This study develops a novel transdermal delivery vehicle for the enhanced delivery of sodium diclofenac (Na-DFC). The system utilizes the advantages of reversed hexagonal lyotropic liquid crystals (H(II)LC), combined with a peptide cell penetration enhancer (CPE), creating together an adaptable system that provides versatile options in the field of transdermal delivery. This enhancer peptide is based on a family of amphipatic peptides that exhibit improved membrane permeability. Franz permeation cell experiments revealed that the peptide enhancer (RALA) improved Na-DFC skin penetration of the liquid crystal 2.2-fold. We studied the structural effects of RALA solubilization on the H(II) mesophase. RALA acts as a chaotropic agent, interfering in the structure of the water, and causes a measurable swelling of the aqueous cylinders by 5A. Small angle X-ray scattering (SAXS) and attenuated total reflectance-Fourier transform infrared (ATR-FTIR) measurements reveal enhanced hydration of the glycerol monooleate (GMO) headgroups and a 6.5% increase in the fraction of non-freezable water resulting from RALA incorporation. RALA caused a gradual increase in the GMO effective headgroup area due to the hydration, leading eventually to a transform of the hexagonal structure towards a lamellar one. Circular dichroism and ATR-FTIR measurements showed a conservation of the peptide structure when incorporated into the H(II) mesophase. The combined H(II)LC-CPE systems can serve as high potential vehicles for a variety of drugs, as they can easily be modified by varying the composition and temperature, according to the required dose and delivery features.

  3. Thermodynamics of cell-penetrating HIV1 TAT peptide insertion into PC/PS/CHOL model bilayers through transmembrane pores: the roles of cholesterol and anionic lipids.

    PubMed

    Hu, Yuan; Patel, Sandeep

    2016-08-10

    Efficient delivery of pharmaceutically active molecules across cellular membranes using cell penetrating peptides (CPPs), such as the cationic human immunodeficiency virus-1 trans-acting activator of transcription peptide (HIV-1 TAT), continues to attract scientific attention in drug design and disease treatment. Experimental results show that the TAT peptide is not only capable of directly penetrating the biological membrane in a passive manner, but also forming physical, membrane-spanning pores that may facilitate transport. Experiments further show that anionic lipids accelerate peptide permeation within a range of mole percentage composition. In this work, we explored the structures and translocation thermodynamics of the cationic TAT peptide across a series of DPPC/DPPS model membranes with the presence of 0-30 mol% cholesterol. We computed the potentials of the mean force by using umbrella sampling molecular dynamics simulations coupled to the Martini coarse-grained force field. We systematically investigated the roles of cholesterol and anionic lipids (membrane surface charge) in TAT peptide translocation. In qualitative agreement with experimental findings, the barrier heights were significantly reduced in the presence of anionic lipids. A toroidal hydrophilic pore was strongly suggested by membrane structure analysis. Cholesterol stabilizes the liquid-ordered (Lo) phase of membranes and increases the elastic stiffness of bilayers. Consequently, it hinders transmembrane pore formation and thus modulates solute permeability, since the liquid-ordered phase suppresses reorientation of the lipid molecules on simulation time scales. Though cholesterol contributes marginally to the total free energy associated with peptide permeation, the coordination of cholesterol to the peptide weakens more favorable peptide-lipid interactions. The addition of the anionic lipid DPPS to the neutral DPPC bilayer leads to the emergence and further enhancement of an interfacially

  4. The impact of cell-penetrating peptides on membrane bilayer structure during binding and insertion.

    PubMed

    Hirst, Daniel J; Lee, Tzong-Hsien; Kulkarni, Ketav; Wilce, Jacqueline A; Aguilar, Marie-Isabel

    2016-08-01

    We have studied the effect of penetratin and a truncated analogue on the bilayer structure using dual polarisation interferometry, to simultaneously measure changes in mass per unit area and birefringence (an optical parameter representing bilayer order) with high sensitivity during the binding and dissociation from the membrane. Specifically, we studied penetratin (RQIKIWFQNRRMKWKK), along with a shortened and biotinylated version known as R8K-biotin (RRMKWKKK(Biotin)-NH2). Overall both peptides bound only weakly to the neutral DMPC and POPC bilayers, while much higher binding was observed for the anionic DMPC/DMPG and POPC/POPG. The binding of penetratin to gel-phase DMPC/DMPG was adequately represented by a two-state model, whereas on the fluid-phase POPC/POPG it exhibited a distinctly different binding pattern, best represented by a three-state kinetic model. However, R8K-biotin did not bind well to DMPC/DMPG and showed a more transitory and superficial binding to POPC/POPG. Comparing the modelling results for both peptides binding to POPC/POPG suggests an important role for a securely bound intermediate prior to penetratin insertion and translocation. Overall these results further elucidate the mechanism of penetratin, and provide another example of the significance of the ability of DPI to measure structural changes and the use of kinetic analysis to investigate the stages of peptide-membrane interactions. PMID:27163492

  5. Human antimicrobial peptide histatin 5 is a cell-penetrating peptide targeting mitochondrial ATP synthesis in Leishmania.

    PubMed

    Luque-Ortega, Juan Román; van't Hof, Wim; Veerman, Enno C I; Saugar, José M; Rivas, Luis

    2008-06-01

    Histatin 5 (Hst5) is a human salivary antimicrobial peptide that targets fungal mitochondria. In the human parasitic protozoa Leishmania, the mitochondrial ATP production is essential, as it lacks the bioenergetic switch between glycolysis and oxidative phosphorylation described in some yeasts. On these premises, Hst5 activity was assayed on both stages of its life cycle, promastigotes and amastigotes (LC(50)=7.3 and 14.4 microM, respectively). In a further step, its lethal mechanism was studied. The main conclusions drawn were as follows: 1) Hst5 causes limited and temporary damage to the plasma membrane of the parasites, as assessed by electron microscopy, depolarization, and entrance of the vital dye SYTOX Green; 2) Hst5 translocates into the cytoplasm of Leishmania in an achiral receptor-independent manner with accumulation into the mitochondrion, as shown by confocal microscopy; and 3) Hst5 produces a bioenergetic collapse of the parasite, caused essentially by the decrease of mitochondrial ATP synthesis through inhibition of F(1)F(0)-ATPase, with subsequent fast ATP exhaustion. By using the Hst5 enantiomer, it was found that the key steps of its lethal mechanism involved no chiral recognition. Hst5 thus constitutes the first leishmanicidal peptide with a defined nonstereospecific intracellular target. The prospects of its development, by its own or as a carrier molecule for other leishmanicidal molecules, into a novel anti-Leishmania drug with a preferential subcellular accumulation are discussed. PMID:18230684

  6. Dual Receptor Recognizing Cell Penetrating Peptide for Selective Targeting, Efficient Intratumoral Diffusion and Synthesized Anti-Glioma Therapy

    PubMed Central

    Liu, Yayuan; Mei, Ling; Xu, Chaoqun; Yu, Qianwen; Shi, Kairong; Zhang, Li; Wang, Yang; Zhang, Qianyu; Gao, Huile; Zhang, Zhirong; He, Qin

    2016-01-01

    Cell penetrating peptides (CPPs) were widely used for drug delivery to tumor. However, the nonselective in vivo penetration greatly limited the application of CPPs-mediated drug delivery systems. And the treatment of malignant tumors is usually followed by poor prognosis and relapse due to the existence of extravascular core regions of tumor. Thus it is important to endue selective targeting and stronger intratumoral diffusion abilities to CPPs. In this study, an RGD reverse sequence dGR was conjugated to a CPP octa-arginine to form a CendR (R/KXXR/K) motif contained tandem peptide R8-dGR (RRRRRRRRdGR) which could bind to both integrin αvβ3 and neuropilin-1 receptors. The dual receptor recognizing peptide R8-dGR displayed increased cellular uptake and efficient penetration ability into glioma spheroids in vitro. The following in vivo studies indicated the active targeting and intratumoral diffusion capabilities of R8-dGR modified liposomes. When paclitaxel was loaded in the liposomes, PTX-R8-dGR-Lip induced the strongest anti-proliferation effect on both tumor cells and cancer stem cells, and inhibited the formation of vasculogenic mimicry channels in vitro. Finally, the R8-dGR liposomal drug delivery system prolonged the medium survival time of intracranial C6 bearing mice by 2.1-fold compared to the untreated group, and achieved an exhaustive anti-glioma therapy including anti-tumor cells, anti-vasculogenic mimicry and anti-brain cancer stem cells. To sum up, all the results demonstrated that R8-dGR was an ideal dual receptor recognizing CPP with selective glioma targeting and efficient intratumoral diffusion, which could be further used to equip drug delivery system for effective glioma therapy. PMID:26877777

  7. Distal phenylalanine modification for enhancing cellular delivery of fluorophores, proteins and quantum dots by cell penetrating peptides.

    PubMed

    Sayers, E J; Cleal, K; Eissa, N G; Watson, P; Jones, A T

    2014-12-10

    For cell penetrating peptides (CPPs) to fulfil their promise as effective delivery vectors we need a better understanding of their mechanisms of cell binding and uptake. This is especially the case when they are linked to different types of cargo. Here we describe new studies based on our previous findings suggesting that, for peptide-CPP chimeras, distal hydrophobic residues upstream of the CPP sequence can have profound effects on the way they interact with cells. We studied peptides bearing an N-terminal Glycine or Phenylalanine linked via a neutral and flexible bridging group, SGSGSGSG, to three well-studied CPPs: octaarginine, penetratin and TP10. Using a combination of flow cytometry, live-cell imaging and image analysis we examined the effects of this single amino acid change on binding and uptake of Alexa488-fluorophore, bovine serum albumin and quantum dot cargoes. The influence of the glycine-phenylalanine switch for fluorophore delivery was most dramatic in TP10, increasing cellular uptake by 4.4 and 9.9 fold in non-adherent and adherent cells, respectively. Only penetratin showed effective uptake of bovine serum albumin with the phenylalanine variant showing an increase of 1.6 fold over the glycine variant. The uptake of quantum dots was most efficiently demonstrated by octaarginine, with the glycine variant increasing uptake 4.8 fold and the phenylalanine variant increasing uptake 9.5 fold over quantum dots alone. Overall the data demonstrate that hydrophobicity distal to the CPP could be utilised to enhance their capacity to bind to the cell membrane and deliver a range of macromolecules to the insides of cells.

  8. Precise quantification of cellular uptake of cell-penetrating peptides using fluorescence-activated cell sorting and fluorescence correlation spectroscopy.

    PubMed

    Rezgui, Rachid; Blumer, Katy; Yeoh-Tan, Gilbert; Trexler, Adam J; Magzoub, Mazin

    2016-07-01

    Cell-penetrating peptides (CPPs) have emerged as a potentially powerful tool for drug delivery due to their ability to efficiently transport a whole host of biologically active cargoes into cells. Although concerted efforts have shed some light on the cellular internalization pathways of CPPs, quantification of CPP uptake has proved problematic. Here we describe an experimental approach that combines two powerful biophysical techniques, fluorescence-activated cell sorting (FACS) and fluorescence correlation spectroscopy (FCS), to directly, accurately and precisely measure the cellular uptake of fluorescently-labeled molecules. This rapid and technically simple approach is highly versatile and can readily be applied to characterize all major CPP properties that normally require multiple assays, including amount taken up by cells (in moles/cell), uptake efficiency, internalization pathways, intracellular distribution, intracellular degradation and toxicity threshold. The FACS-FCS approach provides a means for quantifying any intracellular biochemical entity, whether expressed in the cell or introduced exogenously and transported across the plasma membrane. PMID:27033412

  9. Sensitivity of Saccharomyces cerevisiae to the cell-penetrating antifungal peptide PAF26 correlates with endogenous nitric oxide (NO) production.

    PubMed

    Carmona, Lourdes; Gandía, Mónica; López-García, Belén; Marcos, Jose F

    2012-01-01

    PAF26 is a synthetic fungicidal hexapeptide with cell-penetration properties and non-lytic mode of action. We demonstrate herein the endogenous accumulation of reactive oxygen species (ROS) and nitric oxide (NO) in the model fungus Saccharomyces cerevisiae treated with PAF26. However, the S. cerevisiae deletion mutant of YAP1 - the major inductor of defense to oxidative stress - did not show high sensitivity to PAF26 but rather increased resistance, and its ROS accumulation did not differ from that of the parental strain. Cross-protection experiments suggest that the oxidant H(2)O(2) and PAF26 kill yeast through different pathways. Overall, the data indicate that ROS are not the primary antifungal mechanism of the peptide. On the contrary, the PAF26-induced intracellular production of NO was blocked in two distinct resistant mutants: the above mentioned Δyap1, which had the induction of NO disrupted, and the previously reported Δarg1 from the biosynthetic pathway of arginine, which has reduced basal NO levels. The NO synthase inhibitor l-NAME partially restored yeast growth in the presence of PAF26. These findings correlate antifungal activity of PAF26 with NO production and provide a plausible explanation for the resistance phenotype of Δarg1 through its involvement in NO biosynthesis. PMID:22120633

  10. Dual Targeting of Intracellular Pathogenic Bacteria with a Cleavable Conjugate of Kanamycin and an Antibacterial Cell-Penetrating Peptide.

    PubMed

    Brezden, Anna; Mohamed, Mohamed F; Nepal, Manish; Harwood, John S; Kuriakose, Jerrin; Seleem, Mohamed N; Chmielewski, Jean

    2016-08-31

    Bacterial infection caused by intracellular pathogens, such as Mycobacterium, Salmonella, and Brucella, is a burgeoning global health epidemic that necessitates urgent action. However, the therapeutic value of a number of antibiotics, including aminoglycosides, against intracellular pathogenic bacteria is compromised due to their inability to traverse eukaryotic membranes. For this significant problem to be addressed, a cleavable conjugate of the antibiotic kanamycin and a nonmembrane lytic, broad-spectrum antimicrobial peptide with efficient mammalian cell penetration, P14LRR, was prepared. This approach allows kanamycin to enter mammalian cells as a conjugate linked via a tether that breaks down in the reducing environment within cells. Potent antimicrobial activity of the P14KanS conjugate was demonstrated in vitro, and this reducible conjugate effectively cleared intracellular pathogenic bacteria within macrophages more potently than that of a conjugate lacking the disulfide moiety. Notably, successful clearance of Mycobacterium tuberculosis within macrophages was observed with the dual antibiotic conjugate, and Salmonella levels were significantly reduced in an in vivo Caenorhabditis elegans model.

  11. Harnessing the power of cell-penetrating peptides: activatable carriers for targeting systemic delivery of cancer therapeutics and imaging agents.

    PubMed

    MacEwan, Sarah R; Chilkoti, Ashutosh

    2013-01-01

    Targeted delivery of cancer therapeutics and imaging agents aims to enhance the accumulation of these molecules in a solid tumor while avoiding uptake in healthy tissues. Tumor-specific accumulation has been pursued with passive targeting by the enhanced permeability and retention effect, as well as with active targeting strategies. Active targeting is achieved by functionalization of carriers to allow specific interactions between the carrier and the tumor environment. Functionalization of carriers with ligands that specifically interact with overexpressed receptors on cancer cells represents a classic approach to active tumor targeting. Cell-penetrating peptides (CPPs) provide a non-specific and receptor-independent mechanism to enhance cellular uptake that offers an exciting alternative to traditional active targeting approaches. While the non-specificity of CPP-mediated internalization has the intriguing potential to make this approach applicable to a wide range of tumor types, their promiscuity is, however, a significant barrier to their clinical utility for systemically administered applications. Many approaches have been investigated to selectively turn on the function of systemically delivered CPP-functionalized carriers specifically in tumors to achieve targeted delivery of cancer therapeutics and imaging agents.

  12. Recombinant expression and purification of a MAP30-cell penetrating peptide fusion protein with higher anti-tumor bioactivity.

    PubMed

    Lv, Qiang; Yang, Xu-Zhong; Fu, Long-Yun; Lu, Yv-Ting; Lu, Yan-Hua; Zhao, Jian; Wang, Fu-Jun

    2015-07-01

    MAP30 (Momordica Antiviral Protein 30 Kd), a single-stranded type-I ribosome inactivating protein, possesses versatile biological activities including anti-tumor abilities. However, the low efficiency penetrating into tumor cells hampers the tumoricidal effect of MAP30. This paper describes MAP30 fused with a human-derived cell penetrating peptide HBD which overcome the low uptake efficiency by tumor cells and exhibits higher anti-tumor bioactivity. MAP30 gene was cloned from the genomic DNA of Momordica charantia and the recombinant plasmid pET28b-MAP30-HBD was established and transferred into Escherichia coli BL21 (DE3). The recombinant MAP30-HBD protein (rMAP30-HBD) was expressed in a soluble form after being induced by 0.5mM IPTG for 14h at 15°C. The recombinant protein was purified to greater than 95% purity with Ni-NTA affinity chromatography. The rMAP30-HBD protein not only has topological inactivation and protein translation inhibition activity but also showed significant improvements in cytotoxic activity compared to that of the rMAP30 protein without HBD in the tested tumor cell lines, and induced higher apoptosis rates in HeLa cells analyzed by Annexin V-FITC with FACS. This paper demonstrated a new method for improving MAP30 protein anti-tumor activity and might have potential applications in cancer therapy area.

  13. Synergistic Enhancement of Antitumor Efficacy by PEGylated Multi-walled Carbon Nanotubes Modified with Cell-Penetrating Peptide TAT

    NASA Astrophysics Data System (ADS)

    Hu, Shanshan; Wang, Tong; Pei, Xibo; Cai, He; Chen, Junyu; Zhang, Xin; Wan, Qianbing; Wang, Jian

    2016-10-01

    In the present study, a cell-penetrating peptide, the transactivating transcriptional factor (TAT) domain from HIV, was linked to PEGylated multi-walled carbon nanotubes (MWCNTs) to develop a highly effective antitumor drug delivery system. FITC was conjugated on MWCNTs-polyethylene glycol (PEG) and MWCNTs-PEG-TAT to provide fluorescence signal for tracing the cellular uptake of the nanocarrier. After loaded with an anticancer agent, doxorubicin (DOX) via π - π stacking interaction, the physicochemical characteristics, release profile and biological evaluation of the obtained nano-sized drug carrier were investigated. The DOX loaded MWCNTs-PEG and MWCNTs-PEG-TAT drug carriers both displayed appropriate particle size, excellent stability, high drug loading, and pH-dependent drug release profile. Nevertheless, compared with DOX-MWCNTs-PEG, DOX-MWCNTs-PEG-TAT showed improved cell internalization, intracellular distribution and potentiated anticancer efficacy due to the TAT-mediated membrane translocation, endosomal escape and nuclear targeting. Furthermore, the therapeutic efficacy of DOX was not compromised after being conjugated with MWCNTs-PEG-TAT and the proposed nanocarrier was also confirmed to have a good biocompatibility. In conclusion, our results suggested that the unique combination of TAT and MWCNTs as a multifunctional drug delivery system might be a powerful tool for improved anticancer drug development.

  14. A novel cell penetrating peptide carrier for the delivery of nematocidal proteins drug

    NASA Astrophysics Data System (ADS)

    Kim, Jea Hyun

    Nematodes have recently become a primary source of harmful diseases to the environment that inflict harsh damages to pine trees and marine species. However, nematodes cannot be killed by normal pesticides or chemicals due to their thick outer protective layer mainly composed of collagen and cuticles. Thus, a novel approach to trigger intracellular delivery of chemicals through the layers of nematodes is required. In this study, the selection of the novel CPP was carefully progressed through protein database and serial digested fragmentation, internalization of each amino sequence was analyzed through flow cytometry and confocal microscope. As one of the most effective CPP material, JH 1.6 was compared with other major CPPs and its cellular toxicity was investigated. Furthermore, JH 1.6 was attached to various RNA, DNA, and proteins and internalization efficiency was evaluated for mammalian cells. To examine its effects on nematodes in vivo, JH 1.6 was conjugated with nematocidal protein - botulinum neurotoxin (BnT) and treated in C.elegans as a model animal. The results showed that JH 1.6 had high relative internalization rate and low cellular toxicity compared to other major CPP such as TAT and GV1001 peptides.

  15. Cell-penetrating peptides, targeting the regulation of store-operated channels, slow decay of the progesterone-induced [Ca2+]i signal in human sperm

    PubMed Central

    Morris, Jennifer; Jones, Sarah; Howl, John; Lukanowska, Monika; Lefievre, Linda; Publicover, Stephen

    2015-01-01

    Previous work has provided evidence for involvement of store-operated channels (SOCs) in [Ca2+]i signalling of human sperm, including a contribution to the transient [Ca2+]i elevation that occurs upon activation of CatSper, a sperm-specific cation channel localized to the flagellum, by progesterone. To further investigate the potential involvement of SOCs in the generation of [Ca2+]i signals in human sperm, we have used cell-penetrating peptides containing the important basic sequence KIKKK, part of the STIM–Orai activating region/CRAC activating domain (SOAR/CAD) of the regulatory protein stromal interaction molecule 1. SOAR/CAD plays a key role in controlling the opening of SOCs, which occurs upon mobilization of stored Ca2+. Resting [Ca2+]i temporarily decreased upon application of KIKKK peptide (3–4 min), but scrambled KIKKK peptide had a similar effect, indicating that this action was not sequence-specific. However, in cells pretreated with KIKKK, the transient [Ca2+]i elevation induced by stimulation with progesterone decayed significantly more slowly than in parallel controls and in cells pretreated with scrambled KIKKK peptide. Examination of single-cell responses showed that this effect was due, at least in part, to an increase in the proportion of cells in which the initial transient was maintained for an extended period, lasting up to 10 min in a subpopulation of cells. We hypothesize that SOCs contribute to the progesterone-induced [Ca2+]i transient, and that interference with the regulatory mechanisms of SOC delays their closure, causing a prolongation of the [Ca2+]i transient. PMID:25882543

  16. S4(13)-PV cell-penetrating peptide induces physical and morphological changes in membrane-mimetic lipid systems and cell membranes: implications for cell internalization.

    PubMed

    Cardoso, Ana M S; Trabulo, Sara; Cardoso, Ana L; Lorents, Annely; Morais, Catarina M; Gomes, Paula; Nunes, Cláudia; Lúcio, Marlene; Reis, Salette; Padari, Kärt; Pooga, Margus; Pedroso de Lima, Maria C; Jurado, Amália S

    2012-03-01

    The present work aims to gain insights into the role of peptide-lipid interactions in the mechanisms of cellular internalization and endosomal escape of the S4(13)-PV cell-penetrating peptide, which has been successfully used in our laboratory as a nucleic acid delivery system. A S4(13)-PV analogue, S4(13)-PVscr, displaying a scrambled amino acid sequence, deficient cell internalization and drug delivery inability, was used in this study for comparative purposes. Differential scanning calorimetry, fluorescence polarization and X-ray diffraction at small and wide angles techniques showed that both peptides interacted with anionic membranes composed of phosphatidylglycerol or a mixture of this lipid with phosphatidylethanolamine, increasing the lipid order, shifting the phase transition to higher temperatures and raising the correlation length between the bilayers. However, S4(13)-PVscr, in contrast to the wild-type peptide, did not promote lipid domain segregation and induced the formation of an inverted hexagonal lipid phase instead of a cubic phase in the lipid systems assayed. Electron microscopy showed that, as opposed to S4(13)-PVscr, the wild-type peptide induced the formation of a non-lamellar organization in membranes of HeLa cells. We concluded that lateral phase separation and destabilization of membrane lamellar structure without compromising membrane integrity are on the basis of the lipid-driven and receptor-independent mechanism of cell entry of S4(13)-PV peptide. Overall, our results can contribute to a better understanding of the role of peptide-lipid interactions in the mechanisms of cell-penetrating peptide membrane translocation, helping in the future design of more efficient cell-penetrating peptide-based drug delivery systems.

  17. A Cancer Specific Cell-Penetrating Peptide, BR2, for the Efficient Delivery of an scFv into Cancer Cells

    PubMed Central

    Lim, Ki Jung; Sung, Bong Hyun; Shin, Ju Ri; Lee, Young Woong; Kim, Da Jung; Yang, Kyung Seok; Kim, Sun Chang

    2013-01-01

    Cell-penetrating peptides (CPPs) have proven very effective as intracellular delivery vehicles for various therapeutics. However, there are some concerns about non-specific penetration and cytotoxicity of CPPs for effective cancer treatments. Herein, based on the cell-penetrating motif of an anticancer peptide, buforin IIb, we designed several CPP derivatives with cancer cell specificity. Among the derivatives, a 17-amino acid peptide (BR2) was found to have cancer-specificity without toxicity to normal cells. After specifically targeting cancer cells through interaction with gangliosides, BR2 entered cells via lipid-mediated macropinocytosis. Moreover, BR2 showed higher membrane translocation efficiency than the well-known CPP Tat (49–57). The capability of BR2 as a cancer-specific drug carrier was demonstrated by fusion of BR2 to a single-chain variable fragment (scFv) directed toward a mutated K-ras (G12V). BR2-fused scFv induced a higher degree of apoptosis than Tat-fused scFv in K-ras mutated HCT116 cells. These results suggest that the novel cell-penetrating peptide BR2 has great potential as a useful drug delivery carrier with cancer cell specificity. PMID:23776609

  18. Targeting Multidrug-resistant Staphylococci with an anti-rpoA Peptide Nucleic Acid Conjugated to the HIV-1 TAT Cell Penetrating Peptide.

    PubMed

    Abushahba, Mostafa Fn; Mohammad, Haroon; Seleem, Mohamed N

    2016-01-01

    Staphylococcus aureus infections present a serious challenge to healthcare practitioners due to the emergence of resistance to numerous conventional antibiotics. Due to their unique mode of action, peptide nucleic acids are novel alternatives to traditional antibiotics to tackle the issue of bacterial multidrug resistance. In this study, we designed a peptide nucleic acid covalently conjugated to the HIV-TAT cell penetrating peptide (GRKKKRRQRRRYK) in order to target the RNA polymerase α subunit gene (rpoA) required for bacterial genes transcription. We explored the antimicrobial activity of the anti-rpoA construct (peptide nucleic acid-TAT) against methicillin-resistant S. aureus, vancomycin-intermediate S. aureus, vancomycin-resistant S. aureus, linezolid-resistant S. aureus, and methicillin-resistant S. epidermidis in pure culture, infected mammalian cell culture, and in an in vivo Caenorhabditis elegans infection model. The anti-rpoA construct led to a concentration-dependent inhibition of bacterial growth (at micromolar concentrations) in vitro and in both infected cell culture and in vivo in C. elegans. Moreover, rpoA gene silencing resulted in suppression of its message as well as reduced expression of two important methicillin-resistant S. aureus USA300 toxins (α-hemolysin and Panton-Valentine leukocidin). This study confirms that rpoA gene is a potential target for development of novel antisense therapeutics to treat infections caused by methicillin-resistant S. aureus. PMID:27434684

  19. Enhanced oral bioavailability of insulin using PLGA nanoparticles co-modified with cell-penetrating peptides and Engrailed secretion peptide (Sec).

    PubMed

    Zhu, Siqi; Chen, Shuangxi; Gao, Yuan; Guo, Feng; Li, Fengying; Xie, Baogang; Zhou, Jianliang; Zhong, Haijun

    2016-07-01

    Biodegradable polymer nanoparticle drug carriers are an attractive strategy for oral delivery of peptide and protein drugs. However, their ability to cross the intestinal epithelium membrane is largely limited. Therefore, in the present study, cell-penetrating peptides (R8, Tat, penetratin) and a secretion peptide (Sec) with N-terminal stearylation were introduced to modify nanoparticles (NPs) on the surface to improve oral bioavailability of peptide and protein drugs. In vitro studies conducted in Caco-2 cells showed the value of the apparent permeability coefficient (Papp) of the nanoparticles co-modified with Sec and penetratin (Sec-Pen-NPs) was about two-times greater than that of the nanoparticles modified with only penetratin (Pen-NPs), while the increase of transcellular transport of nanoparticles modified together with Sec and R8 (Sec-R8-NPs), or Sec and Tat (Sec-Tat-NPs), was not significant compared with nanoparticles modified with only R8 (R8-NPs) or Tat (Tat-NPs). Using insulin as the model drug, in vivo studies performed on rats indicated that compared to Pen-NPs, the relative bioavailability of insulin for Sec-Pen-NPs was 1.71-times increased after ileal segments administration, and stronger hypoglycemic effects was also observed. Therefore, the nanoparticles co-modified with penetratin and Sec could act as attractive carriers for oral delivery of insulin.

  20. Region-Dependent Role of Cell-Penetrating Peptides in Insulin Absorption Across the Rat Small Intestinal Membrane.

    PubMed

    Khafagy, El-Sayed; Iwamae, Ruisha; Kamei, Noriyasu; Takeda-Morishita, Mariko

    2015-11-01

    We have reported that the cell-penetrating peptide (CPP) penetratin acts as a potential absorption enhancer in oral insulin delivery systems and that this action occurs through noncovalent intermolecular interactions. However, the region-dependent role of CPPs in intestinal insulin absorption has not been clarified. To identify the intestinal region where CPPs have the most effect in increasing insulin absorption, the region-dependent action of penetratin was investigated using in situ closed intestinal loops in rats. The order of the insulin area under the insulin concentration-time curve (AUC) increase effect by L-penetratin was ileum > jejunum > duodenum > colon. By contrast, the AUC order after coadministration of insulin with D-penetratin was colon > duodenum ≥ jejunum and ileum. We also compared the effects of the L- and D-forms of penetratin, R8, and PenetraMax on ileal insulin absorption. Along with the CPPs used in this study, L- and D-PenetraMax produced the largest insulin AUCs. An absorption study using ilea pretreated with CPPs showed that PenetraMax had no irreversible effect on the intestinal epithelial membrane. The degradation of insulin in the presence of CPPs was assessed in rat intestinal enzymatic fluid. The half-life (t 1/2) of insulin increased from 14.5 to 23.7 and 184.7 min in the presence of L- and D-PenetraMax, respectively. These enzymatic degradation-resistant effects might contribute partly to the increased ileal absorption of insulin induced by D-PenetraMax. In conclusion, this study demonstrated that the ability of the L- and D-forms of penetratin to increase intestinal insulin absorption was maximal in the ileum and the colon, respectively, and that D-PenetraMax is a powerful but transient enhancer of oral insulin absorption.

  1. Performance of cell-penetrating peptide-linked polymers physically mixed with poorly membrane-permeable molecules on cell membranes.

    PubMed

    Sakuma, Shinji; Suita, Masaya; Yamamoto, Takafumi; Masaoka, Yoshie; Kataoka, Makoto; Yamashita, Shinji; Nakajima, Noriko; Shinkai, Norihiro; Yamauchi, Hitoshi; Hiwatari, Ken-Ichiro; Hashizume, Akio; Tachikawa, Hiroyuki; Kimura, Ryoji; Ishimaru, Yuki; Kasai, Atsushi; Maeda, Sadaaki

    2012-05-01

    We are investigating a new class of penetration enhancers that enable poorly membrane-permeable molecules physically mixed with them to effectively penetrate cell membranes without their concomitant cellular uptake. Since we previously revealed that poly(N-vinylacetamide-co-acrylic acid) modified with d-octaarginine, which is a typical cell-penetrating peptide, significantly enhanced the nasal absorption of insulin, we examined the performance of the polymers on cell membranes. When Caco-2 cells were incubated with 5(6)-carboxyfluorescein (CF) for 30 min, approximately 0.1% of applied CF was internalized into the cells. This poor membrane permeability was dramatically enhanced by d-octaarginine-linked polymers; a 25-fold increase in the cellular uptake of CF was observed when the polymer concentration was adjusted to 0.2mg/mL. None of the individual components, for example, d-octaarginine, had any influence on CF uptake, demonstrating that only d-octaarginine anchored chemically to the polymeric platform enhanced the membrane permeation of CF. The polymer-induced CF uptake was consistently high even when the incubation time was extended to 120 min. Confocal laser scanning microphotographs of cells incubated with d-octaarginine-linked polymers bearing rhodamine red demonstrated that the cell outline was stained with red fluorescence. The polymer-induced CF uptake was significantly suppressed by 5-(N-ethyl-N-isopropyl)amiloride, which is an inhibitor of macropinocytosis. Results indicated that d-octaarginine-linked polymers remained on the cell membrane and poorly membrane-permeable CF was continuously internalized into cells mainly via macropinocytosis repeated for the individual peptidyl branches in the polymer backbone.

  2. Effect of cell-penetrating peptide-coated nanostructured lipid carriers on the oral absorption of tripterine

    PubMed Central

    Chen, Yan; Yuan, Ling; Zhou, Lei; Zhang, Zhen-hai; Cao, Wei; Wu, Qingqing

    2012-01-01

    Purpose To develop nanostructured-lipid carriers (NLCs) coated with cell-penetrating peptides (CPP) for improving the oral bioavailability of tripterine. Methods We prepared CPP-coated tripterine-loaded NLCs (CT-NLCs) by using a solvent evaporation method, and determined their physical properties. In vitro drug release was determined by using a dialysis bag diffusion technique, and intestinal toxicity was evaluated by performing MTT assay using Caco-2 cells. In vivo absorption was studied in an in situ rat intestinal perfusion model, and oral bioavailability was examined in beagles. Results The average particle size, zeta potential, and encapsulation efficiency of the optimized CT-NLCs were 126.7 ± 9.2 nm, 28.7 ± 3.4 mV, and 72.64% ± 1.37%, respectively. The CT-NLCs showed a controlled release profile in vitro and had significantly lower intestinal cytotoxicity than the tripterine solution (P < 0.05). The absorption levels of tripterine from the CT-NLCs in the rat duodenum and jejunum were markedly higher than with tripterine-loaded NLCs without the CPP coating (T-NLCs), and with tripterine solution. Pharmacokinetic study showed that the maximum concentration of the CT-NLCs was greater than that of the T-NLCs and tripterine suspension, and that the time to maximum concentration of the CT-NLCs as well as the T-NLCs, was longer than that of the tripterine suspension. The relative oral bioavailability of the CT-NLCs compared to that of tripterine suspension and T-NLCs were 484.75% and 149.91% respectively. Conclusion The oral bioavailability of tripterine is dramatically increased by CT-NLCs. Therefore, CT-NLCs seem to be a promising carrier for oral delivery of tripterine. PMID:22942642

  3. Membrane Surface-Associated Helices Promote Lipid Interactions and Cellular Uptake of Human Calcitonin-Derived Cell Penetrating Peptides

    PubMed Central

    Herbig, Michael E.; Weller, Kathrin; Krauss, Ulrike; Beck-Sickinger, Annette G.; Merkle, Hans P.; Zerbe, Oliver

    2005-01-01

    hCT(9-32) is a human calcitonin (hCT)-derived cell-penetrating peptide that has been shown to translocate the plasma membrane of mammalian cells. It has been suggested as a cellular carrier for drugs, green fluorescent protein, and plasmid DNA. Because of its temperature-dependent cellular translocation resulting in punctuated cytoplasmatic distribution, its uptake is likely to follow an endocytic pathway. To gain insight into the molecular orientation of hCT(9-32) when interacting with lipid models, and to learn more about its mode of action, various biophysical techniques from liposome partitioning to high-resolution NMR spectroscopy were utilized. Moreover, to establish the role of individual residues for the topology of its association with the lipid membrane, two mutants of hCT(9-32), i.e., W30-hCT(9-32) and A23-hCT(9-32), were also investigated. Although unstructured in aqueous solution, hCT(9-32) adopted two short helical stretches when bound to dodecylphosphocholine micelles, extending from Thr10 to Asn17 and from Gln24 to Val29. A23-hCT(9-32), in which the helix-breaking Pro23 was replaced by Ala, displayed a continuous α-helix extending from residue 12 to 26. Probing with the spin label 5-doxylstearate revealed that association with dodecylphosphocholine micelles was such that the helix engaged in parallel orientation to the micelle surface. Moreover, the Gly to Trp exchange in W30-hCT(9-32) resulted in a more stable anchoring of the C-terminal segment close to the interface, as reflected by a twofold increase in the partition coefficient in liposomes. Interestingly, tighter binding to model membranes was associated with an increase in the in vitro uptake in human cervix epithelial andenocarcinoma cell line cells. Liposome leakage studies excluded pore formation, and the punctuated fluorescence pattern of internalized peptide indicated vesicular localization and, in conclusion, strongly suggested an endocytic pathway of translocation. PMID:16183886

  4. Comparison of the efficiency of complexes based on S4(13)-PV cell-penetrating peptides in plasmid DNA and siRNA delivery.

    PubMed

    Cardoso, Ana M; Trabulo, Sara; Cardoso, Ana L; Maia, Sílvia; Gomes, Paula; Jurado, Amália S; Pedroso de Lima, Maria C

    2013-07-01

    The successful application of gene therapy approaches is highly dependent on the efficient delivery of nucleic acids into target cells. In the present study, new peptide-based nonviral systems were developed to enhance plasmid DNA and siRNA delivery, aiming at generating appropriate gene delivery and gene silencing tools for preclinical and clinical application. For this purpose, a new cell-penetrating peptide derived from the wild-type S4(13)-PV peptide was synthesized through the addition of a five-histidine tail to its N-terminus (H5-S4(13)-PV), and its ability to mediate gene expression and gene silencing was evaluated and compared to that of the wild-type peptide. The histidine-enriched peptide, H5-S4(13)-PV, proved to be generally more efficient and less toxic than the wild-type peptide in the delivery of plasmid DNA. In addition, complexes of H5-S4(13)-PV with siRNAs, but not of S4(13)-PV, were efficiently internalized by cells and presented high knockdown activity (63%). Interestingly, systems containing the S4(13)-PV or the H5-S4(13)-PV peptide exhibited superior biological activity when compared to those containing the reverse NLS or scrambled peptides, suggesting that both the cell-penetrating sequence and the NLS of the S4(13)-PV peptide influence the competence of binary and ternary complexes to accomplish nucleic acid delivery. In order to unravel the cancer therapeutic potential of formulations with the histidine-enriched peptide, their efficiency to mediate silencing of the oncogenic protein survivin was evaluated. As opposed to complexes with the wild-type peptide, H5-S4(13)-PV complexes showed the ability to promote a high survivin knockdown at the level of both protein (44%) and mRNA (73%), in HT1080 cells.

  5. Hierarchy of Specific Lipid-Peptide Interactions Produces the Activity of Cell-penetrating and Cell-permeating Peptides

    NASA Astrophysics Data System (ADS)

    Davis, Matthew; Parente, Daniel; Gordon, Vernita; Mishra, Abhijit; Schmidt, Nathan; Yang, Lihua; Coridan, Robert; Som, Abhigyan; Tew, Gregory; Wong, Gerard

    2008-03-01

    Protein transduction domains can cross cell membranes with high efficiency, even when carrying a variety of cargos, and thus has strong biotechnological potential. The molecular mechanism of entry, however, is not well understood. We use small-angle x-ray scattering (SAXS) and confocal microscopy to systematically study the interaction of the TAT and ANTP PTD with model membranes of variable composition. Their membrane transduction activity requires the presence of both PE and PS lipids in the membrane. Antimicrobial peptides (AMP's) are cationic amphiphiles that comprise a key component of innate immunity. Synthetic analogs of AMP's, such as the family of phenylene ethynylene antimicrobial oligomers (AMO's), recently demonstrated broad-spectrum antimicrobial activity, but the underlying molecular mechanism is unknown. PE lipid greatly enhances permeating activity of AMO in these membranes, showing the importance of specific lipid composition for the activity of cell-permeating peptides. Since bacterial cell membranes are richer in PE lipids than are eukaryotic cell membranes, this may indicate a mechanism for antimicrobial specificity.

  6. Design and mechanism of action of a novel bacteria-selective antimicrobial peptide from the cell-penetrating peptide Pep-1

    SciTech Connect

    Zhu, W.L.; Lan Hongliang; Park, Il-Seon; Kim, Jae Il; Jin, H.Z.; Hahm, Kyung-Soo; Shin, S.Y. . E-mail: syshin@chosun.ac.kr

    2006-10-20

    Here, we report the successful design of a novel bacteria-selective antimicrobial peptide, Pep-1-K (KKTWWKTWWTKWSQPKKKRKV). Pep-1-K was designed by replacing Glu-2, Glu-6, and Glu-11 in the cell-penetrating peptide Pep-1 with Lys. Pep-1-K showed strong antibacterial activity against reference strains (MIC = 1-2 {mu}M) of Gram-positive and Gram-negative bacteria as well as against clinical isolates (MIC = 1-8 {mu}M) of methicillin-resistant Staphylococcus aureus and multidrug-resistant Pseudomonas aeruginosa. In contrast, Pep-1-K did not cause hemolysis of human erythrocytes even at 200 {mu}M. These results indicate that Pep-1-K may be a good candidate for antimicrobial drug development, especially as a topical agent against antibiotic-resistant microorganisms. Tryptophan fluorescence studies indicated that the lack of hemolytic activity of Pep-1-K correlated with its weak ability to penetrate zwitterionic phosphatidylcholine/cholesterol (10:1, w/w) vesicles, which mimic eukaryotic membranes. Furthermore, Pep-1-K caused little or no dye leakage from negatively charged phosphatidylethanolamine/phosphatidylglycerol (7:3, w/w) vesicles, which mimic bacterial membranes but had a potent ability to cause depolarization of the cytoplasmic membrane potential of intact S. aureus cells. These results suggested that Pep-1-K kills microorganisms by not the membrane-disrupting mode but the formation of small channels that permit transit of ions or protons but not molecules as large as calcein.

  7. Quantification of Cell-Penetrating Peptide Associated with Polymeric Nanoparticles Using Isobaric-Tagging and MALDI-TOF MS/MS

    NASA Astrophysics Data System (ADS)

    Chiu, Jasper Z. S.; Tucker, Ian G.; McDowell, Arlene

    2016-09-01

    High sensitivity quantification of the putative cell-penetrating peptide di-arginine-histidine (RRH) associated with poly (ethyl-cyanoacrylate) (PECA) nanoparticles was achieved without analyte separation, using a novel application of isobaric-tagging and high matrix-assisted laser desorption/ionization coupled to time-of-flight (MALDI-TOF) mass spectrometry. Isobaric-tagging reaction equilibrium was reached after 5 min, with 90% or greater RRH peptide successfully isobaric-tagged after 60 min. The accuracy was greater than 90%, which indicates good reliability of using isobaric-tagged RRH as an internal standard for RRH quantification. The sample intra- and inter-spot coefficients of variations were less than 11%, which indicate good repeatability. The majority of RRH peptides in the nanoparticle formulation were physically associated with the nanoparticles (46.6%), whereas only a small fraction remained unassociated (13.7%). The unrecovered RRH peptide (~40%) was assumed to be covalently associated with PECA nanoparticles.

  8. Quantification of Cell-Penetrating Peptide Associated with Polymeric Nanoparticles Using Isobaric-Tagging and MALDI-TOF MS/MS

    NASA Astrophysics Data System (ADS)

    Chiu, Jasper Z. S.; Tucker, Ian G.; McDowell, Arlene

    2016-11-01

    High sensitivity quantification of the putative cell-penetrating peptide di-arginine-histidine (RRH) associated with poly (ethyl-cyanoacrylate) (PECA) nanoparticles was achieved without analyte separation, using a novel application of isobaric-tagging and high matrix-assisted laser desorption/ionization coupled to time-of-flight (MALDI-TOF) mass spectrometry. Isobaric-tagging reaction equilibrium was reached after 5 min, with 90% or greater RRH peptide successfully isobaric-tagged after 60 min. The accuracy was greater than 90%, which indicates good reliability of using isobaric-tagged RRH as an internal standard for RRH quantification. The sample intra- and inter-spot coefficients of variations were less than 11%, which indicate good repeatability. The majority of RRH peptides in the nanoparticle formulation were physically associated with the nanoparticles (46.6%), whereas only a small fraction remained unassociated (13.7%). The unrecovered RRH peptide (~40%) was assumed to be covalently associated with PECA nanoparticles.

  9. Effects of pyrenebutyrate on the translocation of arginine-rich cell-penetrating peptides through artificial membranes: recruiting peptides to the membranes, dissipating liquid-ordered phases, and inducing curvature.

    PubMed

    Katayama, Sayaka; Nakase, Ikuhiko; Yano, Yoshiaki; Murayama, Tomo; Nakata, Yasushi; Matsuzaki, Katsumi; Futaki, Shiroh

    2013-09-01

    Arginine-rich cell-penetrating peptides, including octaarginine (R8) and HIV-1 TAT peptides, have the ability to translocate through cell membranes and transport exogenous bioactive molecules into cells. Hydrophobic counteranions such as pyrenebutyrate (PyB) have been reported to markedly promote the membrane translocation of these peptides. In this study, using model membranes having liquid-ordered (Lo) and liquid-disordered (Ld) phases, we explored the effects of PyB on the promotion of R8 translocation. Confocal microscopic observations of giant unilamellar vesicles (GUVs) showed that PyB significantly accelerated the accumulation of R8 on membranes containing negatively charged lipids, leading to the internalization of R8 without collapse of the GUV structures. PyB displayed an alternative activity, increasing the fluidity of the negatively charged membranes, which diminished the distinct Lo/Ld phase separation on GUVs. This was supported by the decrease in fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH). Additionally, PyB induced membrane curvature, which has been suggested as a possible mechanism of membrane translocation for R8. Taken together, our results indicate that PyB may have multiple effects that promote R8 translocation through cell membranes.

  10. Folic Acid-Targeted and Cell Penetrating Peptide-Mediated Theranostic Nanoplatform for High-Efficiency Tri-Modal Imaging-Guided Synergistic Anticancer Phototherapy.

    PubMed

    Li, Na; Li, Tingting; Liu, Chen; Ye, Shiyi; Liang, Jiangong; Han, Heyou

    2016-05-01

    A novel nanomaterial with precisely-defined size and shape, biocompatible composition, and excellent stability, which can integrate multi modal targeted imaging and therapy into a single system for visualized therapeutics, has recently attracted significant research interest. Here, we developed a multifunctional nanoplatform based on silica-coated 4-mercaptobenzoic acid-modified gold nanorods (Au NRs) decorated with gold nanoclusters rich in the photosensitizer Ce6 (Au-Ce6 NCs). The nanoparticles also comprised folic acid and cell penetrating peptide molecules anchored on the surface, obtaining the Au@SiO2@Au-cell penetrating peptide nanocomposite. The Au-Ce6 NCs enhanced the photophysical stability, provided numerous bonding sites and offered a large surface-area and interior space to achieve a high drug loading efficiency (up to 55%). The anchored folic acid and cell penetrating peptide synergistically enhanced the efficiency of uptake of nanocomposites by HeLa cells (up to 70.7%) and improved therapeutic efficacy. The nanocomposite also has good water-solubility, excellent biocompatibility, and long-term stability against illumination and exposure to pH 3-12, thus facilitating their bioapplications in cancer theranostics. Here, the nanocomposite was established for high-resolution and noninvasive tri-modal surface-enhanced Raman spectrum/dark-field/fluorescence imaging-guided high-efficiency synergistic photodynamic/photothermal therapy of cancer. Our studies demonstrate that the multifunctional nanocomposite has the potential as a novel and sensitive contrast agent for complementary and synergistic theranostics in the clinic.

  11. Folic Acid-Targeted and Cell Penetrating Peptide-Mediated Theranostic Nanoplatform for High-Efficiency Tri-Modal Imaging-Guided Synergistic Anticancer Phototherapy.

    PubMed

    Li, Na; Li, Tingting; Liu, Chen; Ye, Shiyi; Liang, Jiangong; Han, Heyou

    2016-05-01

    A novel nanomaterial with precisely-defined size and shape, biocompatible composition, and excellent stability, which can integrate multi modal targeted imaging and therapy into a single system for visualized therapeutics, has recently attracted significant research interest. Here, we developed a multifunctional nanoplatform based on silica-coated 4-mercaptobenzoic acid-modified gold nanorods (Au NRs) decorated with gold nanoclusters rich in the photosensitizer Ce6 (Au-Ce6 NCs). The nanoparticles also comprised folic acid and cell penetrating peptide molecules anchored on the surface, obtaining the Au@SiO2@Au-cell penetrating peptide nanocomposite. The Au-Ce6 NCs enhanced the photophysical stability, provided numerous bonding sites and offered a large surface-area and interior space to achieve a high drug loading efficiency (up to 55%). The anchored folic acid and cell penetrating peptide synergistically enhanced the efficiency of uptake of nanocomposites by HeLa cells (up to 70.7%) and improved therapeutic efficacy. The nanocomposite also has good water-solubility, excellent biocompatibility, and long-term stability against illumination and exposure to pH 3-12, thus facilitating their bioapplications in cancer theranostics. Here, the nanocomposite was established for high-resolution and noninvasive tri-modal surface-enhanced Raman spectrum/dark-field/fluorescence imaging-guided high-efficiency synergistic photodynamic/photothermal therapy of cancer. Our studies demonstrate that the multifunctional nanocomposite has the potential as a novel and sensitive contrast agent for complementary and synergistic theranostics in the clinic. PMID:27305812

  12. Using the peptide BP100 as a cell-penetrating tool for the chemical engineering of actin filaments within living plant cells.

    PubMed

    Eggenberger, Kai; Mink, Christian; Wadhwani, Parvesh; Ulrich, Anne S; Nick, Peter

    2011-01-01

    The delivery of externally applied macromolecules or nanoparticles into living cells still represents a critically limiting step before the full capabilities of chemical engineering can be explored. Molecular transporters such as cell-penetrating peptides, peptoids, and other mimetics can be used to carry cargo across the cellular membrane, but it is still difficult to find suitable sequences that operate efficiently for any particular type of cell. Here we report that BP100 (KKLFKKILKYL-amide), originally designed as an antimicrobial peptide against plant pathogens, can be employed as a fast and efficient cell-penetrating agent to transport fluorescent test cargoes into the cytosol of walled plant cells. The uptake of BP100 proceeds slightly more slowly than the endocytosis of fluorescent dextranes, but BP100 accumulates more efficiently and to much higher levels (by an order of magnitude). The entry of BP100 can be efficiently blocked by latrunculin B; this suggests that actin filaments are essential to the uptake mechanism. To test whether this novel transporter can also be used to deliver functional cargoes, we designed a fusion construct of BP100 with the actin-binding Lifeact peptide (MGVADLIKKFESISKEE). We demonstrated that the short BP100 could transport the attached 17-residue sequence quickly and efficiently into tobacco cells. The Lifeact construct retained its functionality as it successfully labeled the actin bundles that tether the nucleus in the cell center.

  13. Structural parameters modulating the cellular uptake of disulfide-rich cyclic cell-penetrating peptides: MCoTI-II and SFTI-1.

    PubMed

    D'Souza, Charlotte; Henriques, Sónia Troeira; Wang, Conan K; Craik, David J

    2014-12-17

    Peptides are emerging as a new class of therapeutics due to their high potency and specificity for a range of targets, including the inhibition of protein-protein interactions. Disulfide-rich cyclic peptides, in particular, have attracted much attention in drug design due to their ultra-stable structure. Moreover, some of them have been shown to internalize into cells, which makes them potential scaffolds to deliver pharmaceutically bioactive sequences to intracellular targets. Here we examined the effects of structural modifications on the cell-penetrating properties of two disulfide-rich cyclic cell-penetrating peptides, Momordica cochinchinensis trypsin inhibitor II (MCoTI-II) and sunflower trypsin inhibitor-1 (SFTI-1). We found that the cellular uptake of MCoTI-II can be improved by increasing the overall positive charge of the native sequence. On the other hand, mutations to SFTI-1 did not significantly influence its cellular uptake, suggesting a non-specific endocytosis-dependent mechanism of cellular uptake. This study provides an understanding of the structural features affecting the internalization of MCoTI-II and SFTI-1, and hence provides a guide for the development of these disulfide-rich cyclic scaffolds into potential drug leads. PMID:24985034

  14. Structural parameters modulating the cellular uptake of disulfide-rich cyclic cell-penetrating peptides: MCoTI-II and SFTI-1.

    PubMed

    D'Souza, Charlotte; Henriques, Sónia Troeira; Wang, Conan K; Craik, David J

    2014-12-17

    Peptides are emerging as a new class of therapeutics due to their high potency and specificity for a range of targets, including the inhibition of protein-protein interactions. Disulfide-rich cyclic peptides, in particular, have attracted much attention in drug design due to their ultra-stable structure. Moreover, some of them have been shown to internalize into cells, which makes them potential scaffolds to deliver pharmaceutically bioactive sequences to intracellular targets. Here we examined the effects of structural modifications on the cell-penetrating properties of two disulfide-rich cyclic cell-penetrating peptides, Momordica cochinchinensis trypsin inhibitor II (MCoTI-II) and sunflower trypsin inhibitor-1 (SFTI-1). We found that the cellular uptake of MCoTI-II can be improved by increasing the overall positive charge of the native sequence. On the other hand, mutations to SFTI-1 did not significantly influence its cellular uptake, suggesting a non-specific endocytosis-dependent mechanism of cellular uptake. This study provides an understanding of the structural features affecting the internalization of MCoTI-II and SFTI-1, and hence provides a guide for the development of these disulfide-rich cyclic scaffolds into potential drug leads.

  15. The potent antimicrobial properties of cell penetrating peptide-conjugated silver nanoparticles with excellent selectivity for Gram-positive bacteria over erythrocytes

    NASA Astrophysics Data System (ADS)

    Liu, Lihong; Yang, Jun; Xie, Jianping; Luo, Zhentao; Jiang, Jiang; Yang, Yi Yan; Liu, Shaomin

    2013-04-01

    Silver nanoparticles are of great interest for use as antimicrobial agents. Studies aimed at producing potent nano-silver biocides have focused on manipulation of particle size, shape, composition and surface charge. Here, we report the cell penetrating peptide catalyzed formation of antimicrobial silver nanoparticles in N,N-dimethylformamide. The novel nano-composite demonstrated a distinctly enhanced biocidal effect toward bacteria (Gram-positive Bacillus subtilis, Gram-negative Escherichia coli) and pathogenic yeast (Candida albicans), as compared to triangular and extremely small silver nanoparticles. In addition, a satisfactory biocompatibility was verified by a haemolysis test. Our results provide a paradigm in developing strategies that can maximize the silver nanoparticle application potentials while minimizing the toxic effects.Silver nanoparticles are of great interest for use as antimicrobial agents. Studies aimed at producing potent nano-silver biocides have focused on manipulation of particle size, shape, composition and surface charge. Here, we report the cell penetrating peptide catalyzed formation of antimicrobial silver nanoparticles in N,N-dimethylformamide. The novel nano-composite demonstrated a distinctly enhanced biocidal effect toward bacteria (Gram-positive Bacillus subtilis, Gram-negative Escherichia coli) and pathogenic yeast (Candida albicans), as compared to triangular and extremely small silver nanoparticles. In addition, a satisfactory biocompatibility was verified by a haemolysis test. Our results provide a paradigm in developing strategies that can maximize the silver nanoparticle application potentials while minimizing the toxic effects. Electronic supplementary information (ESI) available. See DOI: 10.1039/c3nr34254a

  16. Discovery of a non-cationic cell penetrating peptide derived from membrane-interacting human proteins and its potential as a protein delivery carrier.

    PubMed

    Young Kim, Hyo; Young Yum, Soo; Jang, Goo; Ahn, Dae-Ro

    2015-01-01

    Cell penetrating peptides (CPPs) are peptides that can be translocated into cells and used as a carrier platform for the intracellular uptake of cargo molecules. Subject to the source of CPP sequences and their positively charged nature, the cytotoxicity and immunogenicity of conventional CPPs needs to be optimized to expand their utility for biomedical applications. In addition to these safety issues, the stability of CPPs needs to be addressed since their positively charged residues are prone to interact with the biological milieu. As an effort to overcome these limitations of the current CPP technology, we isolated CPP candidate sequences and synthesized peptides from twelve isoforms of annexin, a family of membrane-interacting human proteins. The candidate screen returned a CPP rich in hydrophobic residues that showed more efficient cellular uptake than TAT-CPP. We then investigated the uptake mechanism, subcellular localization, and biophysical properties of the newly found CPP, verifying low cytotoxicity, long-term serum stability, and non-immunogenicity. Finally, model proteins conjugated to this peptide were successfully delivered into mammalian cells both in vitro and in vivo, indicating a potential use of the peptide as a carrier for the delivery of macromolecular cargos. PMID:26114640

  17. Multivalent presentation of the cell-penetrating peptide nona-arginine on a linear scaffold strongly increases its membrane-perturbing capacity.

    PubMed

    Chakrabarti, Alokta; Witsenburg, J Joris; Sinzinger, Michael D; Richter, Martin; Wallbrecher, Rike; Cluitmans, Judith C; Verdurmen, Wouter P R; Tanis, Sabine; Adjobo-Hermans, Merel J W; Rademann, Jörg; Brock, Roland

    2014-12-01

    Arginine-rich cell-penetrating peptides (CPP) are widely employed as delivery vehicles for a large variety of macromolecular cargos. As a mechanism-of-action for induction of uptake cross-linking of heparan sulfates and interaction with lipid head groups have been proposed. Here, we employed a multivalent display of the CPP nona-arginine (R9) on a linear dextran scaffold to assess the impact of heparan sulfate and lipid interactions on uptake and membrane perturbation. Increased avidity through multivalency should potentiate molecular phenomena that may only play a minor role if only individual peptides are used. To this point, the impact of multivalency has only been explored for dendrimers, CPP-decorated proteins and nanoparticles. We reasoned that multivalency on a linear scaffold would more faithfully mimic the arrangement of peptides at the membrane at high local peptide concentrations. On average, five R9 were coupled to a linear dextran backbone. The conjugate displayed a direct cytoplasmic uptake similar to free R9 at concentrations higher than 10μM. However, this uptake was accompanied by an increased membrane disturbance and cellular toxicity that was independent of the presence of heparan sulfates. In contrast, for erythrocytes, the multivalent conjugate induced aggregation, however, showed only limited membrane perturbation. Overall, the results demonstrate that multivalency of R9 on a linear scaffold strongly increases the capacity to interact with the plasma membrane. However, the induction of membrane perturbation is a function of the cellular response to peptide binding. PMID:25109936

  18. The cell-penetrating peptide domain from human heparin-binding epidermal growth factor-like growth factor (HB-EGF) has anti-inflammatory activity in vitro and in vivo

    SciTech Connect

    Lee, Jue-Yeon; Seo, Yoo-Na; Park, Hyun-Jung; Park, Yoon-Jeong; Chung, Chong-Pyoung

    2012-03-23

    Highlights: Black-Right-Pointing-Pointer HBP sequence identified from HB-EGF has cell penetration activity. Black-Right-Pointing-Pointer HBP inhibits the NF-{kappa}B dependent inflammatory responses. Black-Right-Pointing-Pointer HBP directly blocks phosphorylation and degradation of I{kappa}B{alpha}. Black-Right-Pointing-Pointer HBP inhibits nuclear translocation of NF-{kappa}B p65 subunit. -- Abstract: A heparin-binding peptide (HBP) sequence from human heparin-binding epidermal growth factor-like growth factor (HB-EGF) was identified and was shown to exhibit cell penetration activity. This cell penetration induced an anti-inflammatory reaction in lipopolysaccharide (LPS)-treated RAW 264.7 macrophages. HBP penetrated the cell membrane during the 10 min treatment and reduced the LPS-induced production of nitric oxide (NO), inducible nitric oxide synthase (iNOS), and cytokines (TNF-{alpha} and IL-6) in a concentration-dependent manner. Additionally, HBP inhibited the LPS-induced upregulation of cytokines, including TNF-{alpha} and IL-6, and decreased the interstitial infiltration of polymorphonuclear leukocytes in a lung inflammation model. HBP inhibited NF-{kappa}B-dependent inflammatory responses by directly blocking the phosphorylation and degradation of I{kappa}B{alpha} and by subsequently inhibiting the nuclear translocation of the p65 subunit of NF-{kappa}B. Taken together, this novel HBP may be potentially useful candidate for anti-inflammatory treatments and can be combined with other drugs of interest to transport attached molecules into cells.

  19. A novel cell-penetrating peptide derived from WT1 enhances p53 activity, induces cell senescence and displays antimelanoma activity in xeno- and syngeneic systems☆

    PubMed Central

    Massaoka, Mariana H.; Matsuo, Alisson L.; Figueiredo, Carlos R.; Girola, Natalia; Faria, Camyla F.; Azevedo, Ricardo A.; Travassos, Luiz R.

    2014-01-01

    The Wilms tumor protein 1 (WT1) transcription factor has been associated in malignant melanoma with cell survival and metastasis, thus emerging as a candidate for targeted therapy. A lysine–arginine rich peptide, WT1-pTj, derived from the ZF domain of WT1 was evaluated as an antitumor agent against A2058 human melanoma cells and B16F10-Nex2 syngeneic murine melanoma. Peptide WT1-pTj quickly penetrated human melanoma cells and induced senescence, recognized by increased SA-β-galactosidase activity, enhanced transcriptional activity of p53, and induction of the cell cycle inhibitors p21 and p27. Moreover, the peptide bound to p53 and competed with WT1 protein for binding to p53. WT1-pTj treatment led to sustained cell growth suppression, abrogation of clonogenicity and G2/M cell cycle arrest. Notably, in vivo studies showed that WT1-pTj inhibited both the metastases and subcutaneous growth of murine melanoma in syngeneic mice, and prolonged the survival of nude mice challenged with human melanoma cells. The 27-amino acid cell-penetrating WT1-derived peptide, depends on C3 and H16 for effective antimelanoma activity, inhibits proliferation of WT1-expressing human tumor cell lines, and may have an effective role in the treatment of WT1-expressing malignancies. PMID:24490140

  20. Structural Elucidation of the Cell-Penetrating Penetratin Peptide in Model Membranes at the Atomic Level: Probing Hydrophobic Interactions in the Blood-Brain Barrier.

    PubMed

    Bera, Swapna; Kar, Rajiv K; Mondal, Susanta; Pahan, Kalipada; Bhunia, Anirban

    2016-09-01

    Cell-penetrating peptides (CPPs) have shown promise in nonpermeable therapeutic drug delivery, because of their ability to transport a variety of cargo molecules across the cell membranes and their noncytotoxicity. Drosophila antennapedia homeodomain-derived CPP penetratin (RQIKIWFQNRRMKWKK), being rich in positively charged residues, has been increasingly used as a potential drug carrier for various purposes. Penetratin can breach the tight endothelial network known as the blood-brain barrier (BBB), permitting treatment of several neurodegenerative maladies, including Alzheimer's disease, Parkinson's disease, and Huntington's disease. However, a detailed structural understanding of penetratin and its mechanism of action is lacking. This study defines structural features of the penetratin-derived peptide, DK17 (DRQIKIWFQNRRMKWKK), in several model membranes and describes a membrane-induced conformational transition of the DK17 peptide in these environments. A series of biophysical experiments, including high-resolution nuclear magnetic resonance spectroscopy, provides the three-dimensional structure of DK17 in different membranes mimicking the BBB or total brain lipid extract. Molecular dynamics simulations support the experimental results showing preferential binding of DK17 to particular lipids at atomic resolution. The peptide conserves the structure of the subdomain spanning residues Ile6-Arg11, despite considerable conformational variation in different membrane models. In vivo data suggest that the wild type, not a mutated sequence, enters the central nervous system. Together, these data highlight important structural and functional attributes of DK17 that could be utilized in drug delivery for neurodegenerative disorders.

  1. Enhanced Cellular Uptake of Albumin-Based Lyophilisomes when Functionalized with Cell-Penetrating Peptide TAT in HeLa Cells

    PubMed Central

    van Bracht, Etienne; Versteegden, Luuk R. M.; Stolle, Sarah; Verdurmen, Wouter P. R.; Woestenenk, Rob; Raavé, René; Hafmans, Theo; Oosterwijk, Egbert; Brock, Roland; van Kuppevelt, Toin H.; Daamen, Willeke F.

    2014-01-01

    Lyophilisomes are a novel class of biodegradable proteinaceous nano/micrometer capsules with potential use as drug delivery carrier. Cell-penetrating peptides (CPPs) including the TAT peptide have been successfully implemented for intracellular delivery of a broad variety of cargos including various nanoparticulate pharmaceutical carriers. In the present study, lyophilisomes were modified using CPPs in order to achieve enhanced cellular uptake. Lyophilisomes were prepared by a freezing, annealing, and lyophilization method and a cystein-elongated TAT peptide was conjugated to the lyophilisomes using a heterobifunctional linker. Fluorescent-activated cell sorting (FACS) was utilized to acquire a lyophilisome population with a particle diameter smaller than 1000 nm. Cultured HeLa, OVCAR-3, Caco-2 and SKOV-3 cells were exposed to unmodified lyophilisomes and TAT-conjugated lyophilisomes and examined with FACS. HeLa cells were investigated in more detail using a trypan blue quenching assay, confocal microscopy, and transmission electron microscopy. TAT-conjugation strongly increased binding and cellular uptake of lyophilisomes in a time-dependent manner in vitro, as assessed by FACS. These results were confirmed by confocal microscopy. Transmission electron microscopy indicated rapid cellular uptake of TAT-conjugated lyophilisomes via phagocytosis and/or macropinocytosis. In conclusion, TAT-peptides conjugated to albumin-based lyophilisomes are able to enhance cellular uptake of lyophilisomes in HeLa cells. PMID:25369131

  2. Mechanisms of cell penetration and cytotoxicity of ultrasmall Au nanoparticles conjugated to doxorubicin and/or targeting peptides

    NASA Astrophysics Data System (ADS)

    Nadeau, Jay; Poon, Wilson; Zhang, Xuan

    2015-03-01

    The goals of this work were to determine whether conjugation of any of four selected peptides to Au nanoparticles improved their delivery to B16 melanoma in vitro and in vivo. In in vitro cytotoxicity assays, peptides and conjugates were endocytosed but did not escape from endosomes. None of the peptides showed any cytotoxicity, with or without conjugation to the nanoparticles. The combination of peptides and doxorubicin did not improve upon the cytotoxicity of gold-doxorubicin alone. We then tested targeting in vivo using inductively coupled plasma mass spectrometry to quantify the concentration of Au in the organs of B16 tumor-bearing mice 4, 24, and 72 h after intravenous Au nanoparticle injection. These experiments showed that in some cases, peptide conjugation improved upon the enhanced permeability and retention (EPR) effect. A peptide based upon the myxoma virus and the cyclic RGD peptide were both effective at tumor targeting; myxoma was more effective with un-PEGylated particles, and cRGD with PEGylated particles. The FREG and melanocyte stimulating hormone (MSH) peptides did not improve targeting. These results suggest that these peptides may improve delivery of Au particles to tumors, but also may prevent entry of particles into cell nuclei.

  3. An efficient PEGylated liposomal nanocarrier containing cell-penetrating peptide and pH-sensitive hydrazone bond for enhancing tumor-targeted drug delivery

    PubMed Central

    Ding, Yuan; Sun, Dan; Wang, Gui-Ling; Yang, Hong-Ge; Xu, Hai-Feng; Chen, Jian-Hua; Xie, Ying; Wang, Zhi-Qiang

    2015-01-01

    Cell-penetrating peptides (CPPs) as small molecular transporters with abilities of cell penetrating, internalization, and endosomal escape have potential prospect in drug delivery systems. However, a bottleneck hampering their application is the poor specificity for cells. By utilizing the function of hydration shell of polyethylene glycol (PEG) and acid sensitivity of hydrazone bond, we constructed a kind of CPP-modified pH-sensitive PEGylated liposomes (CPPL) to improve the selectivity of these peptides for tumor targeting. In CPPL, CPP was directly attached to liposome surfaces via coupling with stearate (STR) to avoid the hindrance of PEG as a linker on the penetrating efficiency of CPP. A PEG derivative by conjugating PEG with STR via acid-degradable hydrazone bond (PEG2000-Hz-STR, PHS) was synthesized. High-performance liquid chromatography and flow cytometry demonstrated that PHS was stable at normal neutral conditions and PEG could be completely cleaved from liposome surface to expose CPP under acidic environments in tumor. An optimal CPP density on liposomes was screened to guaranty a maximum targeting efficiency on tumor cells as well as not being captured by normal cells that consequently lead to a long circulation in blood. In vitro and in vivo studies indicated, in 4 mol% CPP of lipid modified system, that CPP exerted higher efficiency on internalizing the liposomes into targeted subcellular compartments while remaining inactive and free from opsonins at a maximum extent in systemic circulation. The 4% CPPL as a drug delivery system will have great potential in the clinical application of anticancer drugs in future. PMID:26491292

  4. An efficient PEGylated liposomal nanocarrier containing cell-penetrating peptide and pH-sensitive hydrazone bond for enhancing tumor-targeted drug delivery.

    PubMed

    Ding, Yuan; Sun, Dan; Wang, Gui-Ling; Yang, Hong-Ge; Xu, Hai-Feng; Chen, Jian-Hua; Xie, Ying; Wang, Zhi-Qiang

    2015-01-01

    Cell-penetrating peptides (CPPs) as small molecular transporters with abilities of cell penetrating, internalization, and endosomal escape have potential prospect in drug delivery systems. However, a bottleneck hampering their application is the poor specificity for cells. By utilizing the function of hydration shell of polyethylene glycol (PEG) and acid sensitivity of hydrazone bond, we constructed a kind of CPP-modified pH-sensitive PEGylated liposomes (CPPL) to improve the selectivity of these peptides for tumor targeting. In CPPL, CPP was directly attached to liposome surfaces via coupling with stearate (STR) to avoid the hindrance of PEG as a linker on the penetrating efficiency of CPP. A PEG derivative by conjugating PEG with STR via acid-degradable hydrazone bond (PEG2000-Hz-STR, PHS) was synthesized. High-performance liquid chromatography and flow cytometry demonstrated that PHS was stable at normal neutral conditions and PEG could be completely cleaved from liposome surface to expose CPP under acidic environments in tumor. An optimal CPP density on liposomes was screened to guaranty a maximum targeting efficiency on tumor cells as well as not being captured by normal cells that consequently lead to a long circulation in blood. In vitro and in vivo studies indicated, in 4 mol% CPP of lipid modified system, that CPP exerted higher efficiency on internalizing the liposomes into targeted subcellular compartments while remaining inactive and free from opsonins at a maximum extent in systemic circulation. The 4% CPPL as a drug delivery system will have great potential in the clinical application of anticancer drugs in future. PMID:26491292

  5. Tumor-Homing Cell-Penetrating Peptide Linked to Colloidal Mesoporous Silica Encapsulated (-)-Epigallocatechin-3-gallate as Drug Delivery System for Breast Cancer Therapy in Vivo.

    PubMed

    Ding, Jie; Yao, Jing; Xue, Jingjing; Li, Rong; Bao, Bo; Jiang, Liping; Zhu, Jun-jie; He, Zhiwei

    2015-08-19

    Chemotherapy is the use of chemical drugs to prevent cancer cell proliferation, invasion, and metastasis, but a serious obstacle is that chemotherapeutics strikes not only on cancerous cells, but also on normal cells. Thus, anticancer drugs without side effects should be developed and extracted. (-)-Epigallocatechin-3-gallate (EGCG), a major ingredient of green tea, possesses excellent medicinal values, such as anticancer effects, DNA-protective effects, etc. However, EGCG will be mostly metabolized if it is directly orally ingested. Here, we report a drug delivery system (DDS) for loading EGCG to enhance its stability, promising target and anticancer effects in vitro and in vivo. The designed DDS is composed of three main moieties: anticancer drug, EGCG; drug vector, colloidal mesoporous silica (CMS); target ligand, breast tumor-homing cell-penetrating peptide (PEGA-pVEC peptide). Based on the results of CCK-8 assay, confocal imaging, cell cycle analysis, and Western blot, the anticancer effect of EGCG was increased by loading of EGCG into CMS and CMS@peptide. In vivo treatment displayed that CMS had a not obvious influence on breast tumor bearing mice, but CMS@peptide@EGCG showed the greatest tumor inhibition rate, with about 89.66%. H&E staining of organs showed no tissue injury in all experimental groups. All the above results prove that EGCG is an excellent anticancer drug without side effects and CMS@peptide could greatly promote the efficacy of EGCG on breast tumors by targeted accumulation and release, which provide much evidence for the CMS@peptide as a promising and targeting vector for DDS. PMID:26225796

  6. Inhibition of malaria parasite Plasmodium falciparum development by crotamine, a cell penetrating peptide from the snake venom.

    PubMed

    El Chamy Maluf, S; Dal Mas, C; Oliveira, E B; Melo, P M; Carmona, A K; Gazarini, M L; Hayashi, M A F

    2016-04-01

    We show here that crotamine, a polypeptide from the South American rattlesnake venom with cell penetrating and selective anti-fungal and anti-tumoral properties, presents a potent anti-plasmodial activity in culture. Crotamine inhibits the development of the Plasmodium falciparum parasites in a dose-dependent manner [IC50 value of 1.87 μM], and confocal microscopy analysis showed a selective internalization of fluorescent-labeled crotamine into P. falciparum infected erythrocytes, with no detectable fluorescence in uninfected healthy erythrocytes. In addition, similarly to the crotamine cytotoxic effects, the mechanism underlying the anti-plasmodial activity may involve the disruption of parasite acidic compartments H(+) homeostasis. In fact, crotamine promoted a reduction of parasites organelle fluorescence loaded with the lysosomotropic fluorochrome acridine orange, in the same way as previously observed mammalian tumoral cells. Taken together, we show for the first time crotamine not only compromised the metabolism of the P. falciparum, but this toxin also inhibited the parasite growth. Therefore, we suggest this snake polypeptide as a promising lead molecule for the development of potential new molecules, namely peptidomimetics, with selectivity for infected erythrocytes and ability to inhibit the malaria infection by its natural affinity for acid vesicles. PMID:26806200

  7. Synthesis, characterization and applications of carboxylated and polyethylene-glycolated bifunctionalized InP/ZnS quantum dots in cellular internalization mediated by cell-penetrating peptides.

    PubMed

    Liu, Betty R; Winiarz, Jeffrey G; Moon, Jong-Sik; Lo, Shih-Yen; Huang, Yue-Wern; Aronstam, Robert S; Lee, Han-Jung

    2013-11-01

    Semiconductor nanoparticles, also known as quantum dots (QDs), are widely used in biomedical imaging studies and pharmaceutical research. Cell-penetrating peptides (CPPs) are a group of small peptides that are able to traverse cell membrane and deliver a variety of cargoes into living cells. CPPs deliver QDs into cells with minimal nonspecific absorption and toxic effect. In this study, water-soluble, monodisperse, carboxyl-functionalized indium phosphide (InP)/zinc sulfide (ZnS) QDs coated with polyethylene glycol lipids (designated QInP) were synthesized for the first time. The physicochemical properties (optical absorption, fluorescence and charging state) and cellular internalization of QInP and CPP/QInP complexes were characterized. CPPs noncovalently interact with QInP in vitro to form stable CPP/QInP complexes, which can then efficiently deliver QInP into human A549 cells. The introduction of 500nM of CPP/QInP complexes and QInP at concentrations of less than 1μM did not reduce cell viability. These results indicate that carboxylated and polyethylene-glycolylated (PEGylated) bifunctionalized QInP are biocompatible nanoparticles with potential for use in biomedical imaging studies and drug delivery applications. PMID:23792556

  8. Identification of a Short Cell-Penetrating Peptide from Bovine Lactoferricin for Intracellular Delivery of DNA in Human A549 Cells.

    PubMed

    Liu, Betty R; Huang, Yue-Wern; Aronstam, Robert S; Lee, Han-Jung

    2016-01-01

    Cell-penetrating peptides (CPPs) have been shown to deliver cargos, including protein, DNA, RNA, and nanomaterials, in fully active forms into live cells. Most of the CPP sequences in use today are based on non-native proteins that may be immunogenic. Here we demonstrate that the L5a CPP (RRWQW) from bovine lactoferricin (LFcin), stably and noncovalently complexed with plasmid DNA and prepared at an optimal nitrogen/phosphate ratio of 12, is able to efficiently enter into human lung cancer A549 cells. The L5a CPP delivered a plasmid containing the enhanced green fluorescent protein (EGFP) coding sequence that was subsequently expressed in cells, as revealed by real-time PCR and fluorescent microscopy at the mRNA and protein levels, respectively. Treatment with calcium chloride increased the level of gene expression, without affecting CPP-mediated transfection efficiency. Zeta-potential analysis revealed that positively electrostatic interactions of CPP/DNA complexes correlated with CPP-mediated transport. The L5a and L5a/DNA complexes were not cytotoxic. This biomimetic LFcin L5a represents one of the shortest effective CPPs and could be a promising lead peptide with less immunogenic for DNA delivery in gene therapy. PMID:26942714

  9. Identification of a Short Cell-Penetrating Peptide from Bovine Lactoferricin for Intracellular Delivery of DNA in Human A549 Cells

    PubMed Central

    Liu, Betty R.; Huang, Yue-Wern; Aronstam, Robert S.; Lee, Han-Jung

    2016-01-01

    Cell-penetrating peptides (CPPs) have been shown to deliver cargos, including protein, DNA, RNA, and nanomaterials, in fully active forms into live cells. Most of the CPP sequences in use today are based on non-native proteins that may be immunogenic. Here we demonstrate that the L5a CPP (RRWQW) from bovine lactoferricin (LFcin), stably and noncovalently complexed with plasmid DNA and prepared at an optimal nitrogen/phosphate ratio of 12, is able to efficiently enter into human lung cancer A549 cells. The L5a CPP delivered a plasmid containing the enhanced green fluorescent protein (EGFP) coding sequence that was subsequently expressed in cells, as revealed by real-time PCR and fluorescent microscopy at the mRNA and protein levels, respectively. Treatment with calcium chloride increased the level of gene expression, without affecting CPP-mediated transfection efficiency. Zeta-potential analysis revealed that positively electrostatic interactions of CPP/DNA complexes correlated with CPP-mediated transport. The L5a and L5a/DNA complexes were not cytotoxic. This biomimetic LFcin L5a represents one of the shortest effective CPPs and could be a promising lead peptide with less immunogenic for DNA delivery in gene therapy. PMID:26942714

  10. Identification of a Short Cell-Penetrating Peptide from Bovine Lactoferricin for Intracellular Delivery of DNA in Human A549 Cells.

    PubMed

    Liu, Betty R; Huang, Yue-Wern; Aronstam, Robert S; Lee, Han-Jung

    2016-01-01

    Cell-penetrating peptides (CPPs) have been shown to deliver cargos, including protein, DNA, RNA, and nanomaterials, in fully active forms into live cells. Most of the CPP sequences in use today are based on non-native proteins that may be immunogenic. Here we demonstrate that the L5a CPP (RRWQW) from bovine lactoferricin (LFcin), stably and noncovalently complexed with plasmid DNA and prepared at an optimal nitrogen/phosphate ratio of 12, is able to efficiently enter into human lung cancer A549 cells. The L5a CPP delivered a plasmid containing the enhanced green fluorescent protein (EGFP) coding sequence that was subsequently expressed in cells, as revealed by real-time PCR and fluorescent microscopy at the mRNA and protein levels, respectively. Treatment with calcium chloride increased the level of gene expression, without affecting CPP-mediated transfection efficiency. Zeta-potential analysis revealed that positively electrostatic interactions of CPP/DNA complexes correlated with CPP-mediated transport. The L5a and L5a/DNA complexes were not cytotoxic. This biomimetic LFcin L5a represents one of the shortest effective CPPs and could be a promising lead peptide with less immunogenic for DNA delivery in gene therapy.

  11. Cell-penetration by Co(III)cyclen-based peptide-cleaving catalysts selective for pathogenic proteins of amyloidoses.

    PubMed

    Chei, Woo Suk; Lee, Joo-Won; Kim, Jae Bum; Suh, Junghun

    2010-07-15

    Derivatives of the Co(III) complex of 1,4,7,10-tetraazacyclododecane (cyclen) with various organic pendants have been reported as target-selective peptide-cleaving catalysts, which can be exploited as catalytic drugs. In order to provide a firm basis for the catalytic drugs based on Co(III)cyclen, the ability of the Co(III)cyclen-containing peptide-cleaving catalysts to penetrate animal cells such as mouse fibroblast NIH-3T 3 or human embryonic kidney (HEK) 293 cells is demonstrated in the present study. Since the catalysts destroy pathogenic proteins for amyloidoses, results of the present study are expected to initiate extensive efforts to obtain therapeutically safe catalytic drugs for amyloidoses such as Alzheimer's disease, type 2 diabetes mellitus, Parkinson's disease, Huntington's disease, mad cow disease, and so on. PMID:20542701

  12. Anti-inflammatory Effect of a Cell-Penetrating Peptide Targeting the Nrf2/Keap1 Interaction

    PubMed Central

    2012-01-01

    Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is increasingly recognized as a central regulator of multiple signaling pathways in inflammation and cancer, and the ability to use chemical biological tools to investigate its biological effects is very attractive. A peptide comprising a TAT-conjugated Nrf2 sequence is shown to activate Nrf2 and its downstream target gene heme-oxygenase-1 (HO-1) in a dose-dependent manner in intact human THP-1 monocytes. Levels of Nrf2 protein peak after 3 h, whereas HO-1 mRNA and protein peak after 6 and 12 h, respectively. The peptide is also shown to inhibit the production of the pro-inflammatory cytokine TNF. The TAT-14mer constitutes a useful chemical biology tool with potential therapeutic applications. PMID:22582137

  13. Cell-penetrating peptide-doxorubicin conjugate loaded NGR-modified nanobubbles for ultrasound triggered drug delivery.

    PubMed

    Lin, Wen; Xie, Xiangyang; Deng, Jianping; Liu, Hui; Chen, Ying; Fu, Xudong; Liu, Hong; Yang, Yang

    2016-01-01

    A new drug-targeting system for CD13(+) tumors has been developed, based on ultrasound-sensitive nanobubbles (NBs) and cell-permeable peptides (CPPs). Here, the CPP-doxorubicin conjugate (CPP-DOX) was entrapped in the asparagine-glycine-arginine (NGR) peptide modified NB (CPP-DOX/NGR-NB) and the penetration of CPP-DOX was temporally masked; local ultrasound stimulation could trigger the CPP-DOX release from NB and activate its penetration. The CPP-DOX/NGR-NBs had particle sizes of about 200 nm and drug entrapment efficiency larger than 90%. In vitro release results showed that over 85% of the encapsulated DOX or CPP-DOX would release from NBs in the presence of ultrasound, while less than 1.5% of that (30 min) without ultrasound. Cell experiments showed the higher cellular CPP-DOX uptake of CPP-DOX/NGR-NB among the various NB formulations in Human fibrosarcoma cells (HT-1080, CD13(+)). The CPP-DOX/NGR-NB with ultrasound treatment exhibited an increased cytotoxic activity than the one without ultrasound. In nude mice xenograft of HT-1080 cells, CPP-DOX/NGR-NB with ultrasound showed a higher tumor inhibition effect (3.1% of T/C%, day 24), longer median survival time (50 days) and excellent body safety compared with the normal DOX injection group. These results indicate that the constructed vesicle would be a promising drug delivery system for specific cancer treatment.

  14. Cell penetrating synthetic antimicrobial peptides (SAMPs) exhibiting potent and selective killing of mycobacterium by targeting its DNA.

    PubMed

    Sharma, Aashish; Pohane, Amol Arunrao; Bansal, Sandhya; Bajaj, Avinash; Jain, Vikas; Srivastava, Aasheesh

    2015-02-23

    Naturally occurring antimicrobial peptides (AMPs) are powerful defence tools to tackle pathogenic microbes. However, limited natural production and high synthetic costs in addition to poor selectivity limit large-scale use of AMPs in clinical settings. Here, we present a series of synthetic AMPs (SAMPs) that exhibit highly selective and potent killing of Mycobacterium (minimum inhibitory concentration <20 μg mL(-1)) over E. coli or mammalian cells. These SAMPs are active against rapidly multiplying as well as growth saturated Mycobacterium cultures. These SAMPs are not membrane-lytic in nature, and are readily internalized by Mycobacterium and mammalian cells; whereas in E. coli, the lipopolysaccharide layer inhibits their cellular uptake, and hence, their antibacterial action. Upon internalization, these SAMPs interact with the unprotected genomic DNA of mycobacteria, and impede DNA-dependent processes, leading to bacterial cell death.

  15. Novel pH-sensitive charge-reversal cell penetrating peptide conjugated PEG-PLA micelles for docetaxel delivery: in vitro study.

    PubMed

    Ouahab, Ammar; Cheraga, Nihad; Onoja, Vitus; Shen, Yan; Tu, Jiasheng

    2014-05-15

    In order to create a pH-sensitive charge-reversal system for cell penetrating peptides (CPP) to prevent non-specific internalization of the drug; and concomitantly enhance the physical stability and tumor targetability of poly(ethylene glycol)-poly(d,l-lactide) (PEG-PLA) micelles, two sets of novel PEG-PLA micelles were developed. Cell penetrating decapeptide arginine-glycine (RG)5 and a pH-sensitive masking decapeptide histidine-glutamic acid (HE)5 were conjugated at the PEG free end to produce pH sensitive with peptides outside micelles (PHPO), while the pH sensitive with peptides inside micelles (PHPI) are the micelles obtained with the two peptides conjugated to the free end of the PLA block. The polymers were successfully synthesized and characterized by (1)H NMR and GPC. The mixed micelles were prepared and characterized for their loading efficiency, particle size and zeta potential. The surface charge of PHPO was greatly affected by the pH of the solution and (RG)5:(HE)5 ratio at the surface. The pH value of the solution at which the surface charge of PHPO reversed could be manipulated by the feed ratio of (RG)5-PEG-PLA (RGO) and (HE)5-PEG-PLA (HEO), hence, HEO:RGO molar ratio of 45:55 was selected for tumor targeting. Docetaxel (DTX) was sufficiently solubilized by DTX-PHPO with a loading efficiency of 90.18 ± 1.65%. At pH 7.4, DTX loaded mPEG-PLA (DTX-PM) (41.2 ± 0.3 nm), DTX-PHPO (195.3 ± 1.9 nm) and DTX-PHPI (190.9 ± 4.5 nm) showed sustained DTX release of less than 55% within 48 h. However, at pH 6.8 DTX-PHPI released 87.29 ± 0.24%, while DTX-PHPO released 70.49 ± 0.39% of the initial DTX amount within 48 h. Moreover, the physical stability of DTX-PHPO was increased due to the electrostatic interaction of the two peptides. The cellular uptake of DTX-PHPO in SGC-7901 cells and the cell killing effect tested on MCF-7 cells were enhanced by 2 folds at pH 6.8 compared to pH 7.4. Hence, DTX-PHPO is highly pH-sensitive in mildly acidic pH and exhibited

  16. Novel cell-penetrating peptide-loaded nanobubbles synergized with ultrasound irradiation enhance EGFR siRNA delivery for triple negative Breast cancer therapy.

    PubMed

    Jing, Hui; Cheng, Wen; Li, Shouqiang; Wu, Bolin; Leng, Xiaoping; Xu, Shouping; Tian, Jiawei

    2016-10-01

    The lack of safe and effective gene delivery strategies remains a bottleneck for cancer gene therapy. Here, we describe the synthesis, characterization, and application of cell-penetrating peptide (CPP)-loaded nanobubbles (NBs), which are characterized by their safety, strong penetrating power and high gene loading capability for gene delivery. An epidermal growth factor receptor (EGFR)-targeted small interfering RNA (siEGFR) was transfected into triple negative breast cancer (TNBC) cells via prepared CPP-NBs synergized with ultrasound-targeted microbubble destruction (UTMD) technology. Fluorescence microscopy showed that siEGFR and CPP were loaded on the shells of the NBs. The transfection efficiency and cell proliferation levels were evaluated by FACS and MTT assays, respectively. In addition, in vivo experiments showed that the expression of EGFR mRNA and protein could be efficiently downregulated and that the growth of a xenograft tumor derived from TNBC cells could be inhibited. Our results indicate that CPP-NBs carrying siEGFR could potentially be used as a promising non-viral gene vector that can be synergized with UTMD technology for efficient TNBC therapy. PMID:27388967

  17. A novel cell-penetrating peptide suppresses breast tumorigenesis by inhibiting β-catenin/LEF-1 signaling

    PubMed Central

    Hsieh, Tsung-Hua; Hsu, Chia-Yi; Tsai, Cheng-Fang; Chiu, Chien-Chih; Liang, Shih-Shin; Wang, Tsu-Nai; Kuo, Po-Lin; Long, Cheng-Yu; Tsai, Eing-Mei

    2016-01-01

    The inhibition of β-catenin/LEF-1 signaling is an emerging strategy in cancer therapy. However, clinical targeted treatment of the β-catenin/LEF-1 complex remains relatively ineffective. Therefore, development of specific molecular targets is a key approach for identifying new cancer therapeutics. Thus, we attempted to synthesize a peptide (TAT-NLS-BLBD-6) that could interfere with the interaction of β-catenin and LEF-1 at nuclei in human breast cancer cells. TAT-NLS-BLBD-6 directly interacted with β-catenin and inhibited breast cancer cell growth, invasion, migration, and colony formation as well as increased arrest of sub-G1 phase and apoptosis; it also suppressed breast tumor growth in nude mouse and zebrafish xenotransplantation models, showed no signs of toxicity, and did not affect body weight. Furthermore, the human global gene expression profiles and Ingenuity Pathway Analysis software showed that the TAT-NLS-BLBD-6 downstream target genes were associated with the HER-2 and IL-9 signaling pathways. TAT-NLS-BLBD-6 commonly down-regulated 27 candidate genes in MCF-7 and MDA-MB-231 cells, which are concurrent with Wnt downstream target genes in human breast cancer. Our study suggests that TAT-NLS-BLBD-6 is a promising drug candidate for the development of effective therapeutics specific for Wnt/β-catenin signaling inhibition. PMID:26750754

  18. Highly efficient delivery of functional cargoes by the synergistic effect of GAG binding motifs and cell-penetrating peptides

    PubMed Central

    Dixon, James E.; Osman, Gizem; Morris, Gavin E.; Markides, Hareklea; Rotherham, Michael; Bayoussef, Zahia; El Haj, Alicia J.; Denning, Chris; Shakesheff, Kevin M.

    2016-01-01

    Protein transduction domains (PTDs) are powerful nongenetic tools that allow intracellular delivery of conjugated cargoes to modify cell behavior. Their use in biomedicine has been hampered by inefficient delivery to nuclear and cytoplasmic targets. Here we overcame this deficiency by developing a series of novel fusion proteins that couple a membrane-docking peptide to heparan sulfate glycosaminoglycans (GAGs) with a PTD. We showed that this GET (GAG-binding enhanced transduction) system could deliver enzymes (Cre, neomycin phosphotransferase), transcription factors (NANOG, MYOD), antibodies, native proteins (cytochrome C), magnetic nanoparticles (MNPs), and nucleic acids [plasmid (p)DNA, modified (mod)RNA, and small inhibitory RNA] at efficiencies of up to two orders of magnitude higher than previously reported in cell types considered hard to transduce, such as mouse embryonic stem cells (mESCs), human ESCs (hESCs), and induced pluripotent stem cells (hiPSCs). This technology represents an efficient strategy for controlling cell labeling and directing cell fate or behavior that has broad applicability for basic research, disease modeling, and clinical application. PMID:26733682

  19. Enhanced intracellular translocation and biodistribution of gold nanoparticles functionalized with a cell-penetrating peptide (VG-21) from vesicular stomatitis virus.

    PubMed

    Tiwari, Pooja Munnilal; Eroglu, Erdal; Bawage, Swapnil Subhash; Vig, Komal; Miller, Michael E; Pillai, Shreekumar; Dennis, Vida A; Singh, Shree Ram

    2014-11-01

    Reduced toxicity and ease of modification make gold nanoparticles (GNPs) suitable for targeted delivery, bioimaging and theranostics by conjugating cell-penetrating peptides (CPPs). This study presents the biodistribution and enhanced intracellular uptake of GNPs functionalized with VG-21, a CPP derived from vesicular stomatitis virus glycoprotein (G). Cell penetrating efficiency of VG-21 was demonstrated using CellPPD web server, conjugated to GNPs and were characterized using, UV-visible and FTIR spectroscopy, transmission electron microscopy, dynamic light scattering and zeta potential. Uptake of VG-21 functionalized GNPs (fGNPs) was tested in eukaryotic cell lines, HEp-2, HeLa, Vero and Cos-7, using flow cytometry, fluorescence and transmission electron microscopy (TEM), and inductively coupled plasmon optical emission spectroscopy (ICP-OES). The effects of nanoparticles on stress and toxicity related genes were studied in HEp-2 cells. Cytokine response to fGNPs was studied in vitro and in vivo. Biodistribution of nanoparticles was studied in BALB/c mice using TEM and ICP-OES. VG-21, GNPs and fGNPs had little to no effect on cell viability. Upon exposure to fGNPs, HEp-2 cells revealed minimal down regulation of stress response genes. fGNPs displayed higher uptake than GNPs in all cell lines with highest internalization by HEp-2, HeLa and Cos-7 cells, in endocytotic vesicles and nuclei. Cytokine ELISA showed that mouse J774 cells exposed to fGNPs produced less IL-6 than did GNP-treated macrophage cells, whereas TNF-α levels were low in both treatment groups. Biodistribution studies in BALB/c mice revealed higher accumulation of fGNPs than GNPs in the liver and spleen. Histopathological analyses showed that fGNP-treated mice accumulated 35 ng/mg tissue and 20 ng/mg tissue gold in spleen and liver respectively, without any adverse effects. Likewise, serum cytokines were low in both GNP- and fGNP-treated mice. Thus, VG-21-conjugated GNPs have enhanced cellular

  20. Comparative density functional theory and density functional tight binding study of arginine and arginine-rich cell penetrating peptide TAT adsorption on anatase TiO2.

    PubMed

    Li, Wenxuan; Kotsis, Konstantinos; Manzhos, Sergei

    2016-07-20

    We present a comparative density functional theory (DFT) and density functional tight binding (DFTB) study of geometries and electronic structures of arginine (Arg), arginine adsorbed on the anatase (101) surface of titania in several adsorption configurations, and of an arginine-rich cell penetrating peptide TAT and its adsorption on the surface of TiO2. Two DFTB parameterizations are considered, tiorg-0-1/mio-1-1 and matsci-0-3. While there is good agreement in the structures and relative energies of Arg and peptide conformers between DFT and DFTB, both adsorption geometries and energies are noticeably different for Arg adsorbed on TiO2. The tiorg-0-1/mio-1-1 parameterization performs better than matsci-0-3. We relate this difference to the difference in electronic structures resulting from the two methods (DFT and DFTB) and specifically to the band alignment between the molecule and the oxide. We show that the band alignment of TAT and TiO2 modeled with DFTB is qualitatively correct but that with DFT using the PBE functional is not. This is specific to the modeling of large molecules where the HOMO is close to the conduction band of the oxide. We therefore report a case where the approximate DFT-based method - DFTB (with which the correct band structure can be effectively obtained) - performs better than the DFT itself with a functional approximation feasible for the modeling of large bio-inorganic interfaces, i.e. GGA (as opposed to hybrid functionals which are impractical at such a scale). Our results highlight the utility of the DFTB method for the modeling of bioinorganic interfaces not only from the CPU cost perspective but also from the accuracy point of view.

  1. Cellular delivery of small interfering RNA by a non-covalently attached cell-penetrating peptide: quantitative analysis of uptake and biological effect

    PubMed Central

    Veldhoen, Sandra; Laufer, Sandra D.; Trampe, Alexander; Restle, Tobias

    2006-01-01

    Cell-penetrating peptides (CPPs) have evolved as promising new tools to deliver nucleic acids into cells. So far, the majority of these delivery systems require a covalent linkage between carrier and cargo. To exploit the higher flexibility of a non-covalent strategy, we focused on the characterisation of a novel carrier peptide termed MPGα, which spontaneously forms complexes with nucleic acids. Using a luciferase-targeted small interfering RNA (siRNA) as cargo, we optimised the conditions for MPGα-mediated transfection of mammalian cells. In this system, reporter gene activity could be inhibited up to 90% with an IC50 value in the sub-nanomolar range. As a key issue, we addressed the cellular uptake mechanism of MPGα/siRNA complexes applying various approaches. First, transfection of HeLa cells with MPGα/siRNA complexes in the presence of several inhibitors of endocytosis showed a significant reduction of the RNA interference (RNAi) effect. Second, confocal laser microscopy revealed a punctual intracellular pattern rather than a diffuse distribution of fluorescently labelled RNA-cargo. These data provide strong evidence of an endocytotic pathway contributing significantly to the uptake of MPGα/siRNA complexes. Finally, we quantified the intracellular number of siRNA molecules after MPGα-mediated transfection. The amount of siRNA required to induce half maximal RNAi was 10 000 molecules per cell. Together, the combination of methods provided allows for a detailed side by side quantitative analysis of cargo internalisation and related biological effects. Thus, the overall efficiency of a given delivery technique as well as the mechanism of uptake can be assessed. PMID:17135188

  2. Prolongation of life in rats with malignant glioma by intranasal siRNA/drug codelivery to the brain with cell-penetrating peptide-modified micelles.

    PubMed

    Kanazawa, Takanori; Morisaki, Kazuki; Suzuki, Shohei; Takashima, Yuuki

    2014-05-01

    New therapeutic strategies are required to develop candidate drugs and ensure efficient delivery of these drugs to the brain and the central nervous system (CNS). Small interfering RNA (siRNA)-based therapies have been investigated as potential novel approaches for the treatment of brain disorders. Previously, we showed that Tat, a cell-penetrating peptide derived from HIV-Tat, and the modified block copolymers (MPEG-PCL-Tat) can form stable complexes with siRNA or can be loaded with an anticancer drug and efficiently deliver the drugs to the brain tissue via intranasal delivery. In this study, to develop a novel, efficient, and safe therapeutic strategy for managing brain disorders, we used MPEG-PCL-Tat micelles with a nose-to-brain delivery system to investigate its therapeutic effects on a rat model of malignant glioma using siRNA with a Raf-1 (siRaf-1)/camptothecin (CPT) codelivery system. MPEG-PCL-Tat and CPT-loaded MPEG-PCL-Tat can form a stable complex with siRNA with a particle size from 60 to 200 nm and a positive charge at N/P ratios up to 5. Additionally, MPEG-PCL-Tat/siRaf-1 and CPT-loaded MPEG-PCL-Tat/siRaf-1 have fostered cell death in rat glioma cells after the high cellular uptake of siRaf-1/drug by the MPEG-PCL-Tat carrier. Furthermore, compared to the unloaded MPEG-PCL-Tat/siRaf-1 complex, a CPT-loaded MPEG-PCL-Tat/siRaf-1 complex achieved the high therapeutic effect because of the additive effects of CPT and siRaf-1. These results indicate that drug/siRNA codelivery using MPEG-PCL-Tat nanomicelles with nose-to-brain delivery is an excellent therapeutic approach for brain and CNS diseases.

  3. Development of a novel nanoparticle by dual modification with the pluripotential cell-penetrating peptide PepFect6 for cellular uptake, endosomal escape, and decondensation of an siRNA core complex.

    PubMed

    Mitsueda, Asako; Shimatani, Yuri; Ito, Masahiro; Ohgita, Takashi; Yamada, Asako; Hama, Susumu; Gräslund, Astrid; Lindberg, Staffan; Langel, Ülo; Harashima, Hideyoshi; Nakase, Ikuhiko; Futaki, Shiroh; Kogure, Kentaro

    2013-11-01

    Development of novel devices for effective nucleotide release from nanoparticles is required to improve the functionality of nonviral delivery systems, because decondensation of nucleotide/polycation complexes is considered as a key step for cytoplasmic delivery of nucleotides. Previously, PepFect6 (PF6) comprised chloroquine analog moieties and a stearylated cell-penetrating peptide to facilitate endosomal escape and cellular uptake, respectively, was developed as a device for efficient siRNA delivery. As PF6 contains bulky chloroquine analog moieties, the polyplexes are expected to be loose structure, which facilitates decondensation. In the present study, siRNA was electrostatically condensed by PF6, and the PF6/siRNA complexes were coated with lipid membranes. The surface of the nanoparticles encapsulating the PF6/siRNA core (PF6-NP) was modified with PF6 for endosomal escape (PF6/PF6-NP). The RNAi effect of PF6/PF6-NP was compared with those of stearylated cell-penetrating peptide octaarginine (R8)-modified PF6-NP, R8-modified nanoparticles encapsulating the R8/siRNA core (R8-NP) and PF6-modified R8-NP. Nanoparticles encapsulating the PF6 polyplex, especially PF/PF-NP, showed a significant knockdown effect on luciferase activity of B16-F1 cells stably expressing luciferase. siRNA was widely distributed within the cytoplasm after transfection of the nanoparticles encapsulating the PF6 polyplex, while siRNA encapsulated in the R8-presenting nanoparticles was localized within the nuclei. Thus, the siRNA distribution was dependent on the manner of peptide-modification. In conclusion, we have successfully developed PF6/PF6-NP exhibiting a potent RNAi effect resulting from high cellular uptake, efficient endosomal escape and decondensation of the polyplexes based on the multifunctional cell penetrating peptide PF6. PF6 is therefore a useful pluripotential device for siRNA delivery. PMID:23893316

  4. Membrane damage as first and DNA as the secondary target for anti-candidal activity of antimicrobial peptide P7 derived from cell-penetrating peptide ppTG20 against Candida albicans.

    PubMed

    Li, Lirong; Song, Fengxia; Sun, Jin; Tian, Xu; Xia, Shufang; Le, Guowei

    2016-06-01

    P7, a peptide analogue derived from cell-penetrating peptide ppTG20, possesses antibacterial and antitumor activities without significant hemolytic activity. In this study, we investigated the antifungal effect of P7 and its anti-Candida acting mode in Candida albicans. P7 displayed antifungal activity against the reference C. albicans (MIC = 4 μM), Aspergilla niger (MIC = 32 μM), Aspergillus flavus (MIC = 8 μM), and Trichopyton rubrum (MIC = 16 μM). The effect of P7 on the C. albicans cell membrane was examined by investigating the calcein leakage from fungal membrane models made of egg yolk l-phosphatidylcholine/ergosterol (10 : 1, w/w) liposomes. P7 showed potent leakage effects against fungal liposomes similar to Melittin-treated cells. C. albicans protoplast regeneration assay demonstrated that P7 interacted with the C. albicans plasma membrane. Flow cytometry of the plasma membrane potential and integrity of C. albicans showed that P7 caused 60.9 ± 1.8% depolarization of the membrane potential of intact C. albicans cells and caused 58.1 ± 3.2% C. albicans cell membrane damage. Confocal laser scanning microscopy demonstrated that part of FITC-P7 accumulated in the cytoplasm. DNA retardation analysis was also performed, which showed that P7 interacted with C. albicans genomic DNA after penetrating the cell membrane, completely inhibiting the migration of genomic DNA above the weight ratio (peptide : DNA) of 6. Our results indicated that the plasma membrane was the primary target, and DNA was the secondary intracellular target of the mode of action of P7 against C. albicans. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd. PMID:27197902

  5. Conjugation of cell-penetrating peptides with poly(lactic-co-glycolic acid)-polyethylene glycol nanoparticles improves ocular drug delivery

    PubMed Central

    Vasconcelos, Aimee; Vega, Estefania; Pérez, Yolanda; Gómara, María J; García, María Luisa; Haro, Isabel

    2015-01-01

    In this work, a peptide for ocular delivery (POD) and human immunodeficiency virus transactivator were conjugated with biodegradable poly(lactic-co-glycolic acid) (PGLA)–polyethylene glycol (PEG)-nanoparticles (NPs) in an attempt to improve ocular drug bioavailability. The NPs were prepared by the solvent displacement method following two different pathways. One involved preparation of PLGA NPs followed by PEG and peptide conjugation (PLGA-NPs-PEG-peptide); the other involved self-assembly of PLGA-PEG and the PLGA-PEG-peptide copolymer followed by NP formulation. The conjugation of the PEG and the peptide was confirmed by a colorimetric test and proton nuclear magnetic resonance spectroscopy. Flurbiprofen was used as an example of an anti-inflammatory drug. The physicochemical properties of the resulting NPs (morphology, in vitro release, cell viability, and ocular tolerance) were studied. In vivo anti-inflammatory efficacy was assessed in rabbit eyes after topical instillation of sodium arachidonate. Of the formulations developed, the PLGA-PEG-POD NPs were the smaller particles and exhibited greater entrapment efficiency and more sustained release. The positive charge on the surface of these NPs, due to the conjugation with the positively charged peptide, facilitated penetration into the corneal epithelium, resulting in more effective prevention of ocular inflammation. The in vitro toxicity of the NPs developed was very low; no ocular irritation in vitro (hen’s egg test–chorioallantoic membrane assay) or in vivo (Draize test) was detected. Taken together, these data demonstrate that PLGA-PEG-POD NPs are promising vehicles for ocular drug delivery. PMID:25670897

  6. The neuroprotective efficacy of cell-penetrating peptides TAT, penetratin, Arg-9, and Pep-1 in glutamic acid, kainic acid, and in vitro ischemia injury models using primary cortical neuronal cultures.

    PubMed

    Meloni, Bruno P; Craig, Amanda J; Milech, Nadia; Hopkins, Richard M; Watt, Paul M; Knuckey, Neville W

    2014-03-01

    Cell-penetrating peptides (CPPs) are small peptides (typically 5-25 amino acids), which are used to facilitate the delivery of normally non-permeable cargos such as other peptides, proteins, nucleic acids, or drugs into cells. However, several recent studies have demonstrated that the TAT CPP has neuroprotective properties. Therefore, in this study, we assessed the TAT and three other CPPs (penetratin, Arg-9, Pep-1) for their neuroprotective properties in cortical neuronal cultures following exposure to glutamic acid, kainic acid, or in vitro ischemia (oxygen-glucose deprivation). Arg-9, penetratin, and TAT-D displayed consistent and high level neuroprotective activity in both the glutamic acid (IC50: 0.78, 3.4, 13.9 μM) and kainic acid (IC50: 0.81, 2.0, 6.2 μM) injury models, while Pep-1 was ineffective. The TAT-D isoform displayed similar efficacy to the TAT-L isoform in the glutamic acid model. Interestingly, Arg-9 was the only CPP that displayed efficacy when washed-out prior to glutamic acid exposure. Neuroprotection following in vitro ischemia was more variable with all peptides providing some level of neuroprotection (IC50; Arg-9: 6.0 μM, TAT-D: 7.1 μM, penetratin/Pep-1: >10 μM). The positive control peptides JNKI-1D-TAT (JNK inhibitory peptide) and/or PYC36L-TAT (AP-1 inhibitory peptide) were neuroprotective in all models. Finally, in a post-glutamic acid treatment experiment, Arg-9 was highly effective when added immediately after, and mildly effective when added 15 min post-insult, while the JNKI-1D-TAT control peptide was ineffective when added post-insult. These findings demonstrate that different CPPs have the ability to inhibit neurodamaging events/pathways associated with excitotoxic and ischemic injuries. More importantly, they highlight the need to interpret neuroprotection studies when using CPPs as delivery agents with caution. On a positive note, the cytoprotective properties of CPPs suggests they are ideal carrier molecules to

  7. Development of an antisense RNA delivery system using conjugates of the MS2 bacteriophage capsids and HIV-1 TAT cell-penetrating peptide.

    PubMed

    Wei, Baojun; Wei, Yuxiang; Zhang, Kuo; Wang, Jing; Xu, Ruihuan; Zhan, Sien; Lin, Guigao; Wang, Wei; Liu, Min; Wang, Lunan; Zhang, Rui; Li, Jinming

    2009-05-01

    RNA-based therapeutic strategies are used widely due to their highly specific mode of action. However, the major obstacle in any RNA-based therapy is cellular delivery and stability in the cells. The self-assembly of the MS2 bacteriophage capsids has been used to develop virus-like particles (VLPs) for drug delivery. In this study, we utilized the heterobifunctional crosslinker, sulfosuccinimidyl-4-(p-maleimidophenyl)-butyrate (sulfo-SMPB), to conjugate the human immunodeficiency virus-1 (HIV-1) Tat peptide and MS2 VLPs; the antisense RNA against the 5'-untranslated region (UTR) and the internal ribosome entry site (IRES) of the hepatitis C virus (HCV) was packaged into these particles by using a two-plasmid coexpression system. The MS2 VLPs conjugated with the Tat peptide were then transferred into Huh-7 cells containing an HCV reporter system. The packaged antisense RNA showed an inhibitory effect on the translation of HCV. This paper describes our initial results with this system using the Tat peptide. PMID:18823738

  8. Identification of a cell-penetrating peptide domain from human beta-defensin 3 and characterization of its anti-inflammatory activity

    PubMed Central

    Lee, Jue Yeon; Suh, Jin Sook; Kim, Jung Min; Kim, Jeong Hwa; Park, Hyun Jung; Park, Yoon Jeong; Chung, Chong Pyoung

    2015-01-01

    Human beta-defensins (hBDs) are crucial factors of intrinsic immunity that function in the immunologic response to a variety of invading enveloped viruses, bacteria, and fungi. hBDs can cause membrane depolarization and cell lysis due to their highly cationic nature. These molecules participate in antimicrobial defenses and the control of adaptive and innate immunity in every mammalian species and are produced by various cell types. The C-terminal 15-mer peptide within hBD3, designated as hBD3-3, was selected for study due to its cell- and skin-penetrating activity, which can induce anti-inflammatory activity in lipopolysaccharide-treated RAW 264.7 macrophages. hBD3-3 penetrated both the outer membrane of the cells and mouse skin within a short treatment period. Two other peptide fragments showed poorer penetration activity compared to hBD3-3. hBD3-3 inhibited the lipopolysaccharide-induced production of inducible nitric oxide synthase, nitric oxide, and secretory cytokines, such as interleukin-6 and tumor necrosis factor in a concentration-dependent manner. Moreover, hBD3-3 reduced the interstitial infiltration of polymorphonuclear leukocytes in a lung inflammation model. Further investigation also revealed that hBD3-3 downregulated nuclear factor kappa B-dependent inflammation by directly suppressing the degradation of phosphorylated-IκBα and by downregulating active nuclear factor kappa B p65. Our findings indicate that hBD3-3 may be conjugated with drugs of interest to ensure their proper translocation to sites, such as the cytoplasm or nucleus, as hBD3-3 has the ability to be used as a carrier, and suggest a potential approach to effectively treat inflammatory diseases. PMID:26347021

  9. Optimization of the cyclotide framework to improve cell penetration properties

    PubMed Central

    Huang, Yen-Hua; Chaousis, Stephanie; Cheneval, Olivier; Craik, David J.; Henriques, Sónia T.

    2015-01-01

    Cell penetrating peptides have been regarded as promising vectors to deliver hydrophilic molecules inside cells. Although they are great tools for research and have high potential as drug delivery systems, their application as drugs is impaired by their low stability in serum. Cyclotides, cyclic disulfide-rich peptides from plants, are ultra-stable molecules that have inspired applications in drug design as they can be used as scaffolds to stabilize linear bioactive sequences. Recently, they have also been shown to possess cell-penetrating properties. The combination of their remarkable stability and cell-penetrating properties opens new avenues for the application of peptides to bind to and inhibit intracellular proteins. Nevertheless, for a broader application of these molecules as vectors is of utmost importance to improve their cellular internalization efficiency. In this study we successfully modified MCoTI-II, one of the most widely studied cyclotide scaffolds in drug design, and improved its internalization properties. The internalization of the newly designed MCoTI-II is as efficient as the gold standard cell-penetrating peptide (CPP) TAT and maintains all the required features as a template to graft desired bioactivities. PMID:25709580

  10. Optimization of the cyclotide framework to improve cell penetration properties.

    PubMed

    Huang, Yen-Hua; Chaousis, Stephanie; Cheneval, Olivier; Craik, David J; Henriques, Sónia T

    2015-01-01

    Cell penetrating peptides have been regarded as promising vectors to deliver hydrophilic molecules inside cells. Although they are great tools for research and have high potential as drug delivery systems, their application as drugs is impaired by their low stability in serum. Cyclotides, cyclic disulfide-rich peptides from plants, are ultra-stable molecules that have inspired applications in drug design as they can be used as scaffolds to stabilize linear bioactive sequences. Recently, they have also been shown to possess cell-penetrating properties. The combination of their remarkable stability and cell-penetrating properties opens new avenues for the application of peptides to bind to and inhibit intracellular proteins. Nevertheless, for a broader application of these molecules as vectors is of utmost importance to improve their cellular internalization efficiency. In this study we successfully modified MCoTI-II, one of the most widely studied cyclotide scaffolds in drug design, and improved its internalization properties. The internalization of the newly designed MCoTI-II is as efficient as the gold standard cell-penetrating peptide (CPP) TAT and maintains all the required features as a template to graft desired bioactivities. PMID:25709580

  11. Thermal and magnetic dual-responsive liposomes with a cell-penetrating peptide-siRNA conjugate for enhanced and targeted cancer therapy.

    PubMed

    Yang, Yanfang; Xie, Xiangyang; Xu, Xueqing; Xia, Xuejun; Wang, Hongliang; Li, Lin; Dong, Wujun; Ma, Panpan; Yang, Yang; Liu, Yuling; Mei, Xingguo

    2016-10-01

    Due to the absence of effective in vivo delivery systems, the employment of small interfering RNA (siRNA) in the clinic has been hindered. Here, we describe a novel siRNA targeting system that combines features of biological (cell-permeable peptides, CPPs) and physical (magnetic) siRNA targeting for use in magnetic hyperthermia-triggered release. A siRNA-CPPs conjugate (siRNA-CPPs) was loaded into thermal and magnetic dual-responsive liposomes (TML) (siRNA-CPPs/TML), and in vitro siRNA-CPPs thermosensitive release activity, targeted cellular uptake, gene silencing efficiency, in vivo targeted delivery and in vivo antitumor activity were determined. The results demonstrated that siRNA-CPPs/TML exhibited good physicochemical properties, effective cellular uptake, endosomal escape and a significant gene silencing efficiency in MCF-7 cells in vitro. Additionally, in the in vivo study, siRNA-CPPs/TML under an alternating current (AC) magnetic field displayed a superior in vivo targeted delivery efficacy, antitumor efficacy and gene silencing efficiency in a MCF-7 xenograft murine model. In conclusion, the application of siRNA-CPPs/TML under an AC magnetic field represents a new strategy for the selective and efficient delivery of siRNA. PMID:27429294

  12. A cell-penetrating peptide analogue, P7, exerts antimicrobial activity against Escherichia coli ATCC25922 via penetrating cell membrane and targeting intracellular DNA.

    PubMed

    Li, Lirong; Shi, Yonghui; Cheng, Xiangrong; Xia, Shufang; Cheserek, Maureen Jepkorir; Le, Guowei

    2015-01-01

    The antibacterial activities and mechanism of a new P7 were investigated in this study. P7 showed antimicrobial activities against five harmful microorganisms which contaminate and spoil food (MIC=4-32 μM). Flow cytometry and scanning electron microscopy analyses demonstrated that P7 induced pore-formation on the cell surface and led to morphological changes but did not lyse cell. Confocal fluorescence microscopic observations and flow cytometry analysis expressed that P7 could penetrate the Escherichia coli cell membrane and accumulate in the cytoplasm. Moreover, P7 possessed a strong DNA binding affinity. Further cell cycle analysis and change in gene expression analysis suggested that P7 induced a decreased expression in the genes involved in DNA replication. Up-regulated expression genes encoding DNA damage repair. This study suggests that P7 could be applied as a candidate for the development of new food preservatives as it exerts its antibacterial activities by penetrating cell membranes and targets intracellular DNA.

  13. Peptides Interfering 3A Protein Dimerization Decrease FMDV Multiplication

    PubMed Central

    de la Torre, Beatriz G.; Valle, Javier; Andreu, David; Sobrino, Francisco

    2015-01-01

    Nonstructural protein 3A is involved in relevant functions in foot-and-mouth disease virus (FMDV) replication. FMDV 3A can form homodimers and preservation of the two hydrophobic α-helices (α1 and α2) that stabilize the dimer interface is essential for virus replication. In this work, small peptides mimicking residues involved in the dimer interface were used to interfere with dimerization and thus gain insight on its biological function. The dimer interface peptides α1, α2 and that spanning the two hydrophobic α-helices, α12, impaired in vitro dimer formation of a peptide containing the two α-helices, this effect being higher with peptide α12. To assess the effect of dimer inhibition in cultured cells, the interfering peptides were N-terminally fused to a heptaarginine (R7) sequence to favor their intracellular translocation. Thus, when fused to R7, interference peptides (100 μM) were able to inhibit dimerization of transiently expressed 3A, the higher inhibitions being found with peptides α1 and α12. The 3A dimerization impairment exerted by the peptides correlated with significant, specific reductions in the viral yield recovered from peptide-treated FMDV infected cells. In this case, α2 was the only peptide producing significant reductions at concentrations lower than 100 μM. Thus, dimer interface peptides constitute a tool to understand the structure-function relationship of this viral protein and point to 3A dimerization as a potential antiviral target. PMID:26505190

  14. Liquid Crystalline Nanodispersions Functionalized with Cell-Penetrating Peptides for Topical Delivery of Short-Interfering RNAs: A Proposal for Silencing a Pro-Inflammatory Cytokine in Cutaneous Diseases.

    PubMed

    Petrilli, R; Eloy, J O; Praça, F S G; Del Ciampo, J O; Fantini, M A C; Fonseca, M J V; Bentley, M V L B

    2016-05-01

    Short-interfering RNAs (siRNAs) are a potential strategy for the treatment of cutaneous diseases. In this context, liquid crystalline nanoparticles functionalized with specific proteins and peptide-transduction domains (PTDs), which act as penetration enhancers, are a promising carrier for siRNA delivery through the skin. Herein, hexagonal phase liquid crystal nanoparticles based on monoolein (MO) and/or oleic acid (OA) containing (or lacking) the cationic polymer polyethylenimine (PEI) and the cationic lipid oleylamine (OAM) were functionalized with the membrane transduction peptides transcriptional activator (TAT) or penetratin (PNT). These nanoparticles were complexed with siRNA and characterized by particle size, polydispersity, zeta potential, complexation efficiency and siRNA release. The formulations containing cationic agents presented positive zeta potentials, sizes on the nanometer scale, and complexed siRNAs at concentrations of 10 μM; these agents were successfully released in a heparin competition assay. Cell culture studies demonstrated that nanoparticles composed of MO:OA:PEI functionalized with TAT were the most efficient at transfecting L929 cells, and the uptake efficiency was enhanced by TAT peptide functionalization. Thereafter, the selected formulations were evaluated for in vivo skin irritation, penetration and in vivo efficacy using a chemically induced inflammatory animal model. These nanoparticles did not irritate the skin and provided higher siRNA penetration and delivery into the skin than control formulations. Additionally, efficacy studies in the animal model showed that the association of TAT with the nanodispersion provided higher suppression of tumor necrosis factor (TNF)-α. Thus, the development of liquid crystalline nanodispersions containing TAT may lead to improved topical siRNA delivery for the treatment of inflammatory skin diseases. PMID:27305826

  15. Decreasing brain natriuretic peptide levels after treatment for hyperthyroidism.

    PubMed

    Ertugrul, Derun Taner; Yavuz, Bunyamin; Ata, Naim; Yalcin, Ahmet Arif; Kucukazman, Metin; Algul, Beyza; Dal, Kursad; Akin, Kadir Okhan; Deveci, Onur Sinan; Canbolat, Nihat; Ure, Oznur Sari; Tutal, Emre

    2009-01-01

    BNP are produced in ventricular cardiomyocytes, and secreted in response to volume expansion or pressure overload. The purpose of this study was to assess BNP levels in patients with hyperthyroidism before specific treatment for hyperthyroidism and after euthyroidism was achieved. The study was performed in a prospective design. The study population consisted of 48 consecutive newly diagnosed untreated overt hyper-thyroid patients who had not been treated any anti-thyroid medications before. All subjects underwent transt-horacic echocardiography. Levels of fT3, fT4, TSH and BNP were measured before the onset of the treatment and after euthyroidism was achieved. A significant decrease in BNP (102.5 (6.7-1769) ng/L vs. 5.0 (0.1-87.0) ng/L p< 0.001) levels were observed, after euthyroidism was achieved. The decrease in BNP levels was posi-tively correlated with the decrease in fT3 (r=0.298; p=0.049) and fT4 (r=0.313; p=0.030). There was no cor-relation between BNP levels and TSH levels (p=NS). We conclude that hyperthyroidism may cause high BNP measurements which can lead to misdiagnosis of congestive heart failure. We suggest that thyroid hormones should be checked in patients with high levels of BNP.

  16. Cell-penetrating conjugates of pentaglutamylated methotrexate as potential anticancer drugs against resistant tumor cells.

    PubMed

    Szabó, Ildikó; Orbán, Erika; Schlosser, Gitta; Hudecz, Ferenc; Bánóczi, Zoltán

    2016-06-10

    The emerging resistance of tumor cells against methotrexate (MTX) is one of the major limitations of the MTX treatment of tumorous diseases. The disturbance in the polyglutamation which is a main step in the mechanism of methotrexate action is often the reason of the resistance. Delivery of polyglutamylated MTX into cells may evade the mechanisms that are responsible for drug resistance. In this study conjugates of methotrexate and its pentaglutamylated derivatives with cell-penetrating peptides - penetratin and octaarginine - were investigated. The cellular-uptake and in vitro cytostatic activity of conjugates were examined on breast cancer cell cultures (MDA-MB-231 as resistant and MCF-7 as sensitive cell culture). These cell cultures showed very different behaviour towards the conjugates. Although the presence of pentaglutamyl moiety significantly decreased the internalisation of conjugates, some of them were significantly active in vitro. All of the conjugates were able to penetrate in some extent into both cell types, but only the conjugates of penetratin showed in vitro cytostatic activity. The most effective conjugates were the MTX-Glu5-Penetratin(desMet) and MTX-Glu5-GFLG-Penetratin(desMet). The latter was effective on both cell cultures while the former was active only on the resistant tumor cells. Our results suggest that the translocation of polyglutamylated MTX may be a new way to treat sensitive and more importantly resistant tumors. While both penetratin and octaarginine peptides were successfully used to deliver several kinds of cargos earlier in our case the activity of penetratin conjugates was more pronounced. PMID:27031212

  17. Crotamine, a small basic polypeptide myotoxin from rattlesnake venom with cell-penetrating properties.

    PubMed

    Rádis-Baptista, Gandhi; Kerkis, Irina

    2011-12-01

    Crotamine, a low molecular weight cationic polypeptide from the venom of the South American rattlesnake Crotalus durissus terrificus is a natural cell-penetrating peptide with functional versatility. The presence of nine lysine residues and three disulfide bonds renders crotamine highly compact, stable and positively charged. Topologically, crotamine adopts an ancient β-defensin fold that is found in diverse families of endogenous and venom polypeptides dedicated to host defense. Crotamine is unique among several classes of bioactive peptides because it possesses both cell penetrating and antimicrobial activities and selective biological action toward some cell types at a given cell cycle phase. Because it can rapidly and efficiently translocate into actively proliferating cells, crotamine is being investigated for labeling highly replicating cells and for use as a chemotherapeutic adjuvant. Peptides derived from crotamine, nucleolar targeting peptides (NrTPs), have been designed and are being studied. NrTPs retain some crotamine properties, such as efficient cellular uptake and preferential nuclear localization whereas they improve upon other properties. For example, NrTPs are smaller than crotamine, show higher preferential nucleolar localization, and better facilitate ZIP-code localization of therapeutic proteins. PMID:22204433

  18. Enthalpy-driven interactions with sulfated glycosaminoglycans promote cell membrane penetration of arginine peptides.

    PubMed

    Takechi-Haraya, Yuki; Nadai, Ryo; Kimura, Hitoshi; Nishitsuji, Kazuchika; Uchimura, Kenji; Sakai-Kato, Kumiko; Kawakami, Kohsaku; Shigenaga, Akira; Kawakami, Toru; Otaka, Akira; Hojo, Hironobu; Sakashita, Naomi; Saito, Hiroyuki

    2016-06-01

    The first step of cell membrane penetration of arginine peptides is thought to occur via electrostatic interactions between positive charges of arginine residues and negative charges of sulfated glycosaminoglycans (GAGs) on the cell surface. However, the molecular interaction of arginine peptides with GAG still remains unclear. Here, we compared the interactions of several arginine peptides of Tat, R8, and Rev and their analogues with heparin in relation to the cell membrane penetration efficiency. The high-affinity binding of arginine peptides to heparin was shown to be driven by large favorable enthalpy contributions, possibly reflecting multidentate hydrogen bondings of arginine residues with sulfate groups of heparin. Interestingly, the lysine peptides in which all arginine residues are substituted with lysine residues exhibited negligible binding enthalpy despite of their considerable binding to heparin. In CHO-K1 cells, arginine peptides exhibited a great cell-penetrating ability whereas their corresponding lysine peptides did not penetrate into cells. The degree of cell penetration of arginine peptides markedly decreased by the chlorate treatment of cells which prevents the sulfation of GAG chains. Significantly, the cell penetration efficiency of arginine peptides was found to be correlated with the favorable enthalpy of binding to heparin. These results suggest that the enthalpy-driven strong interaction with sulfated GAGs such as heparan sulfate plays a critical role in the efficient cell membrane penetration of arginine peptides. PMID:27003128

  19. Enthalpy-driven interactions with sulfated glycosaminoglycans promote cell membrane penetration of arginine peptides.

    PubMed

    Takechi-Haraya, Yuki; Nadai, Ryo; Kimura, Hitoshi; Nishitsuji, Kazuchika; Uchimura, Kenji; Sakai-Kato, Kumiko; Kawakami, Kohsaku; Shigenaga, Akira; Kawakami, Toru; Otaka, Akira; Hojo, Hironobu; Sakashita, Naomi; Saito, Hiroyuki

    2016-06-01

    The first step of cell membrane penetration of arginine peptides is thought to occur via electrostatic interactions between positive charges of arginine residues and negative charges of sulfated glycosaminoglycans (GAGs) on the cell surface. However, the molecular interaction of arginine peptides with GAG still remains unclear. Here, we compared the interactions of several arginine peptides of Tat, R8, and Rev and their analogues with heparin in relation to the cell membrane penetration efficiency. The high-affinity binding of arginine peptides to heparin was shown to be driven by large favorable enthalpy contributions, possibly reflecting multidentate hydrogen bondings of arginine residues with sulfate groups of heparin. Interestingly, the lysine peptides in which all arginine residues are substituted with lysine residues exhibited negligible binding enthalpy despite of their considerable binding to heparin. In CHO-K1 cells, arginine peptides exhibited a great cell-penetrating ability whereas their corresponding lysine peptides did not penetrate into cells. The degree of cell penetration of arginine peptides markedly decreased by the chlorate treatment of cells which prevents the sulfation of GAG chains. Significantly, the cell penetration efficiency of arginine peptides was found to be correlated with the favorable enthalpy of binding to heparin. These results suggest that the enthalpy-driven strong interaction with sulfated GAGs such as heparan sulfate plays a critical role in the efficient cell membrane penetration of arginine peptides.

  20. A single cell penetration system by ultrasonic driving

    NASA Astrophysics Data System (ADS)

    Zhou, Zhaoying; Xiao, Mingfei; Yang, Xing; Wu, Ting

    2008-12-01

    The researches of single cell's control and operation are the hotspots in whole world. Among the various technologies, the transmission of ectogenic genetic materials between cell membrane is very significant. Imitating the Chinese traditional acupuncture therapy, a new ultrasonic resonance driving method, is imported to drive a cell's penetration probe. A set of the single cell penetration system was established to perform this function. This system includes four subsystems: driving part, micromanipulation part, observation and measurement part, and actuation part. Some fish egg experiments indicate that this system is workable and effective.

  1. SAP(E) - A cell-penetrating polyproline helix at lipid interfaces.

    PubMed

    Franz, Johannes; Lelle, Marco; Peneva, Kalina; Bonn, Mischa; Weidner, Tobias

    2016-09-01

    Cell-penetrating peptides (CPPs) are short membrane-permeating amino acid sequences that can be used to deliver cargoes, e.g. drugs, into cells. The mechanism for CPP internalization is still subject of ongoing research. An interesting family of CPPs is the sweet arrow peptides - SAP(E) - which are known to adopt a polyproline II helical secondary structure. SAP(E) peptides stand out among CPPs because they carry a net negative charge while most CPPs are positively charged, the latter being conducive to electrostatic interaction with generally negatively charged membranes. For SAP(E)s, an internalization mechanism has been proposed, based on polypeptide aggregation on the cell surface, followed by an endocytic uptake. However, this process has not yet been observed directly - since peptide-membrane interactions are inherently difficult to monitor on a molecular scale. Here, we use sum frequency generation (SFG) vibrational spectroscopy to investigate molecular interactions of SAP(E) with differently charged model membranes, in both mono- and bi-layer configurations. The data suggest that the initial binding mechanism is accompanied by structural changes of the peptide. Also, the peptide-model membrane interaction depends on the charge of the lipid headgroup with phosphocholine being a favorable binding site. Moreover, while direct penetration has also been observed for some CPPs, the spectroscopy reveals that for SAP(E), its interaction with model membranes remains limited to the headgroup region, and insertion into the hydrophobic core of the lipid layer does not occur.

  2. Interaction of β(3) /β(2) -peptides, consisting of Val-Ala-Leu segments, with POPC giant unilamellar vesicles (GUVs) and white blood cancer cells (U937)--a new type of cell-penetrating peptides, and a surprising chain-length dependence of their vesicle- and cell-lysing activity.

    PubMed

    Kolesinska, Beata; Eyer, Klaus; Robinson, Tom; Dittrich, Petra S; Beck, Albert K; Seebach, Dieter; Walde, Peter

    2015-05-01

    Many years ago, β(2) /β(3) -peptides, consisting of alternatively arranged β(2) - and β(3) h-amino-acid residues, have been found to undergo folding to a unique type of helix, the 10/12-helix, and to exhibit non-polar, lipophilic properties (Helv. Chim. Acta 1997, 80, 2033). We have now synthesized such 'mixed' hexa-, nona-, dodeca-, and octadecapeptides, consisting of Val-Ala-Leu triads, with N-terminal fluorescein (FAM) labels, i.e., 1-4, and studied their interactions with POPC (=1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) giant unilamellar vesicles (GUVs) and with human white blood cancer cells U937. The methods used were microfluidic technology, fluorescence correlation spectroscopy (FCS), a flow-cytometry assay, a membrane-toxicity assay with the dehydrogenase G6PDH as enzymatic reporter, and visual microscopy observations. All β(3) /β(2) -peptide derivatives penetrate the GUVs and/or the cells. As shown with the isomeric β(3) /β(2) -, β(3) -, and β(2) -nonamers, 2, 5, and 6, respectively, the derivatives 5 and 6 consisting exclusively of β(3) - or β(2) -amino-acid residues, respectively, interact neither with the vesicles nor with the cells. Depending on the method of investigation and on the pretreatment of the cells, the β(3) /β(2) -nonamer and/or the β(3) /β(2) -dodecamer derivative, 2 and/or 3, respectively, cause a surprising disintegration or lysis of the GUVs and cells, comparable with the action of tensides, viral fusion peptides, and host-defense antimicrobial peptides. Possible sources of the chain-length-dependent destructive potential of the β(3) /β(2) -nona- and β(3) /β(2) -dodecapeptide derivatives, and a possible relationship with the phosphate-to-phosphate and hydrocarbon thicknesses of GUVs, and eukaryotic cells are discussed. Further investigations with other types of GUVs and of eukaryotic or prokaryotic cells will be necessary to elucidate the mechanism(s) of interaction of 'mixed' β(3) /β(2) -peptides with

  3. Interaction of β(3) /β(2) -peptides, consisting of Val-Ala-Leu segments, with POPC giant unilamellar vesicles (GUVs) and white blood cancer cells (U937)--a new type of cell-penetrating peptides, and a surprising chain-length dependence of their vesicle- and cell-lysing activity.

    PubMed

    Kolesinska, Beata; Eyer, Klaus; Robinson, Tom; Dittrich, Petra S; Beck, Albert K; Seebach, Dieter; Walde, Peter

    2015-05-01

    Many years ago, β(2) /β(3) -peptides, consisting of alternatively arranged β(2) - and β(3) h-amino-acid residues, have been found to undergo folding to a unique type of helix, the 10/12-helix, and to exhibit non-polar, lipophilic properties (Helv. Chim. Acta 1997, 80, 2033). We have now synthesized such 'mixed' hexa-, nona-, dodeca-, and octadecapeptides, consisting of Val-Ala-Leu triads, with N-terminal fluorescein (FAM) labels, i.e., 1-4, and studied their interactions with POPC (=1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) giant unilamellar vesicles (GUVs) and with human white blood cancer cells U937. The methods used were microfluidic technology, fluorescence correlation spectroscopy (FCS), a flow-cytometry assay, a membrane-toxicity assay with the dehydrogenase G6PDH as enzymatic reporter, and visual microscopy observations. All β(3) /β(2) -peptide derivatives penetrate the GUVs and/or the cells. As shown with the isomeric β(3) /β(2) -, β(3) -, and β(2) -nonamers, 2, 5, and 6, respectively, the derivatives 5 and 6 consisting exclusively of β(3) - or β(2) -amino-acid residues, respectively, interact neither with the vesicles nor with the cells. Depending on the method of investigation and on the pretreatment of the cells, the β(3) /β(2) -nonamer and/or the β(3) /β(2) -dodecamer derivative, 2 and/or 3, respectively, cause a surprising disintegration or lysis of the GUVs and cells, comparable with the action of tensides, viral fusion peptides, and host-defense antimicrobial peptides. Possible sources of the chain-length-dependent destructive potential of the β(3) /β(2) -nona- and β(3) /β(2) -dodecapeptide derivatives, and a possible relationship with the phosphate-to-phosphate and hydrocarbon thicknesses of GUVs, and eukaryotic cells are discussed. Further investigations with other types of GUVs and of eukaryotic or prokaryotic cells will be necessary to elucidate the mechanism(s) of interaction of 'mixed' β(3) /β(2) -peptides with

  4. Quantitative evaluation of the cell penetrating properties of an iodinated Tyr-L-maurocalcine analog.

    PubMed

    Tisseyre, Céline; Ahmadi, Mitra; Bacot, Sandrine; Dardevet, Lucie; Perret, Pascale; Ronjat, Michel; Fagret, Daniel; Usson, Yves; Ghezzi, Catherine; De Waard, Michel

    2014-10-01

    L-Maurocalcine (L-MCa) is the first reported animal cell-penetrating toxin. Characterizing its cell penetration properties is crucial considering its potential as a vector for the intracellular delivery of drugs. Radiolabeling is a sensitive and quantitative method to follow the cell accumulation of a molecule of interest. An L-MCa analog containing an additional N-terminal tyrosine residue (Tyr-L-MCa) was synthesized, shown to fold and oxidize properly, and successfully radioiodinated to (125)I-Tyr-L-MCa. Using various microscopy techniques, the average volume of the rat line F98 glioma cells was evaluated at 8.9 to 18.9×10(-7)μl. (125)I-Tyr-L-MCa accumulates within cells with a dose-dependency similar to the one previously published using 5,6-carboxyfluorescein-L-MCa. According to subcellular fractionation of F98 cells, plasma membranes keep less than 3% of the peptide, regardless of the extracellular concentration, while the nucleus accumulates over 75% and the cytosol around 20% of the radioactive material. Taking into account both nuclear and cytosolic fractions, cells accumulate intracellular concentrations of the peptide that are equal to the extracellular concentrations. Estimation of (125)I-Tyr-L-MCa cell entry kinetics indicate a first rapid phase with a 5min time constant for the plasma membrane followed by slower processes for the cytoplasm and the nucleus. Once inside cells, the labeled material no longer escapes from the intracellular environment since 90% of the radioactivity remains 24h after washout. Dead cells were found to have a lower uptake than live ones. The quantitative information gained herein will be useful for better framing the use of L-MCa in biotechnological applications. This article is part of a Special Issue entitled: Calcium Signaling in Health and Disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau. PMID:24667409

  5. Mitofusin-2 knockdown increases ER-mitochondria contact and decreases amyloid β-peptide production.

    PubMed

    Leal, Nuno Santos; Schreiner, Bernadette; Pinho, Catarina Moreira; Filadi, Riccardo; Wiehager, Birgitta; Karlström, Helena; Pizzo, Paola; Ankarcrona, Maria

    2016-09-01

    Mitochondria are physically and biochemically in contact with other organelles including the endoplasmic reticulum (ER). Such contacts are formed between mitochondria-associated ER membranes (MAM), specialized subregions of ER, and the outer mitochondrial membrane (OMM). We have previously shown increased expression of MAM-associated proteins and enhanced ER to mitochondria Ca(2+) transfer from ER to mitochondria in Alzheimer's disease (AD) and amyloid β-peptide (Aβ)-related neuronal models. Here, we report that siRNA knockdown of mitofusin-2 (Mfn2), a protein that is involved in the tethering of ER and mitochondria, leads to increased contact between the two organelles. Cells depleted in Mfn2 showed increased Ca(2+) transfer from ER to mitchondria and longer stretches of ER forming contacts with OMM. Interestingly, increased contact resulted in decreased concentrations of intra- and extracellular Aβ40 and Aβ42 . Analysis of γ-secretase protein expression, maturation and activity revealed that the low Aβ concentrations were a result of impaired γ-secretase complex function. Amyloid-β precursor protein (APP), β-site APP-cleaving enzyme 1 and neprilysin expression as well as neprilysin activity were not affected by Mfn2 siRNA treatment. In summary, our data shows that modulation of ER-mitochondria contact affects γ-secretase activity and Aβ generation. Increased ER-mitochondria contact results in lower γ-secretase activity suggesting a new mechanism by which Aβ generation can be controlled. PMID:27203684

  6. Culture on Electrospun Polyurethane Scaffolds Decreases Atrial Natriuretic Peptide Expression by Cardiomyocytes In Vitro

    PubMed Central

    Rockwood, Danielle N.; Akins, Robert E.; Parrag, Ian; Woodhouse, Kimberly A.; Rabolt, John F.

    2008-01-01

    The function of the mammalian heart depends on the functional alignment of cardiomyocytes, and controlling cell alignment is an important consideration in biomaterial design for cardiac tissue engineering and research. The physical cues that guide functional cell alignment in vitro and the impact of substrate-imposed alignment on cell phenotype, however, are only partially understood. In this report, primary cardiac ventricular cells were grown on electrospun, biodegradable polyurethane (ES-PU) with either aligned or unaligned microfibers. ES-PU scaffolds supported high-density cultures, and cell subpopulations remained intact over two weeks in culture. ES-PU cultures contained electrically-coupled cardiomyocytes with connexin-43 localized to points of cell:cell contact. Multi-cellular organization correlated with microfiber orientation, and aligned materials yielded highly oriented cardiomyocyte groupings. Atrial natriuretic peptide, a molecular marker that has decreasing expression during ventricular cell maturation, was significantly lower in cultures grown on ES-PU scaffolds than in those grown on tissue culture polystyrene. Cells grown on aligned ES-PU had significantly lower steady state levels of ANP and constitutively released less ANP over time indicating that scaffold-imposed cell organization resulted in a shift in cell phenotype to a more mature state. We conclude that the physical organization of microfibers in ES-PU scaffolds impacts both multi-cellular architecture and cardiac cell phenotype in vitro. PMID:18823659

  7. Transplanted Human Bone Marrow Mesenchymal Stem Cells Seeded onto Peptide Hydrogel Decrease Alveolar Bone Loss

    PubMed Central

    Karlström, Erik; Cedervall, Jessica; Wendel, Mikael

    2012-01-01

    Abstract Alveolar bone loss can be caused by periodontitis or periodontal trauma. We have evaluated the effects of transplanted undifferentiated human mesenchymal stem cells (hMSCs) on alveolar bone reaction and periodontal ligament healing in an experimental periodontal wound model. The hMSCs seeded onto a self-assembling peptide hydrogel in combination with collagen sponge were implanted into the right mandible of 12 rats and followed for 1 (n=6) or 4 weeks (n=6) postoperatively. The other 12 sham-treated rats were used as controls. Histological and histomorphometrical methods were used to assess the periodontal tissue reaction. The alveolar bone volume density was significantly higher at 1 week after surgery, and the osteoclast number was significantly lower at both 1 week and 4 weeks postoperatively in the mandibles treated with hMSCs. The implanted cells were detected only at 1 week after surgery. In conclusion, transplanted hMSCs can contribute to alveolar bone preservation after a periodontal surgical trauma at least by decreasing local osteoclast number. PMID:23514848

  8. Cell-penetrating and neurotargeting dendritic siRNA nanostructures.

    PubMed

    Brunner, Korbinian; Harder, Johannes; Halbach, Tobias; Willibald, Julian; Spada, Fabio; Gnerlich, Felix; Sparrer, Konstantin; Beil, Andreas; Möckl, Leonhard; Bräuchle, Christoph; Conzelmann, Karl-Klaus; Carell, Thomas

    2015-02-01

    We report the development of dendritic siRNA nanostructures that are able to penetrate even difficult to transfect cells such as neurons with the help of a special receptor ligand. The nanoparticles elicit strong siRNA responses, despite the dendritic structure. An siRNA dendrimer directed against the crucial rabies virus (RABV) nucleoprotein (N protein) and phosphoprotein (P protein) allowed the suppression of the virus titer in neurons below the detection limit. The cell-penetrating siRNA dendrimers, which were assembled using click chemistry, open up new avenues toward finding novel molecules able to cure this deadly disease.

  9. Cell-penetrating compounds preferentially bind glycosaminoglycans over plasma membrane lipids in a charge density- and stereochemistry-dependent manner.

    PubMed

    Prevette, Lisa E; Benish, Nicolas C; Schoenecker, Amber R; Braden, Kristin J

    2015-12-01

    Cell-penetrating compounds (CPCs) are often conjugated to drugs and genes to facilitate cellular uptake. We hypothesize that the electrostatic interaction between the positively charged amines of the cell-penetrating compounds and the negatively charged glycosaminoglycans (GAGs) extending from cell surfaces is the initiating step in the internalization process. The interactions of generation 5 PAMAM dendrimer, Tat peptide and 25 kDa linear PEI with four different GAGs have been studied using isothermal titration calorimetry to elucidate structure-function relationships that could lead to improved drug and gene delivery methods to a wide variety of cell types. Detailed thermodynamic analysis has determined that CPC-GAG binding constants range from 8.7×10(3) to 2.4×10(6)M(-1) and that affinity is dependent upon GAG charge density and stereochemistry and CPC molecular weight. The effect of GAG composition on affinity is likely due to hydrogen bonding between CPC amines and amides and GAG hydroxyl and amine groups. These results were compared to the association of CPCs with lipid vesicles of varying composition as model plasma membranes to finally clarify the relative importance of each cell surface component in initial cell recognition. CPC-lipid affinity increases with anionic lipid content, but GAG affinity is higher for all cell-penetrating compounds, confirming the role these heterogeneous polysaccharides play in cellular association and clustering.

  10. Endosomolytic Nano-Polyplex Platform Technology for Cytosolic Peptide Delivery To Inhibit Pathological Vasoconstriction

    PubMed Central

    2016-01-01

    A platform technology has been developed and tested for delivery of intracellular-acting peptides through electrostatically complexed nanoparticles, or nano-polyplexes, formulated from an anionic endosomolytic polymer and cationic therapeutic peptides. This delivery platform has been initially tested and optimized for delivery of two unique vasoactive peptides, a phosphomimetic of heat shock protein 20 and an inhibitor of MAPKAP kinase II, to prevent pathological vasoconstriction (i.e., vasospasm) in human vascular tissue. These peptides inhibit vasoconstriction and promote vasorelaxation by modulating actin dynamics in vascular smooth muscle cells. Formulating these peptides into nano-polyplexes significantly enhances peptide uptake and retention, facilitates cytosolic delivery through a pH-dependent endosomal escape mechanism, and enhances peptide bioactivity in vitro as measured by inhibition of F-actin stress fiber formation. In comparison to treatment with the free peptides, which were endowed with cell-penetrating sequences, the nano-polyplexes significantly increased vasorelaxation, inhibited vasoconstriction, and decreased F-actin formation in the human saphenous vein ex vivo. These results suggest that these formulations have significant potential for treatment of conditions such as cerebral vasospasm following subarachnoid hemorrhage. Furthermore, because many therapeutic peptides include cationic cell-penetrating segments, this simple and modular platform technology may have broad applicability as a cost-effective approach for enhancing the efficacy of cytosolically active peptides. PMID:26004140

  11. Endosomolytic Nano-Polyplex Platform Technology for Cytosolic Peptide Delivery To Inhibit Pathological Vasoconstriction.

    PubMed

    Evans, Brian C; Hocking, Kyle M; Kilchrist, Kameron V; Wise, Eric S; Brophy, Colleen M; Duvall, Craig L

    2015-06-23

    A platform technology has been developed and tested for delivery of intracellular-acting peptides through electrostatically complexed nanoparticles, or nano-polyplexes, formulated from an anionic endosomolytic polymer and cationic therapeutic peptides. This delivery platform has been initially tested and optimized for delivery of two unique vasoactive peptides, a phosphomimetic of heat shock protein 20 and an inhibitor of MAPKAP kinase II, to prevent pathological vasoconstriction (i.e., vasospasm) in human vascular tissue. These peptides inhibit vasoconstriction and promote vasorelaxation by modulating actin dynamics in vascular smooth muscle cells. Formulating these peptides into nano-polyplexes significantly enhances peptide uptake and retention, facilitates cytosolic delivery through a pH-dependent endosomal escape mechanism, and enhances peptide bioactivity in vitro as measured by inhibition of F-actin stress fiber formation. In comparison to treatment with the free peptides, which were endowed with cell-penetrating sequences, the nano-polyplexes significantly increased vasorelaxation, inhibited vasoconstriction, and decreased F-actin formation in the human saphenous vein ex vivo. These results suggest that these formulations have significant potential for treatment of conditions such as cerebral vasospasm following subarachnoid hemorrhage. Furthermore, because many therapeutic peptides include cationic cell-penetrating segments, this simple and modular platform technology may have broad applicability as a cost-effective approach for enhancing the efficacy of cytosolically active peptides.

  12. Glucagon-like peptide 1 decreases lipotoxicity in non-alcoholic steatohepatitis

    PubMed Central

    Armstrong, Matthew J.; Hull, Diana; Guo, Kathy; Barton, Darren; Hazlehurst, Jonathan M.; Gathercole, Laura L.; Nasiri, Maryam; Yu, Jinglei; Gough, Stephen C.; Newsome, Philip N.; Tomlinson, Jeremy W.

    2016-01-01

    Background & Aims Insulin resistance and lipotoxicity are pathognomonic in non-alcoholic steatohepatitis (NASH). Glucagon-like peptide-1 (GLP-1) analogues are licensed for type 2 diabetes, but no prospective experimental data exists in NASH. This study determined the effect of a long-acting GLP-1 analogue, liraglutide, on organ-specific insulin sensitivity, hepatic lipid handling and adipose dysfunction in biopsy-proven NASH. Methods Fourteen patients were randomised to 1.8 mg liraglutide or placebo for 12-weeks of the mechanistic component of a double-blind, randomised, placebo-controlled trial (ClinicalTrials.gov-NCT01237119). Patients underwent paired hyperinsulinaemic euglycaemic clamps, stable isotope tracers, adipose microdialysis and serum adipocytokine/metabolic profiling. In vitro isotope experiments on lipid flux were performed on primary human hepatocytes. Results Liraglutide reduced BMI (−1.9 vs. +0.04 kg/m2; p <0.001), HbA1c (−0.3 vs. +0.3%; p <0.01), cholesterol-LDL (−0.7 vs. +0.05 mmol/L; p <0.01), ALT (−54 vs. −4.0 IU/L; p <0.01) and serum leptin, adiponectin, and CCL-2 (all p <0.05). Liraglutide increased hepatic insulin sensitivity (−9.36 vs. −2.54% suppression of hepatic endogenous glucose production with low-dose insulin; p <0.05). Liraglutide increased adipose tissue insulin sensitivity enhancing the ability of insulin to suppress lipolysis both globally (−24.9 vs. +54.8 pmol/L insulin required to ½ maximally suppress serum non-esterified fatty acids; p <0.05), and specifically within subcutaneous adipose tissue (p <0.05). In addition, liraglutide decreased hepatic de novo lipogenesis in vivo (−1.26 vs. +1.30%; p <0.05); a finding endorsed by the effect of GLP-1 receptor agonist on primary human hepatocytes (24.6% decrease in lipogenesis vs. untreated controls; p <0.01). Conclusions Liraglutide reduces metabolic dysfunction, insulin resistance and lipotoxicity in the key metabolic organs in the pathogenesis of

  13. Improved affinity at the cost of decreased specificity: a recurring theme in PDZ-peptide interactions

    PubMed Central

    Karlsson, O. Andreas; Sundell, Gustav N.; Andersson, Eva; Ivarsson, Ylva; Jemth, Per

    2016-01-01

    The E6 protein from human papillomavirus (HPV) plays an important role during productive infection and is a potential drug target. We have previously designed a high affinity bivalent protein binder for the E6 protein, a fusion between a helix from the E6 associated protein and PDZØ9, an engineered variant (L391F/K392M) of the second PDZ domain from synapse associated protein 97 (SAP97 PDZ2). How the substitutions improve the affinity of SAP97 PDZ2 for HPV E6 is not clear and it is not known to what extent they affect the specificity for cellular targets. Here, we explore the specificity of wild type SAP97 PDZ2 and PDZØ9 through proteomic peptide phage display. In addition, we employ a double mutant cycle of SAP97 PDZ2 in which the binding kinetics for nine identified potential cellular peptide ligands are measured and compared with those for the C-terminal E6 peptide. The results demonstrate that PDZØ9 has an increased affinity for all peptides, but at the cost of specificity. Furthermore, there is a peptide dependent coupling free energy between the side chains at positions 391 and 392. This corroborates our previous allosteric model for PDZ domains, involving sampling of intramolecular energetic pathways. PMID:27694853

  14. The Taming of the Cell Penetrating Domain of the HIV Tat: Myths and Realities

    PubMed Central

    Chauhan, Ashok; Tikoo, Akshay; Kapur, Arvinder K.; Singh, Mahavir

    2007-01-01

    Protein transduction with cell penetrating peptides over the past several years has been shown to be an effective way of delivering proteins in vitro and now several reports have also shown valuable in vivo applications in correcting disease states. An impressive bioinspired phenomenon of crossing biological barriers came from HIV transactivator Tat protein. Specifically, the protein transduction domain of HIV-Tat has been shown to be a potent pleiotropic peptide in protein delivery. Various approaches such as molecular modeling, arginine guanidinium head group structural strategy, multimerization of PTD sequence and phage display system have been applied for taming of the PTD. This has resulted in identification of PTD variants which are efficient in cell membrane penetration and cytoplasmic delivery. Inspite of these state of the art technologies, the dilemma of low protein transduction efficiency and target specific delivery of PTD fusion proteins remains unsolved. Moreover, some misconceptions about PTD of Tat in the literature require considerations. We have assembled critical information on secretory, plasma membrane penetration and transcellular properties of Tat and PTD using molecular analysis and available experimental evidences. PMID:17196289

  15. Discovery and characterization of a new cell-penetrating protein.

    PubMed

    Simeon, Rudo L; Chamoun, Ana Maria; McMillin, Thomas; Chen, Zhilei

    2013-12-20

    We describe a new cell-penetrating protein, B1, capable of delivering conjugated proteins and nucleic acids into mammalian cells. B1 is a 244-amino-acid product of a single-base frameshift in the gene encoding enhanced green fluorescent protein (eGFP). The molecule has a net positive charge of 43 and a very high charge-to-mass ratio of 1.5. eGFP-fused B1 potently penetrates both adherent and suspension cells with >80% of cells taking up the protein when exposed to concentrations as low as 1 μM. The protein was found to cluster in the paranuclear region of TZM-bl cells. Most importantly, we show that B1 not only facilitates cellular uptake but allows biomolecular cargo to reach sites of biological relevance. For example, baby hamster kidney cells underwent DNA recombination when exposed to B1-tagged Cre recombinase at protein concentrations as low as 2.5 μM, indicating potent nuclear delivery of functional protein cargos. Additionally, B1 delivers noncovalently conjugated RNA and DNA across the cell membrane to cytosolic and nuclear sites accessible to the cellular translation and transcription machinery, as gauged by detection of encoded reporter functions, with efficiency comparable to commercially available cationic lipid reagents. B1 appears to utilize cell-surface glycans and multiple competing endocytic pathways to enter and traffic through cells. These studies provide both a new tool for intracellular delivery of biomolecules and insights that could aid in the design of more effective cell penetrating proteins.

  16. Discovery and Characterization of a New Cell-Penetrating Protein

    PubMed Central

    Simeon, Rudo L.; Chamoun, Ana Maria; McMillin, Thomas; Chen, Zhilei

    2013-01-01

    We describe a new cell-penetrating protein, B1, capable of delivering conjugated proteins and nucleic acids into mammalian cells. B1 is a 244-amino-acid product of a single-base frameshift in the gene encoding enhanced green fluorescent protein (eGFP). The molecule has a net positive charge of 43 and a very high charge-to-mass ratio of 1.5. eGFP-fused B1 potently penetrates both adherent and suspension cells with >80% of cells taking up the protein when exposed to concentrations as low as 1 μM. The protein was found to cluster in the paranuclear region of TZM-bl cells. Most importantly, we show that B1 not only facilitates cellular uptake, but allows biomolecular cargo to reach sites of biological relevance. For example, baby hamster kidney cells underwent DNA recombination when exposed to B1-tagged Cre recombinase at protein concentrations as low as 2.5 μM, indicating potent nuclear delivery of functional protein cargos. Additionally, B1 delivers non-covalently conjugated RNA and DNA across the cell membrane to cytosolic and nuclear sites accessible to the cellular translation and transcription machinery, as gauged by detection of encoded reporter functions, with efficiency comparable to commercially available cationic lipid reagents. B1 appears to utilize cell-surface glycans and multiple competing endocytic pathways to enter and traffic through cells. These studies provide both a new tool for intracellular delivery of biomolecules and insights that could aid in the design of more effective cell penetrating proteins. PMID:24047285

  17. Genetic Decreases in Atrial Natriuretic Peptide and Salt-Sensitive Hypertension

    NASA Astrophysics Data System (ADS)

    John, Simon W. M.; Krege, John H.; Oliver, Paula M.; Hagaman, John R.; Hodgin, Jeffrey B.; Pang, Stephen C.; Flynn, T. Geoffrey; Smithies, Oliver

    1995-02-01

    To determine if defects in the atrial natriuretic peptide (ANP) system can cause hypertension, mice were generated with a disruption of the proANP gene. Homozygous mutants had no circulating or atrial ANP, and their blood pressures were elevated by 8 to 23 millimeters of mercury when they were fed standard (0.5 percent sodium chloride) and intermediate (2 percent sodium chloride) salt diets. On standard salt diets, heterozygotes had normal amounts of circulating ANP and normal blood pressures. However, on high (8 percent sodium chloride) salt diets they were hypertensive, with blood pressures elevated by 27 millimeters of mercury. These results demonstrate that genetically reduced production of ANP can lead to salt-sensitive hypertension.

  18. Cell penetrable humanized-VH/V(H)H that inhibit RNA dependent RNA polymerase (NS5B) of HCV.

    PubMed

    Thueng-in, Kanyarat; Thanongsaksrikul, Jeeraphong; Srimanote, Potjanee; Bangphoomi, Kunan; Poungpair, Ornnuthchar; Maneewatch, Santi; Choowongkomon, Kiattawee; Chaicumpa, Wanpen

    2012-01-01

    NS5B is pivotal RNA dependent RNA polymerase (RdRp) of HCV and NS5B function interfering halts the virus infective cycle. This work aimed to produce cell penetrable humanized single domain antibodies (SdAb; VH/V(H)H) that interfere with the RdRp activity. Recombinant NS5BΔ55 of genotype 3a HCV with de novo RNA synthetic activity was produced and used in phage biopanning for selecting phage clones that displayed NS5BΔ55 bound VH/V(H)H from a humanized-camel VH/V(H)H display library. VH/V(H)H from E. coli transfected with four selected phage clones inhibited RdRp activity when tested by ELISA inhibition using 3'di-cytidylate 25 nucleotide directed in vitro RNA synthesis. Deduced amino acid sequences of two clones showed V(H)H hallmark and were designated V(H)H6 and V(H)H24; other clones were conventional VH, designated VH9 and VH13. All VH/V(H)H were linked molecularly to a cell penetrating peptide, penetratin. The cell penetrable VH9, VH13, V(H)H6 and V(H)H24 added to culture of Huh7 cells transfected with JHF-1 RNA of genotype 2a HCV reduced the amounts of RNA intracellularly and in culture medium implying that they inhibited the virus replication. VH/V(H)H mimotopes matched with residues scattered on the polymerase fingers, palm and thumb which were likely juxtaposed to form conformational epitopes. Molecular docking revealed that the antibodies covered the RdRp catalytic groove. The transbodies await further studies for in vivo role in inhibiting HCV replication.

  19. Enhanced anticancer activity of nanopreparation containing an MMP2-sensitive PEG-drug conjugate and cell-penetrating moiety.

    PubMed

    Zhu, Lin; Wang, Tao; Perche, Federico; Taigind, Anton; Torchilin, Vladimir P

    2013-10-15

    In response to the challenges of cancer chemotherapeutics, including poor physicochemical properties, low tumor targeting, insufficient tumor cell internalization/bioavailability, and side effects, we developed a unique tumor-targeted micellar drug-delivery platform. Using paclitaxel as a model therapeutic, a nanopreparation composed of a matrix metalloproteinase 2 (MMP2)-sensitive self-assembly PEG 2000-paclitaxel conjugate (as a prodrug and MMP 2-sensitive moiety), transactivating transcriptional activator peptide-PEG1000-phosphoethanolamine (PE) (a cell-penetrating enhancer), and PEG1000-PE (a nanocarrier building block) was prepared. Several major drug delivery strategies, including self-assembly, PEGylation, the enhanced permeability and retention effect, stimulus sensitivity, a cell-penetrating moiety, and the concept of prodrug, were used in design of this nanoparticle in a collaborative manner. The nanopreparation allowed superior cell internalization, cytotoxicity, tumor targeting, and antitumor efficacy in vitro and in vivo over its nonsensitive counterpart, free paclitaxel and conventional micelles. This uniquely engineered nanoparticle has potential for effective intracellular delivery of drug into cancer cells. PMID:24062440

  20. Enhanced anticancer activity of nanopreparation containing an MMP2-sensitive PEG-drug conjugate and cell-penetrating moiety

    PubMed Central

    Zhu, Lin; Wang, Tao; Perche, Federico; Taigind, Anton; Torchilin, Vladimir P.

    2013-01-01

    In response to the challenges of cancer chemotherapeutics, including poor physicochemical properties, low tumor targeting, insufficient tumor cell internalization/bioavailability, and side effects, we developed a unique tumor-targeted micellar drug-delivery platform. Using paclitaxel as a model therapeutic, a nanopreparation composed of a matrix metalloproteinase 2 (MMP2)-sensitive self-assembly PEG 2000-paclitaxel conjugate (as a prodrug and MMP 2-sensitive moiety), transactivating transcriptional activator peptide-PEG1000-phosphoethanolamine (PE) (a cell-penetrating enhancer), and PEG1000-PE (a nanocarrier building block) was prepared. Several major drug delivery strategies, including self-assembly, PEGylation, the enhanced permeability and retention effect, stimulus sensitivity, a cell-penetrating moiety, and the concept of prodrug, were used in design of this nanoparticle in a collaborative manner. The nanopreparation allowed superior cell internalization, cytotoxicity, tumor targeting, and antitumor efficacy in vitro and in vivo over its nonsensitive counterpart, free paclitaxel and conventional micelles. This uniquely engineered nanoparticle has potential for effective intracellular delivery of drug into cancer cells. PMID:24062440

  1. Astrocytic adenosine A2A receptors control the amyloid-β peptide-induced decrease of glutamate uptake.

    PubMed

    Matos, Marco; Augusto, Elisabete; Machado, Nuno J; dos Santos-Rodrigues, Alexandre; Cunha, Rodrigo A; Agostinho, Paula

    2012-01-01

    Alzheimer's disease (AD) is characterized by a progressive cognitive impairment tightly correlated with the accumulation of amyloid-β (Aβ) peptides (mainly Aβ(1-42)). There is a precocious disruption of glutamatergic synapses in AD, in line with an ability of Aβ to decrease astrocytic glutamate uptake. Accumulating evidence indicates that caffeine prevents the burden of AD, likely through the antagonism of A(2A) receptors (A(2A)R) which attenuates Aβ-induced memory impairment and synaptotoxicity. Since A(2A)R also modulate astrocytic glutamate uptake, we now tested if A(2A)R blockade could prevent the decrease of astrocytic glutamate uptake caused by Aβ. In cultured astrocytes, Aβ(1-42). (1 μM for 24 hours) triggered an astrogliosis typified by an increased density of GFAP, which was mimicked by the A(2A)R agonist, CGS 26180 (30 nM), and prevented by the A(2A)R antagonist, SCH 58261 (100 nM). Aβ1-42 also decreased D-aspartate uptake by 28 ± 4%, an effect abrogated upon genetic inactivation or pharmacological blockade of A(2A)R. In accordance with the long term control of glutamate transporter expression by A(2A)R, Aβ(1-42). enhanced the expression and density of astrocytic A(2A)R and decreased GLAST and GLT-I expression in astrocytes from wild type, but not from A(2A)R knockout mice. This impact of Aβ(1-42). on glutamate transporters and uptake, dependent on A(2A)R function, was also confirmed in an ex vivo astrocyte preparation (gliosomes) from rats intracerebroventricularly (icv) injected with Aβ(1-42). . These results provide the first demonstration for a direct key role of astrocytic A(2A)R in the ability of Aβ-induced impairment of glutamate uptake, which may underlie glutamatergic synaptic dysfunction and excitotoxicity in AD.

  2. Weight loss during oligofructose supplementation is associated with decreased ghrelin and increased peptide YY in overweight and obese adults2

    PubMed Central

    Parnell, Jill A; Reimer, Raylene A

    2013-01-01

    Background Rodent studies show that oligofructose promotes weight loss, stimulates satiety hormone secretion, reduces energy intake, and improves lipid profiles. Objective Our objective was to examine the effects of oligofructose supplementation on body weight and satiety hormone concentrations in overweight and obese adults. Design This study was a randomized, double-blind, placebo-controlled trial. Forty-eight otherwise healthy adults with a body mass index (in kg/m2) > 25 were randomly assigned to receive 21 g oligo-fructose/d or a placebo (maltodextrin) for 12 wk. Body composition (by dual-energy X-ray absorptiometry); meal tolerance tests, including satiety hormone response; food intake; and subjective appetite ratings were determined. Results There was a reduction in body weight of 1.03 ±0.43 kg with oligofructose supplementation, whereas the control group experienced an increase in body weight of 0.45 ± 0.31 kg over 12 wk (P = 0.01). A lower area under the curve (AUC) for ghrelin (P = 0.004) and a higher AUC for peptide YY (PYY) with oligofructose (P = 0.03) coincided with a reduction in self-reported caloric intake (P ≤ 0.05). Glucose decreased in the oligofructose group and increased in the control group between initial and final tests (P ≤ 0.05). Insulin concentrations mirrored this pattern (P ≤ 0.05). Oligofructose supplementation did not affect plasma active glucagon-like peptide 1 secretion. According to a visual analog scale designed to assess side effects, oligofructose was well tolerated. Conclusions Independent of other lifestyle changes, oligofructose supplementation has the potential to promote weight loss and improve glucose regulation in overweight adults. Suppressed ghrelin and enhanced PYY may contribute in part to the reduction in energy intake. The trial was registered at clinicaltrials.gov as NCT00522353. PMID:19386741

  3. Effect of decreasing the affinity of the class II-associated invariant chain peptide on the MHC class II peptide repertoire in the presence or absence of H-2M.

    PubMed

    Honey, Karen; Forbush, Katherine; Jensen, Peter E; Rudensky, Alexander Y

    2004-04-01

    The class II-associated invariant chain peptide (CLIP) region of the invariant chain (Ii) directly influences MHC class II presentation by occupying the MHC class II peptide-binding groove, thereby preventing premature loading of peptides. Different MHC class II alleles exhibit distinct affinities for CLIP, and a low affinity interaction has been associated with decreased dependence upon H-2M and increased susceptibility to rheumatoid arthritis, suggesting that decreased CLIP affinity alters the MHC class II-bound peptide repertoire, thereby promoting autoimmunity. To examine the role of CLIP affinity in determining the MHC class II peptide repertoire, we generated transgenic mice expressing either wild-type human Ii or human Ii containing a CLIP region of low affinity for MHC class II. Our data indicate that although degradation intermediates of Ii containing a CLIP region with decreased affinity for MHC class II do not remain associated with I-A(b), this does not substantially alter the peptide repertoire bound by MHC class II or increase autoimmune susceptibility in the mice. This implies that the affinity of the CLIP:MHC class II interaction is not a strong contributory factor in determining the probability of developing autoimmunity. In contrast, in the absence of H-2M, MHC class II peptide repertoire diversity is enhanced by decreasing the affinity of CLIP for MHC class II, although MHC class II cell surface expression is reduced. Thus, we show clearly, in vivo, the critical chaperone function of H-2M, which preserves MHC class II molecules for high affinity peptide binding upon dissociation of Ii degradation intermediates. PMID:15034026

  4. Interference of HCV replication by cell penetrable human monoclonal scFv specific to NS5B polymerase

    PubMed Central

    Thueng-in, Kanyarat; Thanongsaksrikul, Jeeraphong; Jittavisutthikul, Surasak; Seesuay, Watee; Chulanetra, Monrat; Sakolvaree, Yuwaporn; Srimanote, Potjanee; Chaicumpa, Wanpen

    2014-01-01

    A new class of hepatitis C virus (HCV)-targeted therapeutics that is safe, broadly effective and can cope with virus mutations is needed. The HCV's NS5B is highly conserved and different from human protein, and thus it is an attractive target for anti-HCV therapeutics development. In this study, NS5B bound-phage clones selected from a human single chain variable antibody fragment (scFv) phage display library were used to transform appropriate E. coli bacteria. Two scFv inhibiting HCV polymerase activity were selected. The scFvs were linked to a cell penetrating peptide to make cell penetrable scFvs. The transbodies reduced the HCV RNA and infectious virus particles released into the culture medium and inside hepatic cells transfected with a heterologous HCV replicon. They also rescued the innate immune response of the transfected cells. Phage mimotope search and homology modeling/molecular docking revealed the NS5B subdomains and residues bound by the scFvs. The scFv mimotopes matched residues of the NS5B, which are important for nucleolin binding during HCV replication, as well as residues that interconnect the fingers and thumb domains for forming a polymerase active groove. Both scFvs docked on several residues at the thumb armadillo-like fold that could be the polymerase interactive sites of other viral/host proteins for the formation of the replication complex and replication initiation. In conclusion, human transbodies that inhibited HCV RdRp activity and HCV replication and restored the host innate immune response were produced. They are potentially future interferon-free anti-HCV candidates, particularly in combination with other cognates that are specific to NS5B epitopes and other HCV enzymes. PMID:25517317

  5. Interference of HCV replication by cell penetrable human monoclonal scFv specific to NS5B polymerase.

    PubMed

    Thueng-in, Kanyarat; Thanongsaksrikul, Jeeraphong; Jittavisutthikul, Surasak; Seesuay, Watee; Chulanetra, Monrat; Sakolvaree, Yuwaporn; Srimanote, Potjanee; Chaicumpa, Wanpen

    2014-01-01

    A new class of hepatitis C virus (HCV)-targeted therapeutics that is safe, broadly effective and can cope with virus mutations is needed. The HCV's NS5B is highly conserved and different from human protein, and thus it is an attractive target for anti-HCV therapeutics development. In this study, NS5B bound-phage clones selected from a human single chain variable antibody fragment (scFv) phage display library were used to transform appropriate E. coli bacteria. Two scFv inhibiting HCV polymerase activity were selected. The scFvs were linked to a cell penetrating peptide to make cell penetrable scFvs. The transbodies reduced the HCV RNA and infectious virus particles released into the culture medium and inside hepatic cells transfected with a heterologous HCV replicon. They also rescued the innate immune response of the transfected cells. Phage mimotope search and homology modeling/molecular docking revealed the NS5B subdomains and residues bound by the scFvs. The scFv mimotopes matched residues of the NS5B, which are important for nucleolin binding during HCV replication, as well as residues that interconnect the fingers and thumb domains for forming a polymerase active groove. Both scFvs docked on several residues at the thumb armadillo-like fold that could be the polymerase interactive sites of other viral/host proteins for the formation of the replication complex and replication initiation. In conclusion, human transbodies that inhibited HCV RdRp activity and HCV replication and restored the host innate immune response were produced. They are potentially future interferon-free anti-HCV candidates, particularly in combination with other cognates that are specific to NS5B epitopes and other HCV enzymes. PMID:25517317

  6. Increased proliferation and decreased membrane permeability as defense mechanisms of Fusobacterium nucleatum against human neutrophilic peptide-1.

    PubMed

    Keskin, Mutlu; Könönen, Eija; Söderling, Eva; Isik, Gülden; Firatli, Erhan; Uitto, Veli-Jukka; Gürsoy, Ulvi Kahraman

    2014-12-01

    Human neutrophilic peptides (HNPs) constitute a class of host defense molecules, which contribute to the non-oxidative killing of bacteria and other microorganisms. Since the adaptability is crucial to bacterial survival in changing environments, it is of interest to know how Fusobacterium nucleatum, the major bridge organism connecting early and late colonizers in dental biofilms, defends itself against HNPs. This study aimed to examine the planktonic growth, membrane permeability, and biofilm formation characteristics as defense mechanisms of F. nucleatum against HNP-1. In all experiments, the type strain of F. nucleatum (ssp. nucleatum ATCC 25586) and two clinical strains (ssp. nucleatum AHN 9508 and ssp. polymorphum AHN 9910) were used. Planktonic growth (measured in colony forming units), capsular polysaccharide production (visualized by Ziehl-Neelsen stain), membrane permeability (demonstrated as N-phenyl-1-naphthylamine uptake), biofilm formation, and established biofilm development (measured as total mass and polysaccharide levels) were analyzed in the presence of 0 μg/ml (control), 1 μg/ml, 5 μg/ml, and 10 μg/ml of HNP-1. Planktonic growth of the strains AHN 9508 and ATCC 25586 were significantly (p<0.05) increased in the presence of HNP-1, while their membrane permeability decreased (p<0.005) in the planktonic form. HNP-1 decreased the biofilm formation of the strains ATCC 25586 and AHN 9910, whereas it increased the growth of the strain AHN 9508 in established biofilms. Capsule formation and polysaccharide production were not observed in any strain. We conclude that the inhibition of the membrane permeability and the increase in planktonic and established biofilm growth could act as bacterial defense mechanisms against neutrophilic defensins. In addition, this strain-dependent survival ability against HNP-1 may explain the variation in the virulence of different F. nucleatum strains.

  7. Bioreducible polymers with cell penetrating and endosome buffering functionality for gene delivery systems.

    PubMed

    Kim, Tae-il; Rothmund, Thomas; Kissel, Thomas; Kim, Sung Wan

    2011-05-30

    Bioreducible cationic polymers (p(DAH(a)-R/API(b))s) composed of different ratios (a:b=2:1, 1:1, 1:2) between arginine-grafted diaminohexane (DAH-R) (cell penetrating functionality) and 1-(3-aminopropyl) imidazole (API) (endosome buffering functionality) monomers were synthesized by Michael reaction of N,N'-cystaminebisacrylamide (CBA) with them, in order to study the effect of endosome buffering moiety on arginine-grafted bioreducible polymeric gene carriers. Several experiments displayed a distinct correlation between monomer composition ratios of p(DAH-R/API)s and the polymer features. Increased endosome buffering capacities proportional to API portions was evaluated for p(DAH-R/API)s due to the imidazole group (pKa=6) of API. Increased portions of API non-ionized at physiological pH and resultant decrease of arginine residues also reduced cytotoxicities of the polymers due to less interaction of cellular compartments with less positively charged polymers but decreased pDNA condensing abilities, Zeta-potential values, cellular uptakes of polyplexes, and finally transfection efficiencies as well. Thus, the predominance of arginine residues over endosome buffering moieties was revealed regarding efficient gene delivery for p(DAH-R/API)s. From transfection results with chloroquine or nigericin, it can be deduced that the endosomal escape of p(DAH-R/API) polyplexes occurs by direct endosome membrane penetration of arginine moieties as well as endosome buffering of the polymers after cellular uptake, which emphasizes the importance of arginine moieties for polymeric gene delivery systems.

  8. Penetratin-Membrane Association: W48/R52/W56 Shield the Peptide from the Aqueous Phase

    PubMed Central

    Lensink, M. F.; Christiaens, B.; Vandekerckhove, J.; Prochiantz, A.; Rosseneu, M.

    2005-01-01

    Using molecular dynamics simulations, we studied the mode of association of the cell-penetrating peptide penetratin with both a neutral and a charged bilayer. The results show that the initial peptide-lipid association is a fast process driven by electrostatic interactions. The homogeneous distribution of positively charged residues along the axis of the helical peptide, and especially residues K46, R53, and K57, contribute to the association of the peptide with lipids. The bilayer enhances the stability of the penetratin helix. Oriented parallel to the lipid-water interface, the subsequent insertion of the peptide through the bilayer headgroups is significantly slower. The presence of negatively charged lipids considerably enhances peptide binding. Lateral side-chain motion creates an opening for the helix into the hydrophobic core of the membrane. The peptide aromatic residues form a π-stacking cluster through W48/R52/W56 and F49/R53, protecting the peptide from the water phase. Interaction with the penetratin peptide has only limited effect on the overall membrane structure, as it affects mainly the conformation of the lipids which interact directly with the peptide. Charge matching locally increases the concentration of negatively charged lipids, lateral lipid diffusion locally decreases. Lipid disorder increases, through decreased order parameters of the lipids interacting with the penetratin side chains. Penetratin molecules at the membrane surface do not seem to aggregate. PMID:15542560

  9. Specific targeting of the IL-23 receptor, using a novel small peptide noncompetitive antagonist, decreases the inflammatory response.

    PubMed

    Quiniou, Christiane; Domínguez-Punaro, Maria; Cloutier, Frank; Erfani, Atefeh; Ennaciri, Jamila; Sivanesan, Durgajini; Sanchez, Mélanie; Chognard, Gaëlle; Hou, Xin; Rivera, José Carlos; Beauchamp, Claudine; Charron, Guy; Vilquin, Marie; Kuchroo, Vijay; Michnick, Stephen; Rioux, John D; Lesage, Sylvie; Chemtob, Sylvain

    2014-11-15

    IL-23 is part of the IL-12 family of cytokines and is composed of the p19 subunit specific to IL-23 and the p40 subunit shared with IL-12. IL-23 specifically contributes to the inflammatory process of multiple chronic inflammatory autoimmune disorders, including psoriasis, multiple sclerosis, inflammatory bowel disease, and rheumatoid arthritis. So far, one antibody targeting the shared p40 subunit of IL-12 and IL-23, Ustekinumab, is approved clinically to treat psoriasis. However, there are no treatments inhibiting specifically the IL-23 proinflammatory response. We have developed small IL-23R-specific antagonists by designing all D-peptides arising from flexible regions of IL-23R. Of these peptides, we selected 2305 (teeeqqly), since in addition to its soluble properties, it inhibited IL-23-induced STAT3 phosphorylation in spleen cells. Peptide 2305 specifically binds to IL-23R/IL-12Rβ1-expressing HEK-293 cells and not to cells devoid of the receptor. Peptide 2305 showed functional selectivity by modulating IL-23-induced gene expression in IL-23R/IL-12Rβ1-expressing cells and in Jurkat cells; 2305 does not inhibit IL-12-induced cytokine expression in IL-12Rβ-IL-12Rβ2-HEK-293 cells. Finally, compared with anti-p40 treatment, 2305 effectively and selectively inhibits IL-23-induced inflammation in three in vivo mouse models: IL-23-induced ear inflammation, anti-CD40-induced systemic inflammatory response, and collagen-induced arthritis. We, hereby, describe the discovery and characterization of a potent IL-23R small-peptide modulator, 2305 (teeeqqly), that is effective in vivo. 2305 may be more convenient, less cumbersome, less costly, and most importantly, more specific than current biologics for the treatment of inflammatory conditions, and conceivably complement the actual therapies for these chronic and debilitating inflammatory diseases. PMID:25354400

  10. Peptides that influence membrane topology

    NASA Astrophysics Data System (ADS)

    Wong, Gerard C. L.

    2014-03-01

    We examine the mechanism of a range of polypeptides that influence membrane topology, including antimicrobial peptides, cell penetrating peptides, viral fusion peptides, and apoptosis proteins, and show how a combination of geometry, coordination chemistry, and soft matter physics can be used to approach a unified understanding. We will also show how such peptides can impact biomedical problems such as auto-immune diseases (psoriasis, lupus), infectious diseases (viral and bacterial infections), and mitochondrial pathologies (under-regulated apoptosis leads to neurodegenerative diseases whereas over-regulated apoptosis leads to cancer.)

  11. Egg ovotransferrin‐derived ACE inhibitory peptide IRW increases ACE2 but decreases proinflammatory genes expression in mesenteric artery of spontaneously hypertensive rats

    PubMed Central

    Majumder, Kaustav; Liang, Guanxiang; Chen, Yanhong; Guan, LeLuo; Davidge, Sandra T.

    2015-01-01

    Scope Egg ovotransferrin‐derived angiotensin converting enzyme (ACE) inhibitory peptide IRW was previously shown to reduce blood pressure in spontaneously hypertensive rats through reduced vascular inflammation and increased nitric oxide‐mediated vasorelaxation. The main objective of the present study was to investigate the molecular mechanism of this peptide through transcriptome analysis by RNAseq technique. Methods and results Total RNA was extracted from kidney and mesenteric arteries; the RNAseq libraries (from untreated and IRW‐treated groups) were constructed and subjected to sequence using HiSeq 2000 system (Illumina) system. A total of 12 764 and 13 352 genes were detected in kidney and mesenteric arteries, respectively. The differentially expressed (DE) genes between untreated and IRW‐treated groups were identified and the functional analysis through ingenuity pathway analysis revealed a greater role of DE genes identified from mesenteric arteries than that of kidney in modulating various cardiovascular functions. Subsequent qPCR analysis further confirmed that IRW significantly increased the expression of ACE‐2, ABCB‐1, IRF‐8, and CDH‐1 while significantly decreased the expression ICAM‐1 and VCAM‐1 in mesenteric arteries. Conclusion Our research showed for the first time that ACE inhibitory peptide IRW could contribute to its antihypertensive activity through increased ACE2 and decreased proinflammatory genes expression. PMID:26016560

  12. An Altered gp100 Peptide Ligand with Decreased Binding by TCR and CD8α Dissects T Cell Cytotoxicity from Production of Cytokines and Activation of NFAT.

    PubMed

    Schaft, Niels; Coccoris, Miriam; Drexhage, Joost; Knoop, Christiaan; de Vries, I Jolanda M; Adema, Gosse J; Debets, Reno

    2013-01-01

    Altered peptide ligands (APLs) provide useful tools to study T cell activation and potentially direct immune responses to improve treatment of cancer patients. To better understand and exploit APLs, we studied the relationship between APLs and T cell function in more detail. Here, we tested a broad panel of gp100280-288 APLs with respect to T cell cytotoxicity, production of cytokines, and activation of Nuclear Factor of Activated T cells (NFAT) by human T cells gene-engineered with a gp100-HLA-A2-specific TCRαβ. We demonstrated that gp100-specific cytotoxicity, production of cytokines, and activation of NFAT were not affected by APLs with single amino acid substitutions, except for an APL with an amino acid substitution at position 3 (APL A3), which did not elicit any T cell response. A gp100 peptide with a double amino acid mutation (APL S4S6) elicited T cell cytotoxicity and production of IFNγ, and to a lesser extent TNFα, IL-4, and IL-5, but not production of IL-2 and IL-10, or activation of NFAT. Notably, T cell receptor (TCR)-mediated functions showed decreases in sensitivities for S4S6 versus gp100 wild-type (wt) peptide, which were minor for cytotoxicity but at least a 1000-fold more prominent for the production of cytokines. TCR-engineered T cells did not bind A3-HLA-A2, but did bind S4S6-HLA-A2 although to a lowered extent compared to wt peptide-HLA-A2. Moreover, S4S6-induced T cell function demonstrated an enhanced dependency on CD8α. Taken together, most gp100 APLs functioned as agonists, but A3 and S4S6 peptides acted as a null ligand and partial agonist, respectively. Our results further suggest that TCR-mediated cytotoxicity can be dissected from production of cytokines and activation of NFAT, and that the agonist potential of peptide mutants relates to the extent of binding by TCR and CD8α. These findings may facilitate the design of APLs to advance the study of T cell activation and their use for therapeutic applications. PMID:24027572

  13. An Altered gp100 Peptide Ligand with Decreased Binding by TCR and CD8α Dissects T Cell Cytotoxicity from Production of Cytokines and Activation of NFAT

    PubMed Central

    Schaft, Niels; Coccoris, Miriam; Drexhage, Joost; Knoop, Christiaan; de Vries, I. Jolanda M.; Adema, Gosse J.; Debets, Reno

    2013-01-01

    Altered peptide ligands (APLs) provide useful tools to study T cell activation and potentially direct immune responses to improve treatment of cancer patients. To better understand and exploit APLs, we studied the relationship between APLs and T cell function in more detail. Here, we tested a broad panel of gp100280–288 APLs with respect to T cell cytotoxicity, production of cytokines, and activation of Nuclear Factor of Activated T cells (NFAT) by human T cells gene-engineered with a gp100-HLA-A2-specific TCRαβ. We demonstrated that gp100-specific cytotoxicity, production of cytokines, and activation of NFAT were not affected by APLs with single amino acid substitutions, except for an APL with an amino acid substitution at position 3 (APL A3), which did not elicit any T cell response. A gp100 peptide with a double amino acid mutation (APL S4S6) elicited T cell cytotoxicity and production of IFNγ, and to a lesser extent TNFα, IL-4, and IL-5, but not production of IL-2 and IL-10, or activation of NFAT. Notably, T cell receptor (TCR)-mediated functions showed decreases in sensitivities for S4S6 versus gp100 wild-type (wt) peptide, which were minor for cytotoxicity but at least a 1000-fold more prominent for the production of cytokines. TCR-engineered T cells did not bind A3-HLA-A2, but did bind S4S6-HLA-A2 although to a lowered extent compared to wt peptide-HLA-A2. Moreover, S4S6-induced T cell function demonstrated an enhanced dependency on CD8α. Taken together, most gp100 APLs functioned as agonists, but A3 and S4S6 peptides acted as a null ligand and partial agonist, respectively. Our results further suggest that TCR-mediated cytotoxicity can be dissected from production of cytokines and activation of NFAT, and that the agonist potential of peptide mutants relates to the extent of binding by TCR and CD8α. These findings may facilitate the design of APLs to advance the study of T cell activation and their use for therapeutic applications. PMID:24027572

  14. A novel cell penetrating aspartic protease inhibitor blocks processing and presentation of tetanus toxoid more efficiently than pepstatin A.

    PubMed

    Zaidi, Nousheen; Burster, Timo; Sommandas, Vinod; Herrmann, Timo; Boehm, Bernhard O; Driessen, Christoph; Voelter, Wolfgang; Kalbacher, Hubert

    2007-12-14

    Selective inhibition of enzymes involved in antigen processing such as cathepsin E and cathepsin D is a valuable tool for investigating the roles of these enzymes in the processing pathway. However, the aspartic protease inhibitors, including the highly potent pepstatin A (PepA), are inefficiently transported across the cell membrane and thus have limited access to antigen processing compartments. Previously described mannose-pepstatin conjugates were efficiently taken up by the cells via receptor mediated uptake. However, cells without mannose receptors are unable to take up these conjugates efficiently. The aim of the present study was to synthesize new cell-permeable aspartic protease inhibitors by conjugating pepstatin A with well-known cell penetrating peptides (CPPs). To achieve this, the most commonly used CPPs namely pAntp(43-58) (penetratin), Tat(49-60), and 9-mer of l-arginine (R9), were synthesized and coupled to pepstatin. The enzyme inhibitory properties of these bioconjugates and their cellular uptake into MCF7 (human breast cancer cell line), Boleths (EBV-transformed B-cell line) and dendritic cells (DC) were the focus of our study. We found that the bioconjugate PepA-penetratin (PepA-P) was the most efficient cell-permeable aspartic protease inhibitor tested, and was more efficient than unconjugated PepA. Additionally, we found that PepA-P efficiently inhibited the tetanus toxoid C-fragment processing in peripheral blood mononuclear cells (PBMC), primary DC and in primary B cells. Therefore, PepA-P can be used in studying the role of intracellular aspartic proteases in the MHC class II antigen processing pathway. Moreover, inhibition of tetanus toxoid C-fragment processing by PepA-P clearly implicates the role of aspartic proteinases in antigen processing.

  15. MAPK3/1 is conducive to luteinizing hormone-mediated C-type natriuretic peptide decrease in bovine granulosa cells

    PubMed Central

    YANG, Lei; WEI, Qiang; GE, Junbang; ZHAO, Xiaoe; MA, Baohua

    2015-01-01

    C-type natriuretic peptide (CNP) plays a role as an oocyte maturation inhibitor (OMI) in many species, including the bovine. However, the effects of luteinizing hormone (LH) on CNP expression and its potential mechanisms have not reported in the bovine. In the present study, we aimed to study the effects of LH on CNP expression and to illuminate the potential molecular mechanism in this process. Our results showed that LH induced epidermal growth factor receptor (EGFR) phosphorylation, mitogen-activated protein kinase3/1 (MAPK3/1) activation and CNP mRNA decrease in cultured bovine granulosa cells. Further study revealed that LH suppressed CNP expression via the MAPK3/1 signaling pathway, which was activated by the EGFR pathway. In conclusion, our research suggested that MAPK3/1 is involved in LH-mediated decrease of CNP and that this process is related to the EGFR and MAPK3/1 signal pathways. PMID:26655567

  16. Improved angiogenic cell penetration in vitro and in vivo in collagen scaffolds with internal channels.

    PubMed

    Yahyouche, Asma; Zhidao, Xia; Triffitt, James T; Czernuszka, Jan T; Clover, A J P

    2013-06-01

    Porous scaffolds are limited in volume due to diffusion constraint and delay of vascular network formation. Channels have the potential to speed up cellular penetration. Their effectiveness in improving angiogenic cell penetration was assessed in vitro and in vivo in 3-D collagen scaffolds. In vitro, channelled and non-channelled scaffolds were seeded with vascular smooth muscle cells. Results demonstrated that the scaffolds supported angiogenic cell ingrowth in culture and the channels improved the depth of cell penetration into the scaffold (P < 0.05). The cells reside mainly around and migrate along the channels. In vivo, channels increased cell migration into the scaffolds (P < 0.05) particularly angiogenic cells (P < 0.05) resulting in a clear branched vascular network of microvessels after 2 weeks in the channelled samples which was not apparent in the non-channelled samples. Channels could aid production of tissue engineered constructs by offering the possibility of rapid blood vessel infiltration into collagen scaffolds.

  17. Synthetic Human TLR9-LRR11 Peptide Attenuates TLR9 Signaling by Binding to and thus Decreasing Internalization of CpG Oligodeoxynucleotides

    PubMed Central

    Pan, Xichun; Li, Bin; Kuang, Mei; Liu, Xin; Cen, Yanyan; Qin, Rongxin; Ding, Guofu; Zheng, Jiang; Zhou, Hong

    2016-01-01

    Toll-like receptor (TLR) 9 is an endosomal receptor recognizing bacterial DNA/CpG-containing oligodeoxynucleotides (CpG ODN). Blocking CpG ODN/TLR9 activity represents a strategy for therapeutic prevention of immune system overactivation. Herein, we report that a synthetic peptide (SP) representing the leucine-rich repeat 11 subdomain of the human TLR9 extracellular domain could attenuate CpG ODN/TLR9 activity in RAW264.7 cells by binding to CpG ODN and decreasing its internalization. Our results demonstrate that preincubation with SP specifically inhibited CpG ODN- but not lipopolysaccharide (LPS)- and lipopeptide (PAM3CSK4)-stimulated TNF-α and IL-6 release. Preincubation of SP with CpG ODN dose-dependently decreased TLR9-driven phosphorylation of IκBα and ERK and activation of NF-κB/p65. Moreover, SP dose-dependently decreased FAM-labeled CpG ODN internalization, whereas non-labeled CpG ODN reversed the inhibition. The KD value of SP-CpG ODN binding was within the micromolar range. Our results demonstrated that SP was a specific inhibitor of CpG ODN/TLR9 activity via binding to CpG ODN, leading to reduced ODN internalization and decreased activation of subsequent pathways within cells. Thus, SP could be used as a potential CpG ODN antagonist to block TLR9 signaling. PMID:26907260

  18. Modifications of Natural Peptides for Nanoparticle and Drug Design

    PubMed Central

    Jallouk, Andrew P.; Palekar, Rohun U.; Pan, Hua; Schlesinger, Paul H.; Wickline, Samuel A.

    2016-01-01

    Natural products serve as an important source of novel compounds for drug development. Recently, peptides have emerged as a new class of therapeutic agents due to their versatility and specificity for biological targets. Yet, their effective application often requires use of a nanoparticle delivery system. In this chapter, we review the role of natural peptides in the design and creation of nanomedicines, with a particular focus on cell-penetrating peptides, antimicrobial peptides, and peptide toxins. The use of natural peptides in conjunction with nanoparticle delivery systems holds great promise for the development of new therapeutic formulations as well as novel platforms for the delivery of various cargoes. PMID:25819276

  19. Short chain fatty acids (propionic and hexanoic) decrease Staphylococcus aureus internalization into bovine mammary epithelial cells and modulate antimicrobial peptide expression.

    PubMed

    Alva-Murillo, Nayeli; Ochoa-Zarzosa, Alejandra; López-Meza, Joel E

    2012-03-23

    Short chain fatty acids (SCFAs) are critical nutrients for ruminants and are mainly obtained from bacterial fermentation of carbohydrates. In addition to their nutrimental function, SCFAs have antimicrobial and anti-inflammatory properties, as well as immunomodulatory roles. It has been reported that sodium butyrate reduces Staphylococcus aureus internalization into bovine mammary epithelial cells (bMEC) and modulates antimicrobial peptide mRNA expression. Nevertheless, it has not been evaluated if sodium propionate (NaP) and sodium hexanoate (NaH) have similar actions. Since they are present in milk, the aim of this study was to determinate the effect of both SCFAs on S. aureus internalization into bMEC and to evaluate their effects on modulation of innate immunity elements. Our data showed that both SCFAs (0.25-5mM) did not affect S. aureus growth and bMEC viability. By gentamicin protection assay (MOI 30:1) we showed that NaP and NaH reduced bacterial internalization into bMEC, which ranged 27-55% and 39-65%, respectively, in relation to non treated controls. Also, both SCFAs up-regulate tracheal antimicrobial peptide (TAP) mRNA expression; however, bovine neutrophil β-defensin 5 (BNBD5) mRNA expression was not modified or was down-regulated. In addition, TAP and BNBD5 expression was up-regulated by S. aureus. Finally, the decrease in bacterial internalization under SCFA treatments is not related to nitric oxide production. In conclusion, NaP and NaH decrease S. aureus internalization into bMEC and modulate TAP gene expression, which may be related to the reduction in bacterial internalization. PMID:21930351

  20. Intracellular delivery of a cell-penetrating SOCS1 that targets IFN-gamma signaling.

    PubMed

    DiGiandomenico, Antonio; Wylezinski, Lukasz S; Hawiger, Jacek

    2009-07-21

    Suppressor of cytokine signaling-1 (SOCS1) is an intracellular inhibitor of the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway that couples interferon-gamma (IFN-gamma) signaling to the nucleus. Because several inflammatory diseases are associated with uncontrolled IFN-gamma signaling, we engineered a recombinant cell-penetrating SOCS1 (CP-SOCS1) to target this pathway. Here, we show that CP-SOCS1, analogous to endogenous SOCS1, interacted with components of the IFN-gamma signaling complex and functionally attenuated the phosphorylation of STAT1, which resulted in the subsequent inhibition of the production of proinflammatory chemokines and cytokines. Thus, controlled, intracellular delivery of recombinant CP-SOCS1 boosted the anti-inflammatory potential of the cell by restoring the homeostatic balance between pro- and anti-inflammatory signaling. This approach to controlling signal transduction has potential use for therapeutic targeting of signaling pathways associated with inflammatory diseases.

  1. Substrate-Initiated Synthesis of Cell-Penetrating Poly(disulfide)s

    PubMed Central

    Molinard, Guillaume; Roux, Aurélien; Sakai, Naomi; Matile, Stefan

    2015-01-01

    Lessons from surface-initiated polymerization are applied to grow cell-penetrating poly(disulfide)s directly on substrates of free choice. Reductive depolymerization after cellular uptake should then release the native substrates and minimize toxicity. In the presence of thiolated substrates, propagators containing a strained disulfide from asparagusic or, preferably, lipoic acid and a guanidinium cation polymerize into poly(disulfide)s in less than 5 min at room temperature at pH 7. Substrate-initiated polymerization of cationic poly(disulfide)s and their depolymerization with dithiothreitol causes the appearance and disappearance of transport activity in fluorogenic vesicles. The same process is further characterized by gel-permeation chromatography and fluorescence resonance energy transfer. PMID:23363440

  2. Effective Design of Multifunctional Peptides by Combining Compatible Functions

    PubMed Central

    Diener, Christian; Garza Ramos Martínez, Georgina; Moreno Blas, Daniel; Castillo González, David A.; Corzo, Gerardo; Castro-Obregon, Susana; Del Rio, Gabriel

    2016-01-01

    Multifunctionality is a common trait of many natural proteins and peptides, yet the rules to generate such multifunctionality remain unclear. We propose that the rules defining some protein/peptide functions are compatible. To explore this hypothesis, we trained a computational method to predict cell-penetrating peptides at the sequence level and learned that antimicrobial peptides and DNA-binding proteins are compatible with the rules of our predictor. Based on this finding, we expected that designing peptides for CPP activity may render AMP and DNA-binding activities. To test this prediction, we designed peptides that embedded two independent functional domains (nuclear localization and yeast pheromone activity), linked by optimizing their composition to fit the rules characterizing cell-penetrating peptides. These peptides presented effective cell penetration, DNA-binding, pheromone and antimicrobial activities, thus confirming the effectiveness of our computational approach to design multifunctional peptides with potential therapeutic uses. Our computational implementation is available at http://bis.ifc.unam.mx/en/software/dcf. PMID:27096600

  3. Effective Design of Multifunctional Peptides by Combining Compatible Functions.

    PubMed

    Diener, Christian; Garza Ramos Martínez, Georgina; Moreno Blas, Daniel; Castillo González, David A; Corzo, Gerardo; Castro-Obregon, Susana; Del Rio, Gabriel

    2016-04-01

    Multifunctionality is a common trait of many natural proteins and peptides, yet the rules to generate such multifunctionality remain unclear. We propose that the rules defining some protein/peptide functions are compatible. To explore this hypothesis, we trained a computational method to predict cell-penetrating peptides at the sequence level and learned that antimicrobial peptides and DNA-binding proteins are compatible with the rules of our predictor. Based on this finding, we expected that designing peptides for CPP activity may render AMP and DNA-binding activities. To test this prediction, we designed peptides that embedded two independent functional domains (nuclear localization and yeast pheromone activity), linked by optimizing their composition to fit the rules characterizing cell-penetrating peptides. These peptides presented effective cell penetration, DNA-binding, pheromone and antimicrobial activities, thus confirming the effectiveness of our computational approach to design multifunctional peptides with potential therapeutic uses. Our computational implementation is available at http://bis.ifc.unam.mx/en/software/dcf.

  4. Dietary peptides from the non-digestible fraction of Phaseolus vulgaris L. decrease angiotensin II-dependent proliferation in HCT116 human colorectal cancer cells through the blockade of the renin-angiotensin system.

    PubMed

    Luna-Vital, Diego A; Liang, Katie; González de Mejía, Elvira; Loarca-Piña, Guadalupe

    2016-05-18

    This study aimed to determine the ability of peptides present in the non-digestible fraction (NDF) of common beans to decrease angiotensin II (AngII) through the blockade of RAS and its effect on the proliferation of HCT116 human colorectal cancer cells. Pure synthesized peptides GLTSK and GEGSGA and the peptide fractions (PF) of cultivars Azufrado Higuera and Bayo Madero were used. The cells were pretreated with pure peptides, PF or AGT at their IC50 or IC25 values, in comparison with the simultaneous treatment of peptides and AGT. For western blot and microscopy analysis, 100 μM and 0.5 mg mL(-1) were used for pure peptides and PF treatments, respectively. According to the ELISA tests, GLTSK and GEGSGA decreased (p < 0.05) the conversion rate of AGT to angiotensin I (AngI) by 38 and 28%, respectively. All the peptides tested reduced (p < 0.05) the conversion rate of AngI to AngII from 38 to 50%. When the cells were pretreated with both pure peptides and PF before exposure to AGT, the effectiveness inhibiting cell proliferation was higher than the simultaneous treatment suggesting their preventive effects. GLTSK and GEGSGA interacted with the catalytic site of renin, the angiotensin-I converting enzyme, and the AngII receptor, mainly through hydrogen bonds, polar, hydrophobic and cation-π interactions according to molecular docking. Through confocal microscopy, it was determined that GLTSK and GEGSGA caused the decrease (p < 0.05) of AngII-dependent STAT3 nuclear activation in HCT116 cells by 66 and 23%, respectively. The results suggest that peptides present in the common bean NDF could potentially ameliorate the effects of RAS overexpression in colorectal cancer. PMID:27156533

  5. Enhanced antitumor effects of the BRBP1 compound peptide BRBP1-TAT-KLA on human brain metastatic breast cancer

    PubMed Central

    Fu, Bo; Long, Wei; Zhang, Ying; Zhang, Aifeng; Miao, Fengqin; Shen, Yuqing; Pan, Ning; Gan, Guangming; Nie, Fang; He, Youji; Zhang, Jianqiong; Teng, Gaojun

    2015-01-01

    Novel molecularly targeted agents that block the development and metastasis of human brain metastatic breast cancer hold great promise for their translational value. In this study, we constructed a novel targeting composite peptide BRBP1-TAT-KLA comprising of three elements: a brain metastatic breast carcinoma cell (231-BR)-binding peptide BRBP1, a cell penetrating peptide TAT, and a proapoptotic peptide KLA. This composite peptide efficiently internalized in 231-BR cells and consequently induced mitochondrial damage and cellular apoptosis. Exposure of 231-BR cells to BRBP1-TAT-KLA significantly decreased cell viability and increased apoptosis compared with the cells treated with the control peptides. In vivo relevance of these findings was further corroborated in the 231-BR tumor-bearing mice that demonstrated significantly delayed tumor development and metastasis following administration of BRBP1-TAT-KLA compared with those treated with TAT-KLA alone. Interestingly, BRBP1-TAT-KLA inhibited the formation of both large and micro-metastases, while TAT-KLA alone failed to significantly reduce micro-metastases in the breast cancer brain metastasis mice. BRBP1-TAT-KLA selectively homed to the tumors in vivo where it induced cellular apoptosis without significant toxicity on non-tumor tissues. Our findings therefore demonstrated the enhanced antitumor effects of the BRBP1 compound peptide BRBP1-TAT-KLA, providing insights toward development of a potential therapeutic strategy for brain metastatic breast cancer. PMID:25619721

  6. Enhanced antitumor effects of the BRBP1 compound peptide BRBP1-TAT-KLA on human brain metastatic breast cancer.

    PubMed

    Fu, Bo; Long, Wei; Zhang, Ying; Zhang, Aifeng; Miao, Fengqin; Shen, Yuqing; Pan, Ning; Gan, Guangming; Nie, Fang; He, Youji; Zhang, Jianqiong; Teng, Gaojun

    2015-01-26

    Novel molecularly targeted agents that block the development and metastasis of human brain metastatic breast cancer hold great promise for their translational value. In this study, we constructed a novel targeting composite peptide BRBP1-TAT-KLA comprising of three elements: a brain metastatic breast carcinoma cell (231-BR)-binding peptide BRBP1, a cell penetrating peptide TAT, and a proapoptotic peptide KLA. This composite peptide efficiently internalized in 231-BR cells and consequently induced mitochondrial damage and cellular apoptosis. Exposure of 231-BR cells to BRBP1-TAT-KLA significantly decreased cell viability and increased apoptosis compared with the cells treated with the control peptides. In vivo relevance of these findings was further corroborated in the 231-BR tumor-bearing mice that demonstrated significantly delayed tumor development and metastasis following administration of BRBP1-TAT-KLA compared with those treated with TAT-KLA alone. Interestingly, BRBP1-TAT-KLA inhibited the formation of both large and micro-metastases, while TAT-KLA alone failed to significantly reduce micro-metastases in the breast cancer brain metastasis mice. BRBP1-TAT-KLA selectively homed to the tumors in vivo where it induced cellular apoptosis without significant toxicity on non-tumor tissues. Our findings therefore demonstrated the enhanced antitumor effects of the BRBP1 compound peptide BRBP1-TAT-KLA, providing insights toward development of a potential therapeutic strategy for brain metastatic breast cancer.

  7. The incretin hormone glucagon‐like peptide 1 increases mitral cell excitability by decreasing conductance of a voltage‐dependent potassium channel

    PubMed Central

    Llewellyn‐Smith, Ida J.; Gribble, Fiona; Reimann, Frank; Trapp, Stefan; Fadool, Debra Ann

    2016-01-01

    Key points The gut hormone called glucagon‐like peptide 1 (GLP‐1) is a strong moderator of energy homeostasis and communication between the peripheral organs and the brain.GLP‐1 signalling occurs in the brain; using a newly developed genetic reporter line of mice, we have discovered GLP‐synthesizing cells in the olfactory bulb.GLP‐1 increases the firing frequency of neurons (mitral cells) that encode olfactory information by decreasing activity of voltage‐dependent K channels (Kv1.3).Modifying GLP‐1 levels, either therapeutically or following the ingestion of food, could alter the excitability of neurons in the olfactory bulb in a nutrition or energy state‐dependent manner to influence olfactory detection or metabolic sensing.The results of the present study uncover a new function for an olfactory bulb neuron (deep short axon cells, Cajal cells) that could be capable of modifying mitral cell activity through the release of GLP‐1. This might be of relevance for the action of GLP‐1 mimetics now widely used in the treatment of diabetes. Abstract The olfactory system is intricately linked with the endocrine system where it may serve as a detector of the internal metabolic state or energy homeostasis in addition to its classical function as a sensor of external olfactory information. The recent development of transgenic mGLU‐yellow fluorescent protein mice that express a genetic reporter under the control of the preproglucagon reporter suggested the presence of the gut hormone, glucagon‐like peptide (GLP‐1), in deep short axon cells (Cajal cells) of the olfactory bulb and its neuromodulatory effect on mitral cell (MC) first‐order neurons. A MC target for the peptide was determined using GLP‐1 receptor binding assays, immunocytochemistry for the receptor and injection of fluorescence‐labelled GLP‐1 analogue exendin‐4. Using patch clamp recording of olfactory bulb slices in the whole‐cell configuration, we report that GLP‐1 and its

  8. Technique for internal channelling of hydroentangled nonwoven scaffolds to enhance cell penetration.

    PubMed

    Durham, Elaine R; Ingham, Eileen; Russell, Stephen J

    2013-08-01

    An important requirement in thick, high-porosity scaffolds is to maximise cellular penetration into the interior and avoid necrosis during culture in vitro. Hitherto, reproducible control of the pore structure in nonwoven scaffolds has proved challenging. A new, channelled scaffold manufacturing process is reported based on water jet entanglement of fibres (hydroentangling) around filamentous template to form a coherent scaffold that is subsequently removed. Longitudinally-oriented channels were introduced within the scaffold in controlled proximity using 220 µm diameter cylindrical templates. In this case study, channelled scaffolds composed of poly(l-lactic acid) were manufactured and evaluated in vitro. Environmental scanning electron microscope and µCT (X-ray microtomography) confirmed channel openings in the scaffold cross-section before and after cell culture with human dermal fibroblasts up to 14 weeks. Histology at week 11 indicated that the channels promoted cell penetration and distribution within the scaffold interior. At week 14, cellular matrix deposition was evident in the internal channel walls and the entrances remained unoccluded by cellular matrix suggesting that diffusion conduits for mass transfer of nutrient to the scaffold interior could be maintained.

  9. Biofunctionalized nanoparticles with pH-responsive and cell penetrating blocks for gene delivery

    NASA Astrophysics Data System (ADS)

    Gaspar, V. M.; Marques, J. G.; Sousa, F.; Louro, R. O.; Queiroz, J. A.; Correia, I. J.

    2013-07-01

    Bridging the gap between nanoparticulate delivery systems and translational gene therapy is a long sought after requirement in nanomedicine-based applications. However, recent developments regarding nanoparticle functionalization have brought forward the ability to synthesize materials with biofunctional moieties that mimic the evolved features of viral particles. Herein we report the versatile conjugation of both cell penetrating arginine and pH-responsive histidine moieties into the chitosan polymeric backbone, to improve the physicochemical characteristics of the native material. Amino acid coupling was confirmed by 2D TOCSY NMR and Fourier transform infrared spectroscopy. The synthesized chitosan-histidine-arginine (CH-H-R) polymer complexed plasmid DNA biopharmaceuticals, and spontaneously assembled into stable 105 nm nanoparticles with spherical morphology and positive surface charge. The functionalized delivery systems were efficiently internalized into the intracellular compartment, and exhibited remarkably higher transfection efficiency than unmodified chitosan without causing any cytotoxic effect. Additional findings regarding intracellular trafficking events reveal their preferential escape from degradative lysosomal pathways and nuclear localization. Overall, this assembly of nanocarriers with bioinspired moieties provides the foundations for the design of efficient and customizable materials for cancer gene therapy.

  10. Allosteric Activation of a G Protein-coupled Receptor with Cell-penetrating Receptor Mimetics*

    PubMed Central

    Zhang, Ping; Leger, Andrew J.; Baleja, James D.; Rana, Rajashree; Corlin, Tiffany; Nguyen, Nga; Koukos, Georgios; Bohm, Andrew; Covic, Lidija; Kuliopulos, Athan

    2015-01-01

    G protein-coupled receptors (GPCRs) are remarkably versatile signaling systems that are activated by a large number of different agonists on the outside of the cell. However, the inside surface of the receptors that couple to G proteins has not yet been effectively modulated for activity or treatment of diseases. Pepducins are cell-penetrating lipopeptides that have enabled chemical and physical access to the intracellular face of GPCRs. The structure of a third intracellular (i3) loop agonist, pepducin, based on protease-activated receptor-1 (PAR1) was solved by NMR and found to closely resemble the i3 loop structure predicted for the intact receptor in the on-state. Mechanistic studies revealed that the pepducin directly interacts with the intracellular H8 helix region of PAR1 and allosterically activates the receptor through the adjacent (D/N)PXXYYY motif through a dimer-like mechanism. The i3 pepducin enhances PAR1/Gα subunit interactions and induces a conformational change in fluorescently labeled PAR1 in a very similar manner to that induced by thrombin. As pepducins can potentially be made to target any GPCR, these data provide insight into the identification of allosteric modulators to this major drug target class. PMID:25934391

  11. Technique for internal channelling of hydroentangled nonwoven scaffolds to enhance cell penetration

    PubMed Central

    Durham, Elaine R; Ingham, Eileen; Russell, Stephen J

    2013-01-01

    An important requirement in thick, high-porosity scaffolds is to maximise cellular penetration into the interior and avoid necrosis during culture in vitro. Hitherto, reproducible control of the pore structure in nonwoven scaffolds has proved challenging. A new, channelled scaffold manufacturing process is reported based on water jet entanglement of fibres (hydroentangling) around filamentous template to form a coherent scaffold that is subsequently removed. Longitudinally-oriented channels were introduced within the scaffold in controlled proximity using 220 µm diameter cylindrical templates. In this case study, channelled scaffolds composed of poly(l-lactic acid) were manufactured and evaluated in vitro. Environmental scanning electron microscope and µCT (X-ray microtomography) confirmed channel openings in the scaffold cross-section before and after cell culture with human dermal fibroblasts up to 14 weeks. Histology at week 11 indicated that the channels promoted cell penetration and distribution within the scaffold interior. At week 14, cellular matrix deposition was evident in the internal channel walls and the entrances remained unoccluded by cellular matrix suggesting that diffusion conduits for mass transfer of nutrient to the scaffold interior could be maintained. PMID:22532409

  12. Crocin improved locomotor function and mechanical behavior in the rat model of contused spinal cord injury through decreasing calcitonin gene related peptide (CGRP).

    PubMed

    Karami, Masoume; Bathaie, S Zahra; Tiraihi, Taqi; Habibi-Rezaei, Mehran; Arabkheradmand, Jalil; Faghihzadeh, Soghrat

    2013-12-15

    Various approaches have been offered to alleviate chronic pain resulting from spinal cord injuries (SCIs). Application of herbs and natural products, with potentially lower adverse effects, to cure diseases has been recommended in both traditional and modern medicines. Here, the effect of crocin on chronic pain induced by spinal cord contusion was investigated in an animal model. Female Wistar rats were randomly divided into five groups (5 rats in each); three groups were contused at the L1 level. One group was treated with crocin (150mg/kg) two weeks after spinal cord injury; the second group, control, was treated with vehicle only; and the third group was treated with ketoprofen. Two normal groups were also considered with or without crocin treatment. The mechanical behavioral test, the locomotor recovery test and the thermal behavioral test were applied weekly to evaluate the injury and recovery of rats. Significant improvements (p<0.05) in mechanical behavioral and locomotor recovery tests were seen in the rats treated with crocin. Thermal behavioral test did not show any significant changes due to crocin treatment. Plasma concentration of calcitonin-gene related peptide (CGRP) changed from 780.2±2.3 to 1140.3±4.5pg/ml due to SCI and reached 789.1±2.7pg/ml after crocin treatment. These changes were significant at the level of p<0.05. The present study shows the beneficial effects of crocin treatment on chronic pain induced by SCI, through decreasing CGRP as an important mediator of inflammation and pain.

  13. A Cell-Permeable Phospholipase C[gamma]1-Binding Peptide Transduces Neurons and Impairs Long-Term Spatial Memory

    ERIC Educational Resources Information Center

    Blum, Sonja; Dash, Pramod K.

    2004-01-01

    Growth factor-mediated signaling has emerged as an essential component of memory formation. In this study, we used a phospholipase C gamma 1 (PLC[gamma]1) binding, cell-penetrating peptide to sequester PLC[gamma]1 away from its target, the phosphotyrosine residues within the activated growth factor receptor. Peptides appear to transduce neurons…

  14. Measuring peptide translocation into large unilamellar vesicles.

    PubMed

    Spinella, Sara A; Nelson, Rachel B; Elmore, Donald E

    2012-01-27

    There is an active interest in peptides that readily cross cell membranes without the assistance of cell membrane receptors(1). Many of these are referred to as cell-penetrating peptides, which are frequently noted for their potential as drug delivery vectors(1-3). Moreover, there is increasing interest in antimicrobial peptides that operate via non-membrane lytic mechanisms(4,5), particularly those that cross bacterial membranes without causing cell lysis and kill cells by interfering with intracellular processes(6,7). In fact, authors have increasingly pointed out the relationship between cell-penetrating and antimicrobial peptides(1,8). A firm understanding of the process of membrane translocation and the relationship between peptide structure and its ability to translocate requires effective, reproducible assays for translocation. Several groups have proposed methods to measure translocation into large unilamellar lipid vesicles (LUVs)(9-13). LUVs serve as useful models for bacterial and eukaryotic cell membranes and are frequently used in peptide fluorescent studies(14,15). Here, we describe our application of the method first developed by Matsuzaki and co-workers to consider antimicrobial peptides, such as magainin and buforin II(16,17). In addition to providing our protocol for this method, we also present a straightforward approach to data analysis that quantifies translocation ability using this assay. The advantages of this translocation assay compared to others are that it has the potential to provide information about the rate of membrane translocation and does not require the addition of a fluorescent label, which can alter peptide properties(18), to tryptophan-containing peptides. Briefly, translocation ability into lipid vesicles is measured as a function of the Foster Resonance Energy Transfer (FRET) between native tryptophan residues and dansyl phosphatidylethanolamine when proteins are associated with the external LUV membrane (Figure 1). Cell-penetrating

  15. Oxysterols decrease apical-to-basolateral transport of Aß peptides via an ABCB1-mediated process in an in vitro Blood-brain barrier model constituted of bovine brain capillary endothelial cells.

    PubMed

    Saint-Pol, Julien; Candela, Pietra; Boucau, Marie-Christine; Fenart, Laurence; Gosselet, Fabien

    2013-06-23

    It is known that activation of the liver X receptors (LXRs) by natural or synthetic agonists decreases the amyloid burden and enhances cognitive function in transgenic murine models of Alzheimer's disease (AD). Recent evidence suggests that LXR activation may affect the transport of amyloid ß (Aß) peptides across the blood-brain barrier (the BBB, which isolates the brain from the peripheral circulation). By using a well-characterized in vitro BBB model, we demonstrated that LXR agonists (24S-hydroxycholesterol, 27-hydroxycholesterol and T0901317) modulated the expression of target genes involved in cholesterol homeostasis (such as ATP-binding cassette sub-family A member 1 (ABCA1)) and promoted cellular cholesterol efflux to apolipoprotein A-I and high density lipoproteins. Interestingly, we also observed a decrease in Aß peptide influx across brain capillary endothelial cells, although ABCA1 did not appear to be directly involved in this process. By focusing on others receptors and transporters that are thought to have major roles in Aß peptide entry into the brain, we then demonstrated that LXR stimulation provoked an increase in expression of the ABCB1 transporter (also named P-glycoprotein (P-gp)). Further investigations confirmed ABCB1's involvement in the restriction of Aß peptide influx. Taken as a whole, our results not only reinforce the BBB's key role in cerebral cholesterol homeostasis but also demonstrate the importance of the LXR/ABCB1 axis in Aß peptide influx-highlighting an attractive new therapeutic approach whereby the brain could be protected from peripheral Aß peptide entry.

  16. Matrix stiffness affects endocytic uptake of MK2-inhibitor peptides.

    PubMed

    Brugnano, Jamie L; Panitch, Alyssa

    2014-01-01

    In this study, the role of substrate stiffness on the endocytic uptake of a cell-penetrating peptide was investigated. The cell-penetrating peptide, an inhibitor of mitogen-activated protein kinase activated protein kinase II (MK2), enters a primary mesothelial cell line predominantly through caveolae. Using tissue culture polystyrene and polyacrylamide gels of varying stiffness for cell culture, and flow cytometry quantification and enzyme-linked immunoassays (ELISA) for uptake assays, we showed that the amount of uptake of the peptide is increased on soft substrates. Further, peptide uptake per cell increased at lower cell density. The improved uptake seen on soft substrates in vitro better correlates with in vivo functional studies where 10-100 µM concentrations of the MK2 inhibitor cell penetrating peptide demonstrated functional activity in several disease models. Additional characterization showed actin polymerization did not affect uptake, while microtubule polymerization had a profound effect on uptake. This work demonstrates that cell culture substrate stiffness can play a role in endocytic uptake, and may be an important consideration to improve correlations between in vitro and in vivo drug efficacy.

  17. Matrix Stiffness Affects Endocytic Uptake of MK2-Inhibitor Peptides

    PubMed Central

    Brugnano, Jamie L.; Panitch, Alyssa

    2014-01-01

    In this study, the role of substrate stiffness on the endocytic uptake of a cell-penetrating peptide was investigated. The cell-penetrating peptide, an inhibitor of mitogen-activated protein kinase activated protein kinase II (MK2), enters a primary mesothelial cell line predominantly through caveolae. Using tissue culture polystyrene and polyacrylamide gels of varying stiffness for cell culture, and flow cytometry quantification and enzyme-linked immunoassays (ELISA) for uptake assays, we showed that the amount of uptake of the peptide is increased on soft substrates. Further, peptide uptake per cell increased at lower cell density. The improved uptake seen on soft substrates in vitro better correlates with in vivo functional studies where 10–100 µM concentrations of the MK2 inhibitor cell penetrating peptide demonstrated functional activity in several disease models. Additional characterization showed actin polymerization did not affect uptake, while microtubule polymerization had a profound effect on uptake. This work demonstrates that cell culture substrate stiffness can play a role in endocytic uptake, and may be an important consideration to improve correlations between in vitro and in vivo drug efficacy. PMID:24400117

  18. Decreased Caffeine-Induced Locomotor Activity via Microinjection of CART Peptide into the Nucleus Accumbens Is Linked to Inhibition of the pCaMKIIa-D3R Interaction.

    PubMed

    Fu, Qiang; Zhou, Xiaoyan; Dong, Yun; Huang, Yonghong; Yang, Jianhua; Oh, Ki-Wan; Hu, Zhenzhen

    2016-01-01

    The purpose of this study was to characterize the inhibitory modulation of cocaine- and amphetamine-regulated transcript (CART) peptides, particularly with respect to the function of the D3 dopamine receptor (D3R), which is activated by its interaction with phosphorylated CaMKIIα (pCaMKIIα) in the nucleus accumbens (NAc). After repeated oral administration of caffeine (30 mg/kg) for five days, microinjection of CART peptide (0.08 μM/0.5 μl/hemisphere) into the NAc affected locomotor behavior. The pCaMKIIα-D3R interaction, D3R phosphorylation and cAMP/PKA/phosphorylated CREB (pCREB) signaling pathway activity were measured in NAc tissues, and Ca2+ influx and pCaMKIIα levels were measured in cultured NAc neurons. We found that CART attenuated the caffeine-mediated enhancement of depolarization-induced Ca2+ influx and CaMKIIα phosphorylation in cultured NAc neurons. Repeated microinjection of CART peptides into the NAc decreased the caffeine-induced enhancement of Ca2+ channels activity, pCaMKIIα levels, the pCaMKIIα-D3R interaction, D3R phosphorylation, cAMP levels, PKA activity and pCREB levels in the NAc. Furthermore, behavioral sensitization was observed in rats that received five-day administration of caffeine following microinjection of saline but not in rats that were treated with caffeine following microinjection of CART peptide. These results suggest that caffeine-induced CREB phosphorylation in the NAc was ameliorated by CART peptide due to its inhibition of D3R phosphorylation. These effects of CART peptides may play a compensatory role by inhibiting locomotor behavior in rats. PMID:27404570

  19. Decreased Caffeine-Induced Locomotor Activity via Microinjection of CART Peptide into the Nucleus Accumbens Is Linked to Inhibition of the pCaMKIIa-D3R Interaction

    PubMed Central

    Fu, Qiang; Zhou, Xiaoyan; Dong, Yun; Huang, Yonghong; Yang, Jianhua; Oh, Ki-Wan; Hu, Zhenzhen

    2016-01-01

    The purpose of this study was to characterize the inhibitory modulation of cocaine- and amphetamine-regulated transcript (CART) peptides, particularly with respect to the function of the D3 dopamine receptor (D3R), which is activated by its interaction with phosphorylated CaMKIIα (pCaMKIIα) in the nucleus accumbens (NAc). After repeated oral administration of caffeine (30 mg/kg) for five days, microinjection of CART peptide (0.08 μM/0.5 μl/hemisphere) into the NAc affected locomotor behavior. The pCaMKIIα-D3R interaction, D3R phosphorylation and cAMP/PKA/phosphorylated CREB (pCREB) signaling pathway activity were measured in NAc tissues, and Ca2+ influx and pCaMKIIα levels were measured in cultured NAc neurons. We found that CART attenuated the caffeine-mediated enhancement of depolarization-induced Ca2+ influx and CaMKIIα phosphorylation in cultured NAc neurons. Repeated microinjection of CART peptides into the NAc decreased the caffeine-induced enhancement of Ca2+ channels activity, pCaMKIIα levels, the pCaMKIIα-D3R interaction, D3R phosphorylation, cAMP levels, PKA activity and pCREB levels in the NAc. Furthermore, behavioral sensitization was observed in rats that received five-day administration of caffeine following microinjection of saline but not in rats that were treated with caffeine following microinjection of CART peptide. These results suggest that caffeine-induced CREB phosphorylation in the NAc was ameliorated by CART peptide due to its inhibition of D3R phosphorylation. These effects of CART peptides may play a compensatory role by inhibiting locomotor behavior in rats. PMID:27404570

  20. Dimerization of 30Kc19 protein in the presence of amphiphilic moiety and importance of Cys-57 during cell penetration.

    PubMed

    Park, Hee Ho; Sohn, Youngsoo; Yeo, Ji Woo; Park, Ju Hyun; Lee, Hong Jai; Ryu, Jina; Rhee, Won Jong; Park, Tai Hyun

    2014-12-01

    Recently, the recombinant 30Kc19 protein, originating from silkworm hemolymph of Bombyx mori has attracted attention due to its cell-penetrating property and potential application as a protein delivery system. However, this observation of penetration across cell membrane has raised questions concerning the interaction of the protein-lipid bilayer. Here, we report a dimerization propensity of the 30Kc19 protein in the presence of amphiphilic moieties; sodium dodecyl sulfate (SDS) or phospholipid. Native PAGE showed that the 30Kc19 monomer formed a dimer when SDS or phospholipid was present. In the glutathione-S-transferase (GST) pull-down assay, supplementation of the 30Kc19 protein to mammalian cell culture medium showed dimerization and penetration; due to phospholipids at the cell membrane, the main components of the lipid bilayer. Mutagenesis was performed, and penetration was observed by 30Kc19 C76A and not 30Kc19 C57A, which meant that the presence of cysteine at position 57 (Cys-57) is involved in dimerization of the 30Kc19 at the cell membrane during penetration. We anticipate application of the native 30Kc19 protein with high cell-penetrating efficiency for delivery of cargos to various cell types. The intracellular cargo delivery using the 30Kc19 protein is a non-virus derived (e.g. TAT) delivery method, which can open up new approaches for the delivery of therapeutics in bioindustries, such as pharma- and cosmeceuticals.

  1. Photoinduced apoptosis using a peptide carrying a photosensitizer.

    PubMed

    Watanabe, Kazunori; Fujiwara, Hayato; Kitamatsu, Mizuki; Ohtsuki, Takashi

    2016-07-01

    A novel molecule, TatBim-Alexa, consisting of the HIV1 Tat cell-penetrating peptide, the Bim apoptosis-inducing peptide, and Alexa Fluor 546 was synthesized for photoinducion of apoptosis. The Alexa Fluor 546 was used as a photosensitizer and covalently attached at the C-terminus of TatBim peptide by the thiol-maleimide reaction. Photo-dependent cytosolic internalization of TatBim-Alexa and photo-dependent apoptosis using TatBim-Alexa were demonstrated in several kinds of mammalian cells including human cancer cell lines. PMID:27165853

  2. A PEPTIDE UNCOUPLING CRMP-2 FROM THE PRESYNAPTIC Ca(2+) CHANNEL COMPLEX DEMONSTRATES EFFICACY IN ANIMAL MODELS OF MIGRAINE AND AIDS THERAPY-INDUCED NEUROPATHY.

    PubMed

    Ripsch, Matthew S; Ballard, Carrie J; Khanna, May; Hurley, Joyce H; White, Fletcher A; Khanna, Rajesh

    2012-03-01

    Biological, genetic, and clinical data provide compelling proof for N-type voltage-gated calcium channels (CaV2.2) as therapeutic targets for chronic pain. While decreasing channel function is ultimately anti-nociceptive, directly targeting the channel can lead to multiple adverse effects. Targeting regulators of channel activity may facilitate improved analgesic properties associated with channel block and afford a broader therapeutic window. Towards this end, we recently identified a short peptide, designated CBD3, derived from collapsin response mediator protein 2 (CRMP-2) that suppressed inflammatory and neuropathic hypersensitivity by inhibiting CRMP-2 binding to CaV2.2 [Brittain et al., Nature Medicine 17:822-829 (2011)]. Rodents administered CBD3 intraperitoneally, fused to the HIV TAT protein cell penetrating domain, exhibited antinociception lasting ~4 hours highlighting potential instability, limited oral bioavailability, and/or rapid elimination of peptide. This report focuses on improving upon the parental CBD3 peptide. Using SPOTScan analysis of synthetic versions of the parental CBD3 peptide, we identified peptides harboring single amino acid mutations that bound with greater affinity to CaV2.2. One such peptide, harboring a phenylalanine instead of glycine (G14F), was tested in rodent models of migraine and neuropathic pain. In vivo laser Doppler blood flowmetry measure of capsaicin-induced meningeal vascular responses related to headache pain was almost completely suppressed by dural application of the G14F peptide. The G14F mutant peptide, administered intraperitoneally, also exhibited greater antinociception in Stavudine (2'-3'-didehydro-2'-3'-dideoxythymidine (d4T)/Zerit®) model of AIDS therapy-induced peripheral neuropathy compared to the parent CBD3 peptide. These results demonstrate the patent translational value of small biologic drugs targeting CaV2.2 for management of clinical pain. PMID:22662308

  3. A PEPTIDE UNCOUPLING CRMP-2 FROM THE PRESYNAPTIC Ca2+ CHANNEL COMPLEX DEMONSTRATES EFFICACY IN ANIMAL MODELS OF MIGRAINE AND AIDS THERAPY-INDUCED NEUROPATHY

    PubMed Central

    Ripsch, Matthew S.; Ballard, Carrie J.; Khanna, May; Hurley, Joyce H.; White, Fletcher A.; Khanna, Rajesh

    2012-01-01

    Biological, genetic, and clinical data provide compelling proof for N-type voltage-gated calcium channels (CaV2.2) as therapeutic targets for chronic pain. While decreasing channel function is ultimately anti-nociceptive, directly targeting the channel can lead to multiple adverse effects. Targeting regulators of channel activity may facilitate improved analgesic properties associated with channel block and afford a broader therapeutic window. Towards this end, we recently identified a short peptide, designated CBD3, derived from collapsin response mediator protein 2 (CRMP-2) that suppressed inflammatory and neuropathic hypersensitivity by inhibiting CRMP-2 binding to CaV2.2 [Brittain et al., Nature Medicine 17:822–829 (2011)]. Rodents administered CBD3 intraperitoneally, fused to the HIV TAT protein cell penetrating domain, exhibited antinociception lasting ~4 hours highlighting potential instability, limited oral bioavailability, and/or rapid elimination of peptide. This report focuses on improving upon the parental CBD3 peptide. Using SPOTScan analysis of synthetic versions of the parental CBD3 peptide, we identified peptides harboring single amino acid mutations that bound with greater affinity to CaV2.2. One such peptide, harboring a phenylalanine instead of glycine (G14F), was tested in rodent models of migraine and neuropathic pain. In vivo laser Doppler blood flowmetry measure of capsaicin-induced meningeal vascular responses related to headache pain was almost completely suppressed by dural application of the G14F peptide. The G14F mutant peptide, administered intraperitoneally, also exhibited greater antinociception in Stavudine (2'-3'-didehydro-2'-3'-dideoxythymidine (d4T)/Zerit®) model of AIDS therapy-induced peripheral neuropathy compared to the parent CBD3 peptide. These results demonstrate the patent translational value of small biologic drugs targeting CaV2.2 for management of clinical pain. PMID:22662308

  4. The Antitumor Peptide CIGB-552 Increases COMMD1 and Inhibits Growth of Human Lung Cancer Cells

    PubMed Central

    Fernández Massó, Julio R.; Oliva Argüelles, Brizaida; Tejeda, Yelaine; Astrada, Soledad; Garay, Hilda; Reyes, Osvaldo; Delgado-Roche, Livan; Bollati-Fogolín, Mariela; Vallespí, Maribel G.

    2013-01-01

    We have demonstrated that the peptide L-2 designed from an alanine scanning of the Limulus-derived LALF32-51 region is a potential candidate for the anticancer therapy and its cell-penetrating capacity is an associated useful property. By the modification in the primary structure of L-2, a second-generation peptide (CIGB-552) was developed. However, the molecular mechanism underlying its cytotoxic activity remains partially unknown. In this study, it was shown that CIGB-552 increases the levels of COMMD1, a protein involved in copper homeostasis, sodium transport, and the NF-κB signaling pathway. We found that CIGB-552 induces ubiquitination of RelA and inhibits the antiapoptotic activity regulated by NF-κB, whereas the knockdown of COMMD1 blocks this effect. We also found that CIGB-552 decreases the antioxidant capacity and induces the peroxidation of proteins and lipids in the tumor cells. Altogether, this study provides new insights into the mechanism of action of the peptide CIGB-552, which could be relevant in the design of future anticancer therapies. PMID:23401744

  5. Structure-function analysis of the glioma targeting NFL-TBS.40-63 peptide corresponding to the tubulin-binding site on the light neurofilament subunit.

    PubMed

    Berges, Raphael; Balzeau, Julien; Takahashi, Masayuki; Prevost, Chantal; Eyer, Joel

    2012-01-01

    We previously reported that a 24 amino acid peptide (NFL-TBS.40-63) corresponding to the tubulin-binding site located on the light neurofilament subunit, selectively enters in glioblastoma cells where it disrupts their microtubule network and inhibits their proliferation. Here, we analyzed the structure-function relationships using an alanine-scanning strategy, in order to identify residues essential for these biological activities. We showed that the majority of modified peptides present a decreased or total loss to penetrate in these cells, or to alter microtubules. Correspondingly, circular dichroism measurements showed that this peptide forms either β-sheet or α-helix structures according to the solvent and that alanine substitution modified or destabilized the structure, in relation with changes in the biological activities. Moreover, substitution of serine residues by phosphoserine or aspartic acid concomitantly decreased the cell penetrating activity and the structure stability. These results indicate the importance of structure for the activities, including selectivity to glioblastoma cells of this peptide, and its regulation by phosphorylation.

  6. The glucagon-like peptide 1 receptor agonist exendin-4 improves reference memory performance and decreases immobility in the forced swim test.

    PubMed

    Isacson, Ruben; Nielsen, Elisabet; Dannaeus, Karin; Bertilsson, Göran; Patrone, Cesare; Zachrisson, Olof; Wikström, Lilian

    2011-01-10

    We have earlier shown that the glucagon-like peptide 1 receptor agonist exendin-4 stimulates neurogenesis in the subventricular zone and excerts anti-parkinsonian behavior. The aim of this study was to assess the effects of exendin-4 treatment on hippocampus-associated cognitive and mood-related behavior in adult rodents. To investigate potential effects of exendin-4 on hippocampal function, radial maze and forced swim test were employed. The time necessary to solve a radial maze task and the duration of immobility in the forced swim test were significantly reduced compared to respective vehicle groups if the animals had received exendin-4 during 1-2weeks before testing. In contrast to the positive control imipramine, single administration of exendin-4 1h before the challenge in the forced swim test had no effect. Immunohistochemical analysis showed that the incorporation of bromodeoxyuridine, a marker for DNA synthesis, as well as doublecortin expression was increased in the hippocampal dentate gyrus following chronic treatment with exendin-4 compared to vehicle-treated controls. The neurogenic effect of exendin-4 on hippocampus was confirmed by quantitative PCR showing an upregulation of mRNA expression for Ki-67, doublecortin and Mash-1. Since exendin-4 significantly improves hippocampus-associated behavior in adult rodents, it may be a candidate for alleviation of mood and cognitive disorders.

  7. Vitamin B12 conjugation of peptide-YY(3-36) decreases food intake compared to native peptide-YY(3-36) upon subcutaneous administration in male rats.

    PubMed

    Henry, Kelly E; Elfers, Clinton T; Burke, Rachael M; Chepurny, Oleg G; Holz, George G; Blevins, James E; Roth, Christian L; Doyle, Robert P

    2015-05-01

    Challenges to peptide-based therapies include rapid clearance, ready degradation by hydrolysis/proteolysis, and poor intestinal uptake and/or a need for blood brain barrier transport. This work evaluates the efficacy of conjugation of vitamin B12 (B12) on sc administered peptide tyrosine tyrosine (PYY)(3-36) function. In the current experiments, a B12-PYY(3-36) conjugate was tested against native PYY(3-36), and an inactive conjugate B12-PYYC36 (null control) in vitro and in vivo. In vitro experiments demonstrated similar agonism for the neuropeptide Y2 receptor by the B12-PYY(3-36) conjugate (EC50 26.5 nM) compared with native PYY(3-36) (EC50 16.0 nM), with the null control having an EC50 of 1.8 μM. In vivo experiments were performed in young adult male Sprague Dawley rats (9 wk). Daily treatments were delivered sc in five 1-hour pulses, each pulse delivering 5-10 nmol/kg, by implanted microinfusion pumps. Increases in hindbrain Fos expression were comparable 90 minutes after B12-PYY(3-36) or PYY3-36 injection relative to saline or B12-PYYC36. Food intake was reduced during a 5-day treatment for both B12-PYY(3-36)- (24%, P = .001) and PYY(3-36)-(13%, P = .008) treated groups relative to baseline. In addition, reduction of food intake after the three dark cycle treatment pulses was more consistent with B12-PYY(3-36) treatment (-26%, -29%, -27%) compared with the PYY(3-36) treatment (-3%, -21%, -16%), and B12-PYY(3-36) generated a significantly longer inhibition of food intake vs. PYY(3-36) treatment after the first two pulses (P = .041 and P = .036, respectively). These findings demonstrate a stronger, more consistent, and longer inhibition of food intake after the pulses of B12-PYY(3-36) conjugate compared with the native PYY(3-36).

  8. Glucagon-like peptide 1 receptor activation regulates cocaine actions and dopamine homeostasis in the lateral septum by decreasing arachidonic acid levels.

    PubMed

    Reddy, I A; Pino, J A; Weikop, P; Osses, N; Sørensen, G; Bering, T; Valle, C; Bluett, R J; Erreger, K; Wortwein, G; Reyes, J G; Graham, D; Stanwood, G D; Hackett, T A; Patel, S; Fink-Jensen, A; Torres, G E; Galli, A

    2016-01-01

    Agonism of the glucagon-like peptide 1 (GLP-1) receptor (GLP-1R) has been effective at treating aspects of addictive behavior for a number of abused substances, including cocaine. However, the molecular mechanisms and brain circuits underlying the therapeutic effects of GLP-1R signaling on cocaine actions remain elusive. Recent evidence has revealed that endogenous signaling at the GLP-1R within the forebrain lateral septum (LS) acts to reduce cocaine-induced locomotion and cocaine conditioned place preference, both considered dopamine (DA)-associated behaviors. DA terminals project from the ventral tegmental area to the LS and express the DA transporter (DAT). Cocaine acts by altering DA bioavailability by targeting the DAT. Therefore, GLP-1R signaling might exert effects on DAT to account for its regulation of cocaine-induced behaviors. We show that the GLP-1R is highly expressed within the LS. GLP-1, in LS slices, significantly enhances DAT surface expression and DAT function. Exenatide (Ex-4), a long-lasting synthetic analog of GLP-1 abolished cocaine-induced elevation of DA. Interestingly, acute administration of Ex-4 reduces septal expression of the retrograde messenger 2-arachidonylglycerol (2-AG), as well as a product of its presynaptic degradation, arachidonic acid (AA). Notably, AA reduces septal DAT function pointing to AA as a novel regulator of central DA homeostasis. We further show that AA oxidation product γ-ketoaldehyde (γ-KA) forms adducts with the DAT and reduces DAT plasma membrane expression and function. These results support a mechanism in which postsynaptic septal GLP-1R activation regulates 2-AG levels to alter presynaptic DA homeostasis and cocaine actions through AA. PMID:27187231

  9. Glucagon-like peptide 1 receptor activation regulates cocaine actions and dopamine homeostasis in the lateral septum by decreasing arachidonic acid levels

    PubMed Central

    Reddy, I A; Pino, J A; Weikop, P; Osses, N; Sørensen, G; Bering, T; Valle, C; Bluett, R J; Erreger, K; Wortwein, G; Reyes, J G; Graham, D; Stanwood, G D; Hackett, T A; Patel, S; Fink-Jensen, A; Torres, G E; Galli, A

    2016-01-01

    Agonism of the glucagon-like peptide 1 (GLP-1) receptor (GLP-1R) has been effective at treating aspects of addictive behavior for a number of abused substances, including cocaine. However, the molecular mechanisms and brain circuits underlying the therapeutic effects of GLP-1R signaling on cocaine actions remain elusive. Recent evidence has revealed that endogenous signaling at the GLP-1R within the forebrain lateral septum (LS) acts to reduce cocaine-induced locomotion and cocaine conditioned place preference, both considered dopamine (DA)-associated behaviors. DA terminals project from the ventral tegmental area to the LS and express the DA transporter (DAT). Cocaine acts by altering DA bioavailability by targeting the DAT. Therefore, GLP-1R signaling might exert effects on DAT to account for its regulation of cocaine-induced behaviors. We show that the GLP-1R is highly expressed within the LS. GLP-1, in LS slices, significantly enhances DAT surface expression and DAT function. Exenatide (Ex-4), a long-lasting synthetic analog of GLP-1 abolished cocaine-induced elevation of DA. Interestingly, acute administration of Ex-4 reduces septal expression of the retrograde messenger 2-arachidonylglycerol (2-AG), as well as a product of its presynaptic degradation, arachidonic acid (AA). Notably, AA reduces septal DAT function pointing to AA as a novel regulator of central DA homeostasis. We further show that AA oxidation product γ-ketoaldehyde (γ-KA) forms adducts with the DAT and reduces DAT plasma membrane expression and function. These results support a mechanism in which postsynaptic septal GLP-1R activation regulates 2-AG levels to alter presynaptic DA homeostasis and cocaine actions through AA. PMID:27187231

  10. The vimentin-tubulin binding site peptide (Vim-TBS.58-81) crosses the plasma membrane and enters the nuclei of human glioma cells.

    PubMed

    Balzeau, Julien; Peterson, Alan; Eyer, Joel

    2012-02-14

    Cell-penetrating peptides (CPPs) can translocate through the plasma membrane and localize in different cell compartments providing a promising delivery system for peptides, proteins, nucleic acids, and other products. Here we describe features of a novel cell-penetrating peptide derived from the intermediate filament protein vimentin, called Vim-TBS.58-81. We show that it enters cells from a glioblastoma line via endocytosis where it distributes throughout the cytoplasm and nucleus. Moreover, when coupled to the pro-apoptogenic peptide P10, it localizes to the nucleus inhibiting cell proliferation. Thus, the Vim-TBS.58-81 peptide represents an effective vector for delivery of peptides and potentially a broad range of cargos to the nucleus.

  11. Cellular uptake and in vivo distribution of polyhistidine peptides.

    PubMed

    Iwasaki, Takashi; Tokuda, Yoshihisa; Kotake, Ayaka; Okada, Hiroyuki; Takeda, Shuji; Kawano, Tsuyoshi; Nakayama, Yuji

    2015-07-28

    Cell-penetrating peptides (CPPs) are arginine/lysine-rich sequences, and they are effectively internalized into cells. In this process, positive charge is crucial. In the present study, we found polyhistidine peptides (PHPs), as the novel CPP, which are efficiently internalized into cells in a positive charge-independent manner. Interestingly, cellular uptake of the PHPs increased as the chain length increased, reaching a maximum uptake at H16 (HHHHHHHHHHHHHHHH-NH2). This H16 peptide showed up to 14.6-fold higher cell-penetrating capacity against HT1080 human fibrosarcoma cells relative to a major CPP, the octa-arginine (RRRRRRRR-NH2) peptide. Cellular uptake of the H16 peptide is mainly due to macropinocytosis and most of the H16 peptide localizes in the lysosome and Golgi apparatus. However, a cytoplasmic pro-apoptotic domain (KLAKLAKKLAKLAK-NH2) conjugated to the H16 peptide showed cytotoxic effects. This indicates that a proportion of the H16 peptide escapes from the macropinosome to the cytoplasm. In a protein transduction study, green fluorescence protein fused to the H16 peptide (GFP-H16) was purified by Ni-NTA chromatography, detected using an anti-His-tag antibody and internalized into cells. This serial process reveals that H16 functions as a His-tag and protein transduction domain. Furthermore, in vivo distribution analysis showed that the H16 peptide accumulates immediately in tumor tissue and is retained up to 132h following injection into the tumor (HT1080 human fibrosarcoma)-bearing mice. This is the first observation of a His-polymer being internalize into cells efficiently. The findings suggest that PHPs are novel CPPs. In particular, the H16 peptide represents a promising drug delivery carrier candidate in medical and biotechnological fields.

  12. Glucocorticoids facilitate astrocytic amyloid-β peptide deposition by increasing the expression of APP and BACE1 and decreasing the expression of amyloid-β-degrading proteases.

    PubMed

    Wang, Yanyan; Li, Maoquan; Tang, Jun; Song, Min; Xu, Xueqing; Xiong, Jiaxiang; Li, Junxia; Bai, Yun

    2011-07-01

    In most cases, the molecular mechanism underlying the pathogenesis of sporadic Alzheimer's disease (AD) is unknown. Elevated basal cortisol levels in AD patients suggest that glucocorticoids (GC) may contribute to the development and/or maintenance of AD. Amyloid plaques are the hallmark of AD, and they are considered to play an early role in the AD process. However, little is known about how their formation is regulated by stress and GC. Astrocyte accumulation is one of the earliest neuropathological changes in AD. Here, we report that GC elevated amyloid-β (Aβ) production in primary cultures of astrocytes by increasing amyloid precursor protein (APP) and β-site APP-cleaving enzyme 1 gene expression. Notably, GC administered to normal, middle-aged mice promoted the expression of APP and β-site APP-cleaving enzyme 1 in astrocytes, as determined by double immunofluorescence. Additionally, confocal microscopy and ELISA revealed that GC markedly reduced Aβ degradation and clearance by astrocytes in vitro, indicating a decreased neuroprotective capacity of the astrocytes. This may have been due to the decrease of several Aβ-degrading proteases, such as insulin-degrading enzyme and matrix metalloproteinase-9. These effects occurred through the activation of GC receptors. Taken together, our results demonstrate that GC can enhance the production of Aβ, reduce its degradation in astrocytes, and provide a molecular mechanism linking stress factors to AD. Our study suggests that GC can facilitate AD pathogenesis and that reducing GC in the elderly and early AD patients would be beneficial.

  13. Development of surface modified biodegradable polymeric nanoparticles to deliver GSE24.2 peptide to cells: a promising approach for the treatment of defective telomerase disorders.

    PubMed

    Egusquiaguirre, Susana P; Manguán-García, Cristina; Pintado-Berninches, Laura; Iarriccio, Laura; Carbajo, Daniel; Albericio, Fernando; Royo, Miriam; Pedraz, José Luís; Hernández, Rosa M; Perona, Rosario; Igartua, Manuela

    2015-04-01

    The aim of the present study was to develop a novel strategy to deliver intracellularly the peptide GSE24.2 for the treatment of Dyskeratosis congenita (DC) and other defective telomerase disorders. For this purpose, biodegradable polymeric nanoparticles using poly(lactic-co-glycolic acid) (PLGA NPs) or poly(lactic-co-glycolic acid)-poly ethylene glycol (PLGA-PEG NPs) attached to either polycations or cell-penetrating peptides (CPPs) were prepared in order to increase their cellular uptake. The particles exhibited an adequate size and zeta potential, with good peptide loading and a biphasic pattern obtained in the in vitro release assay, showing an initial burst release and a later sustained release. GSE24.2 structural integrity after encapsulation was assessed using SDS-PAGE, revealing an unaltered peptide after the NPs elaboration. According to the cytotoxicity results, cell viability was not affected by uncoated polymeric NPs, but the incorporation of surface modifiers slightly decreased the viability of cells. The intracellular uptake exhibited a remarkable improvement of the internalization, when the NPs were conjugated to the CPPs. Finally, the bioactivity, addressed by measuring DNA damage rescue and telomerase reactivation, showed that some formulations had the lowest cytotoxicity and highest biological activity. These results proved that GSE24.2-loaded NPs could be delivered to cells, and therefore, become an effective approach for the treatment of DC and other defective telomerase syndromes. PMID:25660910

  14. Increased peptide YY blood concentrations, not decreased acyl-ghrelin, are associated with reduced hunger and food intake in healthy older women: Preliminary evidence.

    PubMed

    Hickson, Mary; Moss, Charlotte; Dhillo, Waljit S; Bottin, Jeanne; Frost, Gary

    2016-10-01

    With ageing there is frequently a loss of appetite, termed anorexia of ageing, which can result in under-nutrition. We do not know how appetite control alters with ageing. The objective of this study was to investigate whether differences in the release of, and response to, gastrointestinal appetite hormones is altered in young compared to old healthy volunteers. We hypothesised that an increase in PYY and GLP-1 or a decrease ghrelin may result in a decreased appetite. A comparative experimental design, using a cross-sectional sample of ages from a healthy population, matched for sex and BMI was used. The study compared total ghrelin, acyl-ghrelin, PYY, GLP-1 and subjective appetite responses to ingestion of a standardised 2781kj (660 kcal) test meal. 31 female volunteers aged between 21 and 92yrs took part. Multiple linear regression showed that both age and sex had an independent effect on energy intake. Subjective appetite scores showed that hunger, pleasantness to eat, and prospective food intake were significantly lower in the older age groups. PYY incremental area under the curve (IAUC) was greater in the oldest old compared to younger ages f(3,27) = 2.9, p = 0.05. No differences in GLP-1, ghrelin or acyl-ghrelin were observed in the older compared to younger age groups. Our data suggest that there may be increases in postprandial PYY(3-36) levels in female octogenarians, potentially resulting in reduced appetite. There does not appear to be any change in ghrelin or acyl-ghrelin concentrations with ageing. PMID:27264721

  15. Increased peptide YY blood concentrations, not decreased acyl-ghrelin, are associated with reduced hunger and food intake in healthy older women: Preliminary evidence.

    PubMed

    Hickson, Mary; Moss, Charlotte; Dhillo, Waljit S; Bottin, Jeanne; Frost, Gary

    2016-10-01

    With ageing there is frequently a loss of appetite, termed anorexia of ageing, which can result in under-nutrition. We do not know how appetite control alters with ageing. The objective of this study was to investigate whether differences in the release of, and response to, gastrointestinal appetite hormones is altered in young compared to old healthy volunteers. We hypothesised that an increase in PYY and GLP-1 or a decrease ghrelin may result in a decreased appetite. A comparative experimental design, using a cross-sectional sample of ages from a healthy population, matched for sex and BMI was used. The study compared total ghrelin, acyl-ghrelin, PYY, GLP-1 and subjective appetite responses to ingestion of a standardised 2781kj (660 kcal) test meal. 31 female volunteers aged between 21 and 92yrs took part. Multiple linear regression showed that both age and sex had an independent effect on energy intake. Subjective appetite scores showed that hunger, pleasantness to eat, and prospective food intake were significantly lower in the older age groups. PYY incremental area under the curve (IAUC) was greater in the oldest old compared to younger ages f(3,27) = 2.9, p = 0.05. No differences in GLP-1, ghrelin or acyl-ghrelin were observed in the older compared to younger age groups. Our data suggest that there may be increases in postprandial PYY(3-36) levels in female octogenarians, potentially resulting in reduced appetite. There does not appear to be any change in ghrelin or acyl-ghrelin concentrations with ageing.

  16. Gramicidin A-based peptide vector for intracellular protein delivery.

    PubMed

    Stoilova, Tatiana B; Kovalchuk, Sergey I; Egorova, Natalya S; Surovoy, Andrey Y; Ivanov, Vadim T

    2008-10-01

    The development of the peptide-based vectors for the intracellular delivery of biologically active macromolecules has opened new prospects of their application in research and therapy. Earlier the amphipathic cell-penetrating peptide (CPP) Pep-1 was reported to mediate cellular uptake of proteins without covalent binding to them. In this work we studied the ability of a series of membrane-active amphipathic peptides, based on the gramicidin A sequence, to transport a model protein across the eukaryotic cell membrane. Among them the positively charged Cys-containing peptide P10C demonstrated the most effective beta-galactosidase intracellular delivery. Besides, this peptide was shown to form noncovalent associates with beta-galactosidase as judged from electrophoresis and enzymatic activity assays. In addition, a series of new gramicidin analogues were prepared and the effect of N-terminus modification of gramicidin on the protein transduction efficiency was studied.

  17. Membrane Thinning and Thickening Induced by Membrane-Active Amphipathic Peptides.

    PubMed

    Grage, Stephan L; Afonin, Sergii; Kara, Sezgin; Buth, Gernot; Ulrich, Anne S

    2016-01-01

    Membrane thinning has been discussed as a fundamental mechanism by which antimicrobial peptides can perturb cellular membranes. To understand which factors play a role in this process, we compared several amphipathic peptides with different structures, sizes and functions in their influence on the lipid bilayer thickness. PGLa and magainin 2 from X. laevis were studied as typical representatives of antimicrobial cationic amphipathic α-helices. A 1:1 mixture of these peptides, which is known to possess synergistically enhanced activity, allowed us to evaluate whether and how this synergistic interaction correlates with changes in membrane thickness. Other systems investigated here include the α-helical stress-response peptide TisB from E. coli (which forms membrane-spanning dimers), as well as gramicidin S from A. migulanus (a natural antibiotic), and BP100 (designer-made antimicrobial and cell penetrating peptide). The latter two are very short, with a circular β-pleated and a compact α-helical structure, respectively. Solid-state (2)H-NMR and grazing incidence small angle X-ray scattering (GISAXS) on oriented phospholipid bilayers were used as complementary techniques to access the hydrophobic thickness as well as the bilayer-bilayer repeat distance including the water layer in between. This way, we found that magainin 2, gramicidin S, and BP100 induced membrane thinning, as expected for amphiphilic peptides residing in the polar/apolar interface of the bilayer. PGLa, on the other hand, decreased the hydrophobic thickness only at very high peptide:lipid ratios, and did not change the bilayer-bilayer repeat distance. TisB even caused an increase in the hydrophobic thickness and repeat distance. When reconstituted as a mixture, PGLa and magainin 2 showed a moderate thinning effect which was less than that of magainin 2 alone, hence their synergistically enhanced activity does not seem to correlate with a modulation of membrane thickness. Overall, the absence of

  18. Membrane Thinning and Thickening Induced by Membrane-Active Amphipathic Peptides

    PubMed Central

    Grage, Stephan L.; Afonin, Sergii; Kara, Sezgin; Buth, Gernot; Ulrich, Anne S.

    2016-01-01

    Membrane thinning has been discussed as a fundamental mechanism by which antimicrobial peptides can perturb cellular membranes. To understand which factors play a role in this process, we compared several amphipathic peptides with different structures, sizes and functions in their influence on the lipid bilayer thickness. PGLa and magainin 2 from X. laevis were studied as typical representatives of antimicrobial cationic amphipathic α-helices. A 1:1 mixture of these peptides, which is known to possess synergistically enhanced activity, allowed us to evaluate whether and how this synergistic interaction correlates with changes in membrane thickness. Other systems investigated here include the α-helical stress-response peptide TisB from E. coli (which forms membrane-spanning dimers), as well as gramicidin S from A. migulanus (a natural antibiotic), and BP100 (designer-made antimicrobial and cell penetrating peptide). The latter two are very short, with a circular β-pleated and a compact α-helical structure, respectively. Solid-state 2H-NMR and grazing incidence small angle X-ray scattering (GISAXS) on oriented phospholipid bilayers were used as complementary techniques to access the hydrophobic thickness as well as the bilayer-bilayer repeat distance including the water layer in between. This way, we found that magainin 2, gramicidin S, and BP100 induced membrane thinning, as expected for amphiphilic peptides residing in the polar/apolar interface of the bilayer. PGLa, on the other hand, decreased the hydrophobic thickness only at very high peptide:lipid ratios, and did not change the bilayer-bilayer repeat distance. TisB even caused an increase in the hydrophobic thickness and repeat distance. When reconstituted as a mixture, PGLa and magainin 2 showed a moderate thinning effect which was less than that of magainin 2 alone, hence their synergistically enhanced activity does not seem to correlate with a modulation of membrane thickness. Overall, the absence of a

  19. Design and synthesis of a multivalent fluorescent folate-calix[4]arene conjugate: cancer cell penetration and intracellular localization.

    PubMed

    Consoli, Grazia Maria Letizia; Granata, Giuseppe; Fragassi, Giorgia; Grossi, Mauro; Sallese, Michele; Geraci, Corrada

    2015-03-21

    A novel fluorescently labeled folate conjugate in which four folic acid units are covalently conjugated with a 7-nitro-benzofurazan fluorophore by means of a calix[4]arene platform was synthesized by using a Cu-catalyzed azide-alkyne cycloaddition reaction (click chemistry). The synthesized construct (FA-C4-NBD) was characterized by mass spectrometry, NMR and fluorescence spectroscopy. Confocal fluorescence microscopy experiments were carried out to evaluate the cell penetration ability of FA-C4-NBD on normal and cancer cells. The cellular uptake of FA-C4-NBD proceeds via folate receptor-mediated endocytosis. FA-C4-NBD is internalized into HeLa cancer cells which express high levels of folate receptors, whereas the uptake into fibroblast NIH3T3 cells which have very low expression levels of folate receptors is negligible. The involvement of the folate receptor was corroborated by competition tests with free folic acid. Co-localization analysis with different organelle markers indicated that FA-C4-NBD is not eliminated by recycling towards the outside of the cell, but accumulates intracellularly in the endo-lysosomal system.

  20. Antimicrobial Peptides in 2014

    PubMed Central

    Wang, Guangshun; Mishra, Biswajit; Lau, Kyle; Lushnikova, Tamara; Golla, Radha; Wang, Xiuqing

    2015-01-01

    This article highlights new members, novel mechanisms of action, new functions, and interesting applications of antimicrobial peptides reported in 2014. As of December 2014, over 100 new peptides were registered into the Antimicrobial Peptide Database, increasing the total number of entries to 2493. Unique antimicrobial peptides have been identified from marine bacteria, fungi, and plants. Environmental conditions clearly influence peptide activity or function. Human α-defensin HD-6 is only antimicrobial under reduced conditions. The pH-dependent oligomerization of human cathelicidin LL-37 is linked to double-stranded RNA delivery to endosomes, where the acidic pH triggers the dissociation of the peptide aggregate to release its cargo. Proline-rich peptides, previously known to bind to heat shock proteins, are shown to inhibit protein synthesis. A model antimicrobial peptide is demonstrated to have multiple hits on bacteria, including surface protein delocalization. While cell surface modification to decrease cationic peptide binding is a recognized resistance mechanism for pathogenic bacteria, it is also used as a survival strategy for commensal bacteria. The year 2014 also witnessed continued efforts in exploiting potential applications of antimicrobial peptides. We highlight 3D structure-based design of peptide antimicrobials and vaccines, surface coating, delivery systems, and microbial detection devices involving antimicrobial peptides. The 2014 results also support that combination therapy is preferred over monotherapy in treating biofilms. PMID:25806720

  1. Transthyretin stabilization by iododiflunisal promotes amyloid-β peptide clearance, decreases its deposition, and ameliorates cognitive deficits in an Alzheimer's disease mouse model.

    PubMed

    Ribeiro, Carlos A; Oliveira, Sandra Marisa; Guido, Luis F; Magalhães, Ana; Valencia, Gregorio; Arsequell, Gemma; Saraiva, Maria João; Cardoso, Isabel

    2014-01-01

    Alzheimer's disease (AD) is the most common form of dementia and now represents 50-70% of total dementia cases. Over the last two decades, transthyretin (TTR) has been associated with AD and, very recently, a novel concept of TTR stability has been established in vitro as a key factor in TTR/amyloid-β (Aβ) interaction. Small compounds, TTR stabilizers (usually non-steroid anti-inflammatory drugs), bind to the thyroxine (T4) central binding channel, increasing TTR tetrameric stability and TTR/Aβ interaction. In this work, we evaluated in vivo the effects of one of the TTR stabilizers identified as improving TTR/Aβ interaction, iododiflunisal (IDIF), in Aβ deposition and other AD features, using AβPPswe/PS1A246E transgenic mice, either carrying two or just one copy of the TTR gene (AD/TTR+/+ or AD/TTR+/-, respectively), available and characterized in our laboratory. The results showed that IDIF administered orally bound TTR in plasma and stabilized the protein, as assessed by T4 displacement assays, and was able to enter the brain as revealed by mass spectrometry analysis of cerebrospinal fluid. TTR levels, both in plasma and cerebrospinal fluid, were not altered. In AD/TTR+/- mice, IDIF administration resulted not only in decreased brain Aβ levels and deposition but also in improved cognitive function associated with the AD-like neuropathology in this mouse model, although no improvements were detectable in the AD/TTR+/+ animals. Further, in AD/TTR+/- mice, Aβ levels were reduced in plasma suggesting TTR promoted Aβ clearance from the brain and from the periphery. Taken together, these results strengthen the importance of TTR stability in the design of therapeutic drugs, highlighting the capacity of IDIF to be used in AD treatment to prevent and to slow the progression of the disease. PMID:24169237

  2. Lipopolysaccharide (LPS) and tumor necrosis factor alpha (TNFα) blunt the response of Neuropeptide Y/Agouti-related peptide (NPY/AgRP) glucose inhibited (GI) neurons to decreased glucose.

    PubMed

    Hao, Lihong; Sheng, Zhenyu; Potian, Joseph; Deak, Adam; Rohowsky-Kochan, Christine; Routh, Vanessa H

    2016-10-01

    A population of Neuropeptide Y (NPY) neurons which co-express Agouti-related peptide (AgRP) in the arcuate nucleus of the hypothalamus (ARC) are inhibited at physiological levels of brain glucose and activated when glucose levels decline (e.g. glucose-inhibited or GI neurons). Fasting enhances the activation of NPY/AgRP-GI neurons by low glucose. In the present study we tested the hypothesis that lipopolysaccharide (LPS) inhibits the enhanced activation of NPY/AgRP-GI neurons by low glucose following a fast. Mice which express green fluorescent protein (GFP) on their NPY promoter were used to identify NPY/AgRP neurons. Fasting for 24h and LPS injection decreased blood glucose levels. As we have found previously, fasting increased c-fos expression in NPY/AgRP neurons and increased the activation of NPY/AgRP-GI neurons by decreased glucose. As we predicted, LPS blunted these effects of fasting at the 24h time point. Moreover, the inflammatory cytokine tumor necrosis factor alpha (TNFα) blocked the activation of NPY/AgRP-GI neurons by decreased glucose. These data suggest that LPS and TNFα may alter glucose and energy homeostasis, in part, due to changes in the glucose sensitivity of NPY/AgRP neurons. Interestingly, our findings also suggest that NPY/AgRP-GI neurons use a distinct mechanism to sense changes in extracellular glucose as compared to our previous studies of GI neurons in the adjacent ventromedial hypothalamic nucleus.

  3. Liposomes Combined an Integrin αvβ3-Specific Vector with pH-Responsible Cell-Penetrating Property for Highly Effective Antiglioma Therapy through the Blood-Brain Barrier.

    PubMed

    Shi, Kairong; Long, Yang; Xu, Chaoqun; Wang, Yang; Qiu, Yue; Yu, Qianwen; Liu, Yayuan; Zhang, Qianyu; Gao, Huile; Zhang, Zhirong; He, Qin

    2015-09-30

    Glioma, one of the most common aggressive malignancies, has the highest mortality in the present world. Delivery of nanocarriers from the systemic circulation to the glioma sites would encounter multiple physiological and biological barriers, such as blood-brain barrier (BBB) and the poor penetration of nanocarriers into the tumor. To circumvent these hurdles, the paclitaxel-loaded liposomes were developed by conjugating with a TR peptide (PTX-TR-Lip), integrin αvβ3-specific vector with pH-responsible cell-penetrating property, for transporting drug across the BBB and then delivering into glioma. Surface plasmon resonance (SPR) studies confirmed the very high affinity of TR-Lip and integrin αvβ3. In vitro results showed that TR-Lip exhibited strong transport ability across BBB, killed glioma cells and brain cancer stem cells (CSCs), and destroyed the vasculogenic mimicry (VM) channels. In vivo results demonstrated that TR-Lip could better target glioma, and eliminated brain CSCs and the VM channels in tumor tissues. The median survival time of tumor-bearing mice after administering PTX-TR-Lip (45 days) was significantly longer than that after giving free PTX (25.5 days, p < 0.001) or other controls. In conclusion, PTX-TR-Lip would improve the therapeutic efficacy of brain glioma in vitro and in vivo. PMID:26371468

  4. Biodistribution of the cyclotide MCoTI-II, a cyclic disulfide-rich peptide drug scaffold.

    PubMed

    Wang, Conan K; Stalmans, Sofie; De Spiegeleer, Bart; Craik, David J

    2016-05-01

    Disulfide-rich macrocyclic peptides are promising templates for drug design because of their unique topology and remarkable stability. However, little is known about their pharmacokinetics. In this study, we characterize the biodistribution in mice of Momordica cochinchinensis trypsin inhibitor-II (MCoTI-II), a cyclic three-disulfide-containing peptide that has been used in a number of studies as a drug scaffold. The distribution of MCoTI-II was compared with that of chlorotoxin, which is a four-disulfide-containing peptide that has been used to develop brain tumor imaging agents; dermorphin, which is a disulfide-less peptide; and bovine serum albumin, a large protein. Both MCoTI-II and chlorotoxin distributed predominantly to the serum and kidneys, confirming that they are stable in serum and suggesting that they are eliminated from the blood through renal clearance. Although cell-penetrating peptides have been reported to be able to transport across the blood-brain barrier, MCoTI-II, which is a cell-penetrating peptide, showed no uptake into the brain. The uptake of chlorotoxin was higher than that of MCoTI-II but lower than that of dermorphin, which is considered to have low uptake into the brain. This study provides insight into the behavior of disulfide-rich peptides in vivo. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd. PMID:26929247

  5. Biodistribution of the cyclotide MCoTI-II, a cyclic disulfide-rich peptide drug scaffold.

    PubMed

    Wang, Conan K; Stalmans, Sofie; De Spiegeleer, Bart; Craik, David J

    2016-05-01

    Disulfide-rich macrocyclic peptides are promising templates for drug design because of their unique topology and remarkable stability. However, little is known about their pharmacokinetics. In this study, we characterize the biodistribution in mice of Momordica cochinchinensis trypsin inhibitor-II (MCoTI-II), a cyclic three-disulfide-containing peptide that has been used in a number of studies as a drug scaffold. The distribution of MCoTI-II was compared with that of chlorotoxin, which is a four-disulfide-containing peptide that has been used to develop brain tumor imaging agents; dermorphin, which is a disulfide-less peptide; and bovine serum albumin, a large protein. Both MCoTI-II and chlorotoxin distributed predominantly to the serum and kidneys, confirming that they are stable in serum and suggesting that they are eliminated from the blood through renal clearance. Although cell-penetrating peptides have been reported to be able to transport across the blood-brain barrier, MCoTI-II, which is a cell-penetrating peptide, showed no uptake into the brain. The uptake of chlorotoxin was higher than that of MCoTI-II but lower than that of dermorphin, which is considered to have low uptake into the brain. This study provides insight into the behavior of disulfide-rich peptides in vivo. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

  6. Lipopolysaccharide (LPS) and tumor necrosis factor alpha (TNFα) blunt the response of Neuropeptide Y/Agouti-related peptide (NPY/AgRP) glucose inhibited (GI) neurons to decreased glucose.

    PubMed

    Hao, Lihong; Sheng, Zhenyu; Potian, Joseph; Deak, Adam; Rohowsky-Kochan, Christine; Routh, Vanessa H

    2016-10-01

    A population of Neuropeptide Y (NPY) neurons which co-express Agouti-related peptide (AgRP) in the arcuate nucleus of the hypothalamus (ARC) are inhibited at physiological levels of brain glucose and activated when glucose levels decline (e.g. glucose-inhibited or GI neurons). Fasting enhances the activation of NPY/AgRP-GI neurons by low glucose. In the present study we tested the hypothesis that lipopolysaccharide (LPS) inhibits the enhanced activation of NPY/AgRP-GI neurons by low glucose following a fast. Mice which express green fluorescent protein (GFP) on their NPY promoter were used to identify NPY/AgRP neurons. Fasting for 24h and LPS injection decreased blood glucose levels. As we have found previously, fasting increased c-fos expression in NPY/AgRP neurons and increased the activation of NPY/AgRP-GI neurons by decreased glucose. As we predicted, LPS blunted these effects of fasting at the 24h time point. Moreover, the inflammatory cytokine tumor necrosis factor alpha (TNFα) blocked the activation of NPY/AgRP-GI neurons by decreased glucose. These data suggest that LPS and TNFα may alter glucose and energy homeostasis, in part, due to changes in the glucose sensitivity of NPY/AgRP neurons. Interestingly, our findings also suggest that NPY/AgRP-GI neurons use a distinct mechanism to sense changes in extracellular glucose as compared to our previous studies of GI neurons in the adjacent ventromedial hypothalamic nucleus. PMID:27473896

  7. Clickable Cγ-azido(methylene/butylene) peptide nucleic acids and their clicked fluorescent derivatives: synthesis, DNA hybridization properties, and cell penetration studies.

    PubMed

    Jain, Deepak R; Ganesh, Krishna N

    2014-07-18

    Synthesis, characterization, and DNA complementation studies of clickable C(γ)-substituted methylene (azm)/butylene (azb) azido PNAs show that these analogues enhance the stability of the derived PNA:DNA duplexes. The fluorescent PNA oligomers synthesized by their click reaction with propyne carboxyfluorescein are seen to accumulate around the nuclear membrane in 3T3 cells.

  8. Peptide nanotubes.

    PubMed

    Hamley, Ian W

    2014-07-01

    The self-assembly of different classes of peptide, including cyclic peptides, amyloid peptides and surfactant-like peptides into nanotube structures is reviewed. The modes of self-assembly are discussed. Additionally, applications in bionanotechnology and synthetic materials science are summarized.

  9. Development of a Peptide that Selectively Activates Protein Phosphatase-1 in Living Cells**

    PubMed Central

    Chatterjee, Jayanta; Beullens, Monique; Sukackaite, Rasa; Qian, Junbin; Lesage, Bart; Hart, Darren J; Bollen, Mathieu; Köhn, Maja

    2012-01-01

    The first cell-penetrating peptide that activates protein phosphatase-1 (PP1) by disrupting a subset of PP1 complexes in living cells has been developed. Activated PP1 rapidly dephosphorylates its substrates, counteracting kinase activity inside cells. Activation of PP1 can thus be a novel approach to study PP1 function and to counteract Ser/Thr kinase activity under pathologically increased kinase signaling. PMID:22962028

  10. Current status of multiple antigen-presenting peptide vaccine systems: Application of organic and inorganic nanoparticles

    PubMed Central

    2011-01-01

    Many studies are currently investigating the development of safe and effective vaccines to prevent various infectious diseases. Multiple antigen-presenting peptide vaccine systems have been developed to avoid the adverse effects associated with conventional vaccines (i.e., live-attenuated, killed or inactivated pathogens), carrier proteins and cytotoxic adjuvants. Recently, two main approaches have been used to develop multiple antigen-presenting peptide vaccine systems: (1) the addition of functional components, e.g., T-cell epitopes, cell-penetrating peptides, and lipophilic moieties; and (2) synthetic approaches using size-defined nanomaterials, e.g., self-assembling peptides, non-peptidic dendrimers, and gold nanoparticles, as antigen-displaying platforms. This review summarizes the recent experimental studies directed to the development of multiple antigen-presenting peptide vaccine systems. PMID:21861904

  11. A viral peptide for intracellular delivery

    NASA Astrophysics Data System (ADS)

    Falanga, Annarita; Tarallo, Rossella; Cantisani, Marco; Della Pepa, Maria Elena; Galdiero, Massimiliano; Galdiero, Stefania

    2012-10-01

    Biological membranes represent a critical hindrance for administering active molecules which are often unable to reach their designated intracellular target sites. In order to overcome this barrier-like behavior not easily circumvented by many pharmacologically-active molecules, synthetic transporters have been exploited to promote cellular uptake. Linking or complexing therapeutic molecules to peptides that can translocate through the cellular membranes could enhance their internal delivery, and consequently, a higher amount of active compound would reach the site of action. Use of cell penetrating peptides (CPPs) is one of the most promising strategy to efficiently translocate macromolecules through the plasma membrane, and have attracted a lot of attention. New translocating peptides are continuously described and in the present review, we will focus on viral derived peptides, and in particular a peptide (gH625) derived from the herpes simplex virus type 1 (HSV-1) glycoprotein H (gH) that has proved to be a useful delivery vehicle due to its intrinsic properties of inducing membrane perturbation.

  12. Antihypertensive peptides from food proteins.

    PubMed

    Aluko, Rotimi E

    2015-01-01

    Bioactive peptides are encrypted within the primary structure of food proteins where they remain inactive until released by enzymatic hydrolysis. Once released from the parent protein, certain peptides have the ability to modulate the renin-angiotensin system (RAS) because they decrease activities of renin or angiotensin-converting enzyme (ACE), the two main enzymes that regulate mammalian blood pressure. These antihypertensive peptides can also enhance the endothelial nitric oxide synthase (eNOS) pathway to increase nitric oxide (NO) levels within vascular walls and promote vasodilation. The peptides can block the interactions between angiotensin II (vasoconstrictor) and angiotensin receptors, which can contribute to reduced blood pressure. This review focuses on the methods that are involved in antihypertensive peptide production from food sources, including fractionation protocols that are used to enrich bioactive peptide content and enhance peptide activity. It also discusses mechanisms that are believed to be involved in the antihypertensive activity of these peptides.

  13. Peptide translocation through the plasma membrane of human cells: Can oxidative stress be exploited to gain better intracellular access?

    PubMed Central

    Wang, Ting-Yi; Pellois, Jean-Philippe

    2016-01-01

    ABSTRACT Cell-penetrating peptides (CPPs) enter cells primarily by escaping from endosomal compartments or by directly translocating across the plasma membrane. Due to their capability of permeating into the cytosolic space of the cell, CPPs are utilized for the delivery of cell-impermeable molecules. However, the fundamental mechanisms and parameters associated with the penetration of CPPs and their cargos through the lipid bilayer have not been fully determined. This in turn has hampered their usage in biotechnological or therapeutic applications. We have recently reported that the cell penetration activity of poly-arginine CPPs (PACPPs) is dependent on the oxidation status of the plasma membrane of cells. Our data support a model where the positively-charged PACPP binds negatively-charged lipids exposed on the cell surface as a result of oxidative damage. The PACPP then crosses the membrane via formation of inverted micelles with these anionic lipids. This model provides a plausible explanation for the high variability in the cell delivery efficiency of a PACPP often observed in different settings. Notably, taking into account the current literature describing the effects of lipid oxidation, our data point to a highly complex and underappreciated interplay between PACPPs and oxidized membrane species. Overall, a better understanding of oxidation-dependent cell penetration might provide a fundamental basis for development of optimal cell permeable peptides (including cyclic peptides, stapled peptides, peptoids, etc…) and of robust delivery protocols. PMID:27574543

  14. Peptide translocation through the plasma membrane of human cells: Can oxidative stress be exploited to gain better intracellular access?

    PubMed

    Wang, Ting-Yi; Pellois, Jean-Philippe

    2016-01-01

    Cell-penetrating peptides (CPPs) enter cells primarily by escaping from endosomal compartments or by directly translocating across the plasma membrane. Due to their capability of permeating into the cytosolic space of the cell, CPPs are utilized for the delivery of cell-impermeable molecules. However, the fundamental mechanisms and parameters associated with the penetration of CPPs and their cargos through the lipid bilayer have not been fully determined. This in turn has hampered their usage in biotechnological or therapeutic applications. We have recently reported that the cell penetration activity of poly-arginine CPPs (PACPPs) is dependent on the oxidation status of the plasma membrane of cells. Our data support a model where the positively-charged PACPP binds negatively-charged lipids exposed on the cell surface as a result of oxidative damage. The PACPP then crosses the membrane via formation of inverted micelles with these anionic lipids. This model provides a plausible explanation for the high variability in the cell delivery efficiency of a PACPP often observed in different settings. Notably, taking into account the current literature describing the effects of lipid oxidation, our data point to a highly complex and underappreciated interplay between PACPPs and oxidized membrane species. Overall, a better understanding of oxidation-dependent cell penetration might provide a fundamental basis for development of optimal cell permeable peptides (including cyclic peptides, stapled peptides, peptoids, etc…) and of robust delivery protocols. PMID:27574543

  15. Peptide translocation through the plasma membrane of human cells: Can oxidative stress be exploited to gain better intracellular access?

    PubMed

    Wang, Ting-Yi; Pellois, Jean-Philippe

    2016-01-01

    Cell-penetrating peptides (CPPs) enter cells primarily by escaping from endosomal compartments or by directly translocating across the plasma membrane. Due to their capability of permeating into the cytosolic space of the cell, CPPs are utilized for the delivery of cell-impermeable molecules. However, the fundamental mechanisms and parameters associated with the penetration of CPPs and their cargos through the lipid bilayer have not been fully determined. This in turn has hampered their usage in biotechnological or therapeutic applications. We have recently reported that the cell penetration activity of poly-arginine CPPs (PACPPs) is dependent on the oxidation status of the plasma membrane of cells. Our data support a model where the positively-charged PACPP binds negatively-charged lipids exposed on the cell surface as a result of oxidative damage. The PACPP then crosses the membrane via formation of inverted micelles with these anionic lipids. This model provides a plausible explanation for the high variability in the cell delivery efficiency of a PACPP often observed in different settings. Notably, taking into account the current literature describing the effects of lipid oxidation, our data point to a highly complex and underappreciated interplay between PACPPs and oxidized membrane species. Overall, a better understanding of oxidation-dependent cell penetration might provide a fundamental basis for development of optimal cell permeable peptides (including cyclic peptides, stapled peptides, peptoids, etc…) and of robust delivery protocols.

  16. Enhanced Cytotoxicity through Conjugation of a “Clickable” Luminescent Re(I) Complex to a Cell-Penetrating Lipopeptide

    PubMed Central

    2014-01-01

    Re(I) tricarbonyl polypyridine-based complexes are particularly attractive metal complexes in the field of inorganic chemical biology due to their luminescent properties, ease of conjugation to targeting biomolecules, and the possibility to prepare their “hot” 99mTc analogues for radioimaging. In this study, we prepared and characterized a novel, “clickable” complex, [Re(2,2′-bipyridine)(3-ethynylpyridine)(CO)3](BF4) ([Re(CO)3(bipy)(py-alkyne)](BF4)), exhibiting the characteristic luminescent properties and moderate cytotoxicity of this general class of compound. Using Cu(I)-catalyzed “click” chemistry, the complex was efficiently attached to a lipidated peptide known to increase cell permeability, namely, the myristoylated HIV-1 Tat peptide (myr-Tat), to give Re-myr-Tat. Fluorescence microscopy localization in human cervical cancer cells (HeLa) confirmed enhanced cellular uptake of Re-myr-Tat compared with [Re(CO)3(bipy)(py-alkyne)](BF4), and cytotoxicity studies showed that this resulted in an increase in potency to a level comparable with cisplatin (13.0 ± 2.0 μM). PMID:25050170

  17. DNA-histone complexes as ligands amplify cell penetration and nuclear targeting of anti-DNA antibodies via energy-independent mechanisms.

    PubMed

    Zannikou, Markella; Bellou, Sofia; Eliades, Petros; Hatzioannou, Aikaterini; Mantzaris, Michael D; Carayanniotis, George; Avrameas, Stratis; Lymberi, Peggy

    2016-01-01

    We have generated three monoclonal cell-penetrating antibodies (CPAbs) from a non-immunized lupus-prone (NZB × NZW)F1 mouse that exhibited high anti-DNA serum titres. These CPAbs are polyreactive because they bind to DNA and other cellular components, and localize mainly in the nucleus of HeLa cells, albeit with a distinct nuclear labelling profile. Herein, we have examined whether DNA-histone complexes (DHC) binding to CPAbs, before cell entry, could modify the cell penetration of CPAbs or their nuclear staining properties. By applying confocal microscopy and image analysis, we found that extracellular binding of purified CPAbs to DHC significantly enhanced their subsequent cell-entry, both in terms of percentages of positively labelled cells and fluorescence intensity (internalized CPAb amount), whereas there was a variable effect on their nuclear staining profile. Internalization of CPAbs, either alone or bound to DHC, remained unaltered after the addition of endocytosis-specific inhibitors at 37° or assay performance at 4°, suggesting the involvement of energy-independent mechanisms in the internalization process. These findings assign to CPAbs a more complex pathogenetic role in systemic lupus erythematosus where both CPAbs and nuclear components are abundant.

  18. Antimicrobial peptides

    PubMed Central

    2014-01-01

    With increasing antibiotics resistance, there is an urgent need for novel infection therapeutics. Since antimicrobial peptides provide opportunities for this, identification and optimization of such peptides have attracted much interest during recent years. Here, a brief overview of antimicrobial peptides is provided, with focus placed on how selected hydrophobic modifications of antimicrobial peptides can be employed to combat also more demanding pathogens, including multi-resistant strains, without conferring unacceptable toxicity. PMID:24758244

  19. Biological activity of Tat (47-58) peptide on human pathogenic fungi

    SciTech Connect

    Jung, Hyun Jun; Park, Yoonkyung; Hahm, Kyung-Soo; Lee, Dong Gun . E-mail: dglee222@knu.ac.kr

    2006-06-23

    Tat (47-58) peptide, a positively charged Arginine-rich peptide derived from HIV-1 regulatory protein Tat, is known for a peptidic delivery factor as a cell-penetrating peptide on mammalian cells. In this study, antifungal effect and its mode of action of Tat peptide were investigated on fungal cells. The results indicate that Tat peptide exhibits antifungal activity against pathogenic fungal cells without hemolytic effect on human erythrocytes. To understand the mechanism(s) of Tat peptide, the cellular distribution of the peptide was investigated. Tat peptide internalized in the fungal cells without any damage to cell membrane when examined using an artificial liposome (PC/cholesterol; 10:1, w/w). Moreover, flow cytometry analysis exhibited the uptake of Tat peptide by energy- and salt-independent pathway, and confocal scanning microscopy displayed that this peptide accumulated in the nucleus of fungal cells rapidly without any impediment by time or temperature, which generally influence on the viral infections. After penetration into the nuclear, the peptide affected the process of cell cycle of Candida albicans through the arrest at G1 phase.

  20. Synthetic Mimic of Antimicrobial Peptide with Nonmembrane-Disrupting Antibacterial Properties

    PubMed Central

    2008-01-01

    Polyguanidinium oxanorbornene (PGON) was synthesized from norbornene monomers via ring-opening metathesis polymerization. This polymer was observed to be strongly antibacterial against Gram-negative and Gram-positive bacteria as well as nonhemolytic against human red blood cells. Time-kill studies indicated that this polymer is lethal and not just bacteriostatic. In sharp contrast to previously reported SMAMPs (synthetic mimics of antimicrobial peptides), PGON did not disrupt membranes in vesicle-dye leakage assays and microscopy experiments. The unique biological properties of PGON, in same ways similar to cell-penetrating peptides, strongly encourage the examination of other novel guanidino containing macromolecules as powerful and selective antimicrobial agents. PMID:18850741

  1. Vesicular Galectin-3 levels decrease with donor age and contribute to the reduced osteo-inductive potential of human plasma derived extracellular vesicles.

    PubMed

    Weilner, Sylvia; Keider, Verena; Winter, Melanie; Harreither, Eva; Salzer, Benjamin; Weiss, Florian; Schraml, Elisabeth; Messner, Paul; Pietschmann, Peter; Hildner, Florian; Gabriel, Christian; Redl, Heinz; Grillari-Voglauer, Regina; Grillari, Johannes

    2016-01-01

    Aging results in a decline of physiological functions and in reduced repair capacities, in part due to impaired regenerative power of stem cells, influenced by the systemic environment. In particular osteogenic differentiation capacity (ODC) of mesenchymal stem cells (MSCs) has been shown to decrease with age, thereby contributing to reduced bone formation and an increased fracture risk. Searching for systemic factors that might contribute to this age related decline of regenerative capacity led us to investigate plasma-derived extracellular vesicles (EVs). EVs of the elderly were found to inhibit osteogenesis compared to those of young individuals. By analyzing the differences in the vesicular content Galectin-3 was shown to be reduced in elderly-derived vesicles. While overexpression of Galectin-3 resulted in an enhanced ODC of MSCs, siRNA against Galectin-3 reduced osteogenesis. Modulation of intravesicular Galectin-3 levels correlated with an altered osteo-inductive potential indicating that vesicular Galectin-3 contributes to the biological response of MSCs to EVs. By site-directed mutagenesis we identified a phosphorylation-site on Galectin-3 mediating this effect. Finally, we showed that cell penetrating peptides comprising this phosphorylation-site are sufficient to increase ODC in MSCs. Therefore, we suggest that decrease of Galectin-3 in the plasma of elderly contributes to the age-related loss of ODC. PMID:26752347

  2. Vesicular Galectin-3 levels decrease with donor age and contribute to the reduced osteo-inductive potential of human plasma derived extracellular vesicles

    PubMed Central

    Weilner, Sylvia; Keider, Verena; Winter, Melanie; Harreither, Eva; Salzer, Benjamin; Weiss, Florian; Schraml, Elisabeth; Messner, Paul; Pietschmann, Peter; Hildner, Florian; Gabriel, Christian; Redl, Heinz; Grillari-Voglauer, Regina; Grillari, Johannes

    2016-01-01

    Aging results in a decline of physiological functions and in reduced repair capacities, in part due to impaired regenerative power of stem cells, influenced by the systemic environment. In particular osteogenic differentiation capacity (ODC) of mesenchymal stem cells (MSCs) has been shown to decrease with age, thereby contributing to reduced bone formation and an increased fracture risk. Searching for systemic factors that might contribute to this age related decline of regenerative capacity led us to investigate plasma-derived extracellular vesicles (EVs). EVs of the elderly were found to inhibit osteogenesis compared to those of young individuals. By analyzing the differences in the vesicular content Galectin-3 was shown to be reduced in elderly-derived vesicles. While overexpression of Galectin-3 resulted in an enhanced ODC of MSCs, siRNA against Galectin-3 reduced osteogenesis. Modulation of intravesicular Galectin-3 levels correlated with an altered osteo-inductive potential indicating that vesicular Galectin-3 contributes to the biological response of MSCs to EVs. By site-directed mutagenesis we identified a phosphorylation-site on Galectin-3 mediating this effect. Finally, we showed that cell penetrating peptides comprising this phosphorylation-site are sufficient to increase ODC in MSCs. Therefore, we suggest that decrease of Galectin-3 in the plasma of elderly contributes to the age-related loss of ODC. PMID:26752347

  3. Extra- and Intracellular Imaging of Human Matrix Metalloprotease 11 (hMMP-11) with a Cell-penetrating FRET Substrate*

    PubMed Central

    Meyer, B. Sina; Rademann, Jörg

    2012-01-01

    Matrix metalloprotease 11 (MMP-11), a protease associated with invasion and aggressiveness of cancerous tissue, was postulated as a prognostic marker for pancreatic, breast, and colon cancer patients. Expression analysis, however, did not reveal localization and regulation of this protease. Thus, cellular tools for the visualization of MMP-11 are highly desirable to monitor presence and activity and to elucidate the functional role of MMP-11. Therefore, fluorescein-Dabcyl-labeled Foerster resonance energy transfer (FRET) substrates were developed. The design focused on enhanced peptide binding to human MMP-11, employing an unusual amino acid for the specificity pocket P1′. The addition of several arginines resulted in a cell-permeable FRET substrate SM-P124 (Ac-GRRRK(Dabcyl)-GGAANC(MeOBn)RMGG-fluorescein). In vitro evaluation of SM-P124 with human MMP-11 showed a 25-fold increase of affinity (kcat/Km = 9.16 × 103 m−1 s−1, Km = 8 μm) compared with previously published substrates. Incubation of pancreatic adenocarcinoma cell line MIA PaCa-2 and mamma adenocarcinoma cell line MCF-7 with the substrate SM-P124 (5 μm) indicated intra- and extracellular MMP-11 activity. A negative control cell line (Jurkat) showed no fluorescent signal either intra- or extracellularly. Negative control FRET substrate SM-P123 produced only insignificant extracellular fluorescence without any intracellular fluorescence. SM-P124 therefore enabled intra- and extracellular tracking of MMP-11-overexpressing cancers such as pancreatic and breast adenocarcinoma and might contribute to the understanding of the activation pathways leading to MMP-11-mediated invasive processes. PMID:22927434

  4. Substitution of the Lys linker with the β-Ala linker dramatically decreased the renal uptake of 99mTc-labeled Arg-X-Asp-conjugated and X-Ala-Asp-conjugated α-melanocyte stimulating hormone peptides.

    PubMed

    Flook, Adam M; Yang, Jianquan; Miao, Yubin

    2014-11-13

    The purpose of this study was to examine whether the substitution of the Lys linker with the β-Ala could reduce the renal uptake of (99m)Tc-labeled Arg-X-Asp-conjugated and X-Ala-Asp-conjugated α-melanocyte stimulating hormone (α-MSH) peptides. RSD-β-Ala-(Arg(11))CCMSH (1) {c[Arg-Ser-Asp-dTyr-Asp]-β-Ala-Cys-Cys-Glu-His-dPhe-Arg-Trp-Cys-Arg-Pro-Val-NH2}, RTD-β-Ala-(Arg(11))CCMSH (2), RVD-β-Ala-(Arg(11))CCMSH (3), RAD-β-Ala-(Arg(11))CCMSH (4), NAD-β-Ala-(Arg(11))CCMSH (5), and EAD-β-Ala-(Arg(11))CCMSH (6) peptides were synthesized and evaluated for their melanocortin 1 (MC1) receptor binding affinities in B16/F1 melanoma cells. The biodistribution of their (99m)Tc-conjugates were determined in B16/F1 melanoma-bearing C57 mice. The substitution of the Lys linker with β-Ala linker dramatically reduced the renal uptake of all six (99m)Tc-peptides. (99m)Tc-4 exhibited the highest melanoma uptake (15.66 ± 6.19% ID/g) and the lowest kidney uptake (20.18 ± 3.86% ID/g) among these (99m)Tc-peptides at 2 h postinjection. The B16/F1 melanoma lesions could be clearly visualized by single photon emission computed tomography (SPECT)/CT using (99m)Tc-4 as an imaging probe.

  5. Oriented Circular Dichroism: A Method to Characterize Membrane-Active Peptides in Oriented Lipid Bilayers.

    PubMed

    Bürck, Jochen; Wadhwani, Parvesh; Fanghänel, Susanne; Ulrich, Anne S

    2016-02-16

    transition dipole. Compared to conventional CD, OCD data are not only collected in the biologically relevant environment of a highly hydrated planar lipid bilayer (whose composition can be varied at will), but in addition it provides information about the tilt angle of the polypeptide in the membrane. It is the method of choice for screening numerous different conditions, such as peptide concentration, lipid composition, membrane additives, pH, temperature, and sample hydration. All these factors have been found to affect the peptide alignment in membrane, while having little or no influence on conformation. In many cases, the observed realignment could be related to biological action, such as pore formation by antimicrobial and cell-penetrating peptides, or to binding events of transmembrane segments of integral membrane proteins. Likewise, any lipid-induced conversion from α-helix to β-sheeted conformation is readily picked up by OCD and has been interpreted in terms of protein instability or amyloid-formation. PMID:26756718

  6. Chromogranin A-derived peptides are involved in innate immunity.

    PubMed

    Aslam, R; Atindehou, M; Lavaux, T; Haïkel, Y; Schneider, F; Metz-Boutigue, M-H

    2012-01-01

    New endogenous antimicrobial peptides (AMPs) derived from chromogranin A (CgA) are secreted by nervous, endocrine and immune cells during stress. They display antimicrobial activities by lytic effects at micromolar range using a pore-forming mechanism against Gram-positive bacteria, filamentous fungi and yeasts. These AMPs can also penetrate quickly into neutrophils (without lytic effects), where, similarly to "cell penetrating peptides", they interact with cytoplasmic calmodulin, and induce calcium influx via Store Operated Channels therefore triggering neutrophils activation. Staphylococcus aureus and Salmonella enteritis are bacteria responsible for severe infections. We investigated here the effects of S. aureus and S. enteritis bacterial proteases on CgA-derived peptides and evaluated their antimicrobial activities. We showed that the Glu-C protease produced by S. aureus V8 induces the loss of the AMPs antibacterial activities and produces new antifungal peptides. In addition, four antimicrobial CGA-derived peptides (chromofungin, procatestatin, human/bovine catestatin) are degraded when treated with bacterial supernatants from S. aureus and S. enteritis, whereas, cateslytin, the short active form of catestatin, resists to this degradation. Finally, we demonstrate that several antimicrobial CgA-derived peptides are able to act synergistically with antibiotics against bacteria and fungi indicating their roles in innate defense.

  7. Gene introduction into the mitochondria of Arabidopsis thaliana via peptide-based carriers

    PubMed Central

    Chuah, Jo-Ann; Yoshizumi, Takeshi; Kodama, Yutaka; Numata, Keiji

    2015-01-01

    Available methods in plant genetic transformation are nuclear and plastid transformations because similar procedures have not yet been established for the mitochondria. The double membrane and small size of the organelle, in addition to its large population in cells, are major obstacles in mitochondrial transfection. Here we report the intracellular delivery of exogenous DNA localized to the mitochondria of Arabidopsis thaliana using a combination of mitochondria-targeting peptide and cell-penetrating peptide. Low concentrations of peptides were sufficient to deliver DNA into the mitochondria and expression of imported DNA reached detectable levels within a short incubation period (12 h). We found that electrostatic interaction with the cell membrane is not a critical factor for complex internalization, instead, improved intracellular penetration of mitochondria-targeted complexes significantly enhanced gene transfer efficiency. Our results delineate a simple and effective peptide-based method, as a starting point for the development of more sophisticated plant mitochondrial transfection strategies. PMID:25583214

  8. Gene introduction into the mitochondria of Arabidopsis thaliana via peptide-based carriers.

    PubMed

    Chuah, Jo-Ann; Yoshizumi, Takeshi; Kodama, Yutaka; Numata, Keiji

    2015-01-13

    Available methods in plant genetic transformation are nuclear and plastid transformations because similar procedures have not yet been established for the mitochondria. The double membrane and small size of the organelle, in addition to its large population in cells, are major obstacles in mitochondrial transfection. Here we report the intracellular delivery of exogenous DNA localized to the mitochondria of Arabidopsis thaliana using a combination of mitochondria-targeting peptide and cell-penetrating peptide. Low concentrations of peptides were sufficient to deliver DNA into the mitochondria and expression of imported DNA reached detectable levels within a short incubation period (12 h). We found that electrostatic interaction with the cell membrane is not a critical factor for complex internalization, instead, improved intracellular penetration of mitochondria-targeted complexes significantly enhanced gene transfer efficiency. Our results delineate a simple and effective peptide-based method, as a starting point for the development of more sophisticated plant mitochondrial transfection strategies.

  9. Gene introduction into the mitochondria of Arabidopsis thaliana via peptide-based carriers

    NASA Astrophysics Data System (ADS)

    Chuah, Jo-Ann; Yoshizumi, Takeshi; Kodama, Yutaka; Numata, Keiji

    2015-01-01

    Available methods in plant genetic transformation are nuclear and plastid transformations because similar procedures have not yet been established for the mitochondria. The double membrane and small size of the organelle, in addition to its large population in cells, are major obstacles in mitochondrial transfection. Here we report the intracellular delivery of exogenous DNA localized to the mitochondria of Arabidopsis thaliana using a combination of mitochondria-targeting peptide and cell-penetrating peptide. Low concentrations of peptides were sufficient to deliver DNA into the mitochondria and expression of imported DNA reached detectable levels within a short incubation period (12 h). We found that electrostatic interaction with the cell membrane is not a critical factor for complex internalization, instead, improved intracellular penetration of mitochondria-targeted complexes significantly enhanced gene transfer efficiency. Our results delineate a simple and effective peptide-based method, as a starting point for the development of more sophisticated plant mitochondrial transfection strategies.

  10. Intracellular trafficking of superparamagnetic iron oxide nanoparticles conjugated with TAT peptide: 3-dimensional electron tomography analysis

    SciTech Connect

    Nair, Baiju G.; Fukuda, Takahiro; Mizuki, Toru; Hanajiri, Tatsuro; Maekawa, Toru

    2012-05-18

    Highlights: Black-Right-Pointing-Pointer We study the intracellular localisation of TAT-SPIONs using 3-D electron tomography. Black-Right-Pointing-Pointer 3-D images of TAT-SPIONs in a cell are clearly shown. Black-Right-Pointing-Pointer Release of TAT-SPIONs from endocytic vesicles into the cytoplasm is clearly shown. -- Abstract: Internalisation of nanoparticles conjugated with cell penetrating peptides is a promising approach to various drug delivery applications. Cell penetrating peptides such as transactivating transcriptional activator (TAT) peptides derived from HIV-1 proteins are effective intracellular delivery vectors for a wide range of nanoparticles and pharmaceutical agents thanks to their amicable ability to enter cells and minimum cytotoxicity. Although different mechanisms of intracellular uptake and localisation have been proposed for TAT conjugated nanoparticles, it is necessary to visualise the particles on a 3-D plane in order to investigate the actual intracellular uptake and localisation. Here, we study the intracellular localisation and trafficking of TAT peptide conjugated superparamagnetic ion oxide nanoparticles (TAT-SPIONs) using 3-D electron tomography. 3-D tomograms clearly show the location of TAT-SPIONs in a cell and their slow release from the endocytic vesicles into the cytoplasm. The present methodology may well be utilised for further investigations of the behaviours of nanoparticles in cells and eventually for the development of nano drug delivery systems.

  11. Cell-penetrable mouse forkhead box protein 3 alleviates experimental arthritis in mice by up-regulating regulatory T cells.

    PubMed

    Liu, Xia; Ji, Baoju; Sun, Mengyi; Wu, Weijiang; Huang, Lili; Sun, Aihua; Zong, Yangyong; Xia, Sheng; Shi, Liyun; Qian, Hui; Xu, Wenrong; Shao, Qixiang

    2015-07-01

    Regulatory T cells (T(regs)) have potential applications in clinical disease therapy, such as autoimmune diseases and transplant rejection. However, their numbers are limited. Forkhead box protein 3 (FoxP3) is a key transcription factor that controls T(reg) development and function. Here, we generated a cell-permeable fusion protein, protein transduction domain (PTD)-conjugated mouse FoxP3 protein (PTD-mFoxP3), and evaluated whether PTD-mFoxp3 can alleviate rheumatoid arthritis (RA) in the collagen-induced arthritis (CIA) mouse model. As expected, PTD-mFoxP3 was transduced into cells effectively, and inhibited T cell activation and attenuated the cell proliferation. It decreased interleukin (IL) 2 and interferon (IFN)-γ expression, and increased IL-10 expression in activated CD4(+)CD25(-) T cells. PTD-mFoxP3-transduced CD4(+)CD25(-) T cells attenuated proliferation of activated CD4(+)CD25(-) T cells. In addition, PTD-mFoxP3 blocked the Th17 differentiation programme in vitro and down-regulated IL-17 production from T cells by modulating induction and levels of retinoid-related orphan receptor gamma t (RORγt). Intra-articular delivery of PTD-mFoxP3 delayed disease incidence remarkably and alleviated autoimmune symptoms of CIA mice. Moreover, protective effects of PTD-mFoxP3 were associated with regulating the balance of T helper type 17 (Th17) and T(regs). These results suggest that PTD-mFoxP3 may be a candidate for RA therapy. PMID:25809415

  12. Cell-penetrable mouse forkhead box protein 3 alleviates experimental arthritis in mice by up-regulating regulatory T cells.

    PubMed

    Liu, Xia; Ji, Baoju; Sun, Mengyi; Wu, Weijiang; Huang, Lili; Sun, Aihua; Zong, Yangyong; Xia, Sheng; Shi, Liyun; Qian, Hui; Xu, Wenrong; Shao, Qixiang

    2015-07-01

    Regulatory T cells (T(regs)) have potential applications in clinical disease therapy, such as autoimmune diseases and transplant rejection. However, their numbers are limited. Forkhead box protein 3 (FoxP3) is a key transcription factor that controls T(reg) development and function. Here, we generated a cell-permeable fusion protein, protein transduction domain (PTD)-conjugated mouse FoxP3 protein (PTD-mFoxP3), and evaluated whether PTD-mFoxp3 can alleviate rheumatoid arthritis (RA) in the collagen-induced arthritis (CIA) mouse model. As expected, PTD-mFoxP3 was transduced into cells effectively, and inhibited T cell activation and attenuated the cell proliferation. It decreased interleukin (IL) 2 and interferon (IFN)-γ expression, and increased IL-10 expression in activated CD4(+)CD25(-) T cells. PTD-mFoxP3-transduced CD4(+)CD25(-) T cells attenuated proliferation of activated CD4(+)CD25(-) T cells. In addition, PTD-mFoxP3 blocked the Th17 differentiation programme in vitro and down-regulated IL-17 production from T cells by modulating induction and levels of retinoid-related orphan receptor gamma t (RORγt). Intra-articular delivery of PTD-mFoxP3 delayed disease incidence remarkably and alleviated autoimmune symptoms of CIA mice. Moreover, protective effects of PTD-mFoxP3 were associated with regulating the balance of T helper type 17 (Th17) and T(regs). These results suggest that PTD-mFoxP3 may be a candidate for RA therapy.

  13. Peptide identification

    DOEpatents

    Jarman, Kristin H [Richland, WA; Cannon, William R [Richland, WA; Jarman, Kenneth D [Richland, WA; Heredia-Langner, Alejandro [Richland, WA

    2011-07-12

    Peptides are identified from a list of candidates using collision-induced dissociation tandem mass spectrometry data. A probabilistic model for the occurrence of spectral peaks corresponding to frequently observed partial peptide fragment ions is applied. As part of the identification procedure, a probability score is produced that indicates the likelihood of any given candidate being the correct match. The statistical significance of the score is known without necessarily having reference to the actual identity of the peptide. In one form of the invention, a genetic algorithm is applied to candidate peptides using an objective function that takes into account the number of shifted peaks appearing in the candidate spectrum relative to the test spectrum.

  14. Synthetic peptides.

    PubMed

    Francis, M J

    1996-01-01

    Efforts to produce more stable and defined vaccines have concentrated on studying, in detail, the immune response to many infectious diseases in order to identify the antigenic sites on the pathogens that are involved in stimulating protective immumty. Armed with this knowledge, it is possible to mimic such sites by producing short chains of amino acids (peptides) and to use these as the basis for novel vaccines. The earliest documented work on peptide immunization is actually for a plant virus, tobacco mosaic virus. In 1963, Anderer (1) demonstrated that rabbit antibodies to an isolated hexapeptide fragment from the virus-coat protein coupled to bovine serum albumm would neutralize the infectious vn-us in culture. Two years later, he used a synthetically produced copy of the same peptide to confirm this observation. This was pioneering work, and it was over 10 years before the next example of a peptide that elicited antivirus antibody appeared following work by Sela and his colleagues (2) on a virus, MS2 bacteriophage, which infects bacteria. The emergence of more accessible techniques for sequencing proteins in 1977, coupled with the ability to synthesize readily peptides already developed in 1963, heralded a decade of intensive research into experimental peptide vaccines. The first demonstration that peptides could elicit protective immunity in vivo, in addition to neutralizing activity in vitro, was obtained using a peptide from the VP1 coat protein of foot-and-mouth disease virus (FMDV) in 1982, with the guinea pig as a laboratory animal model (3, 4). PMID:21359696

  15. Discovery of Novel Bacterial Cell-Penetrating Phylloseptins in Defensive Skin Secretions of the South American Hylid Frogs, Phyllomedusa duellmani and Phyllomedusa coelestis

    PubMed Central

    Yang, Nan; Li, Lei; Wu, Di; Gao, Yitian; Xi, Xinping; Zhou, Mei; Wang, Lei; Chen, Tianbao; Shaw, Chris

    2016-01-01

    Phylloseptin (PS) peptides, derived from South American hylid frogs (subfamily Phyllomedusinae), have been found to have broad-spectrum antimicrobial activities and relatively low haemolytic activities. Although PS peptides have been identified from several well-known and widely-distributed species of the Phyllomedusinae, there remains merit in their study in additional, more obscure and specialised members of this taxon. Here, we report the discovery of two novel PS peptides, named PS-Du and PS-Co, which were respectively identified for the first time and isolated from the skin secretions of Phyllomedusa duellmani and Phyllomedusa coelestis. Their encoding cDNAs were cloned, from which it was possible to deduce the entire primary structures of their biosynthetic precursors. Reversed-phase high-performance liquid chromatography (RP-HPLC) and tandem mass spectrometry (MS/MS) analyses were employed to isolate and structurally-characterise respective encoded PS peptides from skin secretions. The peptides had molecular masses of 2049.7 Da (PS-Du) and 1972.8 Da (PS-Co). They shared typical N-terminal sequences and C-terminal amidation with other known phylloseptins. The two peptides exhibited growth inhibitory activity against E. coli (NCTC 10418), as a standard Gram-negative bacterium, S. aureus (NCTC 10788), as a standard Gram-positive bacterium and C. albicans (NCPF 1467), as a standard pathogenic yeast, all as planktonic cultures. Moreover, both peptides demonstrated the capability of eliminating S. aureus biofilm. PMID:27589802

  16. Discovery of Novel Bacterial Cell-Penetrating Phylloseptins in Defensive Skin Secretions of the South American Hylid Frogs, Phyllomedusa duellmani and Phyllomedusa coelestis.

    PubMed

    Yang, Nan; Li, Lei; Wu, Di; Gao, Yitian; Xi, Xinping; Zhou, Mei; Wang, Lei; Chen, Tianbao; Shaw, Chris

    2016-01-01

    Phylloseptin (PS) peptides, derived from South American hylid frogs (subfamily Phyllomedusinae), have been found to have broad-spectrum antimicrobial activities and relatively low haemolytic activities. Although PS peptides have been identified from several well-known and widely-distributed species of the Phyllomedusinae, there remains merit in their study in additional, more obscure and specialised members of this taxon. Here, we report the discovery of two novel PS peptides, named PS-Du and PS-Co, which were respectively identified for the first time and isolated from the skin secretions of Phyllomedusa duellmani and Phyllomedusa coelestis. Their encoding cDNAs were cloned, from which it was possible to deduce the entire primary structures of their biosynthetic precursors. Reversed-phase high-performance liquid chromatography (RP-HPLC) and tandem mass spectrometry (MS/MS) analyses were employed to isolate and structurally-characterise respective encoded PS peptides from skin secretions. The peptides had molecular masses of 2049.7 Da (PS-Du) and 1972.8 Da (PS-Co). They shared typical N-terminal sequences and C-terminal amidation with other known phylloseptins. The two peptides exhibited growth inhibitory activity against E. coli (NCTC 10418), as a standard Gram-negative bacterium, S. aureus (NCTC 10788), as a standard Gram-positive bacterium and C. albicans (NCPF 1467), as a standard pathogenic yeast, all as planktonic cultures. Moreover, both peptides demonstrated the capability of eliminating S. aureus biofilm. PMID:27589802

  17. Guanidinium-Rich, Glycerol-Derived Oligocarbonates: A New Class of Cell-Penetrating Molecular Transporters That Complex, Deliver, and Release siRNA

    PubMed Central

    Wender, Paul A.; Huttner, Melanie A.; Staveness, Daryl; Vargas, Jessica R.; Xu, Adele F.

    2015-01-01

    A highly versatile and step-economical route to a new class of guanidinium-rich molecular transporters and evaluation of their ability to complex, deliver, and release siRNA are described. These new drug/probe delivery systems are prepared in only two steps, irrespective of length or composition, using an organocatalytic ring-opening co-oligomerization of glycerol-derived cyclic carbonate monomers incorporating either protected guanidine or lipid side chains. The resultant amphipathic co-oligomers are highly effective vehicles for siRNA delivery, providing an excellent level of target protein suppression (>85%). These new oligocarbonates are nontoxic at levels required for cell penetration and can be tuned for particle size. Relative to the previously reported methyl(trimethylene)carbonate (MTC) scaffold, the ether linkage at C2 in the new transporters markedly enhances the stability of the siRNA/co-oligomer complexes. Both hybrid co-oligomers, containing a mixture of glycerol- and MTC-derived monomers, and co-oligomers containing only glycerol monomers are found to provide tunable control over siRNA complex stability. On the basis of a glycerol and CO2 backbone, these new co-oligomers represent a rapidly tunable and biocompatible siRNA delivery system that is highly effective in suppressing target protein synthesis. PMID:25588140

  18. Peptidic tools applied to redirect alternative splicing events.

    PubMed

    Nancy, Martínez-Montiel; Nora, Rosas-Murrieta; Rebeca, Martínez-Contreras

    2015-05-01

    Peptides are versatile and attractive biomolecules that can be applied to modulate genetic mechanisms like alternative splicing. In this process, a single transcript yields different mature RNAs leading to the production of protein isoforms with diverse or even antagonistic functions. During splicing events, errors can be caused either by mutations present in the genome or by defects or imbalances in regulatory protein factors. In any case, defects in alternative splicing have been related to several genetic diseases including muscular dystrophy, Alzheimer's disease and cancer from almost every origin. One of the most effective approaches to redirect alternative splicing events has been to attach cell-penetrating peptides to oligonucleotides that can modulate a single splicing event and restore correct gene expression. Here, we summarize how natural existing and bioengineered peptides have been applied over the last few years to regulate alternative splicing and genetic expression. Under different genetic and cellular backgrounds, peptides have been shown to function as potent vehicles for splice correction, and their therapeutic benefits have reached clinical trials and patenting stages, emphasizing the use of regulatory peptides as an exciting therapeutic tool for the treatment of different genetic diseases.

  19. Modulation of muscle contraction by a cell-permeable peptide

    PubMed Central

    Tünnemann, Gisela; Karczewski, Peter; Haase, Hannelore

    2007-01-01

    In contrast to immortal cell lines, primary cells are hardly susceptible to intracellular delivery methods such as transfection. In this study, we evaluated the direct delivery of several cell-permeable peptides under noninvasive conditions into living primary adult rat cardiomyocytes. We specifically monitored the functional effects of a cell-permeable peptide containing the 15 amino acid N-terminal peptide from human ventricular light chain-1 (VLC-1) on contraction and intracellular Ca2+ signals after electrical stimulation in primary adult cardiomyocytes. The transducible VLC-1 variant was taken up by cardiomyocytes within 5 min with more than 95% efficiency and localized to sarcomeric structures. Analysis of the functional effects of the cell-permeable VLC-1 revealed an enhancement of the intrinsic contractility of cardiomyocytes without affecting the intracellular Ca2+. Therefore, peptide transduction mediated by cell-penetrating peptides represents not only a unique strategy to enhance heart muscle function with no secondary effect on intracellular Ca2+ but also an invaluable tool for the modulation and manipulation of protein interactions in general and in primary cells. PMID:17717642

  20. Topical application of superoxide dismutase mediated by HIV-TAT peptide attenuates UVB-induced damages in human skin.

    PubMed

    Chen, Xiaochao; Liu, Shutao; Rao, Pingfan; Bradshaw, Jeremy; Weller, Richard

    2016-10-01

    The purpose of this study was to evaluate whether topical application of superoxide dismutase with cell penetrating peptide (HIV-TAT) could protect against skin damage induced by UVB irradiation in humans. The permeability through stratum corneum of large proteins linked to TAT peptide was firstly confirmed by confocal microscopy and tape stripping. Ten healthy volunteers with either Fitzpatrick skin type II or III were recruited in this clinical study. TAT-SOD (300units/cm(2)) and vehicle cream were applied on two symmetric areas of both inner upper arms 1h prior to UVB irradiation. After one hour of pretreatment, subjects received 10 incremental doses of UVB on pretreated areas. 24h later, erythema, blood flow and apoptotic cells were measured. Pretreatment with TAT-SOD 1h prior to UVB radiation promoted a mean minimal erythema dose (MED) increase of 36.6±18.4% (p=0.013<0.05. n=10) compared to vehicle control. The median blood flow values of all subjects following 2 and 3-MED of UVB were 107.8±51.0units and 239.5±88.0units respectively, which account for 26% and 25% decrease with respect to vehicle groups. These data suggest that TAT-SOD significantly suppresses UVB induced erythema formation and blood flow rise. Furthermore, pretreatment with TAT-SOD 1h prior to 2-MED of UVB irradiation reduced the apoptotic sunburn cell formation by 47.6±8.6% (p<0.0001) in all subjects. Evaluating results generated from all measurements, we conclude that topical application of TAT-SOD significantly attenuates UVB-induced skin damage in man. These biological effects of TAT-SOD are probably mediated via its free radical scavenging properties, clearly differentiating it from other physical sunscreen agents. PMID:27460952

  1. Topical application of superoxide dismutase mediated by HIV-TAT peptide attenuates UVB-induced damages in human skin.

    PubMed

    Chen, Xiaochao; Liu, Shutao; Rao, Pingfan; Bradshaw, Jeremy; Weller, Richard

    2016-10-01

    The purpose of this study was to evaluate whether topical application of superoxide dismutase with cell penetrating peptide (HIV-TAT) could protect against skin damage induced by UVB irradiation in humans. The permeability through stratum corneum of large proteins linked to TAT peptide was firstly confirmed by confocal microscopy and tape stripping. Ten healthy volunteers with either Fitzpatrick skin type II or III were recruited in this clinical study. TAT-SOD (300units/cm(2)) and vehicle cream were applied on two symmetric areas of both inner upper arms 1h prior to UVB irradiation. After one hour of pretreatment, subjects received 10 incremental doses of UVB on pretreated areas. 24h later, erythema, blood flow and apoptotic cells were measured. Pretreatment with TAT-SOD 1h prior to UVB radiation promoted a mean minimal erythema dose (MED) increase of 36.6±18.4% (p=0.013<0.05. n=10) compared to vehicle control. The median blood flow values of all subjects following 2 and 3-MED of UVB were 107.8±51.0units and 239.5±88.0units respectively, which account for 26% and 25% decrease with respect to vehicle groups. These data suggest that TAT-SOD significantly suppresses UVB induced erythema formation and blood flow rise. Furthermore, pretreatment with TAT-SOD 1h prior to 2-MED of UVB irradiation reduced the apoptotic sunburn cell formation by 47.6±8.6% (p<0.0001) in all subjects. Evaluating results generated from all measurements, we conclude that topical application of TAT-SOD significantly attenuates UVB-induced skin damage in man. These biological effects of TAT-SOD are probably mediated via its free radical scavenging properties, clearly differentiating it from other physical sunscreen agents.

  2. Cell penetration to nanofibrous scaffolds

    PubMed Central

    Rampichová, Michala; Buzgo, Matej; Chvojka, Jiří; Prosecká, Eva; Kofroňová, Olga; Amler, Evžen

    2014-01-01

    Cell infiltration is a critical parameter for the successful development of 3D matrices for tissue engineering. Application of electrospun nanofibers in tissue engineering has recently attracted much attention. Notwithstanding several of their advantages, small pore size and small thickness of the electrospun layer limit their application for development of 3D scaffolds. Several methods for the pore size and/or electrospun layer thickness increase have been recently developed. Nevertheless, tissue engineering still needs emerging of either novel nanofiber-enriched composites or new techniques for 3D nanofiber fabrication. Forcespinning® seems to be a promising alternative. The potential of the Forcespinning® method is illustrated in preliminary experiment with mesenchymal stem cells. PMID:24429388

  3. pH-Selective Cytotoxicity of pHLIP-Antimicrobial Peptide Conjugates

    PubMed Central

    Burns, Kelly E.; McCleerey, Tanner P.; Thévenin, Damien

    2016-01-01

    Positively charged antimicrobial peptides have become promising agents for the treatment of cancer by inducing apoptosis though their preferential binding and disruption of negatively charged membranes, such as the mitochondrial membrane. (KLAKLAK)2 is such a peptide but due to its polarity, it cannot cross the cellular membrane and therefore relies on the use of a delivery agent. For targeted delivery, previous studies have relied on cell penetrating peptides, nanoparticles or specific biomarkers. Herein, we investigated the first use of pHLIP to selectively target and directly translocate (KLAKLAK)2 into the cytoplasm of breast cancer cells, based on the acidic tumor micro-environment. With the goal of identifying a lead conjugate with optimized selective cytotoxicity towards cancer cells, we analyzed a family of (KLAKLAK)2 analogs with varying size, polarity and charge. We present a highly efficacious pHLIP conjugate that selectively induces concentration- and pH-dependent toxicity in breast cancer cells. PMID:27334357

  4. Solid-phase synthesis of oxaliplatin-TAT peptide bioconjugates.

    PubMed

    Abramkin, Sergey; Valiahdi, Seied M; Jakupec, Michael A; Galanski, Markus; Metzler-Nolte, Nils; Keppler, Bernhard K

    2012-03-14

    Platinum-based drugs play a crucial role in the fight against cancer. Oxaliplatin, which is used in the treatment of colorectal carcinoma, was the last platinum-based agent to be approved worldwide. However, the efficiency of the therapy is limited for example by a low accumulation of the drug in cancer cells. Cell-penetrating peptides (CPPs) are known to ease the cellular membrane transport and are used as vectors for low-molecular-weight drugs and drug carriers; of them, TAT peptides are the best-studied group. In this work, a TAT-peptide fragment (YGRKKRRQRRR) was for the first time conjugated to a platinum(IV) analog of oxaliplatin as a vehicle for membrane penetration. Solid-phase peptide synthesis and subsequent coupling with the platinum complex afforded mono- and difunctionalized conjugates, which were separated by preparative HPLC and characterized by analytical HPLC, ESI-MS, and (1)H NMR spectroscopy. Both conjugates are active in the low micromolar range in CH1 and SW480 human cancer cells, requiring much lower concentrations than the untargeted analogs for equal effects.

  5. Analysis of peptide uptake and location of root hair-promoting peptide accumulation in plant roots.

    PubMed

    Matsumiya, Yoshiki; Taniguchi, Rikiya; Kubo, Motoki

    2012-03-01

    Peptide uptake by plant roots from degraded soybean-meal products was analyzed in Brassica rapa and Solanum lycopersicum. B. rapa absorbed about 40% of the initial water volume, whereas peptide concentration was decreased by 75% after 24 h. Analysis by reversed-phase HPLC showed that number of peptides was absorbed by the roots during soaking in degraded soybean-meal products for 24 h. Carboxyfluorescein-labeled root hair-promoting peptide was synthesized, and its localization, movement, and accumulation in roots were investigated. The peptide appeared to be absorbed by root hairs and then moved to trichoblasts. Furthermore, the peptide was moved from trichoblasts to atrichoblasts after 24 h. The peptide was accumulated in epidermal cells, suggesting that the peptide may have a function in both trichoblasts and atrichoblasts.

  6. Antimicrobial peptides.

    PubMed

    Zhang, Ling-Juan; Gallo, Richard L

    2016-01-11

    Antimicrobial peptides and proteins (AMPs) are a diverse class of naturally occurring molecules that are produced as a first line of defense by all multicellular organisms. These proteins can have broad activity to directly kill bacteria, yeasts, fungi, viruses and even cancer cells. Insects and plants primarily deploy AMPs as an antibiotic to protect against potential pathogenic microbes, but microbes also produce AMPs to defend their environmental niche. In higher eukaryotic organisms, AMPs can also be referred to as 'host defense peptides', emphasizing their additional immunomodulatory activities. These activities are diverse, specific to the type of AMP, and include a variety of cytokine and growth factor-like effects that are relevant to normal immune homeostasis. In some instances, the inappropriate expression of AMPs can also induce autoimmune diseases, thus further highlighting the importance of understanding these molecules and their complex activities. This Primer will provide an update of our current understanding of AMPs. PMID:26766224

  7. Antimicrobial Peptides

    PubMed Central

    Bahar, Ali Adem; Ren, Dacheng

    2013-01-01

    The rapid increase in drug-resistant infections has presented a serious challenge to antimicrobial therapies. The failure of the most potent antibiotics to kill “superbugs” emphasizes the urgent need to develop other control agents. Here we review the history and new development of antimicrobial peptides (AMPs), a growing class of natural and synthetic peptides with a wide spectrum of targets including viruses, bacteria, fungi, and parasites. We summarize the major types of AMPs, their modes of action, and the common mechanisms of AMP resistance. In addition, we discuss the principles for designing effective AMPs and the potential of using AMPs to control biofilms (multicellular structures of bacteria embedded in extracellular matrixes) and persister cells (dormant phenotypic variants of bacterial cells that are highly tolerant to antibiotics). PMID:24287494

  8. Antimicrobial peptides.

    PubMed

    Bahar, Ali Adem; Ren, Dacheng

    2013-11-28

    The rapid increase in drug-resistant infections has presented a serious challenge to antimicrobial therapies. The failure of the most potent antibiotics to kill "superbugs" emphasizes the urgent need to develop other control agents. Here we review the history and new development of antimicrobial peptides (AMPs), a growing class of natural and synthetic peptides with a wide spectrum of targets including viruses, bacteria, fungi, and parasites. We summarize the major types of AMPs, their modes of action, and the common mechanisms of AMP resistance. In addition, we discuss the principles for designing effective AMPs and the potential of using AMPs to control biofilms (multicellular structures of bacteria embedded in extracellular matrixes) and persister cells (dormant phenotypic variants of bacterial cells that are highly tolerant to antibiotics).

  9. Antibacterial Activity of Novel Cationic Peptides against Clinical Isolates of Multi-Drug Resistant Staphylococcus pseudintermedius from Infected Dogs

    PubMed Central

    Mohamed, Mohamed F.; Hammac, G. Kenitra; Guptill, Lynn; Seleem, Mohamed N.

    2014-01-01

    Staphylococcus pseudintermedius is a major cause of skin and soft tissue infections in companion animals and has zoonotic potential. Additionally, methicillin-resistant S. pseudintermedius (MRSP) has emerged with resistance to virtually all classes of antimicrobials. Thus, novel treatment options with new modes of action are required. Here, we investigated the antimicrobial activity of six synthetic short peptides against clinical isolates of methicillin-susceptible and MRSP isolated from infected dogs. All six peptides demonstrated potent anti-staphylococcal activity regardless of existing resistance phenotype. The most effective peptides were RRIKA (with modified C terminus to increase amphipathicity and hydrophobicity) and WR-12 (α-helical peptide consisting exclusively of arginine and tryptophan) with minimum inhibitory concentration50 (MIC50) of 1 µM and MIC90 of 2 µM. RR (short anti-inflammatory peptide) and IK8 “D isoform” demonstrated good antimicrobial activity with MIC50 of 4 µM and MIC90 of 8 µM. Penetratin and (KFF)3K (two cell penetrating peptides) were the least effective with MIC50 of 8 µM and MIC90 of 16 µM. Killing kinetics revealed a major advantage of peptides over conventional antibiotics, demonstrating potent bactericidal activity within minutes. Studies with propidium iodide and transmission electron microscopy revealed that peptides damaged the bacterial membrane leading to leakage of cytoplasmic contents and consequently, cell death. A potent synergistic increase in the antibacterial effect of the cell penetrating peptide (KFF)3K was noticed when combined with other peptides and with antibiotics. In addition, all peptides displayed synergistic interactions when combined together. Furthermore, peptides demonstrated good therapeutic indices with minimal toxicity toward mammalian cells. Resistance to peptides did not evolve after 10 passages of S. pseudintermedius at sub-inhibitory concentration. However, the MICs of amikacin and

  10. The Homeodomain Derived Peptide Penetratin Induces Curvature of Fluid Membrane Domains

    PubMed Central

    Lamazière, Antonin; Wolf, Claude; Lambert, Olivier; Chassaing, Gérard; Trugnan, Germain; Ayala-Sanmartin, Jesus

    2008-01-01

    Background Protein membrane transduction domains that are able to cross the plasma membrane are present in several transcription factors, such as the homeodomain proteins and the viral proteins such as Tat of HIV-1. Their discovery resulted in both new concepts on the cell communication during development, and the conception of cell penetrating peptide vectors for internalisation of active molecules into cells. A promising cell penetrating peptide is Penetratin, which crosses the cell membranes by a receptor and metabolic energy-independent mechanism. Recent works have claimed that Penetratin and similar peptides are internalized by endocytosis, but other endocytosis-independent mechanisms have been proposed. Endosomes or plasma membranes crossing mechanisms are not well understood. Previously, we have shown that basic peptides induce membrane invaginations suggesting a new mechanism for uptake, “physical endocytosis”. Methodology/Principal Findings Herein, we investigate the role of membrane lipid phases on Penetratin induced membrane deformations (liquid ordered such as in “raft” microdomains versus disordered fluid “non-raft” domains) in membrane models. Experimental data show that zwitterionic lipid headgroups take part in the interaction with Penetratin suggesting that the external leaflet lipids of cells plasma membrane are competent for peptide interaction in the absence of net negative charges. NMR and X-ray diffraction data show that the membrane perturbations (tubulation and vesiculation) are associated with an increase in membrane negative curvature. These effects on curvature were observed in the liquid disordered but not in the liquid ordered (raft-like) membrane domains. Conclusions/Significance The better understanding of the internalisation mechanisms of protein transduction domains will help both the understanding of the mechanisms of cell communication and the development of potential therapeutic molecular vectors. Here we showed that

  11. Action of the multifunctional peptide BP100 on native biomembranes examined by solid-state NMR.

    PubMed

    Misiewicz, Julia; Afonin, Sergii; Grage, Stephan L; van den Berg, Jonas; Strandberg, Erik; Wadhwani, Parvesh; Ulrich, Anne S

    2015-04-01

    Membrane composition is a key factor that regulates the destructive activity of antimicrobial peptides and the non-leaky permeation of cell penetrating peptides in vivo. Hence, the choice of model membrane is a crucial aspect in NMR studies and should reflect the biological situation as closely as possible. Here, we explore the structure and dynamics of the short multifunctional peptide BP100 using a multinuclear solid-state NMR approach. The membrane alignment and mobility of this 11 amino acid peptide was studied in various synthetic lipid bilayers with different net charge, fluidity, and thickness, as well as in native biomembranes harvested from prokaryotic and eukaryotic cells. (19)F-NMR provided the high sensitivity and lack of natural abundance background that are necessary to observe a labelled peptide even in protoplast membranes from Micrococcus luteus and in erythrocyte ghosts. Six selectively (19)F-labeled BP100 analogues gave remarkably similar spectra in all of the macroscopically oriented membrane systems, which were studied under quasi-native conditions of ambient temperature and full hydration. This similarity suggests that BP100 has the same surface-bound helical structure and high mobility in the different biomembranes and model membranes alike, independent of charge, thickness or cholesterol content of the system. (31)P-NMR spectra of the phospholipid components did not indicate any bilayer perturbation, so the formation of toroidal wormholes or micellarization can be excluded as a mechanism of its antimicrobial or cell penetrating action. However, (2)H-NMR analysis of the acyl chain order parameter profiles showed that BP100 leads to considerable membrane thinning and thereby local destabilization.

  12. 4(R/S)-Amino/guanidino-substituted proline peptides: design, synthesis and DNA transfection properties.

    PubMed

    Umashankara, M; Nanda, Manaswini; Sonar, Mahesh; Ganesh, Krishna N

    2012-01-01

    Collagen is a major structural protein found in the connective tissues of higher organisms and mammals and its biomechanical properties are related to the high thermal stability of its triple helical structure. The primary structure of collagen is composed of the repeating tripeptide motif of Pro-Hyp-Gly, where Hyp is 4 R -hydroxy proline. Cationic collagen mimetics consisting of [Pro(X)-Pro(Y)-Gly](6) where Pro(X) and Pro(Y) are 4(R/S)-amino/guanidine proline have been synthesized and shown to form triplexes more stable than the unmodified collagen peptide [Pro-Hyp-Gly](6). The origin of hyperstability is due to conformational pre-organization of proline pucker arising from the electronegativity of the cationic group. These cationic collagen peptides are shown to be effective cell penetrating and plasmid DNA transfecting agents. The results have potential for design of new collagen mimetics for biomaterial applications and efficient cell penetrating agents for drug delivery applications.

  13. Peptide-based delivery of nucleic acids: design, mechanism of uptake and applications to splice-correcting oligonucleotides.

    PubMed

    Abes, S; Moulton, H; Turner, J; Clair, P; Richard, J P; Iversen, P; Gait, M J; Lebleu, B

    2007-02-01

    CPPs (cell-penetrating peptides) have given rise to much interest for the delivery of biomolecules such as peptides, proteins or ONs (oligonucleotides). CPPs and their conjugates were initially thought to translocate through the cell membrane by a non-endocytotic mechanism which has recently been re-evaluated. Basic-amino-acid-rich CPPs first interact with cell-surface proteoglycans before being internalized by endocytosis. Sequestration and degradation in endocytotic vesicles severely limits the cytoplasmic and nuclear delivery of the conjugated biomolecules. Accordingly, splicing correction by CPP-conjugated steric-block ON analogues is inefficient in the absence of endosomolytic agents. New arginine-rich CPPs allowing efficient splicing correction by conjugated PNAs (peptide nucleic acids) or PMO (phosphorodiamidate morpholino oligomer) steric blockers in the absence of endosomolytic agents have recently been defined in our group and are currently being characterized. They offer promising leads for the development of efficient cellular delivery vectors for therapeutic steric-block ON analogues.

  14. Development of polymeric–cationic peptide composite nanoparticles, a nanoparticle-in-nanoparticle system for controlled gene delivery

    PubMed Central

    Jain, Arvind K; Massey, Ashley; Yusuf, Helmy; McDonald, Denise M; McCarthy, Helen O; Kett, Vicky L

    2015-01-01

    We report the formulation of novel composite nanoparticles that combine the high transfection efficiency of cationic peptide-DNA nanoparticles with the biocompatibility and prolonged delivery of polylactic acid–polyethylene glycol (PLA-PEG). The cationic cell-penetrating peptide RALA was used to condense DNA into nanoparticles that were encapsulated within a range of PLA-PEG copolymers. The composite nanoparticles produced exhibited excellent physicochemical properties including size <200 nm and encapsulation efficiency >80%. Images of the composite nanoparticles obtained with a new transmission electron microscopy staining method revealed the peptide-DNA nanoparticles within the PLA-PEG matrix. Varying the copolymers modulated the DNA release rate >6 weeks in vitro. The best formulation was selected and was able to transfect cells while maintaining viability. The effect of transferrin-appended composite nanoparticles was also studied. Thus, we have demonstrated the manufacture of composite nanoparticles for the controlled delivery of DNA. PMID:26648722

  15. Development of polymeric-cationic peptide composite nanoparticles, a nanoparticle-in-nanoparticle system for controlled gene delivery.

    PubMed

    Jain, Arvind K; Massey, Ashley; Yusuf, Helmy; McDonald, Denise M; McCarthy, Helen O; Kett, Vicky L

    2015-01-01

    We report the formulation of novel composite nanoparticles that combine the high transfection efficiency of cationic peptide-DNA nanoparticles with the biocompatibility and prolonged delivery of polylactic acid-polyethylene glycol (PLA-PEG). The cationic cell-penetrating peptide RALA was used to condense DNA into nanoparticles that were encapsulated within a range of PLA-PEG copolymers. The composite nanoparticles produced exhibited excellent physicochemical properties including size <200 nm and encapsulation efficiency >80%. Images of the composite nanoparticles obtained with a new transmission electron microscopy staining method revealed the peptide-DNA nanoparticles within the PLA-PEG matrix. Varying the copolymers modulated the DNA release rate >6 weeks in vitro. The best formulation was selected and was able to transfect cells while maintaining viability. The effect of transferrin-appended composite nanoparticles was also studied. Thus, we have demonstrated the manufacture of composite nanoparticles for the controlled delivery of DNA.

  16. Cellular Uptake and Photo-Cytotoxicity of a Gadolinium(III)-DOTA-Naphthalimide Complex "Clicked" to a Lipidated Tat Peptide.

    PubMed

    O'Malley, William I; Rubbiani, Riccardo; Aulsebrook, Margaret L; Grace, Michael R; Spiccia, Leone; Tuck, Kellie L; Gasser, Gilles; Graham, Bim

    2016-01-01

    A new bifunctional macrocyclic chelator featuring a conjugatable alkynyl-naphthalimide fluorophore pendant group has been prepared and its Gd(III) complex coupled to a cell-penetrating lipidated azido-Tat peptide derivative using Cu(I)-catalysed "click" chemistry. The resulting fluorescent conjugate is able to enter CAL-33 tongue squamous carcinoma cells, as revealed by confocal microscopy, producing a very modest anti-proliferative effect (IC50 = 93 µM). Due to the photo-reactivity of the naphthalimide moiety, however, the conjugate's cytotoxicity is significantly enhanced (IC50 = 16 µM) upon brief low-power UV-A irradiation. PMID:26861271

  17. Mechanistic aspects of peptide-membrane interactions determined by optical, dielectric and piezoelectric techniques: an overview.

    PubMed

    Oliveira, Maria D L; Franco, Octavio L; Nascimento, Jessica M; de Melo, Celso P; Andrade, Cesar A S

    2013-11-01

    Antimicrobial peptides (AMPs) have been isolated from a wide variety of organisms that include microorganisms, plants, insects, frogs and mammals. As part of the innate immune system expressed in many tissues, AMPs are able to provide protection against invasion of foreign microorganisms and exhibit a broad spectrum of activity against bacteria, fungi and/or virus. Non-AMPs cell-penetrating peptides have been used as carriers for overcoming the membrane barrier and helping in the delivery of various molecules into the cell. Physicochemical peptide-lipid interactions studies can provide us with reliable molecular information about microbe defense response, including the elucidation of the prevailing mechanisms of its action, such as the barrel-stave, toroidal pore, carpet and detergent-like models. In this paper, we present an overview of the peptide-lipid mechanisms of interaction as well as discuss alternative techniques that could help to elucidate the peptides functionality. Quartz crystal microbalance (QCM), surface plasmon resonance (SPR) spectroscopy and electrochemical impedance spectroscopy (EIS) are useful techniques to investigate in details of the peptide-membrane interaction. The techniques here discussed could also offer specific and low-cost methods that can to shed some light over the different modes of action of AMPs, contributing to the development of drugs against infectious diseases.

  18. Peptide aptamers: The versatile role of specific protein function inhibitors in plant biotechnology.

    PubMed

    Colombo, Monica; Mizzotti, Chiara; Masiero, Simona; Kater, Martin M; Pesaresi, Paolo

    2015-11-01

    In recent years, peptide aptamers have emerged as novel molecular tools that have attracted the attention of researchers in various fields of basic and applied science, ranging from medicine to analytical chemistry. These artificial short peptides are able to specifically bind, track, and inhibit a given target molecule with high affinity, even molecules with poor immunogenicity or high toxicity, and represent a remarkable alternative to antibodies in many different applications. Their use is on the rise, driven mainly by the medical and pharmaceutical sector. Here we discuss the enormous potential of peptide aptamers in both basic and applied aspects of plant biotechnology and food safety. The different peptide aptamer selection methods available both in vivo and in vitro are introduced, and the most important possible applications in plant biotechnology are illustrated. In particular, we discuss the generation of broad-based virus resistance in crops, "reverse genetics" and aptasensors in bioassays for detecting contaminations in food and feed. Furthermore, we suggest an alternative to the transfer of peptide aptamers into plant cells via genetic transformation, based on the use of cell-penetrating peptides that overcome the limits imposed by both crop transformation and Genetically Modified Organism commercialization.

  19. Manipulation of pro-inflammatory cytokine production by the bacterial cell-penetrating effector protein YopM is independent of its interaction with host cell kinases RSK1 and PRK2

    PubMed Central

    Höfling, Sabrina; Scharnert, Julia; Cromme, Christoph; Bertrand, Jessica; Pap, Thomas; Schmidt, M Alexander; Rüter, Christian

    2014-01-01

    The effector protein Yersinia outer protein M (YopM) of Yersinia enterocolitica has previously been identified and characterized as the first bacterial cell-penetrating protein (CPP). We found that recombinant YopM (rYopM) enters different eukaryotic cell types and downregulates the expression of several pro-inflammatory cytokines (e.g., tumor necrosis factor-α [TNF-α]) after autonomous translocation. After infection with Y. enterocolitica or transfection of host cells, YopM interacts with isoforms of the two kinases ribosomal S6 protein kinase (RSK) and protein kinase C-related kinase (PRK). This interaction caused sustained RSK activation due to interference with dephosphorylation.   Here we demonstrate by co-immunoprecipitation that rYopM interacts with RSK and PRK following cell-penetration. We show that autonomously translocated rYopM forms a trimeric complex with different RSK and PRK isoforms. Furthermore, we constructed a series of truncated versions of rYopM to map the domain required for the formation of the complex. The C-terminus of rYopM was identified to be essential for the interaction with RSK1, whereas any deletion in rYopM’s leucin-rich repeat domains abrogated PRK2 binding. Moreover, we found that the interaction of cell-penetrating rYopM with RSK led to enhanced autophosphorylation of this kinase at serine 380. Finally, we investigated whether downstream signaling of the trimeric rYopM-RSK/PRK complex modulates the expression of pro-inflammatory TNF-α. Here, we could exclude that interaction with RSK1 and PRK2 is essential for the anti-inflammatory effects of rYopM. PMID:25513777

  20. C-Peptide Test

    MedlinePlus

    ... C-peptide is a useful marker of insulin production. The following are some purposes of C-peptide ... it nearly impossible to directly evaluate endogenous insulin production. In these cases, C-peptide measurement is a ...

  1. pACC1 peptide loaded chitosan nanoparticles induces apoptosis via reduced fatty acid synthesis in MDA-MB-231 cells

    NASA Astrophysics Data System (ADS)

    Kaliaperumal, Jagatheesh; Hari, Natarajan; Pavankumar, Padarthi; Elangovan, Namasivayam

    2016-06-01

    The development of formulations with therapeutic peptides has been restricted to poor cell penetration and in this attempt; we developed pACC1 peptide loaded chitosan nanoparticles. The prepared nanoparticles were characterized with FT-IR, XRD, SEM and TEM. In addition, the suitable formulation was evaluated for hemocompatibility, plasma stability and embryo toxicity using Danio rerio embryo model. The results showed that pACC1 peptide loaded chitosan nanoparticles were compatible with plasma. They possess sustained release pattern and also found to be safe up to 300 mg/L in embryo toxicity tests. Cytotoxicity assays with MDA-MB-231 cell lines suggested that, pACC1 peptide loaded chitosan nanoparticles were capable of enhanced cellular penetration and reduced palmitic acid content, which was confirmed by H1 NMR. Hence, these nanoparticles could be employed as excellent adjuvant therapeutics while treating solid tumors with multi-drug resistance.

  2. Branched dimerization of Tat peptide improves permeability to HeLa and hippocampal neuronal cells.

    PubMed

    Monreal, I Abrrey; Liu, Qian; Tyson, Katherine; Bland, Tyler; Dalisay, Doralyn S; Adams, Erin V; Wayman, Gary A; Aguilar, Hector C; Saludes, Jonel P

    2015-03-28

    A dimeric branched peptide TATp-D designed as an analogue of the HIV-Tat protein transduction domain (TATp), a prototypical cell penetrating peptide (CPP), demonstrates significantly enhanced cell uptake at 0.25 to 2.5 μM. Live cell confocal laser scanning microscopy revealed that multivalency dramatically improved the permeation potency of TATp-D to HeLa and primary hippocampal neuronal cells. The observed enhanced ability of TATp-D to translocate through the membrane is highlighted by a non-linear dependence on concentration, exhibiting the greatest uptake at sub-micromolar concentrations as compared to TATp. Multimerization via bis-Fmoc Lysine offered a synthetically straightforward method to investigate the effects of multivalent CPPs while offering orthogonal handles for cargo attachment, increasing the utility of CPPs at significantly lower concentrations. PMID:25733181

  3. Factors Affecting Peptide Interactions with Surface-Bound Microgels.

    PubMed

    Nyström, Lina; Nordström, Randi; Bramhill, Jane; Saunders, Brian R; Álvarez-Asencio, Rubén; Rutland, Mark W; Malmsten, Martin

    2016-02-01

    Effects of electrostatics and peptide size on peptide interactions with surface-bound microgels were investigated with ellipsometry, confocal microscopy, and atomic force microscopy (AFM). Results show that binding of cationic poly-L-lysine (pLys) to anionic, covalently immobilized, poly(ethyl acrylate-co-methacrylic acid) microgels increased with increasing peptide net charge and microgel charge density. Furthermore, peptide release was facilitated by decreasing either microgel or peptide charge density. Analogously, increasing ionic strength facilitated peptide release for short peptides. As a result of peptide binding, the surface-bound microgels displayed pronounced deswelling and increased mechanical rigidity, the latter quantified by quantitative nanomechanical mapping. While short pLys was found to penetrate the entire microgel network and to result in almost complete charge neutralization, larger peptides were partially excluded from the microgel network, forming an outer peptide layer on the microgels. As a result of this difference, microgel flattening was more influenced by the lower Mw peptide than the higher. Peptide-induced deswelling was found to be lower for higher Mw pLys, the latter effect not observed for the corresponding microgels in the dispersed state. While the effects of electrostatics on peptide loading and release were similar to those observed for dispersed microgels, there were thus considerable effects of the underlying surface on peptide-induced microgel deswelling, which need to be considered in the design of surface-bound microgels as carriers of peptide loads, for example, in drug delivery or in functionalized biomaterials. PMID:26750986

  4. The NFL-TBS.40-63 anti-glioblastoma peptide enters selectively in glioma cells by endocytosis.

    PubMed

    Lépinoux-Chambaud, Claire; Eyer, Joël

    2013-10-01

    Glioblastoma are the most frequent and aggressive tumour of the nervous system despite surgical resection associated with chemotherapy and radiotherapy. Recently, we showed that the NFL-TBS.40-63 peptide corresponding to the sequence of a tubulin-binding site of neurofilaments, enters selectively in glioblastoma cells where it blocks microtubule polymerization, inhibits their proliferation, and reduces tumour development in rats bearing glioblastoma (Bocquet et al., 2009; Berges et al., 2012a). Here, we characterized the molecular mechanism responsible for the uptake of NFL-TBS.40-63 peptide by glioblastoma cells. Unlike other cell penetrating peptides (CPPs), which use a balance between endocytosis and direct translocation, the NFL-TBS.40-63 peptide is unable to translocate directly through the membrane when incubated with giant plasma membrane vesicles. Then, using a panel of markers and inhibitors, flow cytometry and confocal microscopy investigations showed that the uptake occurs mainly through endocytosis. Moreover, glycosaminoglycans and αVβ3 integrins are not involved in the NFL-TBS.40-63 peptide recognition and internalization by glioblastoma cells. Finally, the signalling of tyrosine kinase receptors is involved in the peptide uptake, especially via EGFR overexpressed in tumour cells, indicating that the uptake of NFL-TBS.40-63 peptide by glioblastoma cells is related to their abnormally high proliferative activity.

  5. The NFL-TBS.40-63 anti-glioblastoma peptide enters selectively in glioma cells by endocytosis.

    PubMed

    Lépinoux-Chambaud, Claire; Eyer, Joël

    2013-10-01

    Glioblastoma are the most frequent and aggressive tumour of the nervous system despite surgical resection associated with chemotherapy and radiotherapy. Recently, we showed that the NFL-TBS.40-63 peptide corresponding to the sequence of a tubulin-binding site of neurofilaments, enters selectively in glioblastoma cells where it blocks microtubule polymerization, inhibits their proliferation, and reduces tumour development in rats bearing glioblastoma (Bocquet et al., 2009; Berges et al., 2012a). Here, we characterized the molecular mechanism responsible for the uptake of NFL-TBS.40-63 peptide by glioblastoma cells. Unlike other cell penetrating peptides (CPPs), which use a balance between endocytosis and direct translocation, the NFL-TBS.40-63 peptide is unable to translocate directly through the membrane when incubated with giant plasma membrane vesicles. Then, using a panel of markers and inhibitors, flow cytometry and confocal microscopy investigations showed that the uptake occurs mainly through endocytosis. Moreover, glycosaminoglycans and αVβ3 integrins are not involved in the NFL-TBS.40-63 peptide recognition and internalization by glioblastoma cells. Finally, the signalling of tyrosine kinase receptors is involved in the peptide uptake, especially via EGFR overexpressed in tumour cells, indicating that the uptake of NFL-TBS.40-63 peptide by glioblastoma cells is related to their abnormally high proliferative activity. PMID:23603097

  6. [SYNTHETIC PEPTIDE VACCINES].

    PubMed

    Sergeyev, O V; Barinsky, I F

    2016-01-01

    An update on the development and trials of synthetic peptide vaccines is reviewed. The review considers the successful examples of specific protection as a result of immunization with synthetic peptides using various protocols. The importance of conformation for the immunogenicity of the peptide is pointed out. An alternative strategy of the protection of the organism against the infection using synthetic peptides is suggested.

  7. Antimicrobial Peptides from Marine Proteobacteria

    PubMed Central

    Desriac, Florie; Jégou, Camille; Balnois, Eric; Brillet, Benjamin; Le Chevalier, Patrick; Fleury, Yannick

    2013-01-01

    After years of inadequate use and the emergence of multidrug resistant (MDR) strains, the efficiency of “classical” antibiotics has decreased significantly. New drugs to fight MDR strains are urgently needed. Bacteria hold much promise as a source of unusual bioactive metabolites. However, the potential of marine bacteria, except for Actinomycetes and Cyanobacteria, has been largely underexplored. In the past two decades, the structures of several antimicrobial compounds have been elucidated in marine Proteobacteria. Of these compounds, polyketides (PKs), synthesised by condensation of malonyl-coenzyme A and/or acetyl-coenzyme A, and non-ribosomal peptides (NRPs), obtained through the linkage of (unusual) amino acids, have recently generated particular interest. NRPs are good examples of naturally modified peptides. Here, we review and compile the data on the antimicrobial peptides isolated from marine Proteobacteria, especially NRPs. PMID:24084784

  8. The uptake of HIV Tat peptide proceeds via two pathways which differ from macropinocytosis.

    PubMed

    Ben-Dov, Nadav; Korenstein, Rafi

    2015-03-01

    Cell penetrating peptides (CPPs) have been extensively studied as vectors for cellular delivery of therapeutic molecules, yet the identity of their uptake routes remained unclear and is still under debate. In this study we provide new insights into CPP entry routes by quantitatively measuring the intracellular uptake of FAM-labeled Tat-peptide under rigorous kinetic and thermal conditions. The uptake of Tat-peptide between 4 and 15°C corresponds to Q10=1.1, proceeding through a prompt (<5 min), temperature-independent process, suggesting direct membrane translocation. At longer durations, Tat rate of uptake shows linear dependence on temperature with Q10=1.44, accompanied by activation energy Ea=4.45 Kcal/mole. These values are significantly lower than those we found for the macropinocytosis probe dextran (Q10=2.2 and Ea=7.2 Kcal/mole) which possesses an exponential dependence on temperature, characteristic of endocytosis processes. Tat-peptide and dextran do not interfere with each other's uptake rate and the ratio of Tat-peptide uptake to its extracellular concentration is ~15 times higher than that for dextran. In addition, Phloretin, a modulator of cell membrane dipole potential, is shown to increase dextran uptake but to reduce that of Tat. We conclude that the uptake of Tat differs from that of dextran in all parameters. Tat uptake proceeds by dual entry routes which differ by their energy dependence. PMID:25542781

  9. A nanotechnological, molecular-modeling, and immunological approach to study the interaction of the anti-tumorigenic peptide p28 with the p53 family of proteins.

    PubMed

    Coppari, Emilia; Yamada, Tohru; Bizzarri, Anna Rita; Beattie, Craig W; Cannistraro, Salvatore

    2014-01-01

    p28 is an anionic, amphipathic, cell-penetrating peptide derived from the cupredoxin azurin that binds to the DNA-binding domain (DBD) of the tumor suppressor protein, p53, and induces a post-translational increase in the level of wild type and mutated p53 in a wide variety of human cancer cells. As p63 and p73, additional members of the p53 superfamily of proteins, also appear to be involved in the cellular response to cancer therapy and are reportedly required for p53-induced apoptosis, we asked whether p28 also binds to p63 and p73. Atomic force spectroscopy demonstrates that p28 forms a stable, high-affinity complex with full-length p63, the DBD of p63, and full-length p73. Exposure to p28 decreased the level of TAp63α and ΔNp63α, the truncated form of p63, in p53 wild type and mutated human breast cancer cells, respectively. p28 increased the level of TAp73α, but not ΔNp73α, in the same breast cancer cell lines. In contrast, p28 increased the level of the TA and ΔN isoforms of p63 in p53 wild type, but not in p53 mutated melanoma cells, while decreasing TA p73α in p53 wild type and mutated human melanoma cells. All changes were mirrored by an associated change in the expression of the HECT E3 ligases Itch/AIP4, AIP5, and the RING E3 ligase Pirh2, but not in the receptor for activated C kinase or the RING E3 ligases Mdm2 and Cop1. Collectively, the data suggest that molecules such as p28 bind with high affinity to the DBD of p63 and p73 and alter their expression independent of the Mdm2 and Cop1 pathways.

  10. Intracellular delivery of peptide cargos using polyhydroxybutyrate based biodegradable nanoparticles: Studies on antitumor efficacy of BCL-2 converting peptide, NuBCP-9.

    PubMed

    Kapoor, Sumeet; Gupta, Dikishi; Kumar, Manoj; Sharma, Sapna; Gupta, Amit K; Misro, M M; Singh, Harpal

    2016-09-25

    Faster biodegradation, immunogenicity and lack of cell penetrative capabilities are hurdles in development of peptidyl drugs for cancer therapy. Polymeric carriers can be used to overcome these problems. The present study is focused on the use of polyhydroxybutyrate as a potential nanovehicle for the delivery of anticancer peptides. PHB (72kDa) was produced by thermal treatment of high molecular weight PHB (300kDa) under melt conditions and then conjugated with PEG (4kDa) by Steglich esterification reaction. Anticancer peptide NuBCP-9 (FSRSLHSLL) encapsulated PHB(72K)-PEG(4K) NPs were prepared by double emulsion solvent evaporation method. PHB(72K)-PEG(4K) NPs showed encapsulation efficiency of 61% and exhibited sustained release of peptide over a period of 26days at physiological pH. NuBCP-9 loaded PHB(72K)-PEG(4K) NPs showed an IC50 value of 2.2μM & 1.6μM in MCF-7 cells in 48h and 72h respectively. Confocal laser microscopy confirmed efficient cellular uptake and induction of apoptosis by peptide loaded NPs in a time dependent manner. In vivo intraperitonial administration of 20mg/kg NuBCP-9/NPs twice a week for three weeks triggered 90% tumor regression in Ehrlich syngeneic mouse model. Our results illustrated the potential of PHB(72K)-PEG(4K) based nanoformulation as a tool for targeting intracellular proteins. PMID:27492021

  11. Simultaneous imaging and restoration of cell function using cell permeable peptide probe.

    PubMed

    Suh, Jin Sook; Lee, Jue Yeon; Lee, Gene; Chung, Chong Pyoung; Park, Yoon Jeong

    2014-08-01

    Targeting tissues/cells using probing materials to detect diseases such as cancer and inflammatory disease has been attempted with some success. Most of the molecular targets used in diagnosis and therapy were identified through the discovery of intracellular signaling pathways. Among intracellular signaling processes, the ubiquitination of proteins, and thereby their proteasomal degradation, is important because it plays a role in most diseases involving alterations to a component of the ubiquitination system, particularly E3 ligases, which have selective target-binding affinity and are key to the success of regulating the disorder. The regulation and monitoring of E3 ligases can be achieved using peptides containing protein-protein binding motifs. We generated a human protein-derived peptide that could target Smurf1, a member of the E3 ligase family, by competitively binding to osteo-Smads. To effectively deliver it into cells, the peptide was further modified with a cell-penetrating peptide. The peptide contains two fluorescent dyes: fluorescein isothiocyanate (FITC; absorbance/emission wavelengths: 495/519 nm) as a fluorophore and black hole quencher-1 (BHQ-1) as a fluorescence quencher. When the target Smurf1 combined with complementary sequences in the peptide probe, the distance between the fluorophore and BHQ-1 increased via a conformational change, resulting in the recovery of the fluorescence signal. Simultaneously, the degradation of Smad1/5/8 was blocked by the binding of the peptide probe to Smurf1, leading to the potentiation of the osteogenic pathway, which was reflected by an increase in the expression of osteoinductive genes, such as alkaline phosphatase and osteocalcin. Possible future applications of the peptide probe include its integration into imaging tools for the diagnosis of Smurf1-overexpressing diseases. PMID:24831974

  12. Topical and Targeted Delivery of siRNAs to Melanoma Cells Using a Fusion Peptide Carrier.

    PubMed

    Ruan, Renquan; Chen, Ming; Sun, Sijie; Wei, Pengfei; Zou, Lili; Liu, Jing; Gao, Dayong; Wen, Longping; Ding, Weiping

    2016-01-01

    Topical application of siRNAs through the skin is a potentially effective strategy for the treatment of melanoma tumors. In this study, we designed a new and safe fusion peptide carrier SPACE-EGF to improve the skin and cell penetration function of the siRNAs and their targeting ability to B16 cells, such that the apoptosis of B16 cells can be induced. The results show that the carrier is stable and less toxic. The EGF motif does not affect the skin and cell penetration function of the SPACE. Because EGF can strongly bind EGFR, which is overexpressed in cancer cells, the targeting ability of the SPACE-EGF-siRNA complex is increased. In vitro experiments indicate that GAPDH siRNAs conjugated with SPACE-EGF can significantly reduce the GAPDH concentration in B16 cells, and c-Myc siRNAs can cause the gene silencing of c-Myc and thus the apoptosis of cells. In vivo experiments show that the topical application of c-Myc siRNAs delivered by SPACE-EGF through the skin can significantly inhibit the growth of melanoma tumors. This work may provide insight into the development of new transdermal drug carriers to treat a variety of skin disorders. PMID:27374619

  13. Topical and Targeted Delivery of siRNAs to Melanoma Cells Using a Fusion Peptide Carrier

    PubMed Central

    Ruan, Renquan; Chen, Ming; Sun, Sijie; Wei, Pengfei; Zou, Lili; Liu, Jing; Gao, Dayong; Wen, Longping; Ding, Weiping

    2016-01-01

    Topical application of siRNAs through the skin is a potentially effective strategy for the treatment of melanoma tumors. In this study, we designed a new and safe fusion peptide carrier SPACE-EGF to improve the skin and cell penetration function of the siRNAs and their targeting ability to B16 cells, such that the apoptosis of B16 cells can be induced. The results show that the carrier is stable and less toxic. The EGF motif does not affect the skin and cell penetration function of the SPACE. Because EGF can strongly bind EGFR, which is overexpressed in cancer cells, the targeting ability of the SPACE-EGF-siRNA complex is increased. In vitro experiments indicate that GAPDH siRNAs conjugated with SPACE-EGF can significantly reduce the GAPDH concentration in B16 cells, and c-Myc siRNAs can cause the gene silencing of c-Myc and thus the apoptosis of cells. In vivo experiments show that the topical application of c-Myc siRNAs delivered by SPACE-EGF through the skin can significantly inhibit the growth of melanoma tumors. This work may provide insight into the development of new transdermal drug carriers to treat a variety of skin disorders. PMID:27374619

  14. Peptides and Food Intake

    PubMed Central

    Sobrino Crespo, Carmen; Perianes Cachero, Aránzazu; Puebla Jiménez, Lilian; Barrios, Vicente; Arilla Ferreiro, Eduardo

    2014-01-01

    The mechanisms for controlling food intake involve mainly an interplay between gut, brain, and adipose tissue (AT), among the major organs. Parasympathetic, sympathetic, and other systems are required for communication between the brain satiety center, gut, and AT. These neuronal circuits include a variety of peptides and hormones, being ghrelin the only orexigenic molecule known, whereas the plethora of other factors are inhibitors of appetite, suggesting its physiological relevance in the regulation of food intake and energy homeostasis. Nutrients generated by food digestion have been proposed to activate G-protein-coupled receptors on the luminal side of enteroendocrine cells, e.g., the L-cells. This stimulates the release of gut hormones into the circulation such as glucagon-like peptide-1 (GLP-1), oxyntomodulin, pancreatic polypeptides, peptide tyrosine tyrosine, and cholecystokinin, which inhibit appetite. Ghrelin is a peptide secreted from the stomach and, in contrast to other gut hormones, plasma levels decrease after a meal and potently stimulate food intake. Other circulating factors such as insulin and leptin relay information regarding long-term energy stores. Both hormones circulate at proportional levels to body fat content, enter the CNS proportionally to their plasma levels, and reduce food intake. Circulating hormones can influence the activity of the arcuate nucleus (ARC) neurons of the hypothalamus, after passing across the median eminence. Circulating factors such as gut hormones may also influence the nucleus of the tractus solitarius (NTS) through the adjacent circumventricular organ. On the other hand, gastrointestinal vagal afferents converge in the NTS of the brainstem. Neural projections from the NTS, in turn, carry signals to the hypothalamus. The ARC acts as an integrative center, with two major subpopulations of neurons influencing appetite, one of them coexpressing neuropeptide Y and agouti-related protein (AgRP) that increases food

  15. Peptides and food intake.

    PubMed

    Sobrino Crespo, Carmen; Perianes Cachero, Aránzazu; Puebla Jiménez, Lilian; Barrios, Vicente; Arilla Ferreiro, Eduardo

    2014-01-01

    The mechanisms for controlling food intake involve mainly an interplay between gut, brain, and adipose tissue (AT), among the major organs. Parasympathetic, sympathetic, and other systems are required for communication between the brain satiety center, gut, and AT. These neuronal circuits include a variety of peptides and hormones, being ghrelin the only orexigenic molecule known, whereas the plethora of other factors are inhibitors of appetite, suggesting its physiological relevance in the regulation of food intake and energy homeostasis. Nutrients generated by food digestion have been proposed to activate G-protein-coupled receptors on the luminal side of enteroendocrine cells, e.g., the L-cells. This stimulates the release of gut hormones into the circulation such as glucagon-like peptide-1 (GLP-1), oxyntomodulin, pancreatic polypeptides, peptide tyrosine tyrosine, and cholecystokinin, which inhibit appetite. Ghrelin is a peptide secreted from the stomach and, in contrast to other gut hormones, plasma levels decrease after a meal and potently stimulate food intake. Other circulating factors such as insulin and leptin relay information regarding long-term energy stores. Both hormones circulate at proportional levels to body fat content, enter the CNS proportionally to their plasma levels, and reduce food intake. Circulating hormones can influence the activity of the arcuate nucleus (ARC) neurons of the hypothalamus, after passing across the median eminence. Circulating factors such as gut hormones may also influence the nucleus of the tractus solitarius (NTS) through the adjacent circumventricular organ. On the other hand, gastrointestinal vagal afferents converge in the NTS of the brainstem. Neural projections from the NTS, in turn, carry signals to the hypothalamus. The ARC acts as an integrative center, with two major subpopulations of neurons influencing appetite, one of them coexpressing neuropeptide Y and agouti-related protein (AgRP) that increases food

  16. Taurine Boosts Cellular Uptake of Small d-Peptides for Enzyme-Instructed Intracellular Molecular Self-Assembly

    PubMed Central

    2016-01-01

    Due to their biostability, d-peptides are emerging as an important molecular platform for biomedical applications. Being proteolytically resistant, d-peptides lack interactions with endogenous transporters and hardly enter cells. Here we show that taurine, a natural amino acid, drastically boosts the cellular uptake of small d-peptides in mammalian cells by >10-fold, from 118 μM (without conjugating taurine) to >1.6 mM (after conjugating taurine). The uptake of a large amount of the ester conjugate of taurine and d-peptide allows intracellular esterase to trigger intracellular self-assembly of the d-peptide derivative, further enhancing their cellular accumulation. The study on the mechanism of the uptake reveals that the conjugates enter cells via both dynamin-dependent endocytosis and macropinocytosis, but likely not relying on taurine transporters. Differing fundamentally from the positively charged cell-penetrating peptides, the biocompatibility, stability, and simplicity of the enzyme-cleavable taurine motif promise new ways to promote the uptake of bioactive molecules for countering the action of efflux pump and contributing to intracellular molecular self-assembly. PMID:26235707

  17. Taurine Boosts Cellular Uptake of Small D-Peptides for Enzyme-Instructed Intracellular Molecular Self-Assembly.

    PubMed

    Zhou, Jie; Du, Xuewen; Li, Jie; Yamagata, Natsuko; Xu, Bing

    2015-08-19

    Due to their biostability, D-peptides are emerging as an important molecular platform for biomedical applications. Being proteolytically resistant, D-peptides lack interactions with endogenous transporters and hardly enter cells. Here we show that taurine, a natural amino acid, drastically boosts the cellular uptake of small D-peptides in mammalian cells by >10-fold, from 118 μM (without conjugating taurine) to >1.6 mM (after conjugating taurine). The uptake of a large amount of the ester conjugate of taurine and D-peptide allows intracellular esterase to trigger intracellular self-assembly of the D-peptide derivative, further enhancing their cellular accumulation. The study on the mechanism of the uptake reveals that the conjugates enter cells via both dynamin-dependent endocytosis and macropinocytosis, but likely not relying on taurine transporters. Differing fundamentally from the positively charged cell-penetrating peptides, the biocompatibility, stability, and simplicity of the enzyme-cleavable taurine motif promise new ways to promote the uptake of bioactive molecules for countering the action of efflux pump and contributing to intracellular molecular self-assembly.

  18. Taurine Boosts Cellular Uptake of Small D-Peptides for Enzyme-Instructed Intracellular Molecular Self-Assembly.

    PubMed

    Zhou, Jie; Du, Xuewen; Li, Jie; Yamagata, Natsuko; Xu, Bing

    2015-08-19

    Due to their biostability, D-peptides are emerging as an important molecular platform for biomedical applications. Being proteolytically resistant, D-peptides lack interactions with endogenous transporters and hardly enter cells. Here we show that taurine, a natural amino acid, drastically boosts the cellular uptake of small D-peptides in mammalian cells by >10-fold, from 118 μM (without conjugating taurine) to >1.6 mM (after conjugating taurine). The uptake of a large amount of the ester conjugate of taurine and D-peptide allows intracellular esterase to trigger intracellular self-assembly of the D-peptide derivative, further enhancing their cellular accumulation. The study on the mechanism of the uptake reveals that the conjugates enter cells via both dynamin-dependent endocytosis and macropinocytosis, but likely not relying on taurine transporters. Differing fundamentally from the positively charged cell-penetrating peptides, the biocompatibility, stability, and simplicity of the enzyme-cleavable taurine motif promise new ways to promote the uptake of bioactive molecules for countering the action of efflux pump and contributing to intracellular molecular self-assembly. PMID:26235707

  19. Firefly luciferase inhibitor-conjugated peptide quenches bioluminescence: a versatile tool for real time monitoring cellular uptake of biomolecules.

    PubMed

    Poutiainen, Pekka K; Rönkkö, Teemu; Hinkkanen, Ari E; Palvimo, Jorma J; Närvänen, Ale; Turhanen, Petri; Laatikainen, Reino; Weisell, Janne; Pulkkinen, Juha T

    2014-01-15

    In this paper, novel firefly luciferase-specific inhibitor compounds (FLICs) are evaluated as potential tools for cellular trafficking of transporter conjugates. As a proof-of-concept, we designed FLICs that were suitable for solid phase peptide synthesis and could be covalently conjugated to peptides via an amide bond. The spacer between inhibitor and peptide was optimized to gain efficient inhibition of recombinant firefly luciferase (FLuc) without compromising the activity of the model peptides. The hypothesis of using FLICs as tools for cellular trafficking studies was ensured with U87Fluc glioblastoma cells expressing firefly luciferase. Results show that cell penetrating peptide (penetratin) FLIC conjugate 9 inhibited FLuc penetrated cells efficiently (IC50 = 1.6 μM) and inhibited bioluminescence, without affecting the viability of the cells. Based on these results, peptide-FLIC conjugates can be used for the analysis of cellular uptake of biomolecules in a new way that can at the same time overcome some downsides seen with other methods. Thus, FLICs can be considered as versatile tools that broaden the plethora of methods that take advantage of the bioluminescence phenomena.

  20. How Membrane-Active Peptides Get into Lipid Membranes.

    PubMed

    Sani, Marc-Antoine; Separovic, Frances

    2016-06-21

    The structure-function relationship for a family of antimicrobial peptides (AMPs) from the skin of Australian tree frogs is discussed and compared with that of peptide toxins from bee and Australian scorpion venoms. Although these membrane-active peptides induce a similar cellular fate by disrupting the lipid bilayer integrity, their lytic activity is achieved via different modes of action, which are investigated in relation to amino acid sequence, secondary structure, and membrane lipid composition. In order to better understand what structural features govern the interaction between peptides and lipid membranes, cell-penetrating peptides (CPPs), which translocate through the membrane without compromising its integrity, are also discussed. AMPs possess membrane lytic activities that are naturally designed to target the cellular membrane of pathogens or competitors. They are extremely diverse in amino acid composition and often show specificity against a particular strain of microbe. Since our antibiotic arsenal is declining precariously in the face of the rise in multiantibiotic resistance, AMPs increasingly are seen as a promising alternative. In an effort to understand their molecular mechanism, biophysical studies of a myriad of AMPs have been reported, yet no unifying mechanism has emerged, rendering difficult the rational design of drug leads. Similarly, a wide variety of cytotoxic peptides are found in venoms, the best known being melittin, yet again, predicting their activity based on a particular amino acid composition or secondary structure remains elusive. A common feature of these membrane-active peptides is their preference for the lipid environment. Indeed, they are mainly unstructured in solution and, in the presence of lipid membranes, quickly adsorb onto the surface, change their secondary structure, eventually insert into the hydrophobic core of the membrane bilayer, and finally disrupt the bilayer integrity. These steps define the molecular

  1. How Membrane-Active Peptides Get into Lipid Membranes.

    PubMed

    Sani, Marc-Antoine; Separovic, Frances

    2016-06-21

    The structure-function relationship for a family of antimicrobial peptides (AMPs) from the skin of Australian tree frogs is discussed and compared with that of peptide toxins from bee and Australian scorpion venoms. Although these membrane-active peptides induce a similar cellular fate by disrupting the lipid bilayer integrity, their lytic activity is achieved via different modes of action, which are investigated in relation to amino acid sequence, secondary structure, and membrane lipid composition. In order to better understand what structural features govern the interaction between peptides and lipid membranes, cell-penetrating peptides (CPPs), which translocate through the membrane without compromising its integrity, are also discussed. AMPs possess membrane lytic activities that are naturally designed to target the cellular membrane of pathogens or competitors. They are extremely diverse in amino acid composition and often show specificity against a particular strain of microbe. Since our antibiotic arsenal is declining precariously in the face of the rise in multiantibiotic resistance, AMPs increasingly are seen as a promising alternative. In an effort to understand their molecular mechanism, biophysical studies of a myriad of AMPs have been reported, yet no unifying mechanism has emerged, rendering difficult the rational design of drug leads. Similarly, a wide variety of cytotoxic peptides are found in venoms, the best known being melittin, yet again, predicting their activity based on a particular amino acid composition or secondary structure remains elusive. A common feature of these membrane-active peptides is their preference for the lipid environment. Indeed, they are mainly unstructured in solution and, in the presence of lipid membranes, quickly adsorb onto the surface, change their secondary structure, eventually insert into the hydrophobic core of the membrane bilayer, and finally disrupt the bilayer integrity. These steps define the molecular

  2. Brain natriutetic peptide test

    MedlinePlus

    ... medlineplus.gov/ency/article/007509.htm Brain natriuretic peptide test To use the sharing features on this page, please enable JavaScript. Brain natriuretic peptide (BNP) test is a blood test that measures ...

  3. Vasoactive intestinal peptide test

    MedlinePlus

    ... medlineplus.gov/ency/article/003508.htm Vasoactive intestinal peptide test To use the sharing features on this page, please enable JavaScript. Vasoactive intestinal peptide (VIP) is a test that measures the amount ...

  4. [SYNTHETIC PEPTIDE VACCINES].

    PubMed

    Sergeyev, O V; Barinsky, I F

    2016-01-01

    An update on the development and trials of synthetic peptide vaccines is reviewed. The review considers the successful examples of specific protection as a result of immunization with synthetic peptides using various protocols. The importance of conformation for the immunogenicity of the peptide is pointed out. An alternative strategy of the protection of the organism against the infection using synthetic peptides is suggested. PMID:27145593

  5. PH dependent adhesive peptides

    DOEpatents

    Tomich, John; Iwamoto, Takeo; Shen, Xinchun; Sun, Xiuzhi Susan

    2010-06-29

    A novel peptide adhesive motif is described that requires no receptor or cross-links to achieve maximal adhesive strength. Several peptides with different degrees of adhesive strength have been designed and synthesized using solid phase chemistries. All peptides contain a common hydrophobic core sequence flanked by positively or negatively charged amino acids sequences.

  6. Buforins: histone H2A-derived antimicrobial peptides from toad stomach.

    PubMed

    Cho, Ju Hyun; Sung, Bong Hyun; Kim, Sun Chang

    2009-08-01

    Antimicrobial peptides (AMPs) constitute an important component of the innate immune system in a variety of organisms. Buforin I is a 39-amino acid AMP that was first isolated from the stomach tissue of the Asian toad Bufo bufo gargarizans. Buforin II is a 21-amino acid peptide that is derived from buforin I and displays an even more potent antimicrobial activity than its parent AMP. Both peptides share complete sequence identity with the N-terminal region of histone H2A that interacts directly with nucleic acids. Buforin I is generated from histone H2A by pepsin-directed proteolysis in the cytoplasm of gastric gland cells. After secretion into the gastric lumen, buforin I remains adhered to the mucous biofilm that lines the stomach, thus providing a protective antimicrobial coat. Buforins, which house a helix-hinge-helix domain, kill a microorganism by entering the cell without membrane permeabilization and thus binding to nucleic acids. The proline hinge is crucial for the cell penetrating activity of buforins. Buforins also are known to possess anti-endotoxin and anticancer activities, thus making these peptides attractive reagents for pharmaceutical applications. This review describes the role of buforins in innate host defense; future research paradigms; and use of these agents as human therapeutics.

  7. Avidin-biotin interaction mediated peptide assemblies as efficient gene delivery vectors for cancer therapy.

    PubMed

    Qu, Wei; Chen, Wei-Hai; Kuang, Ying; Zeng, Xuan; Cheng, Si-Xue; Zhou, Xiang; Zhuo, Ren-Xi; Zhang, Xian-Zheng

    2013-01-01

    Gene therapy offers a bright future for the treatment of cancers. One of the research highlights focuses on smart gene delivery vectors with good biocompatibility and tumor-targeting ability. Here, a novel gene vector self-assembled through avidin-biotin interaction with optimized targeting functionality, biotinylated tumor-targeting peptide/avidin/biotinylated cell-penetrating peptide (TAC), was designed and prepared to mediate the in vitro and in vivo delivery of p53 gene. TAC exhibited efficient DNA-binding ability and low cytotoxicity. In in vitro transfection assay, TAC/p53 complexes showed higher transfection efficiency and expression amount of p53 protein in MCF-7 cells as compared with 293T and HeLa cells, primarily due to the specific recognition between tumor-targeting peptides and receptors on MCF-7 cells. Additionally, by in situ administration of TAC/p53 complexes into tumor-bearing mice, the expression of p53 gene was obviously upregulated in tumor cells, and the tumor growth was significantly suppressed. This study provides an alternative and unique strategy to assemble functionalized peptides, and the novel self-assembled vector TAC developed is a promising gene vector for cancer therapy.

  8. Cellular uptake of a cystine-knot peptide and modulation of its intracellular trafficking

    PubMed Central

    Gao, Xinxin; Stanger, Karen; Kaluarachchi, Harini; Maurer, Till; Ciepla, Paulina; Chalouni, Cecile; Franke, Yvonne; Hannoush, Rami N.

    2016-01-01

    Cyclotides or cyclic cystine-knot peptides have emerged as a promising class of pharmacological ligands that modulate protein function. Interestingly, very few cyclotides have been shown to enter into cells. Yet, it remains unknown whether backbone cyclization is required for their cellular internalization. In this report, we studied the cellular behavior of EETI-II, a model acyclic cystine-knot peptide. Even though synthetic methods have been used to generate EETI-II, recombinant methods that allow efficient large scale biosynthesis of EETI-II have been lagging. Here, we describe a novel protocol for recombinant generation of folded EETI-II in high yields and to near homogeneity. We also uncover that EETI-II is efficiently uptaken via an active endocytic pathway to early endosomes in mammalian cells, eventually accumulating in late endosomes and lysosomes. Notably, co-incubation with a cell-penetrating peptide enhanced the cellular uptake and altered the trafficking of EETI-II, leading to its evasion of lysosomes. Our results demonstrate the feasibility of modulating the subcellular distribution and intracellular targeting of cystine-knot peptides, and hence enable future exploration of their utility in drug discovery and delivery. PMID:27734922

  9. Cell-permeable peptides induce dose- and length-dependent cytotoxic effects.

    PubMed

    Cardozo, Alessandra K; Buchillier, Valérie; Mathieu, Marc; Chen, Jianhua; Ortis, Fernanda; Ladrière, Laurence; Allaman-Pillet, Nathalie; Poirot, Olivier; Kellenberger, Stephan; Beckmann, Jacques S; Eizirik, Decio L; Bonny, Christophe; Maurer, Fabienne

    2007-09-01

    We have explored the threshold of tolerance of three unrelated cell types to treatments with potential cytoprotective peptides bound to Tat(48-57) and Antp(43-58) cell-permeable peptide carriers. Both Tat(48-57) and Antp(43-58) are well known for their good efficacy at crossing membranes of different cell types, their overall low toxicity, and their absence of leakage once internalised. Here, we show that concentrations of up to 100 microM of Tat(48-57) were essentially harmless in all cells tested, whereas Antp(43-58) was significantly more toxic. Moreover, all peptides bound to Tat(48-57) and Antp(43-58) triggered significant and length-dependent cytotoxicity when used at concentrations above 10 microM in all but one cell types (208F rat fibroblasts), irrespective of the sequence of the cargo. Absence of cytotoxicity in 208F fibroblasts correlated with poor intracellular peptide uptake, as monitored by confocal laser scanning fluorescence microscopy. Our data further suggest that the onset of cytotoxicity correlates with the activation of two intracellular stress signalling pathways, namely those involving JNK, and to a lesser extent p38 mitogen-activated protein kinases. These responses are of particular concern for cells that are especially sensitive to the activation of stress kinases. Collectively, these results indicate that in order to avoid unwanted and unspecific cytotoxicity, effector molecules bound to Tat(48-57) should be designed with the shortest possible sequence and the highest possible affinity for their binding partners or targets, so that concentrations below 10 microM can be successfully applied to cells without harm. Considering that cytotoxicity associated to Tat(48-57)- and Antp(43-58) bound peptide conjugates was not restricted to a particular type of cells, our data provide a general framework for the design of cell-penetrating peptides that may apply to broader uses of intracellular peptide and drug delivery.

  10. Computational peptide vaccinology.

    PubMed

    Söllner, Johannes

    2015-01-01

    Immunoinformatics focuses on modeling immune responses for better understanding of the immune system and in many cases for proposing agents able to modify the immune system. The most classical of these agents are vaccines derived from living organisms such as smallpox or polio. More modern vaccines comprise recombinant proteins, protein domains, and in some cases peptides. Generating a vaccine from peptides however requires technologies and concepts very different from classical vaccinology. Immunoinformatics therefore provides the computational tools to propose peptides suitable for formulation into vaccines. This chapter introduces the essential biological concepts affecting design and efficacy of peptide vaccines and discusses current methods and workflows applied to design successful peptide vaccines using computers.

  11. Antimicrobial Peptide Simulations and the Influence of Force Field on the Free Energy for Pore Formation in Lipid Bilayers.

    PubMed

    Bennett, W F Drew; Hong, Chun Kit; Wang, Yi; Tieleman, D Peter

    2016-09-13

    Due to antimicrobial resistance, the development of new drugs to combat bacterial and fungal infections is an important area of research. Nature uses short, charged, and amphipathic peptides for antimicrobial defense, many of which disrupt the lipid membrane in addition to other possible targets inside the cell. Computer simulations have revealed atomistic details for the interactions of antimicrobial peptides and cell-penetrating peptides with lipid bilayers. Strong interactions between the polar interface and the charged peptides can induce bilayer deformations - including membrane rupture and peptide stabilization of a hydrophilic pore. Here, we performed microsecond-long simulations of the antimicrobial peptide CM15 in a POPC bilayer expecting to observe pore formation (based on previous molecular dynamics simulations). We show that caution is needed when interpreting results of equilibrium peptide-membrane simulations, given the length of time single trajectories can dwell in local energy minima for 100's of ns to microseconds. While we did record significant membrane perturbations from the CM15 peptide, pores were not observed. We explain this discrepancy by computing the free energy for pore formation with different force fields. Our results show a large difference in the free energy barrier (ca. 40 kJ/mol) against pore formation predicted by the different force fields that would result in orders of magnitude differences in the simulation time required to observe spontaneous pore formation. This explains why previous simulations using the Berger lipid parameters reported pores induced by charged peptides, while with CHARMM based models pores were not observed in our long time-scale simulations. We reconcile some of the differences in the distance dependent free energies by shifting the free energy profiles to account for thickness differences between force fields. The shifted curves show that all the models describe small defects in lipid bilayers in a

  12. Integration of antimicrobial peptides with gold nanoparticles as unique non-viral vectors for gene delivery to mesenchymal stem cells with antibacterial activity.

    PubMed

    Peng, Li-Hua; Huang, Yan-Fen; Zhang, Chen-Zhen; Niu, Jie; Chen, Ying; Chu, Yang; Jiang, Zhi-Hong; Gao, Jian-Qing; Mao, Zheng-Wei

    2016-10-01

    Gold nanoparticles (AuNPs) have emerged as attractive non-viral gene vectors. However their application in regenerative medicine is still limited partially due to a lack of an intrinsic capacity to transfect difficult-to-transfect cells such as primary cells or stem cells. In current study, we report the synthesis of antimicrobial peptide conjugated cationic AuNPs (AuNPs@PEP) as highly efficient carriers for gene delivery to stem cells with antibacterial ability. The AuNPs@PEP integrate the advantages of cationic AuNPs and antibacterial peptides: the presence of cationic AuNPs can effectively condense DNA and the antimicrobial peptides are essential for the cellular & nucleus entry enhancement to achieve high transfection efficiency and antibacterial ability. As a result, antimicrobial peptides conjugated AuNPs significantly promoted the gene transfection efficiency in rat mesenchymal stem cells than pristine AuNPs, with a similar extent to those expressed by TAT (a well-known cell-penetrating peptide) modified AuNPs. More interestingly, the combinational system has better antibacterial ability than free antimicrobial peptides in vitro and in vivo, possibly due to the high density of peptides on the surface of AuNPs. Finally we present the concept-proving results that AuPs@PEP can be used as a carrier for in vivo gene activation in tissue regeneration, suggesting its potential as a multifunctional system with both gene delivery and antibacterial abilities in clinic. PMID:27376562

  13. How to unveil self-quenched fluorophores and subsequently map the subcellular distribution of exogenous peptides

    PubMed Central

    Swiecicki, Jean-Marie; Thiebaut, Frédéric; Di Pisa, Margherita; Gourdin -Bertin, Simon; Tailhades, Julien; Mansuy, Christelle; Burlina, Fabienne; Chwetzoff, Serge; Trugnan, Germain; Chassaing, Gérard; Lavielle, Solange

    2016-01-01

    Confocal laser scanning microscopy (CLSM) is the most popular technique for mapping the subcellular distribution of a fluorescent molecule and is widely used to investigate the penetration properties of exogenous macromolecules, such as cell-penetrating peptides (CPPs), within cells. Despite the membrane-association propensity of all these CPPs, the signal of the fluorescently labeled CPPs did not colocalize with the plasma membrane. We studied the origin of this fluorescence extinction and the overall consequence on the interpretation of intracellular localizations from CLSM pictures. We demonstrated that this discrepancy originated from fluorescence self-quenching. The fluorescence was unveiled by a “dilution” protocol, i.e. by varying the ratio fluorescent/non-fluorescent CPP. This strategy allowed us to rank with confidence the subcellular distribution of several CPPs, contributing to the elucidation of the penetration mechanism. More generally, this study proposes a broadly applicable and reliable method to study the subcellular distribution of any fluorescently labeled molecules. PMID:26839211

  14. Chemotactic peptide receptor modulation in polymorphonuclear leukocytes

    PubMed Central

    1980-01-01

    The binding of the chemotactic peptide N- formylnorleucylleucylphenylalanine (FNLLP) to its receptor on rabbit polymorphonuclear leukocytes (PMNs) modulates the number of available peptide receptors. Incubation with FNLLP decreases subsequent binding capacity, a phenomenon that has been termed receptor down regulation. Down regulation of the chemotactic peptide receptor is concentration dependent in both the rate and extent of receptor loss. The dose response parallels that of FNLLP binding to the recptor. The time- course is rapid; even at concentrations of FNLLP as low as 3 x 10(-9) M, the new equilibrium concentration of receptors is reached within 15 min. Down regulation is temperature dependent, but does occur even at 4 degrees C. Concomitant with down regulation, some of the peptide becomes irreversibly cell associated. At 4 degrees C, there is a small accumulation of nondissociable peptide that rapidly reaches a plateau. At higher temperatures, accumulation of nondissociable peptide continues after the rceptor number has reached equilibrium, and the amount accumulated can exceed the initial number of receptors by as much as 300%. The dose response of peptide uptake at 37 degrees C reflects that of binding, suggesting that it is receptor mediated. This uptake may occur via a pinocytosis mechanism. Although PMNs have not been considered to be pinocytic, the addition of FNLLP causes a fourfold stimulation of the rate of pinocytosis as measured by the uptake of [3H]sucrose. PMID:7391138

  15. The effect of functionalizing lipid nanocapsules with NFL-TBS.40-63 peptide on their uptake by glioblastoma cells.

    PubMed

    Balzeau, Julien; Pinier, Maud; Berges, Raphael; Saulnier, Patrick; Benoit, Jean-Pierre; Eyer, Joel

    2013-04-01

    We previously described a neurofilament derived cell-penetrating peptide, NFL-TBS.40-63, that specifically enters in glioblastoma cells where it disturbs the microtubule network both in vitro and in vivo. The aim of this study is to test whether this peptide can increase the targeted uptake by glioblastoma cells of lipid nanocapsules filled with Paclitaxel, and thus can increase their anti-proliferation in vitro and in vivo. Here, using the drop tensiometry we show that approximately 60 NFL-TBS.40-63 peptides can bind to one 50 nm lipid nanocapsule. When nanocapsules are filled with a far-red fluorochrome (DiD) and Paclitaxel, the presence of the NFL-TBS.40-63 peptide increases their uptake by glioblastoma cells in culture as evaluated by FACS analysis, and thus reduces their proliferation. Finally, when such nanocapsules were injected in mice bearing a glioma tumour, they are preferentially targeted to the tumour and reduce its progression. These results show that nanocapsules functionalized with the NFL-TBS.40-63 peptide represent a powerful drug-carrier system for glioma targeted treatment.

  16. A peptide targeted against phosphoprotein and leader RNA interaction inhibits growth of Chandipura virus -- an emerging rhabdovirus.

    PubMed

    Roy, Arunava; Chakraborty, Prasenjit; Polley, Smarajit; Chattopadhyay, Dhrubajyoti; Roy, Siddhartha

    2013-11-01

    The fatal illness caused by Chandipura virus (CHPV), an emerging pathogen, presently lacks any therapeutic option. Previous research suggested that interaction between the virally encoded phosphoprotein (P) and the positive sense leader RNA (le-RNA) may play an important role in the viral lifecycle. In this report, we have identified a β-sheet/loop motif in the C-terminal domain of the CHPV P protein as essential for this interaction. A synthetic peptide encompassing this motif and spanning a continuous stretch of 36 amino acids (Pep208-243) was found to bind the le-RNA in vitro and inhibit CHPV growth in infected cells. Furthermore, a stretch of three amino acid residues at position 217-219 was identified as essential for this interaction, both in vitro and in infected cells. siRNA knockdown-rescue experiments demonstrated that these three amino acid residues are crucial for the leader RNA binding function of P protein in the CHPV life cycle. Mutations of these three amino acid residues render the peptide completely ineffective against CHPV. Effect of inhibition of phosphoprotein-leader RNA interaction on viral replication was assayed. Peptide Pep208-243 tagged with a cell penetrating peptide was found to inhibit CHPV replication as ascertained by real time RT-PCR. The specific inhibition of viral growth observed using this peptide suggests a new possibility for designing of anti-viral agents against Mononegavirale group of human viruses.

  17. Peptides regulate cortical thymocytes differentiation, proliferation, and apoptosis.

    PubMed

    Khavinson, V Kh; Polyakova, V O; Linkova, N S; Dudkov, A V; Kvetnoy, I M

    2011-01-01

    The processes of differentiation, proliferation, and apoptosis were studied in a cell culture of human cortical thymocytes under the influence of short peptides T-32 (Glu-Asp-Ala) and T-38 (Lys-Glu-Asp). Peptides T-32 and T-38 amplified cortical thymocytes differentiation towards regulatory T cells, increased their proliferative activity, and decreased the level of apoptosis. Moreover, peptides under study stimulated proliferative and antiapoptotic activity of the mature regulatory T cells.

  18. Peptides Regulate Cortical Thymocytes Differentiation, Proliferation, and Apoptosis

    PubMed Central

    Khavinson, V. Kh.; Polyakova, V. O.; Linkova, N. S.; Dudkov, A. V.; Kvetnoy, I. M.

    2011-01-01

    The processes of differentiation, proliferation, and apoptosis were studied in a cell culture of human cortical thymocytes under the influence of short peptides T-32 (Glu-Asp-Ala) and T-38 (Lys-Glu-Asp). Peptides T-32 and T-38 amplified cortical thymocytes differentiation towards regulatory T cells, increased their proliferative activity, and decreased the level of apoptosis. Moreover, peptides under study stimulated proliferative and antiapoptotic activity of the mature regulatory T cells. PMID:22312461

  19. Plant peptide hormone signalling.

    PubMed

    Motomitsu, Ayane; Sawa, Shinichiro; Ishida, Takashi

    2015-01-01

    The ligand-receptor-based cell-to-cell communication system is one of the most important molecular bases for the establishment of complex multicellular organisms. Plants have evolved highly complex intercellular communication systems. Historical studies have identified several molecules, designated phytohormones, that function in these processes. Recent advances in molecular biological analyses have identified phytohormone receptors and signalling mediators, and have led to the discovery of numerous peptide-based signalling molecules. Subsequent analyses have revealed the involvement in and contribution of these peptides to multiple aspects of the plant life cycle, including development and environmental responses, similar to the functions of canonical phytohormones. On the basis of this knowledge, the view that these peptide hormones are pivotal regulators in plants is becoming increasingly accepted. Peptide hormones are transcribed from the genome and translated into peptides. However, these peptides generally undergo further post-translational modifications to enable them to exert their function. Peptide hormones are expressed in and secreted from specific cells or tissues. Apoplastic peptides are perceived by specialized receptors that are located at the surface of target cells. Peptide hormone-receptor complexes activate intracellular signalling through downstream molecules, including kinases and transcription factors, which then trigger cellular events. In this chapter we provide a comprehensive summary of the biological functions of peptide hormones, focusing on how they mature and the ways in which they modulate plant functions.

  20. Plant peptide hormone signalling.

    PubMed

    Motomitsu, Ayane; Sawa, Shinichiro; Ishida, Takashi

    2015-01-01

    The ligand-receptor-based cell-to-cell communication system is one of the most important molecular bases for the establishment of complex multicellular organisms. Plants have evolved highly complex intercellular communication systems. Historical studies have identified several molecules, designated phytohormones, that function in these processes. Recent advances in molecular biological analyses have identified phytohormone receptors and signalling mediators, and have led to the discovery of numerous peptide-based signalling molecules. Subsequent analyses have revealed the involvement in and contribution of these peptides to multiple aspects of the plant life cycle, including development and environmental responses, similar to the functions of canonical phytohormones. On the basis of this knowledge, the view that these peptide hormones are pivotal regulators in plants is becoming increasingly accepted. Peptide hormones are transcribed from the genome and translated into peptides. However, these peptides generally undergo further post-translational modifications to enable them to exert their function. Peptide hormones are expressed in and secreted from specific cells or tissues. Apoplastic peptides are perceived by specialized receptors that are located at the surface of target cells. Peptide hormone-receptor complexes activate intracellular signalling through downstream molecules, including kinases and transcription factors, which then trigger cellular events. In this chapter we provide a comprehensive summary of the biological functions of peptide hormones, focusing on how they mature and the ways in which they modulate plant functions. PMID:26374891

  1. The interplay of T1- and T2-relaxation on T1-weighted MRI of hMSCs induced by Gd-DOTA-peptides.

    PubMed

    Cao, Limin; Li, Binbin; Yi, Peiwei; Zhang, Hailu; Dai, Jianwu; Tan, Bo; Deng, Zongwu

    2014-04-01

    Three Gd-DOTA-peptide complexes with different peptide sequence are synthesized and used as T1 contrast agent to label human mesenchymal stem cells (hMSCs) for magnetic resonance imaging study. The peptides include a universal cell penetrating peptide TAT, a linear MSC-specific peptide EM7, and a cyclic MSC-specific peptide CC9. A significant difference in labeling efficacy is observed between the Gd-DOTA-peptides as well as a control Dotarem. All Gd-DOTA-peptides as well as Dotarem induce significant increase in T1 relaxation rate which is in favor of T1-weighted MR imaging. Gd-DOTA-CC9 yields the maximum labeling efficacy but poor T1 contrast enhancement. Gd-DOTA-EM7 yields the minimum labeling efficacy but better T1 contrast enhancement. Gd-DOTA-TAT yields a similar labeling efficacy as Gd-DOTA-CC9 and similar T1 contrast enhancement as Gd-DOTA-EM7. The underlying mechanism that governs T1 contrast enhancement effect is discussed. Our results suggest that T1 contrast enhancement induced by Gd-DOTA-peptides depends not only on the introduced cellular Gd content, but more importantly on the effect that Gd-DOTA-peptides exert on the T1-relaxation and T2-relaxation processes/rates. Both T1 and particularly T2 relaxation rate have to be taken into account to interpret T1 contrast enhancement. In addition, the interpretation has to be based on cellular instead of aqueous longitudinal and transverse relaxivities of Gd-DOTA-peptides.

  2. LL37 and Cationic Peptides Enhance TLR3 Signaling by Viral Double-stranded RNAs

    PubMed Central

    Lai, Yvonne; Adhikarakunnathu, Sreedevi; Bhardwaj, Kanchan; Ranjith-Kumar, C. T.; Wen, Yahong; Jordan, Jarrat L.; Wu, Linda H.; Dragnea, Bogdan; Mateo, Lani San; Kao, C. Cheng

    2011-01-01

    Background Toll-like Receptor 3 (TLR3) detects viral dsRNA during viral infection. However, most natural viral dsRNAs are poor activators of TLR3 in cell-based systems, leading us to hypothesize that TLR3 needs additional factors to be activated by viral dsRNAs. The anti-microbial peptide LL37 is the only known human member of the cathelicidin family of anti-microbial peptides. LL37 complexes with bacterial lipopolysaccharide (LPS) to prevent activation of TLR4, binds to ssDNA to modulate TLR9 and ssRNA to modulate TLR7 and 8. It synergizes with TLR2/1, TLR3 and TLR5 agonists to increase IL8 and IL6 production. This work seeks to determine whether LL37 enhances viral dsRNA recognition by TLR3. Methodology/Principal Findings Using a human bronchial epithelial cell line (BEAS2B) and human embryonic kidney cells (HEK 293T) transiently transfected with TLR3, we found that LL37 enhanced poly(I:C)-induced TLR3 signaling and enabled the recognition of viral dsRNAs by TLR3. The presence of LL37 also increased the cytokine response to rhinovirus infection in BEAS2B cells and in activated human peripheral blood mononuclear cells. Confocal microscopy determined that LL37 could co-localize with TLR3. Electron microscopy showed that LL37 and poly(I:C) individually formed globular structures, but a complex of the two formed filamentous structures. To separate the effects of LL37 on TLR3 and TLR4, other peptides that bind RNA and transport the complex into cells were tested and found to activate TLR3 signaling in response to dsRNAs, but had no effect on TLR4 signaling. This is the first demonstration that LL37 and other RNA-binding peptides with cell penetrating motifs can activate TLR3 signaling and facilitate the recognition of viral ligands. Conclusions/Significance LL37 and several cell-penetrating peptides can enhance signaling by TLR3 and enable TLR3 to respond to viral dsRNA. PMID:22039520

  3. Antimicrobial Peptides in Human Sepsis

    PubMed Central

    Martin, Lukas; van Meegern, Anne; Doemming, Sabine; Schuerholz, Tobias

    2015-01-01

    Nearly 100 years ago, antimicrobial peptides (AMPs) were identified as an important part of innate immunity. They exist in species from bacteria to mammals and can be isolated in body fluids and on surfaces constitutively or induced by inflammation. Defensins have anti-bacterial effects against Gram-positive and Gram-negative bacteria as well as anti-viral and anti-yeast effects. Human neutrophil peptides (HNP) 1–3 and human beta-defensins (HBDs) 1–3 are some of the most important defensins in humans. Recent studies have demonstrated higher levels of HNP 1–3 and HBD-2 in sepsis. The bactericidal/permeability-increasing protein (BPI) attenuates local inflammatory response and decreases systemic toxicity of endotoxins. Moreover, BPI might reflect the severity of organ dysfunction in sepsis. Elevated plasma lactoferrin is detected in patients with organ failure. HNP 1–3, lactoferrin, BPI, and heparin-binding protein are increased in sepsis. Human lactoferrin peptide 1–11 (hLF 1–11) possesses antimicrobial activity and modulates inflammation. The recombinant form of lactoferrin [talactoferrin alpha (TLF)] has been shown to decrease mortality in critically ill patients. A phase II/III study with TLF in sepsis did not confirm this result. The growing number of multiresistant bacteria is an ongoing problem in sepsis therapy. Furthermore, antibiotics are known to promote the liberation of pro-inflammatory cell components and thus augment the severity of sepsis. Compared to antibiotics, AMPs kill bacteria but also neutralize pathogenic factors such as lipopolysaccharide. The obstacle to applying naturally occurring AMPs is their high nephro- and neurotoxicity. Therefore, the challenge is to develop peptides to treat septic patients effectively without causing harm. This overview focuses on natural and synthetic AMPs in human and experimental sepsis and their potential to provide significant improvements in the treatment of critically ill with severe infections

  4. PEPTIDE INHIBITORS OF MK2 SHOW PROMISE FOR INHIBITION OF ABDOMINAL ADHESIONS

    PubMed Central

    Ward, Brian C.; Kavalukas, Sandra; Brugnano, Jamie; Barbul, Adrian; Panitch, Alyssa

    2011-01-01

    Background Abdominal adhesions are a common side effect of surgical procedures with complications including infertility, chronic pain, and bowel obstruction, which may lead to the need for surgical lyses of the adhesions. Mitogen-activated protein kinase-activated protein kinase 2 (MK2) has been implicated in several diseases involving inflammation and fibrosis. Thus, the development of a cell-penetrating peptide (CPP) that modulates MK2 activity may confer therapeutic benefit after abdominal surgery in general and more specifically after bowel anastomosis. Study Design This study evaluated the function of a CPP inhibitor of MK2 in human mesothelial cells and in a rat bowel anastomosis model. To determine IC50 and basic specificity, kinase inhibition was performed using a radiometric assay. Enzyme-Linked Immunoassay (ELISA) was used to evaluate interleukin-6 (IL-6) expression in response to IL-1β and tumor necrosis factor-α (TNF-α) stimulation in vitro to validate MK2 kinase inhibition. Following bowel anastomosis (10 rats for each control and treatment at 4 and 10 days), the rats were evaluated for weight loss, normal healing (colonic burst strength and hydroxyproline content at the anastomosis), and number and density of adhesions. Results The IC50 of the MK2 inhibitor peptide (22µM) was similar to that of the nonspecific small molecule Rottlerin (IC50=5µM). The MK2 inhibitor peptide was effective at suppressing IL-1β and TNF-α stimulated IL-6 expression in mesothelial cells. In vivo, the MK2 inhibitor peptide was effective as suppressing both the density and number of adhesions formed as a result of bowel anastomosis. Importantly, the peptide had no negative effect on normal healing. Conclusions In conclusion, the peptide inhibitor of MK2, MMI-0100, has the potential to significantly reduce inflammation through suppression of inflammatory cytokine expression and showed promise as a therapeutic for abdominal adhesions. PMID:21492875

  5. Surface Decorated Gold Nanoparticles by Linear and Cyclic Peptides as Molecular Transporters

    PubMed Central

    Shirazi, Amir Nasrolahi; Tiwari, Rakesh Kumar; Oh, Donghoon; Sullivan, Brian; McCaffrey, Kellen; Mandal, Dindyal; Parang, Keykavous

    2013-01-01

    Gold nanoparticles (AuNPs) were synthesized in situ in a green and rapid method from the reaction of reducing linear and cyclic peptides containing tryptophan and lysine residues, (KW)5 and cyclic [KW]5, with an aqueous solution of HAuCl4 and were evaluated as cellular nanodrug delivery systems. The cyclic or linear nature of the peptide was found to determine the morphology and size of the formed peptide-AuNPs and their in vitro molecular transporting efficiency. While cyclic [KW]5-AuNPs formed sponge-like agglomerates, linear (KW)5-AuNPs demonstrated ball-shaped structures. A comparative flow cytometry study showed that the cellular uptake of fluorescence-labeled anti-HIV drugs (emtricitabine (FTC) and lamivudine (3TC)) in human Leukemia (CCRF-CEM) cells, and a negatively charged cell-impermeable phosphopeptide (GpYEEI) in human ovarian adecarcinoma (SK-OV-3) cells was significantly higher in the presence of cyclic [KW]5-AuNPs than that of linear (KW)5-AuNPs, parent cyclic [KW]5, and linear (KW)5 peptides. For example, the cellular uptake of F′-GpYEEI was enhanced 12.8-fold by c[KW]5-AuNPs. Confocal microscopy revealed the localization of fluorescence-labeled-3TC in the presence of c[KW]5-AuNPs mostly in nucleus in SK-OV-3 cells after 1 h. On the other hand, l(KW)5-AuNPs delivered fluorescence-labeled-3TC in cytoplasm. These data suggest that non-cell penetrating peptides can be converted to efficient molecular transporters through peptide-capped AuNPs formation. PMID:23834324

  6. Design and evaluation of solid lipid nanoparticles modified with peptide ligand for oral delivery of protein drugs.

    PubMed

    Fan, Tingting; Chen, Chunhui; Guo, Han; Xu, Juan; Zhang, Jian; Zhu, Xi; Yang, Yang; Zhou, Zhou; Li, Lian; Huang, Yuan

    2014-10-01

    Designing feasible and effective peptide ligand modified solid lipid nanoparticles (SLNs) to improve oral bioavailability of protein drugs and evaluating the influence of mucus remains important. In the present work, two kinds of peptide ligand modified SLNs loaded with salmon calcitonin (sCT), namely, sCT CSK-SLNs and sCT IRQ-SLNs, were prepared by coupling the peptide ligand CSKSSDYQC (CSK) which was reported to show affinity with goblet cells, or IRQRRRR (IRQ), a cell penetrating peptide, to polyoxyethylene (40) stearate (SA-PEG2000). Compared with unmodified SLNs, CSK or IRQ modified SLNs with better drug protection ability could facilitate the internalization of drug on Caco-2/HT29-MTX co-cultured cells and permeation in excised rat duodenum mucosa. The internalization mechanism of two kinds of peptide ligand modified SLNs was mainly active transport via both clathrin- and caveolae-dependent endocytosis. Although mucus was an impediment to the transport of SLNs, the peptide ligand modified SLNs still showed improved drug absorption. The absolute bioavailability of sCT CSK-SLNs (12.41 ± 3.65%) and sCT IRQ-SLNs (10.05 ± 5.10%) raised to 2.45-fold and 1.98-fold compared with unmodified SLNs (5.07 ± 0.54%), implying the feasibility and effectiveness of CSK and IRQ peptide modification for the enhancement of the oral bioavailability of protein drugs. In summary, the nanoparticles modified with CSK or IRQ peptide ligand could be the potential carriers for the transport of protein drugs across intestinal barriers.

  7. Antimicrobial Peptides in Reptiles

    PubMed Central

    van Hoek, Monique L.

    2014-01-01

    Reptiles are among the oldest known amniotes and are highly diverse in their morphology and ecological niches. These animals have an evolutionarily ancient innate-immune system that is of great interest to scientists trying to identify new and useful antimicrobial peptides. Significant work in the last decade in the fields of biochemistry, proteomics and genomics has begun to reveal the complexity of reptilian antimicrobial peptides. Here, the current knowledge about antimicrobial peptides in reptiles is reviewed, with specific examples in each of the four orders: Testudines (turtles and tortosises), Sphenodontia (tuataras), Squamata (snakes and lizards), and Crocodilia (crocodilans). Examples are presented of the major classes of antimicrobial peptides expressed by reptiles including defensins, cathelicidins, liver-expressed peptides (hepcidin and LEAP-2), lysozyme, crotamine, and others. Some of these peptides have been identified and tested for their antibacterial or antiviral activity; others are only predicted as possible genes from genomic sequencing. Bioinformatic analysis of the reptile genomes is presented, revealing many predicted candidate antimicrobial peptides genes across this diverse class. The study of how these ancient creatures use antimicrobial peptides within their innate immune systems may reveal new understandings of our mammalian innate immune system and may also provide new and powerful antimicrobial peptides as scaffolds for potential therapeutic development. PMID:24918867

  8. Shuttling gold nanoparticles into tumoral cells with an amphipathic proline-rich peptide.

    PubMed

    Pujals, Sílvia; Bastús, Neus G; Pereiro, Eva; López-Iglesias, Carmen; Puntes, Víctor F; Kogan, Marcelo J; Giralt, Ernest

    2009-04-17

    Cell-penetrating peptides (CPPs) are a potential tool for intracellular delivery of different kinds of cargoes. Because of their growing use in nanobiomedicine, both for diagnostics and for treatment, metal nanoparticles are an interesting cargo for CPPs. Here, gold nanoparticles (AuNps) and the amphipathic proline-rich peptide SAP have been used. Conjugation of the peptide onto the AuNps was achieved by addition of a cysteine to the SAP sequence for thiol chemisorption on gold, and the attachment was confirmed by visible spectroscopy, dynamic light scattering (DLS), zeta-potential (ZP), stability towards ionic strength (as high as 1 M NaCl), X-ray photoelectron spectroscopy (XPS) and high-resolution transmission electron microscopy (HR-TEM) coupled to electron energy loss spectroscopy (EELS). AuNp-C-SAP internalization in HeLa cells was observed by three different microscopy techniques-TEM, confocal laser scanning microscopy (CLSM) and transmission X-ray microscopy (TXM)-and all of them have confirmed the effective intracellular delivery of AuNps by SAP. PMID:19322842

  9. Optimisation of vectorisation property: A comparative study for a secondary amphipathic peptide.

    PubMed

    Konate, Karidia; Lindberg, Mattias F; Vaissiere, Anaïs; Jourdan, Carole; Aldrian, Gudrun; Margeat, Emmanuel; Deshayes, Sébastien; Boisguerin, Prisca

    2016-07-25

    RNA interference provides a powerful technology for specific gene silencing. Therapeutic applications of small interfering RNA (siRNA) however require efficient vehicles for stable complexation and intracellular delivery. In order to enhance their cell delivery, short amphipathic peptides called cell-penetrating peptides (CPPs) have been intensively developed for the last two decades. In this context, the secondary amphipathic peptide CADY has shown to form stable siRNA complexes and to improve their cellular uptake independent of the endosomal pathway. In the present work, we have described the parameters influencing CADY nanoparticle formation (buffers, excipients, presence of serum, etc.), and have followed in details the CPP:siRNA self-assembly. Once optimal conditions were determined, we have compared the ability of seven different CADY analogues to form siRNA-loaded nanoparticles compared to CADY:siRNA. First of all, we were able to show by biophysical methods that structural polymorphism (α-helix) is an important prerequisite for stable nanoparticle formation independently of occurring sequence mutations. Luciferase assays revealed that siRNA complexed to CADY-K (shorter version) shows better knock-down efficiency on Neuro2a-Luc(+) and B16-F10-Luc(+) cells compared to CADY:siRNA. Altogether, CADY-K is an ideal candidate for further application especially with regards to ex vivo or in vivo applications. PMID:27224007

  10. An anti-apoptotic peptide improves survival in lethal total body irradiation.

    PubMed

    McDunn, Jonathan E; Muenzer, Jared T; Dunne, Benjamin; Zhou, Anthony; Yuan, Kevin; Hoekzema, Andrew; Hilliard, Carolyn; Chang, Katherine C; Davis, Christopher G; McDonough, Jacquelyn; Hunt, Clayton; Grigsby, Perry; Piwnica-Worms, David; Hotchkiss, Richard S

    2009-05-15

    Cell penetrating peptides (CPPs) have been used to deliver the anti-apoptotic Bcl-xL-derived BH4 peptide to prevent injury-induced apoptosis both in vitro and in vivo. Here we demonstrate that the nuclear localization sequence (NLS) from the SV40 large T antigen has favorable properties for BH4 domain delivery to lymphocytes compared to sequences based on the HIV-1 TAT sequence. While both TAT-BH4 and NLS-BH4 protected primary human mononuclear cells from radiation-induced apoptotic cell death, TAT-BH4 caused persistent membrane damage and even cell death at the highest concentrations tested (5-10 microM) and correlated with in vivo toxicity as intravenous administration of TAT-BH4 caused rapid death. The NLS-BH4 peptide has significantly attenuated toxicity compared to TAT-BH4 and we established a dosing regimen of NLS-BH4 that conferred a significant survival advantage in a post-exposure treatment model of LD90 total body irradiation.

  11. An anti-apoptotic peptide improves survival in lethal total body irradiation

    SciTech Connect

    McDunn, Jonathan E.; Muenzer, Jared T.; Dunne, Benjamin; Zhou, Anthony; Yuan, Kevin; Hoekzema, Andrew; Hilliard, Carolyn; Chang, Katherine C.; Davis, Christopher G.; McDonough, Jacquelyn; Hunt, Clayton; Grigsby, Perry; Piwnica-Worms, David; Hotchkiss, Richard S.

    2009-05-15

    Cell penetrating peptides (CPPs) have been used to deliver the anti-apoptotic Bcl-xL-derived BH4 peptide to prevent injury-induced apoptosis both in vitro and in vivo. Here we demonstrate that the nuclear localization sequence (NLS) from the SV40 large T antigen has favorable properties for BH4 domain delivery to lymphocytes compared to sequences based on the HIV-1 TAT sequence. While both TAT-BH4 and NLS-BH4 protected primary human mononuclear cells from radiation-induced apoptotic cell death, TAT-BH4 caused persistent membrane damage and even cell death at the highest concentrations tested (5-10 {mu}M) and correlated with in vivo toxicity as intravenous administration of TAT-BH4 caused rapid death. The NLS-BH4 peptide has significantly attenuated toxicity compared to TAT-BH4 and we established a dosing regimen of NLS-BH4 that conferred a significant survival advantage in a post-exposure treatment model of LD90 total body irradiation.

  12. N-terminal peptides from unprocessed prion proteins enter cells by macropinocytosis

    SciTech Connect

    Magzoub, Mazin; Sandgren, Staffan; Lundberg, Pontus; Oglecka, Kamila; Lilja, Johanna; Wittrup, Anders; Goeran Eriksson, L.E.; Langel, Ulo; Belting, Mattias . E-mail: mattias.belting@med.lu.se; Graeslund, Astrid . E-mail: astrid@dbb.su.se

    2006-09-22

    A peptide derived from the N-terminus of the unprocessed bovine prion protein (bPrPp), incorporating the hydrophobic signal sequence (residues 1-24) and a basic domain (KKRPKP, residues 25-30), internalizes into mammalian cells, even when coupled to a sizeable cargo, and therefore functions as a cell-penetrating peptide (CPP). Confocal microscopy and co-localization studies indicate that the internalization of bPrPp is mainly through macropinocytosis, a fluid-phase endocytosis process, initiated by binding to cell-surface proteoglycans. Electron microscopy studies show internalized bPrPp-DNA-gold complexes residing in endosomal vesicles. bPrPp induces expression of a complexed luciferase-encoding DNA plasmid, demonstrating the peptide's ability to transport the cargo across the endosomal membrane and into the cytosol and nucleus. The novel CPP activity of the unprocessed N-terminal domain of PrP could be important for the retrotranslocation of partly processed PrP and for PrP trafficking inside or between cells, with implications for the infectivity associated with prion diseases.

  13. Atomistic Molecular Simulations Suggest a Kinetic Model for Membrane Translocation by Arginine-Rich Peptides.

    PubMed

    Sun, Delin; Forsman, Jan; Woodward, Clifford E

    2015-11-12

    Arginine-rich cell penetrating peptides (ARCPPs) are known to quickly permeate cell membranes through a non-endocytotic pathway. Potential clinical applications of this facility have prompted enormous effort, both experimental and theoretical, to better understand how ARCPPs manage to overcome the prodigious thermodynamic cost of lipid bilayer permeation by these highly charged peptides. In this work we report the results of all-atom simulations, which suggest that a kinetic (rather than thermodynamic) mechanism may explain how ARCPPs are able to achieve this. Our simulations reveal that octaarginine significantly hinders the closing of membrane pores, either individually or via aggregation in the membrane pore, while octalysine (not an ARCPP) lacks this ability. Our proposed mechanism is an alternative to current attempts to explain pore-mediated translocation of ARCPPs. It asserts that ARCPPs need not lower the equilibrium thermodynamic cost of pore formation. Instead, they can achieve rapid bilayer translocation by instead slowing down the kinetics of naturally occurring thermal pores. Linking the pore lifetime to the characteristic time for peptide diffusion out of the pore, ARCPPs are able to cooperatively permeate the membrane pore. PMID:26485313

  14. Insulin C-peptide test

    MedlinePlus

    C-peptide ... the test depends on the reason for the C-peptide measurement. Ask your health care provider if ... C-peptide is measured to tell the difference between insulin the body produces and insulin someone injects ...

  15. Bacteriocin Inducer Peptides

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Novel peptides produced by bacteriocin-producing bacteria stimulate the production of bacteriocins in vitro. The producer bacteria are cultured in the presence of a novel inducer bacteria and a peptide having a carboxy terminal sequence of VKGLT in order to achieve an increase in bacteriocin produc...

  16. Introduction to peptide synthesis.

    PubMed

    Stawikowski, Maciej; Fields, Gregg B

    2012-08-01

    A number of synthetic peptides are significant commercial or pharmaceutical products, ranging from the dipeptide sugar substitute aspartame to clinically used hormones such as oxytocin, adrenocorticotropic hormone, and calcitonin. This unit provides an overview of the field of synthetic peptides and proteins. It discusses selecting the solid support and common coupling reagents. Additional information is provided regarding common side reactions and synthesizing modified residues.

  17. Effects of opioid peptides on thermoregulation

    SciTech Connect

    Clark, W.G.

    1981-11-01

    In a given species, injected opioid peptides usually cause changes in temperature similar to those caused by nonpeptide opioids. The main effect in those species most studied, the cat, rat, and mouse, is an increase in the level about which body temperature is regulated; there is a coordinated change in the activity of thermoregulatory effectors such that hyperthermia is produced in both hot and cold environments. Larger doses may depress thermoregulation, thereby causing body temperature to decrease in the cold. Elicitation of different patterns of response over a range of environmental temperatures and studies with naloxone and naltrexone indicate that stimulation of a number of different receptors by both peptide and nonpeptide opioids can evoke thermoregulatory responses. ..beta..-Endorphin is readily antagonized by naloxone whereas methionine-enkephalin can act on naloxone-insensitive receptors. Moreover, synthetic peptide analogs do not necessarily evoke the same response as does the related endogenous peptide. The lack of effect of naloxone on body temperature of subjects housed at usual laboratory temperature or on pyrogen-induced increases in body temperature indicates that an action of endogenous peptides on naloxone-sensitive receptors plays little, if any, role in normal thermoregulation or in fever. However, there is some evidence that such an action may be involved in responses to restraint or ambient temperature-induced stress. Further evaluation of possible physiological roles of endogenous opioid peptides will be facilitated when specific antagonists at other types of opioid receptors become available.

  18. Antimicrobial Peptides from Fish

    PubMed Central

    Masso-Silva, Jorge A.; Diamond, Gill

    2014-01-01

    Antimicrobial peptides (AMPs) are found widely distributed through Nature, and participate in the innate host defense of each species. Fish are a great source of these peptides, as they express all of the major classes of AMPs, including defensins, cathelicidins, hepcidins, histone-derived peptides, and a fish-specific class of the cecropin family, called piscidins. As with other species, the fish peptides exhibit broad-spectrum antimicrobial activity, killing both fish and human pathogens. They are also immunomodulatory, and their genes are highly responsive to microbes and innate immuno-stimulatory molecules. Recent research has demonstrated that some of the unique properties of fish peptides, including their ability to act even in very high salt concentrations, make them good potential targets for development as therapeutic antimicrobials. Further, the stimulation of their gene expression by exogenous factors could be useful in preventing pathogenic microbes in aquaculture. PMID:24594555

  19. Novel role of Engrailed 1 as a prosurvival transcription factor in basal-like breast cancer and engineering of interference peptides block its oncogenic function

    PubMed Central

    Beltran, A S; Graves, L M; Blancafort, P

    2014-01-01

    Basal-like breast tumors are aggressive cancers associated with high proliferation and metastasis. Chemotherapy is currently the only treatment option; however, resistance often occurs resulting in recurrence and patient death. Some extremely aggressive cancers are also associated with hypoxia, inflammation and high leukocyte infiltration. Herein, we discovered that the neural-specific transcription factor, Engrailed 1 (EN1), is exclusively overexpressed in these tumors. Short hairpin RNA (shRNA)-mediated knockdown of EN1 triggered potent and selective cell death. In contrast, ectopic overexpression of EN1 in normal cells activated survival pathways and conferred resistance to chemotherapeutic agents. Exogenous expression of EN1 cDNA reprogrammed the breast epithelial cells toward a long-lived, neural-like phenotype displaying dopaminergic markers. Gene expression microarrays demonstrated that the EN1 cDNA altered transcription of a high number of inflammatory molecules, notably chemokines and chemokine receptors, which could mediate prosurvival pathways. To block EN1 function, we engineered synthetic interference peptides (iPeps) comprising the EN1-specific sequences that mediate essential protein-protein interactions necessary for EN1 function and an N-terminal cell-penetrating peptide/nuclear localization sequence. These EN1-iPeps rapidly mediated a strong apoptotic response in tumor cells overexpressing EN1, with no toxicity to normal or non EN1-expressing cells. Delivery of EN1-iPeps into basal-like cancer cells significantly decreased the fifty percent inhibitory concentrations (IC50) of chemotherapeutic drugs routinely used to treat breast cancer. Lastly, matrix-assisted laser desorption/ionization-time of flight mass spectrometry and immunoprecipitation assays demonstrated that EN1-iPeps captured targets involved in transcriptional and post-transcriptional regulation. Importantly, the EN1-iPeps bound the glutamyl-prolyl tRNA synthetase (EPRS) target, which

  20. The Role of Hydrophobic Surfaces in Altering Water-Mediated Peptide-Peptide Interactions in an Aqueous Environment

    SciTech Connect

    Yoo, Soohaeng; Xantheas, Sotiris S

    2011-06-16

    Using Born-Oppenheimer molecular dynamics within the density functional framework, we calculated the effective force acting on water-mediated peptide-peptide interaction between antiparallel β-sheets in an aqueous environment and also in the vicinity of a hydrophobic surface. From the magnitude of the effective force (corresponding to the slope of the free energy as a function of the inter-peptide distance) and its sign (a negative value indicating an effective attraction whereas a positive value an effective repulsion) we can elucidate the fundamental differences of the water-mediated peptide-peptide interactions in those two environments. The computed effective forces indicate that the water-mediated interaction between peptides in an aqueous environment is attractive in the range of inter-peptide distance d=7-8 Å when hydrophobic surfaces are not nearby. Due to the stabilization of the water molecules bridging between the two β-sheets, a free energy barrier exists between the direct and indirect (water-mediated) inter-peptide interactions. However, when the peptides are in the proximity of hydrophobic surfaces, this free energy barrier decreases because the hydrophobic surfaces enhance the inter-peptide attraction by the destabilization and ease-to-libration of the bridging water molecules between them. This work was supported by the Chemical Sciences, Geosciences and Biosciences Division, Office of Basic Energy Sciences, US Department of Energy. Battelle operates the Pacific Northwest National Laboratory for the U.S. Department of Energy.

  1. Stability of peptides in high-temperature aqueous solutions

    NASA Astrophysics Data System (ADS)

    Shock, Everett L.

    1992-09-01

    Estimated standard molal thermodynamic properties of aqueous dipeptides and their constituent amino acids indicate that temperature increases correspond to increased stability of peptide bonds relative to hydrolysis reactions. Pressure increases cause slight decreases in peptide bond stability, which are generally offset by greater stability caused by temperature increases along geothermal gradients. These calculations suggest that peptides, polypeptides, and proteins may survive hydrothermal alteration of organic matter depending on the rates of the hydrolysis reactions. Extremely thermophilic organisms may be able to take advantage of the decreased energy required to form peptide bonds in order to maintain structural proteins and enzymes at elevated temperatures and pressures. As the rates of hydrolysis reactions increase with increasing temperature, formation of peptide bonds may become a facile process in hydrothermal systems and deep in sedimentary basins.

  2. Simultaneous Inhibition of Key Growth Pathways in Melanoma Cells and Tumor Regression by a Designed Bidentate Constrained Helical Peptide

    PubMed Central

    Dhar, Amlanjyoti; Mallick, Shampa; Ghosh, Piya; Maiti, Atanu; Ahmed, Israr; Bhattacharyya, Seemana; Mandal, Tapashi; Manna, Asit; Roy, Koushik; Singh, Sandeep; Nayak, Dipak Kumar; Wilder, Paul T.; Markowitz, Joseph; Weber, David J.; Ghosh, Mrinal K.; Chattopadhyay, Samit; Guha, Rajdeep; Konar, Aditya; Bandyopadhyay, Santu; Roy, Siddhartha

    2014-01-01

    Protein-protein interactions are part of a large number of signaling networks and potential targets for drug development. However, discovering molecules that can specifically inhibit such interactions is a major challenge. S100B, a calcium-regulated protein, plays a crucial role in the proliferation of melanoma cells through protein-protein interactions. In this article, we report the design and development of a bidentate conformationally constrained peptide against dimeric S100B based on a natural tight binding peptide, TRTK-12. The helical conformation of the peptide was constrained by substitution of α-amino isobutyric acid----an amino acid having high helical propensity----in positions which do not interact with S100B. A branched bidentate version of the peptide, bound to S100B tightly with a dissociation constant of 8 nM. When conjugated to a cell penetrating peptide, it caused growth inhibition and rapid apoptosis in melanoma cells. The molecule exerts anti-proliferative action through simultaneous inhibition of key growth pathways including reactivation of wild-type p53 and inhibition of Akt and STAT-3 phosphorylation. The apoptosis induced by the bidentate constrained helix is caused by direct migration of p53 to mitochondria. At moderate intravenous dose, the peptide completely inhibits melanoma growth in a mouse model without any significant observable toxicity. The specificity was shown by lack of ability of a double mutant peptide to cause tumor regression at the same dose level. The methodology described here for direct protein-protein interaction inhibition may be effective for rapid development of inhibitors against relatively weak protein-protein interactions for de novo drug development. PMID:24839139

  3. Engineering cyclic peptide toxins.

    PubMed

    Clark, Richard J; Craik, David J

    2012-01-01

    Peptide-based toxins have attracted much attention in recent years for their exciting potential applications in drug design and development. This interest has arisen because toxins are highly potent and selectively target a range of physiologically important receptors. However, peptides suffer from a number of disadvantages, including poor in vivo stability and poor bioavailability. A number of naturally occurring cyclic peptides have been discovered in plants, animals, and bacteria that have exceptional stability and potentially ameliorate these disadvantages. The lessons learned from studies of the structures, stabilities, and biological activities of these cyclic peptides can be applied to the reengineering of toxins that are not naturally cyclic but are amenable to cyclization. In this chapter, we describe solid-phase chemical synthetic methods for the reengineering of peptide toxins to improve their suitability as therapeutic, diagnostic, or imaging agents. The focus is on small disulfide-rich peptides from the venoms of cone snails and scorpions, but the technology is potentially widely applicable to a number of other peptide-based toxins. PMID:22230565

  4. Peptide-functionalized magnetic nanoparticles for cancer therapy applications

    NASA Astrophysics Data System (ADS)

    Hauser, Anastasia Kruse

    Lung cancer is one of the leading causes of cancer deaths in the United States. Radiation and chemotherapy are conventional treatments, but they result in serious side effects and the probability of tumor recurrence remains high. Therefore, there is an increasing need to enhance the efficacy of conventional treatments. Magnetic nanoparticles have been previously studied for a variety of applications such as magnetic resonance imaging contrast agents, anemia treatment, magnetic cell sorting and magnetically mediated hyperthermia (MMH). In this work, dextran coated iron oxide nanoparticles were developed and functionalized with peptides to target the nanoparticles to either the extracellular matrix (ECM) of tumor tissue or to localize the nanoparticles in subcellular regions after cell uptake. The magnetic nanoparticles were utilized for a variety of applications. First, heating properties of the nanoparticles were utilized to administer hyperthermia treatments combined with chemotherapy. The nanoparticles were functionalized with peptides to target fibrinogen in the ECM and extensively characterized for their physicochemical properties, and MMH combined with chemotherapy was able to enhance the toxicity of chemotherapy. The second application of the nanoparticles was magnetically mediated energy delivery. This treatment does not result in a bulk temperature rise upon actuation of the nanoparticles by an alternating magnetic field (AMF) but rather results in intracellular damage via friction from Brownian rotation or nanoscale heating effects from Neel relaxations. The nanoparticles were functionalized with a cell penetrating peptide to facilitate cell uptake and lysosomal escape. The intracellular effects of the internalized nanoparticles alone and with activation by an AMF were evaluated. Iron concentrations in vivo are highly regulated as excess iron can catalyze the formation of the hydroxyl radical through Fenton chemistry. Although often a concern of using iron

  5. Tumor-Penetrating Peptides

    PubMed Central

    Teesalu, Tambet; Sugahara, Kazuki N.; Ruoslahti, Erkki

    2013-01-01

    Tumor-homing peptides can be used to deliver drugs into tumors. Phage library screening in live mice has recently identified homing peptides that specifically recognize the endothelium of tumor vessels, extravasate, and penetrate deep into the extravascular tumor tissue. The prototypic peptide of this class, iRGD (CRGDKGPDC), contains the integrin-binding RGD motif. RGD mediates tumor-homing through binding to αv integrins, which are selectively expressed on various cells in tumors, including tumor endothelial cells. The tumor-penetrating properties of iRGD are mediated by a second sequence motif, R/KXXR/K. This C-end Rule (or CendR) motif is active only when the second basic residue is exposed at the C-terminus of the peptide. Proteolytic processing of iRGD in tumors activates the cryptic CendR motif, which then binds to neuropilin-1 activating an endocytic bulk transport pathway through tumor tissue. Phage screening has also yielded tumor-penetrating peptides that function like iRGD in activating the CendR pathway, but bind to a different primary receptor. Moreover, novel tumor-homing peptides can be constructed from tumor-homing motifs, CendR elements and protease cleavage sites. Pathologies other than tumors can be targeted with tissue-penetrating peptides, and the primary receptor can also be a vascular “zip code” of a normal tissue. The CendR technology provides a solution to a major problem in tumor therapy, poor penetration of drugs into tumors. The tumor-penetrating peptides are capable of taking a payload deep into tumor tissue in mice, and they also penetrate into human tumors ex vivo. Targeting with these peptides specifically increases the accumulation in tumors of a variety of drugs and contrast agents, such as doxorubicin, antibodies, and nanoparticle-based compounds. Remarkably the drug to be targeted does not have to be coupled to the peptide; the bulk transport system activated by the peptide sweeps along any compound that is present in the

  6. Introduction to peptide synthesis.

    PubMed

    Fields, Gregg B

    2002-05-01

    A number of synthetic peptides are significant commercial or pharmaceutical products, ranging from the dipeptide sugar-substitute aspartame to clinically used hormones, such as oxytocin, adrenocorticotropic hormone, and calcitonin. This unit provides an overview of the field of synthetic peptides and proteins, including their purification. It discusses selecting the solid support and common coupling reagents. Additional information is provided regarding common side reactions and synthesizing modified residues.

  7. Introduction to peptide synthesis.

    PubMed

    Fields, Gregg B

    2002-02-01

    A number of synthetic peptides are significant commercial or pharmaceutical products, ranging from the dipeptide sugar-substitute aspartame to clinically used hormones, such as oxytocin, adrenocorticotropic hormone, and calcitonin. This unit provides an overview of the field of synthetic peptides and proteins, including their purification. It discusses selecting the solid support and common coupling reagents. Additional information is provided regarding common side reactions and synthesizing modified residues.

  8. Antimicrobial peptides and plant disease control.

    PubMed

    Montesinos, Emilio

    2007-05-01

    Several diseases caused by viruses, bacteria and fungi affect plant crops, resulting in losses and decreasing the quality and safety of agricultural products. Plant disease control relies mainly on chemical pesticides that are currently subject to strong restrictions and regulatory requirements. Antimicrobial peptides are interesting compounds in plant health because there is a need for new products in plant protection that fit into the new regulations. Living organisms secrete a wide range of antimicrobial peptides produced through ribosomal (defensins and small bacteriocins) or non-ribosomal synthesis (peptaibols, cyclopeptides and pseudopeptides). Several antimicrobial peptides are the basis for the design of new synthetic analogues, have been expressed in transgenic plants to confer disease protection or are secreted by microorganisms that are active ingredients of commercial biopesticides.

  9. Developing a Dissociative Nanocontainer for Peptide Drug Delivery

    PubMed Central

    Kelly, Patrick; Anand, Prachi; Uvaydov, Alexander; Chakravartula, Srinivas; Sherpa, Chhime; Pires, Elena; O’Neil, Alison; Douglas, Trevor; Holford, Mandë

    2015-01-01

    The potency, selectivity, and decreased side effects of bioactive peptides have propelled these agents to the forefront of pharmacological research. Peptides are especially promising for the treatment of neurological disorders and pain. However, delivery of peptide therapeutics often requires invasive techniques, which is a major obstacle to their widespread application. We have developed a tailored peptide drug delivery system in which the viral capsid of P22 bacteriophage is modified to serve as a tunable nanocontainer for the packaging and controlled release of bioactive peptides. Recent efforts have demonstrated that P22 nanocontainers can effectively encapsulate analgesic peptides and translocate them across blood-brain-barrier (BBB) models. However, release of encapsulated peptides at their target site remains a challenge. Here a Ring Opening Metathesis Polymerization (ROMP) reaction is applied to trigger P22 nanocontainer disassembly under physiological conditions. Specifically, the ROMP substrate norbornene (5-Norbornene-2-carboxylic acid) is conjugated to the exterior of a loaded P22 nanocontainer and Grubbs II Catalyst is used to trigger the polymerization reaction leading to nanocontainer disassembly. Our results demonstrate initial attempts to characterize the ROMP-triggered release of cargo peptides from P22 nanocontainers. This work provides proof-of-concept for the construction of a triggerable peptide drug delivery system using viral nanocontainers. PMID:26473893

  10. [The clinical utility of C-peptide measurement in diabetology].

    PubMed

    Otto-Buczkowska, Ewa

    2015-01-01

    C-peptide is produced in equal amounts to insulin and is the best measure of endogenous insulin secretion in patients with diabetes. Measurement of insulin secretion using C-peptide can be helpful in clinical practice: differences in insulin secretion are fundamental to different requirements in the treatment of diabetes. An important clinical role of C-peptide is differentiating between type 1 and type 2 diabetes. Low basal C-peptide can be considered as criterion for transferring the patients, initially diagnosed as type 2 diabetes, in the type 1 diabetes group. C-peptide level may be a good predictor of the clinical partial remission during the first year of type 1 diabetes. Latent autoimmune diabetes in adults (LADA) is a special form of diabetes that is clinically similar to type 2 diabetes but with positivity for pancreatic autoantibodies and lower C-peptide levels. The measurement of C-peptide level and of immunological markers may represent important additional tools for establishing the correct diagnosis. The natural course of these patients shows that C-peptide will decrease with time in parallel with the curve for C-peptide in classical type 1 diabetic patients. Persistence of C-peptide is an important clinical feature of MODY. It is particularly important to identify these patients as they are commonly misdiagnosed as type 1 diabetes and treated with insulin, C-peptide can be used to assist in patient selection for islet cell transplantation and post-transplant monitoring. High uncorrected fasting C-peptid in the presence of hyperglycemia may suggest insulin resistance.

  11. In vivo efficacy of anuran trypsin inhibitory peptides against staphylococcal skin infection and the impact of peptide cyclization.

    PubMed

    Malik, U; Silva, O N; Fensterseifer, I C M; Chan, L Y; Clark, R J; Franco, O L; Daly, N L; Craik, D J

    2015-04-01

    Staphylococcus aureus is a virulent pathogen that is responsible for a wide range of superficial and invasive infections. Its resistance to existing antimicrobial drugs is a global problem, and the development of novel antimicrobial agents is crucial. Antimicrobial peptides from natural resources offer potential as new treatments against staphylococcal infections. In the current study, we have examined the antimicrobial properties of peptides isolated from anuran skin secretions and cyclized synthetic analogues of these peptides. The structures of the peptides were elucidated by nuclear magnetic resonance (NMR) spectroscopy, revealing high structural and sequence similarity with each other and with sunflower trypsin inhibitor 1 (SFTI-1). SFTI-1 is an ultrastable cyclic peptide isolated from sunflower seeds that has subnanomolar trypsin inhibitory activity, and this scaffold offers pharmaceutically relevant characteristics. The five anuran peptides were nonhemolytic and noncytotoxic and had trypsin inhibitory activities similar to that of SFTI-1. They demonstrated weak in vitro inhibitory activities against S. aureus, but several had strong antibacterial activities against S. aureus in an in vivo murine wound infection model. pYR, an immunomodulatory peptide from Rana sevosa, was the most potent, with complete bacterial clearance at 3 mg · kg(-1). Cyclization of the peptides improved their stability but was associated with a concomitant decrease in antimicrobial activity. In summary, these anuran peptides are promising as novel therapeutic agents for treating infections from a clinically resistant pathogen. PMID:25624332

  12. In vivo efficacy of anuran trypsin inhibitory peptides against staphylococcal skin infection and the impact of peptide cyclization.

    PubMed

    Malik, U; Silva, O N; Fensterseifer, I C M; Chan, L Y; Clark, R J; Franco, O L; Daly, N L; Craik, D J

    2015-04-01

    Staphylococcus aureus is a virulent pathogen that is responsible for a wide range of superficial and invasive infections. Its resistance to existing antimicrobial drugs is a global problem, and the development of novel antimicrobial agents is crucial. Antimicrobial peptides from natural resources offer potential as new treatments against staphylococcal infections. In the current study, we have examined the antimicrobial properties of peptides isolated from anuran skin secretions and cyclized synthetic analogues of these peptides. The structures of the peptides were elucidated by nuclear magnetic resonance (NMR) spectroscopy, revealing high structural and sequence similarity with each other and with sunflower trypsin inhibitor 1 (SFTI-1). SFTI-1 is an ultrastable cyclic peptide isolated from sunflower seeds that has subnanomolar trypsin inhibitory activity, and this scaffold offers pharmaceutically relevant characteristics. The five anuran peptides were nonhemolytic and noncytotoxic and had trypsin inhibitory activities similar to that of SFTI-1. They demonstrated weak in vitro inhibitory activities against S. aureus, but several had strong antibacterial activities against S. aureus in an in vivo murine wound infection model. pYR, an immunomodulatory peptide from Rana sevosa, was the most potent, with complete bacterial clearance at 3 mg · kg(-1). Cyclization of the peptides improved their stability but was associated with a concomitant decrease in antimicrobial activity. In summary, these anuran peptides are promising as novel therapeutic agents for treating infections from a clinically resistant pathogen.

  13. In Vivo Efficacy of Anuran Trypsin Inhibitory Peptides against Staphylococcal Skin Infection and the Impact of Peptide Cyclization

    PubMed Central

    Malik, U.; Silva, O. N.; Fensterseifer, I. C. M.; Chan, L. Y.; Clark, R. J.; Franco, O. L.; Daly, N. L.

    2015-01-01

    Staphylococcus aureus is a virulent pathogen that is responsible for a wide range of superficial and invasive infections. Its resistance to existing antimicrobial drugs is a global problem, and the development of novel antimicrobial agents is crucial. Antimicrobial peptides from natural resources offer potential as new treatments against staphylococcal infections. In the current study, we have examined the antimicrobial properties of peptides isolated from anuran skin secretions and cyclized synthetic analogues of these peptides. The structures of the peptides were elucidated by nuclear magnetic resonance (NMR) spectroscopy, revealing high structural and sequence similarity with each other and with sunflower trypsin inhibitor 1 (SFTI-1). SFTI-1 is an ultrastable cyclic peptide isolated from sunflower seeds that has subnanomolar trypsin inhibitory activity, and this scaffold offers pharmaceutically relevant characteristics. The five anuran peptides were nonhemolytic and noncytotoxic and had trypsin inhibitory activities similar to that of SFTI-1. They demonstrated weak in vitro inhibitory activities against S. aureus, but several had strong antibacterial activities against S. aureus in an in vivo murine wound infection model. pYR, an immunomodulatory peptide from Rana sevosa, was the most potent, with complete bacterial clearance at 3 mg · kg−1. Cyclization of the peptides improved their stability but was associated with a concomitant decrease in antimicrobial activity. In summary, these anuran peptides are promising as novel therapeutic agents for treating infections from a clinically resistant pathogen. PMID:25624332

  14. Peptide motifs for insertion of radiolabeled biomolecules into cells and routing to the nucleus for cancer imaging or radiotherapeutic applications.

    PubMed

    Costantini, Danny L; Hu, Meiduo; Reilly, Raymond M

    2008-02-01

    Intracellular compartments, in particular the cytoplasm or nucleus, have generally been poorly accessible or inaccessible to radiolabeled biomolecules (e.g., monoclonal antibodies [mAbs], peptides, or oligonucleotides [ODNs]). However, recently cell-penetrating peptides (CPPs) and nuclear localizing peptide sequences (NLSs) have been shown to have the capability of inserting biomolecules into cells and transporting them to the cell nucleus. This discovery now presents intriguing new opportunities to design radiopharmaceuticals that could potentially probe, through imaging, the expression of key intracellular or intranuclear regulatory proteins that define the tumor phenotype, predict outcome, or act as sensitive reporters of response or resistance to treatment. CPPs could also more efficiently internalize radiolabeled antisense ODNs or peptide nucleic acids (PNAs) into tumor cells to enhance the sensitivity of imaging gene expression at the mRNA level. Perhaps one of the most exciting new developments to emerge is the use of NLS to route mAbs and peptides conjugated to nanometer-micrometer range Auger-electron-emitting radionuclides (e.g., (111)In) to the nucleus of cancer cells following their receptor-mediated internalization. In the nucleus, these electrons are highly potent in causing lethal DNA strand breaks. In some cases, NLSs are present naturally in peptide growth factors or their receptors, where they function to deliver internalized ligands to the nucleus, or alternatively, they can be introduced synthetically. This update reviews the properties of CPPs and NLS and focuses on their use for inserting radiolabeled biomolecules into cancer cells for imaging or targeted Auger electron radiotherapy of malignancies.

  15. Development of stapled helical peptides to perturb the Cdt1-Mcm6 interaction.

    PubMed

    Lee, Jonghan Peter; Liu, Changdong; Li, Tianlu; Zhu, Guang; Li, Xuechen

    2015-07-01

    Six all-hydrocarbon-stapled Cdt1 MBD-derived peptides have been designed and synthesized to perturb the Cdt1-Mcm6 interaction, which is involved in DNA replication. Inconsistency between the helicity of the obtained peptidomimetics and their binding affinity has been observed. The helicity of 13-amino acid stapled peptides increased, while their binding to Mcm6 was decreased. On the other hand, the 30-amino acid stapled peptides exhibited decreased helicity but increased binding affinity.

  16. Intracellular Generation of a Diterpene-Peptide Conjugate that Inhibits 14-3-3-Mediated Interactions.

    PubMed

    Parvatkar, Prakash; Kato, Nobuo; Uesugi, Motonari; Sato, Shin-Ichi; Ohkanda, Junko

    2015-12-23

    Synthetic agents that disrupt intracellular protein-protein interactions (PPIs) are highly desirable for elucidating signaling networks and developing new therapeutics. However, designing cell-penetrating large molecules equipped with the many functional groups necessary for binding to large interfaces remains challenging. Here, we describe a rational strategy for the intracellular oxime ligation-mediated generation of an amphipathic bivalent inhibitor composed of a peptide and diterpene natural product, fusicoccin, which binds 14-3-3 protein with submicromolar affinity. Our results demonstrate that co-treatment of cells with small module molecules, the aldehyde-containing fusicoccin 1 and the aminooxy-containing peptide 2, generates the corresponding conjugate 3 in cells, resulting in significant cytotoxicity. In contrast, chemically synthesized 3 is not cytotoxic, likely due to its inability to penetrate cells. Compound 3, but not 1 or 2, disrupts endogenous 14-3-3/cRaf interactions, suggesting that cell death is caused by inhibition of 14-3-3 activity. These results suggest that intracellular generation of large-sized molecules may serve as a new approach for modulating PPIs.

  17. Use of Peptide Nucleic Acids to Manipulate Gene Expression in the Malaria Parasite Plasmodium falciparum

    PubMed Central

    Naik, Shankar; Yavin, Eylon; Dzikowski, Ron

    2014-01-01

    One of the major concerns in treating malaria by conventional small drug molecules is the rapid emergence of drug resistance. Specific silencing of essential genes by antisense oliogomers has been proposed as an alternative approach that may result in antimalarial activity which is not associated with drug resistance. In addition, such an approach could be an important biological tool for studying many genes' function by reverse genetics. Here we present a novel methodology of using peptide nucleic acids (PNAs) as a useful tool for gene silencing in Plasmodium falciparum. PNAs, designed as specific antisense molecules, were conjugated to a cell penetrating peptide (CPP); namely, octa-D-lysine via the C-terminus, to allow facile delivery through cell membranes. PNAs added to P. falciparum cultures were found exclusively in infected erythrocytes and were eventually localized in nuclei of the parasites at all stages of intra erythrocytic development. We show that these PNAs specifically down regulated both a stably expressed transgene as well as an endogenous essential gene, which significantly reduced parasites' viability. This study paves the way for a simple approach to silence a variety of P. falciparum genes as means of deciphering their function and potentially to develop highly specific and potent antimalarial agents. PMID:24466246

  18. Peptides of the Constant Region of Antibodies Display Fungicidal Activity

    PubMed Central

    Polonelli, Luciano; Ciociola, Tecla; Magliani, Walter; Zanello, Pier Paolo; D'Adda, Tiziana; Galati, Serena; De Bernardis, Flavia; Arancia, Silvia; Gabrielli, Elena; Pericolini, Eva; Vecchiarelli, Anna; Arruda, Denise C.; Pinto, Marcia R.; Travassos, Luiz R.; Pertinhez, Thelma A.; Spisni, Alberto; Conti, Stefania

    2012-01-01

    Synthetic peptides with sequences identical to fragments of the constant region of different classes (IgG, IgM, IgA) of antibodies (Fc-peptides) exerted a fungicidal activity in vitro against pathogenic yeasts, such as Candida albicans, Candida glabrata, Cryptococcus neoformans, and Malassezia furfur, including caspofungin and triazole resistant strains. Alanine-substituted derivatives of fungicidal Fc-peptides, tested to evaluate the critical role of each residue, displayed unaltered, increased or decreased candidacidal activity in vitro. An Fc-peptide, included in all human IgGs, displayed a therapeutic effect against experimental mucosal and systemic candidiasis in mouse models. It is intriguing to hypothesize that some Fc-peptides may influence the antifungal immune response and constitute the basis for devising new antifungal agents. PMID:22470523

  19. Antimicrobial Peptides from Plants.

    PubMed

    Tam, James P; Wang, Shujing; Wong, Ka H; Tan, Wei Liang

    2015-01-01

    Plant antimicrobial peptides (AMPs) have evolved differently from AMPs from other life forms. They are generally rich in cysteine residues which form multiple disulfides. In turn, the disulfides cross-braced plant AMPs as cystine-rich peptides to confer them with extraordinary high chemical, thermal and proteolytic stability. The cystine-rich or commonly known as cysteine-rich peptides (CRPs) of plant AMPs are classified into families based on their sequence similarity, cysteine motifs that determine their distinctive disulfide bond patterns and tertiary structure fold. Cystine-rich plant AMP families include thionins, defensins, hevein-like peptides, knottin-type peptides (linear and cyclic), lipid transfer proteins, α-hairpinin and snakins family. In addition, there are AMPs which are rich in other amino acids. The ability of plant AMPs to organize into specific families with conserved structural folds that enable sequence variation of non-Cys residues encased in the same scaffold within a particular family to play multiple functions. Furthermore, the ability of plant AMPs to tolerate hypervariable sequences using a conserved scaffold provides diversity to recognize different targets by varying the sequence of the non-cysteine residues. These properties bode well for developing plant AMPs as potential therapeutics and for protection of crops through transgenic methods. This review provides an overview of the major families of plant AMPs, including their structures, functions, and putative mechanisms. PMID:26580629

  20. Electron transfer in peptides.

    PubMed

    Shah, Afzal; Adhikari, Bimalendu; Martic, Sanela; Munir, Azeema; Shahzad, Suniya; Ahmad, Khurshid; Kraatz, Heinz-Bernhard

    2015-02-21

    In this review, we discuss the factors that influence electron transfer in peptides. We summarize experimental results from solution and surface studies and highlight the ongoing debate on the mechanistic aspects of this fundamental reaction. Here, we provide a balanced approach that remains unbiased and does not favor one mechanistic view over another. Support for a putative hopping mechanism in which an electron transfers in a stepwise manner is contrasted with experimental results that support electron tunneling or even some form of ballistic transfer or a pathway transfer for an electron between donor and acceptor sites. In some cases, experimental evidence suggests that a change in the electron transfer mechanism occurs as a result of donor-acceptor separation. However, this common understanding of the switch between tunneling and hopping as a function of chain length is not sufficient for explaining electron transfer in peptides. Apart from chain length, several other factors such as the extent of the secondary structure, backbone conformation, dipole orientation, the presence of special amino acids, hydrogen bonding, and the dynamic properties of a peptide also influence the rate and mode of electron transfer in peptides. Electron transfer plays a key role in physical, chemical and biological systems, so its control is a fundamental task in bioelectrochemical systems, the design of peptide based sensors and molecular junctions. Therefore, this topic is at the heart of a number of biological and technological processes and thus remains of vital interest.

  1. Antimicrobial Peptides from Plants

    PubMed Central

    Tam, James P.; Wang, Shujing; Wong, Ka H.; Tan, Wei Liang

    2015-01-01

    Plant antimicrobial peptides (AMPs) have evolved differently from AMPs from other life forms. They are generally rich in cysteine residues which form multiple disulfides. In turn, the disulfides cross-braced plant AMPs as cystine-rich peptides to confer them with extraordinary high chemical, thermal and proteolytic stability. The cystine-rich or commonly known as cysteine-rich peptides (CRPs) of plant AMPs are classified into families based on their sequence similarity, cysteine motifs that determine their distinctive disulfide bond patterns and tertiary structure fold. Cystine-rich plant AMP families include thionins, defensins, hevein-like peptides, knottin-type peptides (linear and cyclic), lipid transfer proteins, α-hairpinin and snakins family. In addition, there are AMPs which are rich in other amino acids. The ability of plant AMPs to organize into specific families with conserved structural folds that enable sequence variation of non-Cys residues encased in the same scaffold within a particular family to play multiple functions. Furthermore, the ability of plant AMPs to tolerate hypervariable sequences using a conserved scaffold provides diversity to recognize different targets by varying the sequence of the non-cysteine residues. These properties bode well for developing plant AMPs as potential therapeutics and for protection of crops through transgenic methods. This review provides an overview of the major families of plant AMPs, including their structures, functions, and putative mechanisms. PMID:26580629

  2. α/β-Peptide Foldamers Targeting Intracellular Protein-Protein Interactions with Activity in Living Cells.

    PubMed

    Checco, James W; Lee, Erinna F; Evangelista, Marco; Sleebs, Nerida J; Rogers, Kelly; Pettikiriarachchi, Anne; Kershaw, Nadia J; Eddinger, Geoffrey A; Belair, David G; Wilson, Julia L; Eller, Chelcie H; Raines, Ronald T; Murphy, William L; Smith, Brian J; Gellman, Samuel H; Fairlie, W Douglas

    2015-09-01

    Peptides can be developed as effective antagonists of protein-protein interactions, but conventional peptides (i.e., oligomers of l-α-amino acids) suffer from significant limitations in vivo. Short half-lives due to rapid proteolytic degradation and an inability to cross cell membranes often preclude biological applications of peptides. Oligomers that contain both α- and β-amino acid residues ("α/β-peptides") manifest decreased susceptibility to proteolytic degradation, and when properly designed these unnatural oligomers can mimic the protein-recognition properties of analogous "α-peptides". This report documents an extension of the α/β-peptide approach to target intracellular protein-protein interactions. Specifically, we have generated α/β-peptides based on a "stapled" Bim BH3 α-peptide, which contains a hydrocarbon cross-link to enhance α-helix stability. We show that a stapled α/β-peptide can structurally and functionally mimic the parent stapled α-peptide in its ability to enter certain types of cells and block protein-protein interactions associated with apoptotic signaling. However, the α/β-peptide is nearly 100-fold more resistant to proteolysis than is the parent stapled α-peptide. These results show that backbone modification, a strategy that has received relatively little attention in terms of peptide engineering for biomedical applications, can be combined with more commonly deployed peripheral modifications such as side chain cross-linking to produce synergistic benefits.

  3. Signal peptide of cellulase.

    PubMed

    Yan, Shaomin; Wu, Guang

    2014-06-01

    Cellulase is an enzyme playing a crucial role in biotechnology industries ranging from textile to biofuel because of tremendous amount of cellulose produced in plant. In order to improve cellulase productivity, huge resource has been spent in search for good cellulases from microorganism in remote areas and in creation of ideal cellulase by engineering. However, not much attention is given to the secretion of cellulases from cell into extracellular space, where a cellulase plays its enzymatic role. In this minireview, the signal peptides, which lead secreted proteins to specific secretion systems and scatter in literature, are reviewed. The patterns of signal peptides are checked against 4,101 cellulases documented in UniProtKB, the largest protein database in the world, to determine how these cellulases are secreted. Simultaneous review on both literature and cellulases from the database not only provides updated knowledge on signal peptides but also indicates the gap in our research.

  4. Synthetic antibiofilm peptides.

    PubMed

    de la Fuente-Núñez, César; Cardoso, Marlon Henrique; de Souza Cândido, Elizabete; Franco, Octavio Luiz; Hancock, Robert E W

    2016-05-01

    Bacteria predominantly exist as multicellular aggregates known as biofilms that are associated with at least two thirds of all infections and exhibit increased adaptive resistance to conventional antibiotic therapies. Therefore, biofilms are major contributors to the global health problem of antibiotic resistance, and novel approaches to counter them are urgently needed. Small molecules of the innate immune system called host defense peptides (HDPs) have emerged as promising templates for the design of potent, broad-spectrum antibiofilm agents. Here, we review recent developments in the new field of synthetic antibiofilm peptides, including mechanistic insights, synergistic interactions with available antibiotics, and their potential as novel antimicrobials against persistent infections caused by biofilms. This article is part of a Special Issue entitled: Antimicrobial peptides edited by Karl Lohner and Kai Hilpert. PMID:26724202

  5. Biomimetic peptide nanosensors.

    PubMed

    Cui, Yue; Kim, Sang N; Naik, Rajesh R; McAlpine, Michael C

    2012-05-15

    The development of a miniaturized sensing platform tailored for sensitive and selective detection of a variety of biochemical analytes could offer transformative fundamental and technological opportunities. Due to their high surface-to-volume ratios, nanoscale materials are extremely sensitive sensors. Likewise, peptides represent robust substrates for selective recognition due to the potential for broad chemical diversity within their relatively compact size. Here we explore the possibilities of linking peptides to nanosensors for the selective detection of biochemical targets. Such systems raise a number of interesting fundamental challenges: What are the peptide sequences, and how can rational design be used to derive selective binders? What nanomaterials should be used, and what are some strategies for assembling hybrid nanosensors? What role does molecular modeling play in elucidating response mechanisms? What is the resulting performance of these sensors, in terms of sensitivity, selectivity, and response time? What are some potential applications? This Account will highlight our early attempts to address these research challenges. Specifically, we use natural peptide sequences or sequences identified from phage display as capture elements. The sensors are based on a variety of nanomaterials including nanowires, graphene, and carbon nanotubes. We couple peptides to the nanomaterial surfaces via traditional surface functionalization methods or self-assembly. Molecular modeling provides detailed insights into the hybrid nanostructure, as well as the sensor detection mechanisms. The peptide nanosensors can distinguish chemically camouflaged mixtures of vapors and detect chemical warfare agents with sensitivities as low as parts-per-billion levels. Finally, we anticipate future uses of this technology in biomedicine: for example, devices based on these sensors could detect disease from the molecular components in human breath. Overall, these results provide a

  6. Invertebrate FMRFamide related peptides.

    PubMed

    Krajniak, Kevin G

    2013-06-01

    In 1977 the neuropeptide FMRFamide was isolated from the clam, Macrocallista nimbosa. Since then several hundred FMRFamide-related peptides (FaRPs) have been isolated from invertebrate animals. Precursors to the FaRPs likely arose in the cnidarians. With the transition to a bilateral body plan FaRPs became a fixture in the invertebrate phyla. They have come to play a critical role as neurotransmitters, neuromodulators, and neurohormones. FaRPs regulate a variety of body functions including, feeding, digestion, circulation, reproduction, movement. The evolution of the molecular form and function of these omnipresent peptides will be considered.

  7. Dicyclopropylmethyl peptide backbone protectant.

    PubMed

    Carpino, Louis A; Nasr, Khaled; Abdel-Maksoud, Adel Ali; El-Faham, Ayman; Ionescu, Dumitru; Henklein, Peter; Wenschuh, Holger; Beyermann, Michael; Krause, Eberhard; Bienert, Michael

    2009-08-20

    The N-dicyclopropylmethyl (Dcpm) residue, introduced into amino acids via reaction of dicyclopropylmethanimine hydrochloride with an amino acid ester followed by sodium cyanoborohydride or triacetoxyborohydride reduction, can be used as an amide bond protectant for peptide synthesis. Examples which demonstrate the amelioration of aggregation effects include syntheses of the alanine decapeptide and the prion peptide (106-126). Avoidance of cyclization to the aminosuccinimide followed substitution of Fmoc-(Dcpm)Gly-OH for Fmoc-Gly-OH in the assembly of sequences containing the sensitive Asp-Gly unit.

  8. Antimicrobial Peptides, Skin Infections and Atopic Dermatitis

    PubMed Central

    Hata, Tissa R.; Gallo, Richard L.

    2008-01-01

    The innate immune system evolved over 2 billion years ago to first recognize pathogens then eradicate them. Several distinct defects in this ancient but rapidly responsive element of human immune defense account for the increased incidence of skin infections in atopics. These defects include abnormalities in the physical barrier of the epidermis, alterations in microbial pattern recognition receptors such as toll receptors and NOD, and a diminished capacity to increase the expression of antimicrobial peptides during inflammation. Several antimicrobial peptides are affected including; cathelicidin, HBD-2, and HBD-3, which are lower in lesional skin of atopics compared to other inflammatory skin diseases, and dermcidin, which is decreased in sweat. Other defects in the immune defense barrier of atopics include a relative deficiency in plasmacytoid dendritic cells. In the future, understanding the cause of these defects may allow therapeutic intervention to reduce the incidence of infection in atopic individuals and potentially decrease the severity of this disorder. PMID:18620136

  9. Brain Peptides and Psychopharmacology

    ERIC Educational Resources Information Center

    Arehart-Treichel, Joan

    1976-01-01

    Proteins isolated from the brain and used as drugs can improve and apparently even transfer mental states and behavior. Much of the pioneering work and recent research with humans and animals is reviewed and crucial questions that are being posed about the psychologically active peptides are related. (BT)

  10. Peptide and peptide library cyclization via bromomethylbenzene derivatives.

    PubMed

    Hacker, David E; Almohaini, Mohammed; Anbazhagan, Aruna; Ma, Zhong; Hartman, Matthew C T

    2015-01-01

    Cyclization confers several advantages to peptides, cumulatively serving to make them more drug-like. In this protocol, cyclic peptides are generated via bis-alkylation of cysteine-containing peptides using α,α'-dibromo-m-xylene. The reactions are robust and high yielding. Multiple reaction platforms for the application of this versatile strategy are described herein: the cyclization of solid-phase-synthesized peptides, both in solution and on resin, as well as the cyclization of in vitro translated mRNA-peptide fusion libraries on oligo(dT) resin.

  11. A multicomponent conjugation strategy to unique N-steroidal peptides: first evidence of the steroidal nucleus as a β-turn inducer in acyclic peptides.

    PubMed

    Rivera, Daniel G; Vasco, Aldrin V; Echemendía, Radell; Concepción, Odette; Pérez, Carlos S; Gavín, José A; Wessjohann, Ludger A

    2014-10-01

    Constraining small peptides into specific secondary structures has been a major challenge in peptide ligand design. So far, the major solution for decreasing the conformational flexibility in small peptides has been cyclization. An alternative is the use of topological templates, which are able to induce and/or stabilize peptide secondary structures by means of covalent attachment to the peptide. Herein a multicomponent strategy and structural analysis of a new type of peptidosteroid architecture having the steroid as N-substituent of an internal amide bond is reported. The approach comprises the one-pot conjugation of two peptide chains (or amino acid derivatives) to aminosteroids by means of the Ugi reaction to give a unique family of N-steroidal peptides. The conjugation efficiency of a variety of peptide sequences and steroidal amines, as well as their consecutive head-to-tail cyclization to produce chimeric cyclopeptide-steroid conjugates, that is, macrocyclic lipopeptides, was assessed. Determination of the three-dimensional structure of an acyclic N-steroidal peptide in solution proved that the bulky, rigid steroidal template is capable of both increasing significantly the conformational rigidity, even in a peptide sequence as short as five amino acid residues, and inducing a β-turn secondary structure even in the all-s-trans isomer. This report provides the first evidence of the steroid skeleton as β-turn inducer in linear peptide sequences.

  12. Tat/HA2 Peptides Conjugated AuNR@pNIPAAm as a Photosensitizer Carrier for Near Infrared Triggered Photodynamic Therapy.

    PubMed

    Ye, Shefang; Kang, Ning; Chen, Min; Wang, Caiding; Wang, Tianxiao; Wang, Yarun; Liu, Yongliang; Li, Donghui; Ren, Lei

    2015-07-01

    To achieve an efficiency of intracellular photosensitizers (PSs) delivery and efficacy of photodynamic therapy, we have developed a novel class of PS formulation for encapsulating sulfonated aluminum phthalocyanine (AlPcS4) by taking advantage of the membrane-disruptive peptides Tat/HA2 and the photothermally triggered delivery system using AuNR@pNIPAAm. The coordinated effects of cell penetrating peptide Tat and fusogenic peptide HA2 could enhance the efficient cellular internalization and endo/lysosome escape of PSs delivery systems. Singlet oxygen generation was inhibited due to the reaction between loaded AlPcS4 and Au nanorods, which indicated that the AlPcS4-loaded, AuNR@pNIPAAm delivery system might be nonphototoxic in the circulatory system. However, this PSs-loaded nanosystem became highly phototoxic as it underwent the near-infrared irradiation by using the combined lights of 808 and 680 nm. Upon irradiation, the Tat/HA2 conjugated AuNR@pNIPAAm-Pc elicited an active photodynamic response against the cancer cells, leading to effective cells killing via mitochondria-associated apoptotic pathway. This study also demonstrated improved PDT therapeutic efficacy after intravenous administration of Tat/HA2-AuNR@pNIPAAm-Pc and the subsequent lights irradiations in tumor-bearing mice. We describe here a strategy for enhanced photodynamic eradication of solid tumors by endo/lysosomal escape and highlight the great promise of peptide-based nanocarriers used for cancer therapy.

  13. Blocking protein phosphatase 2A signaling prevents endothelial-to-mesenchymal transition and renal fibrosis: a peptide-based drug therapy

    NASA Astrophysics Data System (ADS)

    Deng, Yuanjun; Guo, Yanyan; Liu, Ping; Zeng, Rui; Ning, Yong; Pei, Guangchang; Li, Yueqiang; Chen, Meixue; Guo, Shuiming; Li, Xiaoqing; Han, Min; Xu, Gang

    2016-01-01

    Endothelial-to-mesenchymal transition (EndMT) contributes to the emergence of fibroblasts and plays a significant role in renal interstitial fibrosis. Protein phosphatase 2A (PP2A) is a major serine/threonine protein phosphatase in eukaryotic cells and regulates many signaling pathways. However, the significance of PP2A in EndMT is poorly understood. In present study, the role of PP2A in EndMT was evaluated. We demonstrated that PP2A activated in endothelial cells (EC) during their EndMT phenotype acquisition and in the mouse model of obstructive nephropathy (i.e., UUO). Inhibition of PP2A activity by its specific inhibitor prevented EC undergoing EndMT. Importantly, PP2A activation was dependent on tyrosine nitration at 127 in the catalytic subunit of PP2A (PP2Ac). Our renal-protective strategy was to block tyrosine127 nitration to inhibit PP2A activation by using a mimic peptide derived from PP2Ac conjugating a cell penetrating peptide (CPP: TAT), termed TAT-Y127WT. Pretreatment withTAT-Y127WT was able to prevent TGF-β1-induced EndMT. Administration of the peptide to UUO mice significantly ameliorated renal EndMT level, with preserved density of peritubular capillaries and reduction in extracellular matrix deposition. Taken together, these results suggest that inhibiting PP2Ac nitration using a mimic peptide is a potential preventive strategy for EndMT in renal fibrosis.

  14. Peptides and Ageing.

    PubMed

    Khavinson, Vladimir Kh

    2002-01-01

    A technology has been developed for manufacturing of biologically active complex peptide preparations from extracts of different tissues. In particular, the pineal preparation (Epithalamin) augments the in vitro outgrowth of explants from the pineal gland but not from other tissues, the latter being stimulated by peptide preparations from respective tissues. Epithalamin increases melatonin production by the pineal gland of rats, improves immunological parameters in rats and mice, produces anticarcinogenic effects in different experimental models, stimulates antioxidant defenses, and restores the reproductive function in old rats. These effects are combined in the ability of Epithalamin to increase the lifespan in rats, mice, and fruit flies. Many of these effects are reproduced in clinical trials, which have demonstrated the geroprotector activity of Epithalamin in humans. Among the effects of the thymic preparation Thymalin, those related to its ability to stimulate immunity are the most prominent. This ability is associated with anticarcinogenic and geroprotector activities. Clinical trials of the peptide preparations obtained from other organs including the prostate, the cerebral cortex, and the eye retina, have demonstrated beneficial effects reflected by the improvement of the conditions of respective organs. Based on the data about the amino acid compositions of the peptide preparations, novel principles of the design of biologically active short peptides possessing tissue-specific activities has been developed. Dipeptides specific for the thymus and tetrapeptides specific for the heart, liver, brain cortex, and pineal glands stimulate the in vitro outgrowth of explants of respective organs. Interestingly, for eye retina and the pineal gland, a common tetrapeptide Ala-Glu-Asp-Gly (Epitalon) has been designed, probably reflecting the common embryonal origin of these two organs. Epitalon reproduces the effects of Epithalamin including those related to its

  15. Selective enhancement of the uptake and bioactivity of a TAT-conjugated peptide inhibitor of glycogen synthase kinase-3.

    PubMed

    Manceur, Aziza P; Driscoll, Brandon D; Sun, Wei; Audet, Julie

    2009-03-01

    The use of cell-penetrating peptides as transduction vectors is a promising approach to deliver peptides and proteins into cells. However, the uptake and bioavailability of trans-activating transcriptor (TAT)-conjugated molecules vary depending on the cell type and the cargo. This study aimed to determine whether a low-voltage electrical pulse can enhance the TAT-mediated delivery of peptide cargoes in different cell types. In TF-1 and mouse embryonic stem cells, the uptake of a novel detachable TAT-conjugated glycogen synthase kinase-3 (GSK-3) peptide inhibitor was enhanced by an order of magnitude without affecting the cell viability. A similar increase in uptake was achieved in primary mouse bone marrow cells while maintaining >80% of their viability. Interestingly, under these low-voltage conditions, the uptake of a control peptide not conjugated to TAT was not significantly increased. A T-cell factor/lymphoid enhancer factor (TCF/LEF) luciferase reporter assay was also used to assess the bioactivity of the TAT construct. The results indicated that cells loaded with a low-voltage electrical pulse had a twofold increase in TCF/LEF activity, which was equivalent to a level of GSK-3 inhibition similar to that of cells treated with 20 mmol/l lithium or 500 nmol/l (2'Z,3'E)-6-bromoindirubin-3'-oxime. These results demonstrate the usefulness of low-voltage electrical pulses to enhance the uptake and bioactivity of TAT-conjugated molecules in different cell types. PMID:19107119

  16. Peptide vectors for gene delivery: from single peptides to multifunctional peptide nanocarriers.

    PubMed

    Raad, Markus de; Teunissen, Erik A; Mastrobattista, Enrico

    2014-07-01

    The therapeutic use of nucleic acids relies on the availability of sophisticated delivery systems for targeted and intracellular delivery of these molecules. Such a gene delivery should possess essential characteristics to overcome several extracellular and intracellular barriers. Peptides offer an attractive platform for nonviral gene delivery, as several functional peptide classes exist capable of overcoming these barriers. However, none of these functional peptide classes contain all the essential characteristics required to overcome all of the barriers associated with successful gene delivery. Combining functional peptides into multifunctional peptide vectors will be pivotal for improving peptide-based gene delivery systems. By using combinatorial strategies and high-throughput screening, the identification of multifunctional peptide vectors will accelerate the optimization of peptide-based gene delivery systems.

  17. Antagonistic peptide technology for functional dissection of CLE peptides revisited.

    PubMed

    Czyzewicz, Nathan; Wildhagen, Mari; Cattaneo, Pietro; Stahl, Yvonne; Pinto, Karine Gustavo; Aalen, Reidunn B; Butenko, Melinka A; Simon, Rüdiger; Hardtke, Christian S; De Smet, Ive

    2015-08-01

    In the Arabidopsis thaliana genome, over 1000 putative genes encoding small, presumably secreted, signalling peptides can be recognized. However, a major obstacle in identifying the function of genes encoding small signalling peptides is the limited number of available loss-of-function mutants. To overcome this, a promising new tool, antagonistic peptide technology, was recently developed. Here, this antagonistic peptide technology was tested on selected CLE peptides and the related IDA peptide and its usefulness in the context of studies of peptide function discussed. Based on the analyses, it was concluded that the antagonistic peptide approach is not the ultimate means to overcome redundancy or lack of loss-of-function lines. However, information collected using antagonistic peptide approaches (in the broad sense) can be very useful, but these approaches do not work in all cases and require a deep insight on the interaction between the ligand and its receptor to be successful. This, as well as peptide ligand structure considerations, should be taken into account before ordering a wide range of synthetic peptide variants and/or generating transgenic plants.

  18. Biochemical functionalization of peptide nanotubes with phage displayed peptides

    NASA Astrophysics Data System (ADS)

    Swaminathan, Swathi; Cui, Yue

    2016-09-01

    The development of a general approach for the biochemical functionalization of peptide nanotubes (PNTs) could open up existing opportunities in both fundamental studies as well as a variety of applications. PNTs are spontaneously assembled organic nanostructures made from peptides. Phage display has emerged as a powerful approach for identifying selective peptide binding motifs. Here, we demonstrate for the first time the biochemical functionalization of PNTs via peptides identified from a phage display peptide library. The phage-displayed peptides are shown to recognize PNTs. These advances further allow for the development of bifunctional peptides for the capture of bacteria and the self-assembly of silver particles onto PNTs. We anticipate that these results could provide significant opportunities for using PNTs in both fundamental studies and practical applications, including sensors and biosensors nanoelectronics, energy storage devices, drug delivery, and tissue engineering.

  19. Biochemical functionalization of peptide nanotubes with phage displayed peptides.

    PubMed

    Swaminathan, Swathi; Cui, Yue

    2016-09-01

    The development of a general approach for the biochemical functionalization of peptide nanotubes (PNTs) could open up existing opportunities in both fundamental studies as well as a variety of applications. PNTs are spontaneously assembled organic nanostructures made from peptides. Phage display has emerged as a powerful approach for identifying selective peptide binding motifs. Here, we demonstrate for the first time the biochemical functionalization of PNTs via peptides identified from a phage display peptide library. The phage-displayed peptides are shown to recognize PNTs. These advances further allow for the development of bifunctional peptides for the capture of bacteria and the self-assembly of silver particles onto PNTs. We anticipate that these results could provide significant opportunities for using PNTs in both fundamental studies and practical applications, including sensors and biosensors nanoelectronics, energy storage devices, drug delivery, and tissue engineering. PMID:27479451

  20. Antimicrobial peptides: premises and promises.

    PubMed

    Reddy, K V R; Yedery, R D; Aranha, C

    2004-12-01

    Antimicrobial peptides (AMPs) are an important component of the natural defences of most living organisms against invading pathogens. These are relatively small (< 10kDa), cationic and amphipathic peptides of variable length, sequence and structure. During the past two decades several AMPs have been isolated from a wide variety of animals, both vertebrates and invertebrates, and plants as well as from bacteria and fungi. Most of these peptides are obtained from different sources like macrophages, neutrophils, epithelial cells, haemocytes, fat body, reproductive tract, etc. These peptides exhibit broad-spectrum activity against a wide range of microorganisms including Gram-positive and Gram-negative bacteria, protozoa, yeast, fungi and viruses. A few peptides have also been found to be cytotoxic to sperm and tumour cells. AMPs are classified based on the three dimensional structural studies carried out with the help of NMR. The peptides are broadly classified into five major groups namely (a) peptides that form alpha-helical structures, (b) peptides rich in cysteine residues, (c) peptides that form beta-sheet, (d) peptides rich in regular amino acids namely histatin, arginine and proline and (e) peptides composed of rare and modified amino acids. Most of these peptides are believed to act by disrupting the plasma membrane leading to the lysis of the cell. AMPs have been found to be excellent candidates for developing novel antimicrobial agents and a few of these peptides show antimicrobial activity against pathogens causing sexually transmitted infection (STI), including HIV/HSV. Peptides, namely magainin and nisin have been shown to demonstrate contraceptive properties in vitro and in vivo. A few peptides have already entered clinical trials for the treatment of impetigo, diabetic foot ulcers and gastric helicobacter infections. In this review, we discuss the source, structures and mode of action with special reference to therapeutic considerations of various AMPs

  1. A Peptide Filtering Relation Quantifies MHC Class I Peptide Optimization

    PubMed Central

    Goldstein, Leonard D.; Howarth, Mark; Cardelli, Luca; Emmott, Stephen; Elliott, Tim; Werner, Joern M.

    2011-01-01

    Major Histocompatibility Complex (MHC) class I molecules enable cytotoxic T lymphocytes to destroy virus-infected or cancerous cells, thereby preventing disease progression. MHC class I molecules provide a snapshot of the contents of a cell by binding to protein fragments arising from intracellular protein turnover and presenting these fragments at the cell surface. Competing fragments (peptides) are selected for cell-surface presentation on the basis of their ability to form a stable complex with MHC class I, by a process known as peptide optimization. A better understanding of the optimization process is important for our understanding of immunodominance, the predominance of some T lymphocyte specificities over others, which can determine the efficacy of an immune response, the danger of immune evasion, and the success of vaccination strategies. In this paper we present a dynamical systems model of peptide optimization by MHC class I. We incorporate the chaperone molecule tapasin, which has been shown to enhance peptide optimization to different extents for different MHC class I alleles. Using a combination of published and novel experimental data to parameterize the model, we arrive at a relation of peptide filtering, which quantifies peptide optimization as a function of peptide supply and peptide unbinding rates. From this relation, we find that tapasin enhances peptide unbinding to improve peptide optimization without significantly delaying the transit of MHC to the cell surface, and differences in peptide optimization across MHC class I alleles can be explained by allele-specific differences in peptide binding. Importantly, our filtering relation may be used to dynamically predict the cell surface abundance of any number of competing peptides by MHC class I alleles, providing a quantitative basis to investigate viral infection or disease at the cellular level. We exemplify this by simulating optimization of the distribution of peptides derived from Human

  2. Antibody Production with Synthetic Peptides.

    PubMed

    Lee, Bao-Shiang; Huang, Jin-Sheng; Jayathilaka, Lasanthi P; Lee, Jenny; Gupta, Shalini

    2016-01-01

    Peptides (usually 10-20 amino acid residues in length) can be used as effectively as proteins in raising antibodies producing both polyclonal and monoclonal antibodies routinely with titers higher than 20,000. Peptide antigens do not function as immunogens unless they are conjugated to proteins. Production of high quality antipeptide antibodies is dependent upon peptide sequence selection, the success of peptide synthesis, peptide-carrier protein conjugation, the humoral immune response in the host animal, the adjuvant used, the peptide dose administered, the injection method, and the purification of the antibody. Peptide sequence selection is probably the most critical step in the production of antipeptide antibodies. Although the process for designing peptide antigens is not exact, several guidelines and computational B-cell epitope prediction methods can help maximize the likelihood of producing antipeptide antibodies that recognize the protein. Antibodies raised by peptides have become essential tools in life science research. Virtually all phospho-specific antibodies are now produced using phosphopeptides as antigens. Typically, 5-20 mg of peptide is enough for antipeptide antibody production. It takes 3 months to produce a polyclonal antipeptide antibody in rabbits that yields ~100 mL of serum which corresponds to ~8-10 mg of the specific antibody after affinity purification using a peptide column. PMID:27515072

  3. The Use ofa Hydrophobic Binding Peptide Modified Lipid Nanocarrier Improving Tumor Distribution and Antitumor Efficacy.

    PubMed

    Gao, Wei; Yang, Xiucong; Lin, Zhiqiang; Gao, Shanyun; He, Bing; Mei, Bong; Wang, Dan; Yuan, Lan; Zhang, Hua; Dai, Wenbing; Wang, Xueqing; Wang, Jiancheng; Zhang, Xuan; Zhang, Qiang

    2016-06-01

    In addition to showing the specific interaction between a generalized ligand and its receptor and the electrostatic effect between positive cell-penetrating peptides and negative cell membranes, our last study demonstrated the hydrophobic interactivity between a hydrophobic binding peptide (HBP) and biomembranes to be favorable in drug delivery. To yield more evidence for this new strategy and to find more effective HBPs, here we designed and established a novel nanomedicine associated with cyclosporin A (CsA) because this peptide is electrically neutral, highly hydrophobic, very stable in vivo and safe at the given dose. First, isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR) studies showed a strong hydrophobic interaction between the CsA molecules and the lipid membrane. The lactate dehydrogenase release assay proved that CsA exhibited low toxicity to cell membranes. These facts encouraged us to explore the potential application of CsA as an HBP to actualize intracellular delivery of nanomedicines for tumor therapy. When conjugated to lipid nanocarriers, CsA significantly enhanced their binding with cells and,. consequently, increased the internalization of recoded nanomedicines into cells. The in vivo experiments further showed that the CsA-associated nanocarriers could achieve better delivery to tumor tissues and improve the tumor therapy of doxorubicin (DOX) compared to the nonmodified control; these findings were identical to the observations-in cell studies. In conclusion, CsA, a readily obtainable molecule with favorable characteristics, is indeed a good candidate for an HBP, and this study provides solid, novel evidence for the use of HBP-based nanocarriers as effective antitumor drug delivery systems.

  4. The Use ofa Hydrophobic Binding Peptide Modified Lipid Nanocarrier Improving Tumor Distribution and Antitumor Efficacy.

    PubMed

    Gao, Wei; Yang, Xiucong; Lin, Zhiqiang; Gao, Shanyun; He, Bing; Mei, Bong; Wang, Dan; Yuan, Lan; Zhang, Hua; Dai, Wenbing; Wang, Xueqing; Wang, Jiancheng; Zhang, Xuan; Zhang, Qiang

    2016-06-01

    In addition to showing the specific interaction between a generalized ligand and its receptor and the electrostatic effect between positive cell-penetrating peptides and negative cell membranes, our last study demonstrated the hydrophobic interactivity between a hydrophobic binding peptide (HBP) and biomembranes to be favorable in drug delivery. To yield more evidence for this new strategy and to find more effective HBPs, here we designed and established a novel nanomedicine associated with cyclosporin A (CsA) because this peptide is electrically neutral, highly hydrophobic, very stable in vivo and safe at the given dose. First, isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR) studies showed a strong hydrophobic interaction between the CsA molecules and the lipid membrane. The lactate dehydrogenase release assay proved that CsA exhibited low toxicity to cell membranes. These facts encouraged us to explore the potential application of CsA as an HBP to actualize intracellular delivery of nanomedicines for tumor therapy. When conjugated to lipid nanocarriers, CsA significantly enhanced their binding with cells and,. consequently, increased the internalization of recoded nanomedicines into cells. The in vivo experiments further showed that the CsA-associated nanocarriers could achieve better delivery to tumor tissues and improve the tumor therapy of doxorubicin (DOX) compared to the nonmodified control; these findings were identical to the observations-in cell studies. In conclusion, CsA, a readily obtainable molecule with favorable characteristics, is indeed a good candidate for an HBP, and this study provides solid, novel evidence for the use of HBP-based nanocarriers as effective antitumor drug delivery systems. PMID:27319213

  5. Peptide mass fingerprinting.

    PubMed

    Thiede, Bernd; Höhenwarter, Wolfgang; Krah, Alexander; Mattow, Jens; Schmid, Monika; Schmidt, Frank; Jungblut, Peter R

    2005-03-01

    Peptide mass fingerprinting by MALDI-MS and sequencing by tandem mass spectrometry have evolved into the major methods for identification of proteins following separation by two-dimensional gel electrophoresis, SDS-PAGE or liquid chromatography. One main technological goal of proteome analyses beside high sensitivity and automation was the comprehensive analysis of proteins. Therefore, the protein species level with the essential information on co- and post-translational modifications must be achieved. The power of peptide mass fingerprinting for protein identification was described here, as exemplified by the identification of protein species with high molecular masses (spectrin alpha and beta), low molecular masses (elongation factor EF-TU fragments), splice variants (alpha A crystallin), aggregates with disulfide bridges (alkylhydroperoxide reductase), and phosphorylated proteins (heat shock protein 27). Helpful tools for these analyses were the use of the minimal protein identifier concept and the software program MS-Screener to remove mass peaks assignable to contaminants and neighbor spots.

  6. On the Accuracy and Limits of Peptide Fragmentation Spectrum Prediction

    PubMed Central

    Li, Sujun; Arnold, Randy J.; Tang, Haixu; Radivojac, Predrag

    2011-01-01

    We estimated the reproducibility of tandem mass fragmentation spectra for the widely-used collision-induced dissociation (CID) instruments. Using the Pearson correlation coefficient as a measure of spectral similarity, we found that the within-experiment reproducibility of fragment ion intensities is very high (about 0.85). However, across different experiments and instrument types/setups, the correlation decreases by more than 15% (to about 0.70). We further investigated the accuracy of current predictors of peptide fragmentation spectra and found that they are more accurate than the ad-hoc models generally used by search engines (e.g. SEQUEST) and, surprisingly, approaching the empirical upper limit set by the average across-experiment spectral reproducibility (especially for charge +1 and charge +2 precursor ions). These results provide evidence that, in terms of accuracy of modeling, predicted peptide fragmentation spectra provide a viable alternative to spectral libraries for peptide identification, with a higher coverage of peptides and lower storage requirements. Furthermore, using five data sets of proteome digests by two different proteases, we find that PeptideART (a data-driven machine learning approach) is generally more accurate than MassAnalyzer (an approach based on a kinetic model for peptide fragmentation) in predicting fragmentation spectra, but that both models are significantly more accurate than the ad-hoc models. Availability: PeptideART is freely available at www.informatics.indiana.edu/predrag. PMID:21175207

  7. Engineering and Characterization of Peptides and Proteins at Surfaces and Interfaces: A Case Study in Surface-Sensitive Vibrational Spectroscopy.

    PubMed

    Ding, Bei; Jasensky, Joshua; Li, Yaoxin; Chen, Zhan

    2016-06-21

    labeling method with SFG to probe the detailed local structure and microenvironment of peptides at buried interfaces, (2) systematic research on cell membrane associated peptides and proteins including antimicrobial peptides, cell penetrating peptides, G proteins, and other membrane proteins, discussing the factors that influence interfacial peptide and protein structures such as lipid charge, membrane fluidity, and biomolecule solution concentration, and (3) in-depth discussion on solid surface immobilized antimicrobial peptides and enzymes. The effects of immobilization method, substrate surface, immobilization site on the peptide or protein, and surrounding environment are presented. Several examples leading to high impact new research are also briefly introduced: The orientation change of alamethicin detected while varying the model cell membrane potential demonstrates the feasibility to apply SFG to study ion channel protein gating mechanisms. The elucidation of peptide secondary structures at liquid crystal interfaces shows promising results that liquid crystal can detect and recognize different peptides and proteins. The method of retaining the native structure of surface immobilized peptides or proteins in air demonstrates the feasibility to protect and preserve such structures via the use of hydromimetic functionalities when there is no bulk water. We hope that readers in many different disciplines will benefit from the research progress reported in this Account on SFG studies of interfacial structure-function relationships of peptides and proteins and apply this powerful technique to study interfacial biomolecules in the future. PMID:27188920

  8. Engineering and Characterization of Peptides and Proteins at Surfaces and Interfaces: A Case Study in Surface-Sensitive Vibrational Spectroscopy.

    PubMed

    Ding, Bei; Jasensky, Joshua; Li, Yaoxin; Chen, Zhan

    2016-06-21

    labeling method with SFG to probe the detailed local structure and microenvironment of peptides at buried interfaces, (2) systematic research on cell membrane associated peptides and proteins including antimicrobial peptides, cell penetrating peptides, G proteins, and other membrane proteins, discussing the factors that influence interfacial peptide and protein structures such as lipid charge, membrane fluidity, and biomolecule solution concentration, and (3) in-depth discussion on solid surface immobilized antimicrobial peptides and enzymes. The effects of immobilization method, substrate surface, immobilization site on the peptide or protein, and surrounding environment are presented. Several examples leading to high impact new research are also briefly introduced: The orientation change of alamethicin detected while varying the model cell membrane potential demonstrates the feasibility to apply SFG to study ion channel protein gating mechanisms. The elucidation of peptide secondary structures at liquid crystal interfaces shows promising results that liquid crystal can detect and recognize different peptides and proteins. The method of retaining the native structure of surface immobilized peptides or proteins in air demonstrates the feasibility to protect and preserve such structures via the use of hydromimetic functionalities when there is no bulk water. We hope that readers in many different disciplines will benefit from the research progress reported in this Account on SFG studies of interfacial structure-function relationships of peptides and proteins and apply this powerful technique to study interfacial biomolecules in the future.

  9. Protein-peptide interaction: study of heat-induced aggregation and gelation of β-lactoglobulin in the presence of two peptides from its own hydrolysate.

    PubMed

    Kosters, Hans A; Wierenga, Peter A; de Vries, Renko; Gruppen, Harry

    2013-05-01

    Two peptides, [f135-158] and [f135-162]-SH, were used to study the binding of the peptides to native β-lactolobulin, as well as the subsequent effects on aggregation and gelation of β-lactoglobulin. The binding of the peptide [f135-158] to β-lactoglobulin at room temperature was confirmed by SELDI-TOF-MS. It was further illustrated by increased turbidity of mixed solutions of peptide and protein (at pH 7), indicating association of proteins and peptides in larger complexes. At pH below the isoelectric point of the protein, the presence of peptides did not lead to an increased turbidity, showing the absence of complexation. The protein-peptide complexes formed at pH 7 were found to dissociate directly upon heating. After prolonged heating, extensive aggregation was observed, whereas no aggregation was seen for the pure protein or pure peptide solutions. The presence of the free sulfhydryl group in [f135-162]-SH resulted in a 10 times increase in the amount of aggregation of β-lactoglobulin upon heating, illustrating the additional effect of the free sulfhydryl group. Subsequent studies on the gel strength of heat-induced gels also showed a clear difference between these two peptides. The replacement of additional β-lactoglobulin by [f135-158] resulted in a decrease in gel strength, whereas replacement by peptide [f135-162]-SH increased gel strength.

  10. Oxidation of proline decreases immunoreactivity and alters structure of barley prolamin.

    PubMed

    Huang, Xin; Sontag-Strohm, Tuula; Stoddard, Frederick L; Kato, Yoji

    2017-01-01

    Elimination of celiac-toxic prolamin peptides and proteins is essential for Triticeae products to be gluten-free. Instead of enzymatic hydrolysis, in this study we investigated metal-catalyzed oxidation of two model peptides, QQPFP, and PQPQLPY, together with a hordein isolate from barley (Hordeum vulgare L.). We established a multiple reaction monitoring (MRM) LC-MS method to detect and quantify proline oxidation fragments. In addition to fragmentation, aggregation and side chain modifications were identified, including free thiol loss, carbonyl formation, and dityrosine formation. The immunoreactivity of the oxidized hordein isolate was considerably decreased in all metal-catalyzed oxidation systems. Cleavage of peptides or protein fragments at the numerous proline residues partially accounts for the decrease. Metal-catalyzed oxidation can thus be used in the modification and elimination of celiac-toxic peptides and proteins.

  11. Oxidation of proline decreases immunoreactivity and alters structure of barley prolamin.

    PubMed

    Huang, Xin; Sontag-Strohm, Tuula; Stoddard, Frederick L; Kato, Yoji

    2017-01-01

    Elimination of celiac-toxic prolamin peptides and proteins is essential for Triticeae products to be gluten-free. Instead of enzymatic hydrolysis, in this study we investigated metal-catalyzed oxidation of two model peptides, QQPFP, and PQPQLPY, together with a hordein isolate from barley (Hordeum vulgare L.). We established a multiple reaction monitoring (MRM) LC-MS method to detect and quantify proline oxidation fragments. In addition to fragmentation, aggregation and side chain modifications were identified, including free thiol loss, carbonyl formation, and dityrosine formation. The immunoreactivity of the oxidized hordein isolate was considerably decreased in all metal-catalyzed oxidation systems. Cleavage of peptides or protein fragments at the numerous proline residues partially accounts for the decrease. Metal-catalyzed oxidation can thus be used in the modification and elimination of celiac-toxic peptides and proteins. PMID:27507515

  12. α/β-Peptide Foldamers Targeting Intracellular Protein-Protein Interactions with Activity in Living Cells

    PubMed Central

    Checco, James W.; Lee, Erinna F.; Evangelista, Marco; Sleebs, Nerida J.; Rogers, Kelly; Pettikiriarachchi, Anne; Kershaw, Nadia J.; Eddinger, Geoffrey A.; Belair, David G.; Wilson, Julia L.; Eller, Chelcie H.; Raines, Ronald T.; Murphy, William L.; Smith, Brian J.; Gellman, Samuel H.; Fairlie, W. Douglas

    2015-01-01

    Peptides can be developed as effective antagonists of protein-protein interactions, but conventional peptides (i.e., oligomers of L-α-amino acids) suffer from significant limitations in vivo. Short half-lives due to rapid proteolytic degradation and an inability to cross cell membranes often preclude biological applications of peptides. Oligomers that contain both α- and β-amino acid residues (“α/β-peptides”) manifest decreased susceptibility to proteolytic degradation, and when properly designed these unnatural oligomers can mimic the protein-recognition properties of analogous “α-peptides”. This report documents an extension of the α/β-peptide approach to target intracellular protein-protein interactions. Specifically, we have generated α/β-peptides based on a “stapled” Bim BH3 α-peptide, which contains a hydrocarbon crosslink to enhance α-helix stability. We show that a stapled α/β-peptide can structurally and functionally mimic the parent stapled α-peptide in its ability to enter certain types of cells and block protein-protein interactions associated with apoptotic signaling. However, the α/β-peptide is nearly 100-fold more resistant to proteolysis than is the parent α-peptide. These results show that backbone modification, a strategy that has received relatively little attention in terms of peptide engineering for biomedical applications, can be combined with more commonly deployed peripheral modifications such as side chain crosslinking to produce synergistic benefits. PMID:26317395

  13. Synthesis and evaluation of amphiphilic peptides as nanostructures and drug delivery tools

    NASA Astrophysics Data System (ADS)

    Sayeh, Naser Ali

    conjugates although one limitation lies in the effort of controlling the rate of drug release. The encapsulated or complexed drugs tend to be released rapidly (before reaching the target site) and in the dendrimer--drug conjugates, it is the chemical linkage that controls the drug release. Thus, future studies in this field are urgently required to create more efficient and stable biomaterials. Peptides are considered as efficient vectors for achieving optimal cellular uptake. The potential use of peptides as drug delivery vectors received much attention by the discovery of several cell-penetrating peptides (CPPs). The first CPPs discovered in 1988, that were sequences from HIV-1 encoded TAT protein, TAT (48--60), and penetrated very efficiently through cell membranes of cultured mammalian cells. CPPs are a class of diverse peptides, typically with 8--25 amino acids, and unlike most peptides, they can cross the cellular membrane with more efficiency. CPPs have also shown to undergo self-assembly and generate nanostructures. The generation of self-assembled peptides and nanostructures occur through various types of interactions between functional groups of amino acid residues, such as electrostatic, hydrophobic, and hydrogen bonding. Appropriate design and functionalization of peptides are critical for generating nanostructures. Chemically CPPs are classified into two major groups: linear and cyclic peptides. It has been previously reported that linear peptides containing hydrophilic and hydrophobic amino acids could act as membrane protein stabilizers. These compounds are short hydrophilic or amphiphilic peptides that have positively charged amino acids, such as arginine, lysine or histidine, which can interact with the negative charge phospholipids layer on the cell membrane and translocate the cargo into the cells. Conjugation to cationic linear CPPs, such as TAT, penetratin, or oligoarginine efficiently improves the cellular uptake of large hydrophilic molecules, but the

  14. Macrocyclization of Unprotected Peptide Isocyanates.

    PubMed

    Vinogradov, Alexander A; Choo, Zi-Ning; Totaro, Kyle A; Pentelute, Bradley L

    2016-03-18

    A chemistry for the facile two-component macrocyclization of unprotected peptide isocyanates is described. Starting from peptides containing two glutamic acid γ-hydrazide residues, isocyanates can be readily accessed and cyclized with hydrazides of dicarboxylic acids. The choice of a nucleophilic linker allows for the facile modulation of biochemical properties of a macrocyclic peptide. Four cyclic NYAD-1 analogues were synthesized using the described method and displayed a range of biological activities. PMID:26948900

  15. Macrocyclization of Unprotected Peptide Isocyanates.

    PubMed

    Vinogradov, Alexander A; Choo, Zi-Ning; Totaro, Kyle A; Pentelute, Bradley L

    2016-03-18

    A chemistry for the facile two-component macrocyclization of unprotected peptide isocyanates is described. Starting from peptides containing two glutamic acid γ-hydrazide residues, isocyanates can be readily accessed and cyclized with hydrazides of dicarboxylic acids. The choice of a nucleophilic linker allows for the facile modulation of biochemical properties of a macrocyclic peptide. Four cyclic NYAD-1 analogues were synthesized using the described method and displayed a range of biological activities.

  16. Peptide Aptamers: Development and Applications

    PubMed Central

    Reverdatto, Sergey; Burz, David S.; Shekhtman, Alexander

    2015-01-01

    Peptide aptamers are small combinatorial proteins that are selected to bind to specific sites on their target molecules. Peptide aptamers consist of short, 5-20 amino acid residues long sequences, typically embedded as a loop within a stable protein scaffold. Various peptide aptamer scaffolds and in vitro and in vivo selection techniques are reviewed with emphasis on specific biomedical, bioimaging, and bioanalytical applications. PMID:25866267

  17. Degradable polyethylenimine derivate coupled to a bifunctional peptide R13 as a new gene-delivery vector

    PubMed Central

    Liu, Kehai; Wang, Xiaoyu; Fan, Wei; Zhu, Qing; Yang, Jingya; Gao, Jing; Gao, Shen

    2012-01-01

    Background To solve the efficiency versus cytotoxicity and tumor-targeting problems of polyethylenimine (PEI) used as a nonviral gene delivery vector, a degradable PEI derivate coupled to a bifunctional peptide R13 was developed. Methods First, we synthesized a degradable PEI derivate by crosslinking low-molecular-weight PEI with pluronic P123, then used tumor-targeting peptide arginine-glycine-aspartate-cysteine (RGDC), in conjunction with the cell-penetrating peptide Tat (49–57), to yield a bifunctional peptide RGDC-Tat (49–57) named R13, which can improve cell selection and increase cellular uptake, and, lastly, adopted R13 to modify the PEI derivates so as to prepare a new polymeric gene vector (P123-PEI-R13). The new gene vector was characterized in terms of its chemical structure and biophysical parameters. We also investigated the specificity, cytotoxicity, and gene transfection efficiency of this vector in αvβ3-positive human cervical carcinoma Hela cells and murine melanoma B16 cells in vitro. Results The vector showed controlled degradation, strong targeting specificity to αvβ3 receptor, and noncytotoxicity in Hela cells and B16 cells at higher doses, in contrast to PEI 25 KDa. The particle size of P123-PEI-R13/DNA complexes was around 100–250 nm, with proper zeta potential. The nanoparticles can protect plasmid DNA from being digested by DNase I at a concentration of 6 U DNase I/μg DNA. The nanoparticles were resistant to dissociation induced by 50% fetal bovine serum and 600 μg/mL sodium heparin. P123-PEI-R13 also revealed higher transfection efficiency in two cell lines as compared with PEI 25 KDa. Conclusion P123-PEI-R13 is a potential candidate as a safe and efficient gene-delivery carrier for gene therapy. PMID:22412301

  18. Enhanced blood-brain barrier penetration and glioma therapy mediated by a new peptide modified gene delivery system.

    PubMed

    Yao, Hui; Wang, Kaiyuan; Wang, Yi; Wang, Shanshan; Li, Jianfeng; Lou, Jinning; Ye, Liya; Yan, Xueying; Lu, Weiyue; Huang, Rongqin

    2015-01-01

    Successful glioma gene therapy lays on two important factors, the therapeutic genes and efficient delivery vehicles to cross the blood-brain barrier (BBB) and reach gliomas. In this work, a new gene vector was constructed based on dendrigraft poly-l-lysines (DGL) and polyethyleneglycol (PEG), conjugated with a cell-penetrating peptide, the nucleolar translocation signal (NoLS) sequence of the LIM Kinase 2 (LIMK2) protein (LIMK2 NoLS peptide, LNP), yielding DGL-PEG-LNP. Plasmid DNA encoding inhibitor of growth 4 (ING4) was applied as the therapeutic gene. DGL-PEG-LNP/DNA nanoparticles (NPs) were monodispersed, with a mean diameter of 90.6 ± 8.9 nm. The conjugation of LNP significantly enhanced the BBB-crossing efficiency, cellular uptake and gene expression within tumor cells. Mechanism studies suggested the involvement of energy, caveolae-mediated endocytosis and macropinocytosis in cellular uptake of LNP-modified NPs. MTT results showed that no apparent cytotoxicity was observed when cells were treated with synthesized vectors. Furthermore, LNP-modified NPs mediated strongest and most intensive apoptosis on the tumor site, and the longest median survival time of glioma-bearing mice. All the results demonstrated that LNP is a kind of efficient CPPs especially for BBB-crossing application, and DGL-PEG-LNP/DNA is a potential non-viral platform for glioma gene therapy via intravenous administration.

  19. Improving Peptide Applications Using Nanotechnology.

    PubMed

    Narayanaswamy, Radhika; Wang, Tao; Torchilin, Vladimir P

    2016-01-01

    Peptides are being successfully used in various fields including therapy and drug delivery. With advancement in nanotechnology and targeted delivery carrier systems, suitable modification of peptides has enabled achievement of many desirable goals over-riding some of the major disadvantages associated with the delivery of peptides in vivo. Conjugation or physical encapsulation of peptides to various nanocarriers, such as liposomes, micelles and solid-lipid nanoparticles, has improved their in vivo performance multi-fold. The amenability of peptides to modification in chemistry and functionalization with suitable nanocarriers are very relevant aspects in their use and have led to the use of 'smart' nanoparticles with suitable linker chemistries that favor peptide targeting or release at the desired sites, minimizing off-target effects. This review focuses on how nanotechnology has been used to improve the number of peptide applications. The paper also focuses on the chemistry behind peptide conjugation to nanocarriers, the commonly employed linker chemistries and the several improvements that have already been achieved in the areas of peptide use with the help of nanotechnology.

  20. Improving Peptide Applications Using Nanotechnology.

    PubMed

    Narayanaswamy, Radhika; Wang, Tao; Torchilin, Vladimir P

    2016-01-01

    Peptides are being successfully used in various fields including therapy and drug delivery. With advancement in nanotechnology and targeted delivery carrier systems, suitable modification of peptides has enabled achievement of many desirable goals over-riding some of the major disadvantages associated with the delivery of peptides in vivo. Conjugation or physical encapsulation of peptides to various nanocarriers, such as liposomes, micelles and solid-lipid nanoparticles, has improved their in vivo performance multi-fold. The amenability of peptides to modification in chemistry and functionalization with suitable nanocarriers are very relevant aspects in their use and have led to the use of 'smart' nanoparticles with suitable linker chemistries that favor peptide targeting or release at the desired sites, minimizing off-target effects. This review focuses on how nanotechnology has been used to improve the number of peptide applications. The paper also focuses on the chemistry behind peptide conjugation to nanocarriers, the commonly employed linker chemistries and the several improvements that have already been achieved in the areas of peptide use with the help of nanotechnology. PMID:26279082