Science.gov

Sample records for cell-penetrating peptides decreases

  1. Cell Penetrating Peptides and Cationic Antibacterial Peptides

    PubMed Central

    Rodriguez Plaza, Jonathan G.; Morales-Nava, Rosmarbel; Diener, Christian; Schreiber, Gabriele; Gonzalez, Zyanya D.; Lara Ortiz, Maria Teresa; Ortega Blake, Ivan; Pantoja, Omar; Volkmer, Rudolf; Klipp, Edda; Herrmann, Andreas; Del Rio, Gabriel

    2014-01-01

    Cell penetrating peptides (CPP) and cationic antibacterial peptides (CAP) have similar physicochemical properties and yet it is not understood how such similar peptides display different activities. To address this question, we used Iztli peptide 1 (IP-1) because it has both CPP and CAP activities. Combining experimental and computational modeling of the internalization of IP-1, we show it is not internalized by receptor-mediated endocytosis, yet it permeates into many different cell types, including fungi and human cells. We also show that IP-1 makes pores in the presence of high electrical potential at the membrane, such as those found in bacteria and mitochondria. These results provide the basis to understand the functional redundancy of CPPs and CAPs. PMID:24706763

  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 transport therapeutics into cells.

    PubMed

    Ramsey, Joshua D; Flynn, Nicholas H

    2015-10-01

    Nearly 30years ago, certain small, relatively nontoxic peptides were discovered to be capable of traversing the cell membrane. These cell-penetrating peptides, as they are now called, have been shown to not only be capable of crossing the cell membrane themselves but can also carry many different therapeutic agents into cells, including small molecules, plasmid DNA, siRNA, therapeutic proteins, viruses, imaging agents, and other various nanoparticles. Many cell-penetrating peptides have been derived from natural proteins, but several other cell-penetrating peptides have been developed that are either chimeric or completely synthetic. How cell-penetrating peptides are internalized into cells has been a topic of debate, with some peptides seemingly entering cells through an endocytic mechanism and others by directly penetrating the cell membrane. Although the entry mechanism is still not entirely understood, it seems to be dependent on the peptide type, the peptide concentration, the cargo the peptide transports, and the cell type tested. With new intracellular disease targets being discovered, cell-penetrating peptides offer an exciting approach for delivering drugs to these intracellular targets. There are hundreds of cell-penetrating peptides being studied for drug delivery, and ongoing studies are demonstrating their success both in vitro and in vivo. PMID:26210404

  4. Prediction of cell-penetrating peptides with feature selection techniques.

    PubMed

    Tang, Hua; Su, Zhen-Dong; Wei, Huan-Huan; Chen, Wei; Lin, Hao

    2016-08-12

    Cell-penetrating peptides are a group of peptides which can transport different types of cargo molecules such as drugs across plasma membrane and have been applied in the treatment of various diseases. Thus, the accurate prediction of cell-penetrating peptides with bioinformatics methods will accelerate the development of drug delivery systems. The study aims to develop a powerful model to accurately identify cell-penetrating peptides. At first, the peptides were translated into a set of vectors with the same dimension by using dipeptide compositions. Secondly, the Analysis of Variance-based technique was used to reduce the dimension of the vector and explore the optimized features. Finally, the support vector machine was utilized to discriminate cell-penetrating peptides from non-cell-penetrating peptides. The five-fold cross-validated results showed that our proposed method could achieve an overall prediction accuracy of 83.6%. Based on the proposed model, we constructed a free webserver called C2Pred (http://lin.uestc.edu.cn/server/C2Pred). PMID:27291150

  5. Synthesis and studies on cell-penetrating peptides.

    PubMed

    Bertrand, Jean-Remi; Malvy, Claude; Auguste, Tiphanie; Tóth, Gábor K; Kiss-Ivánkovits, Orsolya; Illyés, Eszter; Hollósi, Miklós; Bottka, Sándor; Laczkó, Ilona

    2009-07-01

    The ability of different synthetic cell penetrating peptides, as Antennapedia (wild and Phe(6) mutated penetratins), flock house virus, and integrin peptides to form complexes with a 25mer antisense oligonucleotide was compared and their conformation was determined by circular dichroism spectroscopy. The efficiency for oligonucleotide delivery into cells was measured using peptides labeled with a coumarin derivative showing blue fluorescence and the fluorescein-labeled antisense oligonucleotide showing green fluorescence. Fluorescence due to the excitation energy transfer confirmed the interaction of the antisense oligonucleotide and cell-penetrating peptides. The most efficient oligonucleotide delivery was found for penetratins. Comparison of the two types of penetratins shows that the wild-type penetratin proved to be more efficient than mutated penetratin. The paper also emphasizes that the attachment of a fluorescent label may have an effect on the conformation and flexibility of cell-penetrating peptides that must be taken into consideration when evaluating biological experiments. PMID:19552459

  6. Strategies to stabilize cell penetrating peptides for in vivo applications.

    PubMed

    Fominaya, Jesús; Bravo, Jerónimo; Rebollo, Angelita

    2015-10-01

    In the era of biomedicines and engineered carrier systems, cell penetrating peptides (CPPs) have been established as a promising tool for therapeutic application. Likewise, other therapeutic peptides, successful in vivo application of CPPs will strongly depend on peptide stability, the bottleneck for this type of biodegradable molecules. In this review, the authors describe the current knowledge of the in vivo degradation for known CPPs and the different strategies available to provide a higher resistance to metabolic degradation while preserving cell penetration efficiency. Peptide stability can be improved by different means, either modifying the structure to make it unrecognizable to proteases, or preventing access of proteolytic enzymes by applying conformation restriction or shielding strategies. PMID:26448473

  7. Chemical-Functional Diversity in Cell-Penetrating Peptides

    PubMed Central

    Stalmans, Sofie; Wynendaele, Evelien; Bracke, Nathalie; Gevaert, Bert; D’Hondt, Matthias; Peremans, Kathelijne; Burvenich, Christian; De Spiegeleer, Bart

    2013-01-01

    Cell-penetrating peptides (CPPs) are a promising tool to overcome cell membrane barriers. They have already been successfully applied as carriers for several problematic cargoes, like e.g. plasmid DNA and (si)RNA, opening doors for new therapeutics. Although several hundreds of CPPs are already described in the literature, only a few commercial applications of CPPs are currently available. Cellular uptake studies of these peptides suffer from inconsistencies in used techniques and other experimental conditions, leading to uncertainties about their uptake mechanisms and structural properties. To clarify the structural characteristics influencing the cell-penetrating properties of peptides, the chemical-functional space of peptides, already investigated for cellular uptake, was explored. For 186 peptides, a new cell-penetrating (CP)-response was proposed, based upon the scattered quantitative results for cellular influx available in the literature. Principal component analysis (PCA) and a quantitative structure-property relationship study (QSPR), using chemo-molecular descriptors and our newly defined CP-response, learned that besides typical well-known properties of CPPs, i.e. positive charge and amphipathicity, the shape, structure complexity and the 3D-pattern of constituting atoms influence the cellular uptake capacity of peptides. PMID:23951237

  8. Prediction of cell penetrating peptides by support vector machines.

    PubMed

    Sanders, William S; Johnston, C Ian; Bridges, Susan M; Burgess, Shane C; Willeford, Kenneth O

    2011-07-01

    Cell penetrating peptides (CPPs) are those peptides that can transverse cell membranes to enter cells. Once inside the cell, different CPPs can localize to different cellular components and perform different roles. Some generate pore-forming complexes resulting in the destruction of cells while others localize to various organelles. Use of machine learning methods to predict potential new CPPs will enable more rapid screening for applications such as drug delivery. We have investigated the influence of the composition of training datasets on the ability to classify peptides as cell penetrating using support vector machines (SVMs). We identified 111 known CPPs and 34 known non-penetrating peptides from the literature and commercial vendors and used several approaches to build training data sets for the classifiers. Features were calculated from the datasets using a set of basic biochemical properties combined with features from the literature determined to be relevant in the prediction of CPPs. Our results using different training datasets confirm the importance of a balanced training set with approximately equal number of positive and negative examples. The SVM based classifiers have greater classification accuracy than previously reported methods for the prediction of CPPs, and because they use primary biochemical properties of the peptides as features, these classifiers provide insight into the properties needed for cell-penetration. To confirm our SVM classifications, a subset of peptides classified as either penetrating or non-penetrating was selected for synthesis and experimental validation. Of the synthesized peptides predicted to be CPPs, 100% of these peptides were shown to be penetrating. PMID:21779156

  9. Applications of Cell-Penetrating Peptides for Tumor Targeting and Future Cancer Therapies

    PubMed Central

    Regberg, Jakob; Srimanee, Artita; Langel, Ülo

    2012-01-01

    Cell-penetrating peptides provide a highly promising strategy for intracellular drug delivery. One relevant clinical application of cell-penetrating peptides is cancer therapeutics. Peptide based delivery could increase the uptake of drugs in tumor cells and thereby increase the efficacy of the treatment, either of conventional small molecular drugs or oligonucleotide based therapeutics. This review is focused on the cancer applications of cell penetrating peptides as delivery systems; different aspects of drug loading, cargoes and delivery are discussed together with methods for targeted delivery, activatable cell-penetrating peptides and transducible agents coupled to cell-penetrating peptides. PMID:24280701

  10. 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

  11. 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

  12. 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

  13. 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

  14. Quality control of cationic cell-penetrating peptides.

    PubMed

    Stalmans, Sofie; Gevaert, Bert; Verbeke, Frederick; D'Hondt, Matthias; Bracke, Nathalie; Wynendaele, Evelien; De Spiegeleer, Bart

    2016-01-01

    During fundamental research, it is recommended to evaluate the test compound identity and purity in order to obtain reliable study outcomes. For peptides, quality control (QC) analyses are routinely performed using reversed-phase liquid chromatography coupled to an ultraviolet (UV) detector system. These traditional QC methods, using a C18 column and a linear gradient with formic acid (FA) as acidic modifier in the mobile phase, might not result in optimal chromatographic performance for basic peptides due to their cationic nature and hence may lead to erroneous results. Therefore, the influence of the used chromatographic system on the final QC results of basic peptides was evaluated using five cationic cell-penetrating peptides and five C18-chromatographic systems, differing in the column particle size (high performance liquid chromatography (HPLC) versus ultra-high performance liquid chromatography (UHPLC)), the acidic modifier (FA versus trifluoroacetic acid (TFA)), and the column temperature (30 °C versus 60 °C). Our results indicate that a UHPLC system with the C18 column thermostated at 30 °C and a mobile phase containing TFA, provides the most suitable routine QC analysis method for cationic peptides, outperforming in sensitivity and resolution compared to the other systems. We also demonstrate the use of a single quad mass spectrometry (MS) detector system during QC analysis of (cationic) peptides, allowing identification of the peptide and its impurities, as well as the evaluation of the peak purity. PMID:26397208

  15. Bioportide: an emergent concept of bioactive cell-penetrating peptides.

    PubMed

    Howl, John; Matou-Nasri, Sabine; West, David C; Farquhar, Michelle; Slaninová, Jiřina; Ostenson, Claes-Göran; Zorko, Matjaz; Ostlund, Pernilla; Kumar, Shant; Langel, Ulo; McKeating, Jane; Jones, Sarah

    2012-09-01

    Cell-penetrating peptides (CPPs) have proven utility for the highly efficient intracellular delivery of bioactive cargoes that include peptides, proteins, and oligonucleotides. The many strategies developed to utilize CPPs solely as pharmacokinetic modifiers necessarily requires them to be relatively inert. Moreover, it is feasible to combine one or multiple CPPs with bioactive cargoes either by direct chemical conjugation or, more rarely, as non-covalent complexes. In terms of the message-address hypothesis, this combination of cargo (message) linked to a CPP (address) as a tandem construct conforms to the sychnological organization. More recently, we have introduced the term bioportide to describe monomeric CPPs that are intrinsically bioactive. Herein, we describe the design and biochemical properties of two rhegnylogically organized monometic CPPs that collectively modulate a variety of biological and pathophysiological phenomena. Thus, camptide, a cell-penetrant sequence located within the first intracellular loop of a human calcitonin receptor, regulates cAMP-dependent processes to modulate insulin secretion and viral infectivity. Nosangiotide, a bioportide derived from endothelial nitric oxide synthase, potently inhibits many aspects of the endothelial cell morphology and movement and displays potent anti-angiogenic activity in vivo. We conclude that, due to their capacity to translocate and target intracellular signaling events, bioportides represent an innovative generic class of bioactive agents. PMID:22527714

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

    DOE PAGESBeta

    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

  17. Reinventing Cell Penetrating Peptides Using Glycosylated Methionine Sulfonium Ion Sequences

    PubMed Central

    2015-01-01

    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. PMID:27162954

  18. 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.

  19. Cell-penetrating Peptides as Versatile Vehicles for Oligonucleotide Delivery

    PubMed Central

    Margus, Helerin; Padari, Kärt; Pooga, Margus

    2012-01-01

    Short regulatory oligonucleotides (ONs) have a great therapeutic potential for the modulation of gene expression due to their high specificity and low toxicity. The major obstacles for in vivo clinical applications of ONs are the poor permeability of plasma membrane to nucleic acids and the sensitivity of ONs to enzymatic degradation. Hence, various delivery vehicles have been developed to ensure the transduction of ONs into cells. Among these, the cell-penetrating peptides (CPPs) have gained quickly broadening popularity as promising nonviral transmembrane delivery vectors. For coupling of nucleic acids to CPPs, two distinct strategies may be applied—covalent and noncovalent. The majority of earlier studies have used covalent coupling of CPPs to ONs. However, the number of studies demonstrating very high therapeutic potential of noncovalent complexes of ONs with novel CPP-based delivery vehicles is explosively increasing. In this review, the recent developments in the application of CPP-mediated oligonucleotide delivery by noncovalent strategy will be discussed. PMID:22233581

  20. Cell Penetrating Peptides: How Do They Do It?

    PubMed Central

    Garcia, Angel E.

    2008-01-01

    Cell penetrating peptides consist of short sequences of amino acids containing a large net positive charge that are able to penetrate almost any cell, carrying with them relatively large cargoes such as proteins, oligonucleotides, and drugs. During the 10 years since their discovery, the question of how they manage to translocate across the membrane has remained unanswered. The main discussion has been centered on whether they follow an energy-independent or an energy-dependent pathway. Recently, we have discovered the possibility of an energy-independent pathway that challenges fundamental concepts associated with protein-membrane interactions (Herce and Garcia, PNAS, 104: 20805 (2007) [1]). It involves the translocation of charged residues across the hydrophobic core of the membrane and the passive diffusion of these highly charged peptides across the membrane through the formation of aqueous toroidal pores. The aim of this review is to discuss the details of the mechanism and interpret some experimental results consistent with this view. PMID:19669523

  1. Cell penetrating peptides: how do they do it?

    PubMed

    Herce, Henry D; Garcia, Angel E

    2007-12-01

    Cell penetrating peptides consist of short sequences of amino acids containing a large net positive charge that are able to penetrate almost any cell, carrying with them relatively large cargoes such as proteins, oligonucleotides, and drugs. During the 10 years since their discovery, the question of how they manage to translocate across the membrane has remained unanswered. The main discussion has been centered on whether they follow an energy-independent or an energy-dependent pathway. Recently, we have discovered the possibility of an energy-independent pathway that challenges fundamental concepts associated with protein-membrane interactions (Herce and Garcia, PNAS, 104: 20805 (2007) [1]). It involves the translocation of charged residues across the hydrophobic core of the membrane and the passive diffusion of these highly charged peptides across the membrane through the formation of aqueous toroidal pores. The aim of this review is to discuss the details of the mechanism and interpret some experimental results consistent with this view. PMID:19669523

  2. 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

  3. Translocation of Cell Penetrating Peptide Engrafted Nanoparticles Across Skin Layers

    PubMed Central

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

    2010-01-01

    The objective of the current study was to evaluate the ability of cell penetrating peptides (CPP) to translocate the lipid payload into the skin layers. Fluorescent dye (DID-oil) encapsulated nano lipid crystal nanoparticles (FNLCN) were prepared using Compritol, Miglyol and DOGS-NTA-Ni lipids by hot melt homogenization technique. The FNLCN surface was coated with TAT peptide (FNLCNT) or control YKA peptide (FNLCNY) and in vitro rat skin permeation studies were performed using Franz diffusion cells. Observation of lateral skin sections obtained using cryotome with a confocal microscope demonstrated that skin permeation of FNLCNT was time dependent and after 24 h, fluorescence was observed upto a depth of 120 µm which was localized in the hair follicles and epidermis. In case of FNLCN and FNLCNY formulations fluorescence was mainly observed in the hair follicles. This observation was further supported by confocal Raman spectroscopy where higher fluorescence signal intensity was observed at 80 and 120 µm depth with FNLCNT treated skin and intensity of fluorescence peaks was in the ratio of 2:1:1 and 5:3:1 for FNLCNT, FNLCN, and FNLCNY treated skin sections, respectively. Furthermore, replacement of DID-oil with celecoxib (Cxb), a model lipophilic drug showed similar results and after 24 h, the CXBNT formulation increased the Cxb concentration in SC by 3 and 6 fold and in epidermis by 2 and 3 fold as compared to CXBN and CXBNY formulations respectively. Our results strongly suggest that CPP can translocate nanoparticles with their payloads into deeper skin layers. PMID:20413152

  4. Methods to follow intracellular trafficking of cell-penetrating peptides.

    PubMed

    Pärnaste, Ly; Arukuusk, Piret; Zagato, Elisa; Braeckmans, Kevin; Langel, Ülo

    2016-01-01

    Cell-penetrating peptides (CPPs) are efficient vehicles to transport bioactive molecules into the cells. Despite numerous studies the exact mechanism by which CPPs facilitate delivery of cargo to its intracellular target is still debated. The current work presents methods that can be used for tracking CPP/pDNA complexes through endosomal transport and show the role of endosomal transport in the delivery of cargo. Separation of endosomal vesicles by differential centrifugation enables to pinpoint the localization of delivered cargo without labeling it and gives important quantitative information about pDNA trafficing in certain endosomal compartments. Single particle tracking (SPT) allows following individual CPP/cargo complex through endosomal path in live cells, using fluoresently labled cargo and green fluoresent protein expressing cells. These two different methods show similar results about tested NickFect/pDNA complexes intracellular trafficing. NF51 facilitates rapid internalization of complexes into the cells, prolongs their stay in early endosomes and promotes release to cytosol. NF1 is less capable to induce endosomal release and higher amount of complexes are routed to lysosomes for degradation. Our findings offer potential delivery vector for in vivo applications, NF51, where endosomal entrapment has been allayed. Furthermore, these methods are valuable tools to study other CPP-based delivery systems. PMID:26460120

  5. 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

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

    PubMed Central

    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

  7. pH-responsive PepFect cell-penetrating peptides.

    PubMed

    Regberg, Jakob; Vasconcelos, Luis; Madani, Fatemeh; Langel, Ülo; Hällbrink, Mattias

    2016-03-30

    A series of cell-penetrating PepFect peptide analogues was developed by substitutions of the galanin-derived N-terminal sequence. Histidine modifications were incorporated in order to make the peptides pH-responsive. The peptides were all able to form non-covalent complexes with an oligonucleotide cargo by co-incubation in buffer. The complexes were characterized by dynamic light scattering and circular dichroism, and an assay to evaluate the peptide-cargo affinity was developed. Cellular bioactivity was studied in HeLa cells using a luciferase-based splice correction assay. In addition, the membrane interactions of the peptides in large unilammelar vesicles was studied using a calcein leakage assay. The effects of substitutions were found to be dependent of the non-modified, C-terminal sequence of the peptides; for analogues of PepFect 3 we observed an increase in membrane activity and bioactivity for histidine-containing analogues, whereas the same modifications introduced to PepFect 14 lead to a decreased bioactivity. Peptides modified with a leucine/histidine sequence were found to be pH responsive, complexes formed from these peptides were small at pH 7 and grew under acidic conditions. The most promising of the novel PepFect 3 analogues, PepFect 132 has a significantly higher bioactivity and membrane activity than the parent peptide PepFect 3. PMID:26821060

  8. Sensing lymphoma cells based on a cell-penetrating/apoptosis-inducing/electron-transfer peptide probe.

    PubMed

    Sugawara, Kazuharu; Shinohara, Hiroki; Kadoya, Toshihiko; Kuramitz, Hideki

    2016-06-14

    To electrochemically sense lymphoma cells (U937), we fabricated a multifunctional peptide probe that consists of cell-penetrating/apoptosis-inducing/electron-transfer peptides. Electron-transfer peptides derive from cysteine residue combined with the C-terminals of four tyrosine residues (Y4). A peptide whereby Y4C is bound to the C-terminals of protegrin 1 (RGGRLCYCRRRFCVCVGR-NH2) is known to be an apoptosis-inducing agent against U937 cells, and is referred to as a peptide-1 probe. An oxidation response of the peptide-1 probe has been observed due to a phenolic hydroxyl group, and this response is decreased by the uptake of the peptide probe into the cells. To improve the cell membrane permeability against U937 cells, the RGGR at the N-terminals of the peptide-1 probe was replaced by RRRR (peptide-2 probe). In contrast, RNRCKGTDVQAWY4C (peptide-3 probe), which recognizes ovalbumin, was constructed as a control. Compared with the other probes, the change in the peak current of the peptide-2 probe was the greatest at low concentrations and occurred in a short amount of time. Therefore, the cell membrane permeability of the peptide-2 probe was increased based on the arginine residues and the apoptosis-inducing peptides. The peak current was linear and ranged from 100 to 1000 cells/ml. The relative standard deviation of 600 cells/ml was 5.0% (n = 5). Furthermore, the membrane permeability of the peptide probes was confirmed using fluorescent dye. PMID:27181650

  9. Rational design of a series of novel amphipathic cell-penetrating peptides.

    PubMed

    Regberg, Jakob; Srimanee, Artita; Erlandsson, Mikael; Sillard, Rannar; Dobchev, Dimitar A; Karelson, Mati; Langel, Ulo

    2014-04-10

    A series of novel, amphipathic cell-penetrating peptides was developed based on a combination of the model amphipathic peptide sequence and modifications based on the strategies developed for PepFect and NickFect peptides. The aim was to study the role of amphipathicity for peptide uptake and to investigate if the modifications developed for PepFect peptides could be used to improve the uptake of another class of cell-penetrating peptides. The peptides were synthesized by solid phase peptide synthesis and characterized by circular dichroism spectroscopy. Non-covalent peptide-plasmid complexes were formed by co-incubation of the peptides and plasmids in water solution. The complexes were characterized by dynamic light scattering and cellular uptake of the complexes was studied in a luciferase-based plasmid transfection assay. A quantitative structure-activity relationship (QSAR) model of cellular uptake was developed using descriptors including hydrogen bonding, peptide charge and positions of nitrogen atoms. The peptides were found to be non-toxic and could efficiently transfect cells with plasmid DNA. Cellular uptake data was correlated to QSAR predictions and the predicted biological effects obtained from the model correlated well with experimental data. The QSAR model could improve the understanding of structural requirements for cell penetration, or could potentially be used to predict more efficient cell-penetrating peptides. PMID:24463071

  10. 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

  11. 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 ...

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

    PubMed Central

    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. PMID:26814673

  13. 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.

  14. Glycosylated cell-penetrating peptides and their conjugates to a proapoptotic peptide: preparation by click chemistry and cell viability studies

    PubMed Central

    Dutot, Laurence; Lécorché, Pascaline; Burlina, Fabienne; Marquant, Rodrigue; Point, Vanessa; Sagan, Sandrine; Chassaing, Gérard; Mallet, Jean-Maurice

    2009-01-01

    Cell-penetrating peptides (CPPs), which are usually short basic peptides, are able to cross cell membranes and convey bioactive cargoes inside cells. CPPs have been widely used to deliver inside cells peptides, proteins, and oligonucleotides; however, their entry mechanisms still remain controversial. A major problem concerning CPPs remains their lack of selectivity to target a specific type of cell and/or an intracellular component. We have previously shown that myristoylation of one of these CPPs affected the intracellular distribution of the cargo. We report here on the synthesis of glycosylated analogs of the cell-penetrating peptide (R6/W3): Ac-RRWWRRWRR-NH2. One, two, or three galactose(s), with or without a spacer, were introduced into the sequence of this nonapeptide via a triazole link, the Huisgen reaction being achieved on a solid support. Four of these glycosylated CPPs were coupled via a disulfide bridge to the proapoptotic KLAK peptide, (KLAKLAKKLAKLAK), which alone does not enter into cells. The effect on cell viability and the uptake efficiency of different glycosylated conjugates were studied on CHO cells and were compared to those of the nonglycosylated conjugates: (R6/W3)S-S-KLAK and penetratinS-S-KLAK. We show that glycosylation significantly increases the cell viability of CHO cells compared to the nonglycosylated conjugates and concomitantly decreases the internalization of the KLAK cargo. These results suggest that glycosylation of CPP may be a key point in targeting specific cells. Electronic supplementary material The online version of this article (doi:10.1007/s12154-009-0031-9) contains supplementary material, which is available to authorized users. PMID:19899012

  15. An unusual cell penetrating peptide identified using a plasmid display-based functional selection platform

    PubMed Central

    Gao, Shan; Simon, Melissa J.; Hue, Christopher D.; Morrison, Barclay; Banta, Scott

    2011-01-01

    Cell penetrating peptides (CPPs) have tremendous potential for use in gene and drug delivery applications. The selection of new CPPs with desired capabilities from randomized peptide libraries is challenging, since the CPP phenotype is a complex selection target. Here we report the discovery of an unusual new CPP from a randomized peptide library using a functional selection system based on plasmid display (PD). After four rounds of screening of a 14-mer peptide library over PC12 cells, several peptides were identified and tested for their ability to deliver the green fluorescent protein (GFP). One peptide (SG3) exhibited a cell penetrating phenotype, however unlike other well-known CPPs such as TAT or Penetratin, the newly identified peptide was not highly cationic. The PD protocol necessitated the addition of a cationic lipid (Lipofectamine2000), and in the presence of this compound, the SG3 peptide significantly outperformed the well-known TAT CPP in the delivery of GFP to PC12 cells and primary astrocytes. When the SG3 peptide was fused to the pro-apoptotic BH3 peptide from the Bak protein, significant cell death was induced in cultured primary astrocytes, indicating relevant, intracellular delivery of a functional cargo. The PD platform is a useful method for identifying functional new CPPs from randomized libraries with unique delivery capabilities. PMID:21291271

  16. 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

  17. 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

  18. 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

  19. 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

  20. Transduction of peptides and proteins into live cells by cell penetrating peptides.

    PubMed

    Mussbach, Franziska; Franke, Martin; Zoch, Ansgar; Schaefer, Buerk; Reissmann, Siegmund

    2011-12-01

    Internalization of peptides and proteins into live cells is an essential prerequisite for studies on intracellular signal pathways, for treatment of certain microbial diseases and for signal transduction therapy, especially for cancer treatment. Cell penetrating peptides (CPPs) facilitate the transport of cargo-proteins through the cell membrane into live cells. CPPs which allow formation of non-covalent complexes with the cargo are used primarily in this study due to the relatively easy handling procedure. Efficiency of the protein uptake is estimated qualitatively by fluorescence microscopy and quantitatively by SDS-PAGE. Using the CPP cocktail JBS-Proteoducin, the intracellular concentrations of a secondary antibody and bovine serum albumin can reach the micromolar range. Internalization of antibodies allows mediation of intracellular pathways including knock down of signal transduction. The high specificity and affinity of antibodies makes them potentially more powerful than siRNA. Thus, CPPs represent a significant new possibility to study signal transduction processes in competition or in comparison to the commonly used other techniques. To estimate the highest attainable intracellular concentrations of cargo proteins, the CPPs are tested for cytotoxicity. Cell viability and membrane integrity relative to concentration of CPPs are investigated. Viability as estimated by the reductive activity of mitochondria (MTT-test) is more sensitive to higher concentrations of CPPs versus membrane integrity, as measured by the release of dead cell protease. Distinct differences in uptake efficiency and cytotoxic effects are found using six different CPPs and six different adhesion and suspension cell lines. PMID:21826709

  1. Mechanism of the Cell-Penetrating Peptide Transportan 10 Permeation of Lipid Bilayers

    PubMed Central

    Yandek, Lindsay E.; Pokorny, Antje; Florén, Anders; Knoelke, Kristina; Langel, Ülo; Almeida, Paulo F. F.

    2007-01-01

    The mechanism of the interaction between the cell-penetrating peptide transportan 10 (tp10) and phospholipid membranes was investigated. Tp10 induces graded release of the contents of phospholipid vesicles. The kinetics of peptide association with vesicles and peptide-induced dye efflux from the vesicle lumen were examined experimentally by stopped-flow fluorescence. The experimental kinetics were analyzed by directly fitting to the data the numerical solution of mathematical kinetic models. A very good global fit was obtained using a model in which tp10 binds to the membrane surface and perturbs it because of the mass imbalance thus created across the bilayer. The perturbed bilayer state allows peptide monomers to insert transiently into its hydrophobic core and cross the membrane, until the peptide mass imbalance is dissipated. In that transient state tp10 “catalyzes” dye efflux from the vesicle lumen. These conclusions are consistent with recent reports that used molecular dynamics simulations to study the interactions between peptide antimicrobials and phospholipid bilayers. A thermodynamic analysis of tp10 binding and insertion in the bilayer using water-membrane transfer hydrophobicity scales is entirely consistent with the model proposed. A small bilayer perturbation is both necessary and sufficient to achieve very good agreement with the model, indicating that the role of the lipids must be included to understand the mechanism of cell-penetrating and antimicrobial peptides. PMID:17218466

  2. Photodamage of Lipid Bilayers by Irradiation of a Fluorescently Labeled Cell-Penetrating Peptide

    PubMed Central

    Meerovich, Igor; Muthukrishnan, Nandhini; Johnson, Gregory A.; Erazo-Oliveras, Alfredo; Pellois, Jean-Philippe

    2013-01-01

    Background Fluorescently labeled cell-penetrating peptides can translocate into cells by endocytosis and upon light irradiation, lyse the endocytic vesicles. This photo-inducible endosomolytic activity of Fl-CPPs can be used to efficiently deliver macromolecules such as proteins and nucleic acids and other small organic molecules into the cytosol of live cells. The requirement of a light trigger to induce photolysis provides a more spatial and temporal control to the intracellular delivery process. Methods In this report, we examine the molecular level mechanisms by which cell-penetrating peptides such as TAT when labeled with small organic fluorophore molecules acquire a photo-induced lytic activity using a simplified model of lipid vesicles. Results The peptide TAT labeled with 5(6)-carboxy-tetramethylrhodamine binds to negatively charged phospholipids, thereby bringing the fluorophore in close proximity to the membrane of liposomes. Upon light irradiation, the excited fluorophore produces reactive oxygen species at the lipid bilayer and oxidation of the membrane is achieved. In addition, the fluorescent peptide causes aggregation of photo-oxidized lipids, an activity that requires the presence of arginine residues in the peptide sequence. Conclusions These results suggest that the cell penetrating peptide plays a dual role. On one hand, TAT targets a conjugated fluorophore to membranes. On the other hand, TAT participates directly in the destabilization of photosensitized membranes. Peptide and fluorophore therefore appear to act in synergy to destroy membranes efficiently. General Significance Understanding the mechanism behind Fl-CPP mediated membrane photodamage will help to design optimally photo-endosomolytic compounds. PMID:24135456

  3. Backbone rigidity and static presentation of guanidinium groups increases cellular uptake of arginine-rich cell-penetrating peptides.

    PubMed

    Lättig-Tünnemann, Gisela; Prinz, Manuel; Hoffmann, Daniel; Behlke, Joachim; Palm-Apergi, Caroline; Morano, Ingo; Herce, Henry D; Cardoso, M Cristina

    2011-01-01

    In addition to endocytosis-mediated cellular uptake, hydrophilic cell-penetrating peptides are able to traverse biological membranes in a non-endocytic mode termed transduction, resulting in immediate bioavailability. Here we analysed structural requirements for the non-endocytic uptake mode of arginine-rich cell-penetrating peptides, by a combination of live-cell microscopy, molecular dynamics simulations and analytical ultracentrifugation. We demonstrate that the transduction efficiency of arginine-rich peptides increases with higher peptide structural rigidity. Consequently, cyclic arginine-rich cell-penetrating peptides showed enhanced cellular uptake kinetics relative to their linear and more flexible counterpart. We propose that guanidinium groups are forced into maximally distant positions by cyclization. This orientation increases membrane contacts leading to enhanced cell penetration. PMID:21878907

  4. Backbone rigidity and static presentation of guanidinium groups increases cellular uptake of arginine-rich cell-penetrating peptides

    PubMed Central

    Lättig-Tünnemann, Gisela; Prinz, Manuel; Hoffmann, Daniel; Behlke, Joachim; Palm-Apergi, Caroline; Morano, Ingo; Herce, Henry D.; Cardoso, M. Cristina

    2011-01-01

    In addition to endocytosis-mediated cellular uptake, hydrophilic cell-penetrating peptides are able to traverse biological membranes in a non-endocytic mode termed transduction, resulting in immediate bioavailability. Here we analysed structural requirements for the non-endocytic uptake mode of arginine-rich cell-penetrating peptides, by a combination of live-cell microscopy, molecular dynamics simulations and analytical ultracentrifugation. We demonstrate that the transduction efficiency of arginine-rich peptides increases with higher peptide structural rigidity. Consequently, cyclic arginine-rich cell-penetrating peptides showed enhanced cellular uptake kinetics relative to their linear and more flexible counterpart. We propose that guanidinium groups are forced into maximally distant positions by cyclization. This orientation increases membrane contacts leading to enhanced cell penetration. PMID:21878907

  5. Effect of cell-penetrating peptides on the nasal absorption of insulin.

    PubMed

    Khafagy, El-Sayed; Morishita, Mariko; Isowa, Koichi; Imai, Jun; Takayama, Kozo

    2009-01-19

    The goal of this study was to evaluate whether cell-penetrating peptides (CPPs) affect the nasal absorption of insulin. L- or D-forms of penetratin, or the L- or D-forms of octaarginine (L- or D-R8), was used as first time for nasal insulin delivery. Furthermore, the concentration of lactate dehydrogenase (LDH) in nasal lavage fluid was determined and a histopathological study of nasal respiratory epithelium was conducted. CPPs dramatically increased nasal insulin absorption, and it was more pronounced for L- and D-penetratin than L- or D-R8. L-penetratin was the most effective promoter of insulin absorption compared with others CPPs. A dose-dependent relationship of L-penetratin and insulin bioavailability was statically significant. The pharmacological availability and bioavailability of nasally administered insulin was up to 76.7% and 50.7% relative to the subcutaneous route, respectively. In contrast, increasing the D-penetratin concentration decreased the efficiency of nasal insulin absorption. There was no significant difference in the release of LDH in nasal lavage fluid and the integrity of nasal respiratory epithelium when L-penetratin was present. In conclusion, these data demonstrate that L-penetratin markedly increased the permeability of insulin across the nasal membrane without causing detectable damage to the integrity of cells in the nasal respiratory mucosa. PMID:18930084

  6. 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

  7. A Novel Cell-Penetrating Peptide Derived from Human Eosinophil Cationic Protein

    PubMed Central

    Fang, Shun-lung; Fan, Tan-chi; Fu, Hua-Wen; Chen, Chien-Jung; Hwang, Chi-Shin; Hung, Ta-Jen; Lin, Lih-Yuan; Chang, Margaret Dah-Tsyr

    2013-01-01

    Cell-penetrating peptides (CPPs) are short peptides which can carry various types of molecules into cells; however, although most CPPs rapidly penetrate cells in vitro, their in vivo tissue-targeting specificities are low. Herein, we describe cell-binding, internalization, and targeting characteristics of a newly identified 10-residue CPP, denoted ECP32–41, derived from the core heparin-binding motif of human eosinophil cationic protein (ECP). Besides traditional emphasis on positively charged residues, the presence of cysteine and tryptophan residues was demonstrated to be essential for internalization. ECP32–41 entered Beas-2B and wild-type CHO-K1 cells, but not CHO cells lacking of cell-surface glycosaminoglycans (GAGs), indicating that binding of ECP32–41 to cell-surface GAGs was required for internalization. When cells were cultured with GAGs or pre-treated with GAG-digesting enzymes, significant decreases in ECP32–41 internalization were observed, suggesting that cell-surface GAGs, especially heparan sulfate proteoglycans were necessary for ECP32–41 attachment and penetration. Furthermore, treatment with pharmacological agents identified two forms of energy-dependent endocytosis, lipid-raft endocytosis and macropinocytosis, as the major ECP32–41 internalization routes. ECP32–41 was demonstrated to transport various cargoes including fluorescent chemical, fluorescent protein, and peptidomimetic drug into cultured Beas-2B cells in vitro, and targeted broncho-epithelial and intestinal villi tissues in vivo. Hence this CPP has the potential to serve as a novel vehicle for intracellular delivery of biomolecules or medicines, especially for the treatment of pulmonary or gastrointestinal diseases. PMID:23469189

  8. A cell penetrating peptide-integrated and enediyne-energized fusion protein shows potent antitumor activity.

    PubMed

    Ru, Qin; Shang, Bo-Yang; Miao, Qing-Fang; Li, Liang; Wu, Shu-Ying; Gao, Rui-Juan; Zhen, Yong-Su

    2012-11-20

    Arginine-rich peptides belong to a subclass of cell penetrating peptides that are taken up by living cells and can be detected freely diffusing inside the cytoplasm and nucleoplasm. This phenomenon has been attributed to either an endocytotic mode of uptake and a subsequent release from vesicles or a direct membrane penetration. Lidamycin is an antitumor antibiotic, which consists of an active enediyne chromophore (AE) and a noncovalently bound apoprotein (LDP). In the present study, a fusion protein (Arg)(9)-LDP composed of cell penetrating peptide (Arg)(9) and LDP was prepared by DNA recombination, and the enediyne-energized fusion protein (Arg)(9)-LDP-AE was prepared by molecular reconstitution. The data in fixed cells demonstrated that (Arg)(9)-LDP could rapidly enter cells, and the results based on fluorescence activated cell sorting indicated that the major route for (Arg)(9)-mediated cellular uptake of protein molecules was endocytosis. (Arg)(9)-LDP-AE demonstrated more potent cytotoxicity against different carcinoma cell lines than lidamycin in vitro. In the mouse hepatoma 22 model, (Arg)(9)-LDP-AE (0.3mg/kg) suppressed the tumor growth by 89.2%, whereas lidamycin (0.05 mg/kg) by 74.6%. Furthermore, in the glioma U87 xenograft model in nude mice, (Arg)(9)-LDP-AE at 0.2mg/kg suppressed tumor growth by 88.8%, compared with that of lidamycin by 62.9% at 0.05 mg/kg. No obvious toxic effects were observed in all groups during treatments. The results showed that energized fusion protein (Arg)(9)-LDP-AE was more effective than lidamycin and would be a promising candidate for glioma therapy. In addition, this approach to manufacturing fusion proteins might serve as a technology platform for the development of new cell penetrating peptides-based drugs. PMID:22982402

  9. Enhancing cellular uptake of activable cell-penetrating peptide-doxorubicin conjugate by enzymatic cleavage.

    PubMed

    Shi, Nian-Qiu; Gao, Wei; Xiang, Bai; Qi, Xian-Rong

    2012-01-01

    The use of activable cell-penetrating peptides (ACPPs) as molecular imaging probes is a promising new approach for the visualization of enzymes. The cell-penetrating function of a polycationic cell-penetrating peptide (CPP) is efficiently blocked by intramolecular electrostatic interactions with a polyanionic peptide. Proteolysis of a proteinase-sensitive substrate present between the CPP and polyanionic peptide affords dissociation of both domains and enables the activated CPP to enter cells. This ACPP strategy could also be used to modify antitumor agents for tumor-targeting therapy. Here, we aimed to develop a conjugate of ACPP with antitumor drug doxorubicin (DOX) sensitive to matrix metalloproteinase-2 and -9 (MMP-2/9) for tumor-targeting therapy purposes. The ACPP-DOX conjugate was successfully synthesized. Enzymatic cleavage of ACPP-DOX conjugate by matrix metalloproteinase (MMP)-2/9 indicated that the activation of ACPP-DOX occurred in an enzyme concentration-dependent manner. Flow cytometry and laser confocal microscope studies revealed that the cellular uptake of ACPP-DOX was enhanced after enzymatic-triggered activation and was higher in HT-1080 cells (overexpressed MMPs) than in MCF-7 cells (under-expressed MMPs). The antiproliferative assay showed that ACPP had little toxicity and that ACPP-DOX effectively inhibited HT-1080 cell proliferation. These experiments revealed that the ACPP-DOX conjugate could be triggered by MMP-2/9, which enabled the activated CPP-DOX to enter cells. ACPP-DOX conjugate may be a potential prodrug delivery system used to carry antitumor drugs for MMP-related tumor therapy. PMID:22619516

  10. Oligonucleotide delivery with cell surface binding and cell penetrating Peptide amphiphile nanospheres.

    PubMed

    Mumcuoglu, Didem; Sardan, Melis; Tekinay, Turgay; Guler, Mustafa O; Tekinay, Ayse B

    2015-05-01

    A drug delivery system designed specifically for oligonucleotide therapeutics can ameliorate the problems associated with the in vivo delivery of these molecules. The internalization of free oligonucleotides is challenging, and cytotoxicity is the main obstacle for current transfection vehicles. To develop nontoxic delivery vehicles for efficient transfection of oligonucleotides, we designed a self-assembling peptide amphiphile (PA) nanosphere delivery system decorated with cell penetrating peptides (CPPs) containing multiple arginine residues (R4 and R8), and a cell surface binding peptide (KRSR), and report the efficiency of this system in delivering G-3129, a Bcl-2 antisense oligonucleotide (AON). PA/AON (peptide amphiphile/antisense oligonucleotide) complexes were characterized with regards to their size and secondary structure, and their cellular internalization efficiencies were evaluated. The effect of the number of arginine residues on the cellular internalization was investigated by both flow cytometry and confocal imaging, and the results revealed that uptake efficiency improved as the number of arginines in the sequence increased. The combined effect of cell penetration and surface binding property on the cellular internalization and its uptake mechanism was also evaluated by mixing R8-PA and KRSR-PA. R8 and R8/KRSR decorated PAs were found to drastically increase the internalization of AONs compared to nonbioactive PA control. Overall, the KRSR-decorated self-assembled PA nanospheres were demonstrated to be noncytotoxic delivery vectors with high transfection rates and may serve as a promising delivery system for AONs. PMID:25828697

  11. 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

  12. Mechanisms of antimicrobial, cytolytic, and cell-penetrating peptides: from kinetics to thermodynamics†

    PubMed Central

    Almeida, Paulo F.; Pokorny, Antje

    2009-01-01

    The mechanisms of six different antimicrobial, cytolytic, and cell-penetrating peptides, including some of their variants, are discussed and compared. The specificity of these polypeptides varies, but they all form amphipathic α-helices when bound to membranes, and there are no striking differences in their sequences. We have examined the thermodynamics and kinetics of their interaction with phospholipid vesicles, namely binding and peptide-induced dye efflux. The thermodynamics of binding calculated using the Wimley-White interfacial hydrophobicity scale are in good agreement with the values derived from experiment. The generally accepted view that binding affinity determines functional specificity is also supported by experiment in model membranes. We now propose the hypothesis that it is the thermodynamics of peptide insertion into the membrane, from a surface-bound state, that determines the mechanism. PMID:19655791

  13. Cell Penetrating Peptide Adsorption on Magnetite and Silica Surfaces: A Computational Investigation.

    PubMed

    Grasso, Gianvito; Deriu, Marco A; Prat, Maria; Rimondini, Lia; Vernè, Enrica; Follenzi, Antonia; Danani, Andrea

    2015-07-01

    Magnetic nanoparticles (MNPs) represent one of the most promising materials as they can act as a versatile platform in the field of bionanotechnology for enhanced imaging, diagnosis, and treatment of various diseases. Silica is the most common compound for preparing coated iron oxide NPs since it improves colloidal stability and the binding affinity for various organic molecules. Biomolecules such as cell penetrating peptides (CPPs) might be employed to decorate MNPs, combining their promising physicochemical properties with a cell penetrating ability. In this work, a computational investigation on adsorption of Antennapedia homeodomain-derived penetrating peptide (pAntp) on silica and magnetite (MAG) surfaces is presented. By employing umbrella sampling molecular dynamics, we provided a quantitative estimation of the pAntp-surface adsorption free energy to highlight the influence of surface hydroxylation state on the adsorption mechanism. The interaction between peptide and surface has shown to be mainly driven by electrostatics. In case of MAG surface, also an important contribution of van der Waals (VdW) attraction was observed. Our data suggest that a competitive mechanism between MNPs and cell membrane might partially inhibit the CPP to carry out its membrane penetrating function. PMID:26042722

  14. Computer-Aided Virtual Screening and Designing of Cell-Penetrating Peptides.

    PubMed

    Gautam, Ankur; Chaudhary, Kumardeep; Kumar, Rahul; Raghava, Gajendra Pal Singh

    2015-01-01

    Cell-penetrating peptides (CPPs) have proven their potential as versatile drug delivery vehicles. Last decade has witnessed an unprecedented growth in CPP-based research, demonstrating the potential of CPPs as therapeutic candidates. In the past, many in silico algorithms have been developed for the prediction and screening of CPPs, which expedites the CPP-based research. In silico screening/prediction of CPPs followed by experimental validation seems to be a reliable, less time-consuming, and cost-effective approach. This chapter describes the prediction, screening, and designing of novel efficient CPPs using "CellPPD," an in silico tool. PMID:26202262

  15. Cell-Penetrating HIV1 TAT Peptides Can Generate Pores in Model Membranes

    PubMed Central

    Ciobanasu, Corina; Siebrasse, Jan Peter; Kubitscheck, Ulrich

    2010-01-01

    Abstract Cell-penetrating peptides like the cationic human immunodeficiency virus-1 trans-acting activator of transcription (TAT) peptide have the capability to traverse cell membranes and to deliver large molecular cargoes into the cellular interior. We used optical sectioning and state-of-the-art single-molecule microscopy to examine the passive membrane permeation of fluorescently labeled TAT peptides across the membranes of giant unilamellar vesicles (GUVs). In GUVs formed by phosphatidylcholine and cholesterol only, no translocation of TAT up to a concentration of 2 μM into the GUVs could be observed. At the same peptide concentration, but with 40 mol % of anionic phosphatidylserine in the membrane, rapid translocation of TAT peptides across the bilayers was detected. Efficient translocation of TAT peptides was observed across GUVs containing 20 mol % of phosphatidylethanolamine, which is known to induce a negative curvature into membranes. We discovered that TAT peptides are not only capable of penetrating membranes directly in a passive manner, but they were also able to form physical pores with sizes in the nanometer range, which could be passed by small dye tracer molecules. Lipid topology and anionic charge of the lipid bilayer are decisive parameters for pore formation. PMID:20655843

  16. Cell penetrating peptide delivery of splice directing oligonucleotides as a treatment for Duchenne muscular dystrophy.

    PubMed

    Betts, Corinne A; Wood, Matthew J A

    2013-01-01

    Duchenne muscular dystrophy is a severe, X-linked muscle wasting disorder caused by the absence of an integral structural protein called dystrophin. This is caused by mutations or deletions in the dystrophin gene which disrupt the reading frame, thereby halting the production of a functional protein. A number of potential therapies have been investigated for the treatment of this disease including utrophin upregulation, 'stop-codon read through' aminoglycosides and adeno-associated virus gene replacement as well as stem cell therapy. However, the most promising treatment to date is the use of antisense oligonucleotides which cause exon skipping by binding to a specific mRNA sequence, skipping the desired exon, thereby restoring the reading frame and producing a truncated yet functional protein. The results from recent 2'OMePS and morpholino clinical trials have renewed hope for Duchenne patients; however in vivo studies in a mouse model, mdx, have revealed low systemic distribution and poor delivery of oligonucleotides to affected tissues such as the brain and heart. However a variety of cell penetrating peptides directly conjugated to antisense oligonucleotides have been shown to enhance delivery in Duchenne model systems with improved systemic distribution and greater efficacy compared to 'naked' antisense oligonucleotides. These cell penetrating peptides, combined with an optimised dose and dosing regimen, as well as thorough toxicity profile have the potential to be developed into a promising treatment which may be progressed to clinical trial. PMID:23140454

  17. 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

  18. Xentry, a new class of cell-penetrating peptide uniquely equipped for delivery of drugs

    PubMed Central

    Montrose, Kristopher; Yang, Yi; Sun, Xueying; Wiles, Siouxsie; Krissansen, Geoffrey W.

    2013-01-01

    Here we describe an entirely new class of cell-penetrating peptide (CPP) represented by the short peptide Xentry (LCLRPVG) derived from an N-terminal region of the X-protein of the hepatitis B virus. Xentry permeates adherent cells using syndecan-4 as a portal for entry, and is uniquely restricted from entering syndecan-deficient, non-adherent cells, such as resting blood cells. Intravenous injection of Xentry alone or conjugated to β-galactosidase led to its delivery to most tissues in mice, except circulating blood cells. There was a predilection for uptake by epithelia. Anti-B-raf antibodies and siRNAs linked to Xentry were capable of killing B-raf-dependent melanoma cells. Xentry represents a new class of CPP with properties that are potentially advantageous for life science and therapeutic applications. PMID:23588666

  19. 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

  20. Improving the Endosomal Escape of Cell-Penetrating Peptides and Their Cargos: Strategies and Challenges

    PubMed Central

    Erazo-Oliveras, Alfredo; Muthukrishnan, Nandhini; Baker, Ryan; Wang, Ting-Yi; Pellois, Jean-Philippe

    2012-01-01

    Cell penetrating peptides (CPPs) can deliver cell-impermeable therapeutic cargos into cells. In particular, CPP-cargo conjugates tend to accumulate inside cells by endocytosis. However, they often remain trapped inside endocytic organelles and fail to reach the cytosolic space of cells efficiently. In this review, the evidence for CPP-mediated endosomal escape is discussed. In addition, several strategies that have been utilized to enhance the endosomal escape of CPP-cargos are described. The recent development of branched systems that display multiple copies of a CPP is presented. The use of viral or synthetic peptides that can disrupt the endosomal membrane upon activation by the low pH of endosomes is also discussed. Finally, we survey how CPPs labeled with chromophores can be used in combination with light to stimulate endosomal lysis. The mechanisms and challenges associated with these intracellular delivery methodologies are discussed. PMID:24223492

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

    PubMed

    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

  2. Cell-Penetrating Peptide-Functionized Quantum Dots for Intracellular Delivery

    PubMed Central

    Liu, Betty R.; Huang, Yue-Wern; Chiang, Huey-Jenn; Lee, Han-Jung

    2010-01-01

    Quantum dots (QDs) are luminescent semiconductor nanocrystals that are widely used as fluorescent probes in biomedical applications, including cellular imaging and tumor tracking. Cell-penetrating peptides (CPPs), also called protein transduction domains (PTDs), are short basic peptides that permeate cell membranes and are able to deliver a variety of macromolecule cargoes, such as DNAs, RNAs, proteins, and nanomaterials. Here we review strategies to couple QDs to CPPs, by either covalent linkages or noncovalent interactions, to provide a tool to study intracellular delivery. This facilitated transport of QDs by CPPs into cells is both simple and efficient. Accordingly, CPP-QD nanoparticles are likely to be of broad utility in biological research and advance the development of medical and pharmaceutical therapeutics. PMID:21121277

  3. The cell-type specificity and endosomal escape of cell-penetrating peptides.

    PubMed

    Feng, Jing; Tang, Liling

    2015-01-01

    Cell-penetrating peptides (CPPs) have become a great potential non-invasive carrier candidate for the delivery of various cell-impermeable therapeutic cargoes such as proteins, polypeptides and nucleic acid. However, the lack of tissue specificity and entrapment in the endocytic vesicles is the primary limitation to the application of these peptides in cargo delivery. Emphasis should be placed on developing novel methods to overcome these barriers. In this review, several current strategies to promote tissue specificity and endosomal escape of CPPs will be described, as well as the comparison of different approaches in efficacy and security. Finally, this review will be attributed to new ideas to improve the tissue specificity and cytosolic availability of CPP-cargoes. PMID:25341672

  4. 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

  5. A Cell-penetrating Peptide Suppresses Inflammation by Inhibiting NF-κB Signaling

    PubMed Central

    Wang, Yu Fu; Xu, Xiang; Fan, Xia; Zhang, Chun; Wei, Qiang; Wang, Xi; Guo, Wei; Xing, Wei; Yu, Jian; Yan, Jing-Long; Liang, Hua-Ping

    2011-01-01

    Nuclear factor-κB (NF-κB) is a central regulator of immune response and a potential target for developing anti-inflammatory agents. Mechanistic studies suggest that compounds that directly inhibit NF-κB DNA binding may block inflammation and the associated tissue damage. Thus, we attempted to discover peptides that could interfere with NF-κB signaling based on a highly conserved DNA-binding domain found in all NF-κB members. One such small peptide, designated as anti-inflammatory peptide-6 (AIP6), was characterized in the current study. AIP6 directly interacted with p65 and displayed an intrinsic cell-penetrating property. This peptide demonstrated significant anti-inflammatory effects in vitro and in vivo. In vitro, AIP6 inhibited the DNA-binding and transcriptional activities of the p65 NF-κB subunit as well as the production of inflammatory mediators in macrophages upon stimulation. Local administration of AIP6 significantly inhibited inflammation induced by zymosan in mice. Collectively, our results suggest that AIP6 is a promising lead peptide for the development of specific NF-κB inhibitors as potential anti-inflammatory agents. PMID:21556052

  6. Antibacterial Effects of a Cell-Penetrating Peptide Isolated from Kefir.

    PubMed

    Miao, Jianyin; Guo, Haoxian; Chen, Feilong; Zhao, Lichao; He, Liping; Ou, Yangwen; Huang, Manman; Zhang, Yi; Guo, Baoyan; Cao, Yong; Huang, Qingrong

    2016-04-27

    Kefir is a traditional fermented milk beverage used throughout the world for centuries. A cell-penetrating peptide, F3, was isolated from kefir by Sephadex G-50 gel filtration, DEAE-52 ion exchange, and reverse-phase high-performance liquid chromatography. F3 was determined to be a low molecular weight peptide containing one leucine and one tyrosine with two phosphate radicals. This peptide displayed antimicrobial activity across a broad spectrum of organisms including several Gram-positive and Gram-negative bacteria as well as fungi, with minimal inhibitory concentration (MIC) values ranging from 125 to 500 μg/mL. Cellular penetration and accumulation of F3 were determined by confocal laser scanning microscopy. The peptide was able to penetrate the cellular membrane of Escherichia coli and Staphylococcus aureus. Changes in cell morphology were observed by scanning electron microscopy (SEM). The results indicate that peptide F3 may be a good candidate for use as an effective biological preservative in agriculture and the food industry. PMID:27003578

  7. Wasp Mastoparans Follow the Same Mechanism as the Cell-Penetrating Peptide Transportan 10†

    PubMed Central

    Yandek, Lindsay E.; Pokorny, Antje; Almeida, Paulo F. F.

    2009-01-01

    We have been examining the mechanism and kinetics of the interactions of a selected set of peptides with phospholipid membranes, in a quantitative manner. This set was chosen to cover a broad range of physical-chemical properties and cell specificities. Mastoparan (masL) and mastoparan X (masX) are two similar peptides from the venoms of the wasps Vespula lewisii and Vespa xanthoptera, respectively, and were chosen to complete the set. The rate constants for masX association with, and dissociation from membranes are reported here for the first time. The kinetics of dye efflux induced by both mastoparans from phospholipid vesicles were also examined and quantitatively analyzed. We find that masL and masX follow the same graded kinetic model that we previously proposed for the cell-penetrating peptide transportan 10 (tp10), but with different parameters. This comparison is relevant because tp10 is derived from masL by addition of a mostly nonpolar segment of 7 residues at the N-terminus. Tp10 is more active than the mastoparans toward phosphatidylcholine vesicles, but the mastoparans are more sensitive to the effect of anionic lipids. Furthermore, the Gibbs free energies of binding and insertion of the peptides calculated using the Wimley-White transfer scales are in good agreement with the values derived from our experimental data, and are useful for understanding peptide behavior. PMID:19594111

  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. Inhibiting bladder tumor growth with a cell penetrating R11 peptide derived from the p53 C-terminus.

    PubMed

    Zhang, Tingting; Wu, Kaijie; Ding, Chen; Sun, Kangwei; Guan, Zhenfeng; Wang, Xinyang; Hsieh, Jer-Tsong; He, Dalin; Fan, Jinhai

    2015-11-10

    Urothelial carcinoma of the bladder (UCB) is the most common malignancy of the urinary tract, nearly half of which contains a mutation in TP53 gene. Hence, therapeutic approach by restoring functional p53 protein in cancer cells will be beneficial. Recent studies have demonstrated the inhibition of cancer cell growth by p53 reactivation using a peptide derived from the p53 C-terminus (p53C). However, the outcome of reactivating p53 in controlling bladder cancer development is limited by its efficiency and specificity of peptide delivery, especially in metastatic animal models. Herein, we report that the cell penetrating peptide (polyarginine, R11)-conjugated p53C can exhibit a preferential uptake and growth inhibit of UCB cells expressing either mutant or wild-type TP53 by the activation of p53-dependent pathway. R11-p53C peptide treatment of preclinical orthotopic and metastatic bladder cancer models significantly decreased the tumor burden and increased the lifespan without a significant cytotoxicity. Based on these results, we believe that R11-p53C peptide has therapeutic potential for primary and metastatic bladder cancer, and R11-mediated transduction may be a useful strategy for the therapeutic delivery of large tumor suppressor molecules to tumor cells in vitro and in vivo. PMID:26462022

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

    PubMed

    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

  11. 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

  12. 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

  13. Cell-Penetrating Peptide Induces Leaky Fusion of Liposomes Containing Late Endosome-Specific Anionic Lipid

    PubMed Central

    Yang, Sung-Tae; Zaitseva, Elena; Chernomordik, Leonid V.; Melikov, Kamran

    2010-01-01

    Cationic cell-penetrating peptides (CPPs) are a promising vehicle for the delivery of macromolecular drugs. Although many studies have indicated that CPPs enter cells by endocytosis, the mechanisms by which they cross endosomal membranes remain elusive. On the basis of experiments with liposomes, we propose that CPP escape into the cytosol is based on leaky fusion (i.e., fusion associated with the permeabilization of membranes) of the bis(monoacylglycero)phosphate (BMP)-enriched membranes of late endosomes. In our experiments, prototypic CPP HIV-1 TAT peptide did not interact with liposomes mimicking the outer leaflet of the plasma membrane, but it did induce lipid mixing and membrane leakage as it translocated into liposomes mimicking the lipid composition of late endosome. Both membrane leakage and lipid mixing depended on the BMP content and were promoted at acidic pH, which is characteristic of late endosomes. Substitution of BMP with its structural isomer, phosphatidylglycerol (PG), significantly reduced both leakage of the aqueous probe from liposomes and lipid mixing between liposomes. Although affinity of binding to TAT was similar for BMP and PG, BMP exhibited a higher tendency to support the inverted hexagonal phase than PG. Finally, membrane leakage and peptide translocation were both inhibited by inhibitors of lipid mixing, further substantiating the hypothesis that cationic peptides cross BMP-enriched membranes by inducing leaky fusion between them. PMID:20959093

  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. 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

  16. Plasmid DNA delivery by arginine-rich cell-penetrating peptides containing unnatural amino acids.

    PubMed

    Kato, Takuma; Yamashita, Hiroko; Misawa, Takashi; Nishida, Koyo; Kurihara, Masaaki; Tanaka, Masakazu; Demizu, Yosuke; Oba, Makoto

    2016-06-15

    Cell-penetrating peptides (CPPs) have been developed as drug, protein, and gene delivery tools. In the present study, arginine (Arg)-rich CPPs containing unnatural amino acids were designed to deliver plasmid DNA (pDNA). The transfection ability of one of the Arg-rich CPPs examined here was more effective than that of the Arg nonapeptide, which is the most frequently used CPP. The transfection efficiencies of Arg-rich CPPs increased with longer post-incubation times and were significantly higher at 48-h and 72-h post-incubation than that of the commercially available transfection reagent TurboFect. These Arg-rich CPPs were complexed with pDNA for a long time in cells and effectively escaped from the late endosomes/lysosomes into the cytoplasm. These results will be helpful for designing novel CPPs for pDNA delivery. PMID:27132868

  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. PMID:27576711

  18. In vivo delivery of morpholino oligos by cell-penetrating peptides.

    PubMed

    Moulton, Hong M

    2013-01-01

    Morpholino oligos (Morpholinos) are widely used tools for knocking down gene expression and are currently in a clinical trial for treatment of Duchene muscular dystrophy. A Morpholino analog has been in a clinical trial as a potential anti-bioterrorism agent for inhibiting replication of deadly Marburg viral infection. The cellular uptake of Morpholinos can been greatly increased by conjugation with cell-penetrating peptides (CPP). The use of the CPP-Morpholino conjugates (PPMOs) in vivo has been broadly demonstrated in viral, bacterial, genetic and other diseases. The following aspects of PPMOs will be discussed in this paper including chemistry, stability, antisense specificity, mechanism of cellular uptake, in vivo efficacy, tissue distribution, pharmacokinetics, toxicity and the human clinical trials. PPMOs are powerful research tools for studying gene function in animals and their properties are being improved as potential human therapeutic agents. PMID:23140456

  19. 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

  20. Macropinocytosis: searching for an endocytic identity and role in the uptake of cell penetrating peptides

    PubMed Central

    Jones, Arwyn Tomos

    2007-01-01

    Abstract Macropinocytosis defines a series of events initiated by extensive plasma membrane reorganization or ruffling to form an external macropinocytic structure that is then enclosed and internalized. The process is constitutive in some organisms and cell types but in others it is only pronounced after growth factor stimulation. Internalized macropinosomes share many features with phagosomes and both are distinguished from other forms of pinocytic vesicles by their large size, morphological heterogeneity and lack of coat structures. A paucity of information is available on other distinguishing features for macropinocytosis such as specific marker proteins and drugs that interfere with its mechanism over other endocytic processes. This has hampered efforts to characterize the dynamics of this pathway and to identify regulatory proteins that are expressed in order to allow it to proceed. Upon internalization, macropinosomes acquire regulatory proteins common to other endocytic pathways, suggesting that their identities as unique structures are short-lived. There is however less consensus regarding the overall fate of the macropinosome cargo or its limiting membrane and processes such as fusion, tubulation, recycling and regulated exocytosis have all been implicated in shaping the macropinosome and directing cargo traffic. Macropinocytosis has also been implicated in the internalization of cell penetrating peptides that are of significant interest to researchers aiming to utilize their translocation abilities to deliver therapeutic entities such as genes and proteins into cells. This review focuses on recent findings on the regulation of macropinocytosis, the intracellular fate of the macropinosome and discusses evidence for the role of this pathway as a mechanism of entry for cell penetrating peptides. PMID:17760832

  1. 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

  2. Cellular Internalization of Quantum Dots Noncovalently Conjugated with Arginine-Rich Cell-Penetrating Peptides

    PubMed Central

    Liu, Betty R.; Li, Jheng-Fong; Lu, Shu-Wan; Lee, Han-Jung; Huang, Yue-Wern; Shannon, Katie B.; Aronstam, Robert S.

    2010-01-01

    Protein transduction domains comprised of basic amino acid-rich peptides, can efficiently deliver covalently fused macromolecules into cells. Quantum dots (QDs) are luminescent semiconductor nanocrystals that are finding increasing application in biological imaging. Previous studies showed that protein transduction domains mediate the internalization of covalently attached QDs. In this study, we demonstrate that arginine-rich intracellular delivery peptides (cell-penetrating peptides; CPPs), analogs of naturally-occuring protein transduction domains, deliver noncovalently associated QDs into living cells; CPPs dramatically increase the rate and efficiency of cellular uptake of QD probes. The optimal molecular ratio between arginine-rich CPPs and QD cargoes for cellular internalization is approximately 60:1. Upon entry into cells, the QDs are concentrated in the perinuclear region. There is no cytotoxicity following transport of QDs present at concentrations up to 200 nM. The mechanism for arginine-rich CPP/QD complexes to traverse cell membrane appears to involve a combination of internalization pathways. These results provide insight into the mechanism of arginine-rich CPP delivery of noncovalently attached cargoes, and may provide a powerful tool for imaging in vivo. PMID:21137758

  3. Glycosaminoglycans are required for translocation of amphipathic cell-penetrating peptides across membranes.

    PubMed

    Pae, Janely; Liivamägi, Laura; Lubenets, Dmitri; Arukuusk, Piret; Langel, Ülo; Pooga, Margus

    2016-08-01

    Cell-penetrating peptides (CPPs) are considered as one of the most promising tools to mediate the cellular delivery of various biologically active compounds that are otherwise cell impermeable. CPPs can internalize into cells via two different pathways - endocytosis and direct translocation across the plasma membrane. In both cases, the initial step of internalization requires interactions between CPPs and different plasma membrane components. Despite the extensive research, it is not yet fully understood, which of these cell surface molecules mediate the direct translocation of CPPs across the plasma- and endosomal membrane. In the present study we used giant plasma membrane vesicles (GPMVs) as a model membrane system to elucidate the specific molecular mechanisms behind the internalization and the role of cell surface glycosaminoglycans (GAGs) in the translocation of four well-known CPPs, classified as cationic (nona-arginine, Tat peptide) and amphipathic (transportan and TP10). We demonstrate here that GAGs facilitate the translocation of amphipathic CPPs, but not the internalization of cationic CPPs; and that the uptake is not mediated by a specific GAG class, but rather the overall amount of these polysaccharides is crucial for the internalization of amphipathic peptides. PMID:27117133

  4. Brain delivery of insulin boosted by intranasal coadministration with cell-penetrating peptides.

    PubMed

    Kamei, Noriyasu; Takeda-Morishita, Mariko

    2015-01-10

    Intranasal administration is considered as an alternative route to enable effective drug delivery to the central nervous system (CNS) by bypassing the blood-brain barrier. Several reports have proved that macromolecules can be transferred directly from the nasal cavity to the brain. However, strategies to enhance the delivery of macromolecules from the nasal cavity to CNS are needed because of their low delivery efficiencies via this route in general. We hypothesized that the delivery of biopharmaceuticals to the brain parenchyma can be facilitated by increasing the uptake of drugs by the nasal epithelium including supporting and neuronal cells to maximize the potentiality of the intranasal pathway. To test this hypothesis, the CNS-related model peptide insulin was intranasally coadministered with the cell-penetrating peptide (CPP) penetratin to mice. As a result, insulin coadministered with l- or d-penetratin reached the distal regions of the brain from the nasal cavity, including the cerebral cortex, cerebellum, and brain stem. In particular, d-penetratin could intranasally deliver insulin to the brain with a reduced risk of systemic insulin exposure. Thus, the results obtained in this study suggested that CPPs are potential tools for the brain delivery of peptide- and protein-based pharmaceuticals via intranasal administration. PMID:25445695

  5. Alternative Mechanisms for the Interaction of the Cell-Penetrating Peptides Penetratin and the TAT Peptide with Lipid Bilayers

    PubMed Central

    Yesylevskyy, Semen; Marrink, Siewert-Jan; Mark, Alan E.

    2009-01-01

    Abstract Cell-penetrating peptides (CPPs) have recently attracted much interest due to their apparent ability to penetrate cell membranes in an energy-independent manner. Here molecular-dynamics simulation techniques were used to study the interaction of two CPPs: penetratin and the TAT peptide with 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) phospolipid bilayers shed light on alternative mechanisms by which these peptides might cross biological membranes. In contrast to previous simulation studies of charged peptides interacting with lipid bilayers, no spontaneous formation of transmembrane pores was observed. Instead, the simulations suggest that the peptides may enter the cell by micropinocytosis, whereby the peptides induce curvature in the membrane, ultimately leading to the formation of small vesicles within the cell that encapsulate the peptides. Specifically, multiple peptides were observed to induce large deformations in the lipid bilayer that persisted throughout the timescale of the simulations (hundreds of nanoseconds). Pore formation could be induced in simulations in which an external potential was used to pull a single penetratin or TAT peptide into the membrane. With the use of umbrella-sampling techniques, the free energy of inserting a single penetratin peptide into a DPPC bilayer was estimated to be ∼75 kJmol−1, which suggests that the spontaneous penetration of single peptides would require a timescale of at least seconds to minutes. This work also illustrates the extent to which the results of such simulations can depend on the initial conditions, the extent of equilibration, the size of the system, and the conditions under which the simulations are performed. The implications of this with respect to the current systems and to simulations of membrane-peptide interactions in general are discussed. PMID:19580742

  6. 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

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

    PubMed

    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

  8. Cell-Penetrating Peptide-Mediated Delivery of TALEN Proteins via Bioconjugation for Genome Engineering

    PubMed Central

    Liu, Jia; Gaj, Thomas; Patterson, James T.; Sirk, Shannon J.; Barbas III, Carlos F.

    2014-01-01

    Transcription activator-like (TAL) effector nucleases (TALENs) have enabled the introduction of targeted genetic alterations into a broad range of cell lines and organisms. These customizable nucleases are comprised of programmable sequence-specific DNA-binding modules derived from TAL effector proteins fused to the non-specific FokI cleavage domain. Delivery of these nucleases into cells has proven challenging as the large size and highly repetitive nature of the TAL effector DNA-binding domain precludes their incorporation into many types of viral vectors. Furthermore, viral and non-viral gene delivery methods carry the risk of insertional mutagenesis and have been shown to increase the off-target activity of site-specific nucleases. We previously demonstrated that direct delivery of zinc-finger nuclease proteins enables highly efficient gene knockout in a variety of mammalian cell types with reduced off-target effects. Here we show that conjugation of cell-penetrating poly-Arg peptides to a surface-exposed Cys residue present on each TAL effector repeat imparted cell-penetrating activity to purified TALEN proteins. These modifications are reversible under reducing conditions and enabled TALEN-mediated gene knockout of the human CCR5 and BMPR1A genes at rates comparable to those achieved with transient transfection of TALEN expression vectors. These findings demonstrate that direct protein delivery, facilitated by conjugation of chemical functionalities onto the TALEN protein surface, is a promising alternative to current non-viral and viral-based methods for TALEN delivery into mammalian cells. PMID:24465685

  9. 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.

  10. 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

  11. Cell-Penetrating Peptides as Carriers for Oral Delivery of Biopharmaceuticals.

    PubMed

    Kristensen, Mie; Nielsen, Hanne Mørck

    2016-02-01

    Oral delivery of biopharmaceuticals, for example peptides and proteins, constitutes a great challenge in drug delivery due to their low chemical stability and poor permeation across the intestinal mucosa, to a large extent limiting the mode of administration to injections, which is not favouring patient compliance. Nevertheless, cell-penetrating peptides (CPPs) have shown promising potential as carriers to overcome the epithelium, and this minireview highlights recent knowledge gained within the field of CPP-mediated transepithelial delivery of therapeutic peptides and proteins from the intestine. Two approaches may be pursued: co-administration of the carrier and therapeutic peptide in the form of complexes obtained by simple bulk mixing, or administration of covalent conjugates demanding more advanced production methodologies. These formulation approaches have their pros and cons, and which is to be preferred depends on the physicochemical properties of both the specific CPP and the specific cargo. In addition to the physical epithelial barrier, a metabolic barrier must be overcome in order to obtain CPP-mediated delivery of a cargo drug from the intestine, and a number of strategies have been employed to delay enzymatic degradation of the CPP. The mechanisms by which CPPs translocate across membranes are not fully understood, but possibly involve endocytosis as well as direct translocation, and the CPP-mediated transepithelial delivery of cargo drugs thus likely involves similar mechanisms for the initial membrane interaction and translocation. However, the mechanisms responsible for transcytosis of the cargo drug, if taken up by an endocytic mechanism, or direct translocation across the epithelium are so far not known. PMID:26525297

  12. 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

  13. Mechanism of Cationic Nanoparticles and Cell-Penetrating Peptides Direct Translocate Across Cell Membranes

    NASA Astrophysics Data System (ADS)

    Lin, Jiaqi; Alexander-Katz, Alfredo

    2014-03-01

    Cationic Nanoparticles (NPs) and cell-penetrating peptides (CPPs) are known effective intracellular delivery agents. These positively charged particles can bypass traditional endocytosis route to enter the cytosol, which is known as direct translocation. However, mechanism of direct translocation of both NPs and CPPs is not well understood. Using Coarse-grained (CG) molecular dynamics simulation, we found that gold nanoparticles (AuNPs) as well as HIV-1 Tat peptides can translocate across model biological membranes through nanoscale holes under a transmembrane (TM) potential. After the translocation, the TM is strongly weakened and the holes gradually reseal themselves, while the NPs/CPPs roam freely in the ``intracellular region.'' Both size and shape of the NPs/ CPPs are found to be a determine factor of their translocation behaviour, and the relationship between direct translocation and endocytosis is also discussed. The results provided here establish fundamental rules of direct translocation entry of NPs/CPPs, which may guide the rational design of cationic intracellular nanocarriers.

  14. 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

  15. 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

  16. 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

  17. The many futures for cell-penetrating peptides: how soon is now?

    PubMed

    Howl, J; Nicholl, I D; Jones, S

    2007-08-01

    Studies of CPPs (cell-penetrating peptides), sequences that are also commonly designated as protein transduction domains, now extend to a second decade of exciting and far-reaching discoveries. CPPs are proven vehicles for the intracellular delivery of macromolecules that include oligonucleotides, peptides and proteins, low-molecular-mass drugs, nanoparticles and liposomes. The biochemical properties of different classes of CPP, including various sequences derived from the HIV-1 Tat (transactivator of transcription) [e.g. Tat-(48-60), GRKKRRQRRRPPQ], and the homeodomain of the Drosophila homeoprotein Antennapaedia (residues 43-58, commonly named penetratin, RQIKIWFQNRRMKWKK), also provide novel insights into the fundamental mechanisms of translocation across biological membranes. Thus the efficacy of CPP-mediated cargo delivery continues to provide valuable tools for biomedical research and, as witnessed in 2007, candidate and emerging therapeutics. Thus it is anticipated that the further refinement of CPP technologies will provide drug-delivery vectors, cellular imaging tools, nanoparticulate devices and molecular therapeutics that will have a positive impact on the healthcare arena. The intention of this article is to provide both a succinct overview of current developments and applications of CPP technologies, and to illustrate key developments that the concerted efforts of the many researchers contributing to the Biochemical Society's Focused Meeting in Telford predict for the future. The accompanying papers in this issue of Biochemical Society Transactions provide additional details and appropriate references. Hopefully, the important and eagerly anticipated biomedical and clinical developments within the CPP field will occur sooner rather than later. PMID:17635144

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

    PubMed

    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

  19. Efficient Cargo Delivery into Adult Brain Tissue Using Short Cell-Penetrating Peptides

    PubMed Central

    Thomas, Alvin Kuriakose; Bhattarai, Prabesh; Zhang, Yixin; Brand, Michael

    2015-01-01

    Zebrafish brains can regenerate lost neurons upon neurogenic activity of the radial glial progenitor cells (RGCs) that reside at the ventricular region. Understanding the molecular events underlying this ability is of great interest for translational studies of regenerative medicine. Therefore, functional analyses of gene function in RGCs and neurons are essential. Using cerebroventricular microinjection (CVMI), RGCs can be targeted efficiently but the penetration capacity of the injected molecules reduces dramatically in deeper parts of the brain tissue, such as the parenchymal regions that contain the neurons. In this report, we tested the penetration efficiency of five known cell-penetrating peptides (CPPs) and identified two– polyR and Trans – that efficiently penetrate the brain tissue without overt toxicity in a dose-dependent manner as determined by TUNEL staining and L-Plastin immunohistochemistry. We also found that polyR peptide can help carry plasmid DNA several cell diameters into the brain tissue after a series of coupling reactions using DBCO-PEG4-maleimide-based Michael’s addition and azide-mediated copper-free click reaction. Combined with the advantages of CVMI, such as rapidness, reproducibility, and ability to be used in adult animals, CPPs improve the applicability of the CVMI technique to deeper parts of the central nervous system tissues. PMID:25894337

  20. 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

  1. 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

  2. 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.

  3. 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

  4. 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

  5. Cell penetration and cell-selective drug delivery using α-helix peptides conjugated with gold nanoparticles.

    PubMed

    Park, Hyejin; Tsutsumi, Hiroshi; Mihara, Hisakazu

    2013-07-01

    Cell penetrating peptides (CPPs) have been developed as vectors for molecular delivery into various cells for use in drug delivery, gene therapy and cancer treatment by their property transporting various molecules into cytoplasm. CPPs with high internalization, cell specificity, and low cytotoxicity have been considered to increase the applicability for cell engineering. Gold nanospheres (GNSs) are a useful tool for molecular imaging, because they are non-cytotoxic and have high solubility, ease of synthesis and excellent light scattering property. Here, we investigated the cell penetrability using α-helix peptides of 17-amino acids conjugated to gold nanospheres (P-GNS). Depending on the peptide sequence had the different cell penetrating (CP) activity for three kinds of cell lines. P-GNS showed low cytotoxicity and high selectivity against three cell types, despite just one amino acid difference between the peptide. We studied the cytotoxic activity of an anti-cancer drug doxorubicin (DOX) conjugated to the P-GNS. They showed different cytotoxicity against the three cell lines, depending on the peptide sequence, with a higher efficiency than free DOX at the same concentration. The cytotoxicity by DOX was correlated with the CP activity of the peptides against the three cell lines. These results demonstrated that P-GNS would be a useful tool for the development of a new cell-selective drug delivery system. PMID:23545289

  6. Cell Penetrating Peptide-Mediated Caveolae-Dependent Activation of Lung Endothelial Nitric Oxide Synthase.

    PubMed

    Hutchinson, Tarun E; Hu, Hanbo; Patel, Jawaharlal M

    2016-01-01

    Cell penetrating peptides can be used as therapeutic agents via modulation of selective cell functions. Nitric oxide (NO) generated by vascular endothelial NO synthase (eNOS) plays a critical role in the NO/ cyclic guanosine 5'-monophosphate (cGMP)-mediated pulmonary vascular function. Here we examined whether internalization of a fifteen amino acid (KRFNSISCSSWRRKR) synthetic peptide (P3) enhances the catalytic activity of eNOS via caveolae/eNOS dissociation leading to NO release and increased cGMP production in pulmonary artery endothelial cells (EC). ECs were treated with varying concentrations of P3 and used to monitor internalization, isolation of caveolae-enriched fraction, the catalytic activity of eNOS, NO/cGMP production, and intracellular Ca(2+) release. Confocal images show timedependent internalization of P3 in EC. Treatment of EC with P3, but not scrambled P3, increased the catalytic activity of eNOS in a dose-dependent manner without change in eNOS expression or phosphorylation. Treatment of EC with P3 stimulated intracellular Ca(2+) release, increased the catalytic activity of phospatidylinsositide 3 kinase (PI3K) and resulted in eNOS/caveolae-1 (Cav-1) dissociation leading to translocation of eNOS to intracellular compartment in EC. P3- mediated activation of eNOS was abolished by intracellular Ca(2+) chelator 1,2-bis(2-aminophenooxy)ethane-N,N,N',N'- tertraacetic acid-AM (BAPTA-AM), PI3K inhibition, or by siRNA-mediated Cav-1 suppression. These results demonstrate that exogenous peptide consisting of cationic amino acids can internalize and enhance the catalytic activity of eNOS via modulation of caveolar signaling and intracellular Ca(2+) release in EC. PMID:26655728

  7. Enhancing tumor-specific intracellular delivering efficiency of cell-penetrating peptide by fusion with a peptide targeting to EGFR.

    PubMed

    Nguyen, Long The; Yang, Xu-Zhong; Du, Xuan; Wang, Jia-Wei; Zhang, Rui; Zhao, Jian; Wang, Fu-Jun; Dong, Yang; Li, Peng-Fei

    2015-05-01

    Cell-penetrating peptides (CPPs) are well known as intracellular delivery vectors. However, unsatisfactory delivery efficiency and poor specificity are challenging barriers to CPP applications at the clinical trial stage. Here, we showed that S3, an EGFR-binding domain derived from vaccinia virus growth factor, when fused to a CPP such as HBD or TAT can substantially enhance its internalization efficiency and tumor selectivity. The uptake of S3-HBD (S3H) recombinant molecule by tumor cells was nearly 80 folds increased compared to HBD alone. By contrast, the uptake of S3H by non-neoplastic cells still remained at a low level. The specific recognition between S3 and its receptor, EGFR, as well as between HBD and heparan sulfate proteoglycans on the cell surface was essential for these improvements, suggesting a syngeneic effect between the two functional domains in conjugation. This syngeneic effect is likely similar to that of the heparin-binding epidermal growth factor, which is highly abundant particularly in metastatic tumors. The process that S3H entered cells was dependent on time, dosage, and energy, via macropinocytosis pathway. With excellent cell-penetrating efficacy and a novel tumor-targeting ability, S3H appears as a promising candidate vector for targeted anti-cancer drug delivery. PMID:25655386

  8. 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

  9. 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

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

    PubMed

    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. 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

  13. The molecular mechanism of photochemical internalization of cell penetrating peptide-cargo-photosensitizer conjugates

    PubMed Central

    Ohtsuki, Takashi; Miki, Shunya; Kobayashi, Shouhei; Haraguchi, Tokuko; Nakata, Eiji; Hirakawa, Kazutaka; Sumita, Kensuke; Watanabe, Kazunori; Okazaki, Shigetoshi

    2015-01-01

    In many drug delivery strategies, an inefficient transfer of macromolecules such as proteins and nucleic acids to the cytosol often occurs because of their endosomal entrapment. One of the methods to overcome this problem is photochemical internalization, which is achieved using a photosensitizer and light to facilitate the endosomal escape of the macromolecule. In this study, we examined the molecular mechanism of photochemical internalization of cell penetrating peptide-cargo (macromolecule)-photosensitizer conjugates. We measured the photophysical properties of eight dyes (photosensitizer candidates) and determined the respective endosomal escape efficiencies using these dyes. Correlation plots between these factors indicated that the photogenerated 1O2 molecules from photosensitizers were highly related to the endosomal escape efficiencies. The contribution of 1O2 was confirmed using 1O2 quenchers. In addition, time-lapse fluorescence imaging showed that the photoinduced endosomal escape occurred at a few seconds to a few minutes after irradiation (much longer than 1O2 lifetime), and that the pH increased in the endosome prior to the endosomal escape of the macromolecule. PMID:26686907

  14. Novel Efficient Cell-Penetrating, Peptide-Mediated Strategy for Enhancing Telomerase Inhibitor Oligonucleotides.

    PubMed

    Muñoz-Alarcón, Andrés; Eriksson, Jonas; Langel, Ülo

    2015-12-01

    At present, there are several therapeutic approaches for targeting telomerase in tumors. One in particular, currently undergoing clinical trials, is based on synthetic lipid-modified oligonucleotide antagonists aimed at inhibiting the ribonucleoprotein subunit of human telomerase. However, while enabling efficient uptake, the lipid modifications reduce the potency of the therapeutic oligonucleotides compared to nonmodified oligonucleotides. Moreover, lipid modification may increase oligonucleotide accumulation in the liver causing undesirable hepatotoxicity. Noncovalent complexation strategies for cell-penetrating peptide (CPP)-mediated delivery present an option to circumvent the need for potency-reducing modifications, while allowing for a highly efficient uptake, and could significantly improve the efficiency of telomerase-targeting cancer therapeutics. Delivery of a nonlipidated locked nucleic acid/2'-O-methyl mixmer significantly inhibits the telomerase activity in treated HeLa cells. The inhibitory effect was further improved through addition of a CPP. Furthermore, calculated IC50-values for the oligonucleotide delivered by CPPs into HeLa cells are more than 20 times lower than telomerase inhibitor Imetelstat, currently undergoing clinical trials. These results emphasize the potential of CPP-mediated delivery of future pharmaceuticals and provide means by which to enhance an already promising therapeutic strategy for cancer treatment. PMID:26479411

  15. Cell-Penetrating Peptide-Mediated Therapeutic Molecule Delivery into the Central Nervous System

    PubMed Central

    Zou, Li-Li; Ma, Jie-Lan; Wang, Tao; Yang, Tang-Bin; Liu, Chang-Bai

    2013-01-01

    The blood-brain barrier (BBB), a dynamic and complex barrier formed by endothelial cells, can impede the entry of unwanted substances – pathogens and therapeutic molecules alike – into the central nervous system (CNS) from the blood circulation. Taking into account the fact that CNS-related diseases are the largest and fastest growing unmet medical concern, many potential protein- and nucleic acid-based medicines have been developed for therapeutic purposes. However, due to their poor ability to cross the BBB and the plasma membrane, the above-mentioned bio-macromolecules have limited use in treating neurological diseases. Finding effective, safe, and convenient ways to deliver therapeutic molecules into the CNS is thus urgently required. In recent decades, much effort has been expended in the development of drug delivery technologies, of which cell-penetrating peptides (CPPs) have the most promising potential. The present review covers the latest advances in CPP delivery technology, and provides an update on their use in CNS-targeted drug delivery. PMID:23997754

  16. Concatemerization increases the inhibitory activity of short, cell-penetrating, cationic and tryptophan-rich antifungal peptides.

    PubMed

    López-García, Belén; Harries, Eleonora; Carmona, Lourdes; Campos-Soriano, Lidia; López, José Javier; Manzanares, Paloma; Gandía, Mónica; Coca, María; Marcos, Jose F

    2015-10-01

    There are short cationic and tryptophan-rich antifungal peptides such as the hexapeptide PAF26 (RKKWFW) that have selective toxicity and cell penetration properties against fungal cells. This study demonstrates that concatemeric peptides with tandem repeats of the heptapeptide PAF54 (which is an elongated PAF26 sequence) show increased fungistatic and bacteriostatic activities while maintaining the absence of hemolytic activity of the monomer. The increase in antimicrobial activity of the double-repeated PAF sequences (diPAFs), compared to the nonrepeated PAF, was higher (4-8-fold) than that seen for the triple-repeated sequences (triPAFs) versus the diPAFs (2-fold). However, concatemerization diminished the fungicidal activity against quiescent spores of the filamentous fungus Penicillium digitatum. Peptide solubility and sensitivity to proteolytic degradation were affected by the design of the concatemers: incorporation of the AGPA sequence hinge to separate PAF54 repeats increased solubility while the C-terminal addition of the KDEL sequence decreased in vitro stability. These results led to the design of the triPAF sequence PAF102 of 30 amino acid residues, with increased antimicrobial activity and minimal inhibitory concentration (MIC) value of 1-5 μM depending on the fungus. Further characterization of the mode-of-action of PAF102 demonstrated that it colocalizes first with the fungal cell wall, it is thereafter internalized in an energy dependent manner into hyphal cells of the filamentous fungus Fusarium proliferatum, and finally kills hyphal cells intracellularly. Therefore, PAF102 showed mechanistic properties against fungi similar to the parental PAF26. These observations are of high interest in the future development of PAF-based antimicrobial molecules optimized for their production in biofactories. PMID:25846331

  17. 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

  18. Free Energy of Translocating an Arginine-Rich Cell-Penetrating Peptide across a Lipid Bilayer Suggests Pore Formation

    PubMed Central

    Huang, Kun; García, Angel E.

    2013-01-01

    The molecular mechanism and energetics of the translocation of arginine-rich, cell-penetrating peptides through membranes are still under debate. One possible mechanism involves the formation of a water pore in the membrane such that the hydrophilic residues of the peptide are solvated throughout the translocating process. In this work, employing two different order parameters, we calculate the free energies of translocating a cyclic Arg9 peptide into a lipid bilayer along one path that involves a water-pore formation and another path that does not form a separate pore. The free-energy barrier of translocating the peptide along a pore path is 80 kJ/mol lower than along a pore-free path. This suggests that the peptide translocation is more likely associated with a water-pore formation. PMID:23442863

  19. Cell-Penetrating Peptide-Modified Gold Nanoparticles for the Delivery of Doxorubicin to Brain Metastatic Breast Cancer.

    PubMed

    Morshed, Ramin A; Muroski, Megan E; Dai, Qing; Wegscheid, Michelle L; Auffinger, Brenda; Yu, Dou; Han, Yu; Zhang, Lingjiao; Wu, Meijing; Cheng, Yu; Lesniak, Maciej S

    2016-06-01

    As therapies continue to increase the lifespan of patients with breast cancer, the incidence of brain metastases has steadily increased, affecting a significant number of patients with metastatic disease. However, a major barrier toward treating these lesions is the inability of therapeutics to penetrate into the central nervous system and accumulate within intracranial tumor sites. In this study, we designed a cell-penetrating gold nanoparticle platform to increase drug delivery to brain metastatic breast cancer cells. TAT peptide-modified gold nanoparticles carrying doxorubicin led to improved cytotoxicity toward two brain metastatic breast cancer cell lines with a decrease in the IC50 of at least 80% compared to free drug. Intravenous administration of these particles led to extensive accumulation of particles throughout diffuse intracranial metastatic microsatellites with cleaved caspase-3 activity corresponding to tumor foci. Furthermore, intratumoral administration of these particles improved survival in an intracranial MDA-MB-231-Br xenograft mouse model. Our results demonstrate the promising application of gold nanoparticles for improving drug delivery in the context of brain metastatic breast cancer. PMID:27169484

  20. PepFect15, a novel endosomolytic cell-penetrating peptide for oligonucleotide delivery via scavenger receptors.

    PubMed

    Lindberg, Staffan; Muñoz-Alarcón, Andrés; Helmfors, Henrik; Mosqueira, Diogo; Gyllborg, Daniel; Tudoran, Oana; Langel, Ulo

    2013-01-30

    Gene-regulatory biomolecules such as splice-correcting oligonucleotides and anti-microRNA oligonucleotides are important tools in the struggle to understand and treat genetic disorders caused by defective gene expression or aberrant splicing. However, oligonucleotides generally suffer from low bioavailability, hence requiring efficient and non-toxic delivery vectors to reach their targets. Cell-penetrating peptides constitute a promising category of carrier molecules for intracellular delivery of bioactive cargo. In this study we present a novel cell-penetrating peptide, PepFect15, comprising the previously reported PepFect14 peptide modified with endosomolytic trifluoromethylquinoline moieties to facilitate endosomal escape. Pepfect15 efficiently delivers both splice-correcting oligonucleotides and anti-microRNA oligonucleotides into cells through a non-covalent complexation strategy. To our knowledge this is the first work that describes peptide-mediated anti-microRNA delivery. The peptide and its cargo form stable, negatively charged nanoparticles that are taken up by cells largely through scavenger receptor type A mediated endocytosis. PMID:23200958

  1. 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

  2. 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

  3. Stearylated antimicrobial peptide [D]-K6L9 with cell penetrating property for efficient gene transfer.

    PubMed

    Zhang, Wei; Song, Jingjing; Liang, Ranran; Zheng, Xin; Chen, Jianbo; Li, Guolin; Zhang, Bangzhi; Wang, Kairong; Yan, Xiang; Wang, Rui

    2013-08-01

    Stearyl-cell penetrating peptides (CPPs) have been proved to be efficient nonviral gene vectors. Due to the similarities between antimicrobial peptides and CPPs, we constructed a novel type of gene vectors by introducing stearyl moiety to the N-terminus of antimicrobial peptide [D]-K6L9. In this study, stearyl-[D]-K6L9 delivered plasmids into cells by clathrin- and caveolin-mediated endocytosis. Gratifyingly, stearyl-[D]-K6L9 exhibited high transfection efficiency and almost reached the level of Lipofectamine 2000. Taken together, the combination of the stearyl moiety with [D]-K6L9 provides a novel framework for the development of excellent nonviral gene vectors. PMID:23727033

  4. Effect of crotamine, a cell-penetrating peptide, on blastocyst production and gene expression of in vitro fertilized bovine embryos.

    PubMed

    Campelo, Iana S; Pereira, Alexsandra F; Alcântara-Neto, Agostinho S; Canel, Natalia G; Souza-Fabjan, Joanna M G; Teixeira, Dárcio I A; Camargo, Luiz S A; Melo, Luciana M; Rádis-Baptista, Gandhi; Salamone, Daniel F; Freitas, Vicente J F

    2016-02-01

    The present study investigated the effects of crotamine, a cell-penetrating peptide from rattlesnake venom, at different exposure times and concentrations, on both developmental competence and gene expression (ATP1A1, AQP3, GLUT1 and GLUT3) of in vitro fertilized (IVF) bovine embryos. In Experiment 1, presumptive zygotes were exposed to 0.1 μM crotamine for 6, 12 or 24 h and control groups (vehicle and IVF) were included. In Experiment 2, presumptive zygotes were exposed to 0 (vehicle), 0.1, 1 and 10 μM crotamine for 24 h. Additionally, to visualize crotamine uptake, embryos were exposed to rhodamine B-labelled crotamine and subjected to confocal microscopy. In Experiment 1, no difference (P > 0.05) was observed among different exposure times and control groups for cleavage and blastocyst rates and total cells number per blastocyst. Within each exposure time, mRNA levels were similar (P > 0.05) in embryos cultured with or without crotamine. In Experiment 2, concentrations as high as 10 μM crotamine did not affect (P > 0.05) the blastocyst rate. Crotamine at 0.1 and 10 μM did not alter mRNA levels when compared with the control (P > 0.05). Remarkably, only 1 μM crotamine decreased both ATP1A1 and AQP3 expression levels relative to the control group (P < 0.05). Also, it was possible to visualize the intracellular localization of crotamine. These results indicate that crotamine can translocate intact IVF bovine embryos and its application in the culture medium is possible at concentrations from 0.1-10 μM for 6-24 h. PMID:25532535

  5. 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.

  6. Effect of arginine-rich cell penetrating peptides on membrane pore formation and life-times: a molecular simulation study.

    PubMed

    Sun, Delin; Forsman, Jan; Lund, Mikael; Woodward, Clifford E

    2014-10-14

    The molecular basis for the effectiveness of arginine-rich cell penetrating peptides (ARCPPs) traversing a cell membrane barrier is not well established. The fact that a threshold concentration of ARCPPs is required for efficient translocation in model membranes suggests cooperative action by ARCPPs. We used umbrella sampling simulations to calculate the free energies for membrane pore formation. Membrane-bound octaarginine (ARG8) peptides showed little cooperativity in lowering the free energy barrier to generate membrane pores by direct peptide translocation or by lipid flip-flop. Instead, high concentrations of ARG8 peptides were found to expand the surface area of the lipid bilayer due to the deep partitioning of guanidinium ions into the lipid glycerol regions. Surface-bound ARG8 peptides can also insert an arginine side chain into one existing transient membrane pore, and the lifetime of the transient membrane pore is significantly extended by arginine. This suggests a cooperative kinetic mechanism may act above a threshold adsorption concentration to facilitate the rapid uptake of these peptides. PMID:25166723

  7. 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

  8. 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

  9. 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

  10. Neuropilin-1 and heparan sulfate proteoglycans cooperate in cellular uptake of nanoparticles functionalized by cationic cell-penetrating peptides

    PubMed Central

    Pang, Hong-Bo; Braun, Gary B.; Ruoslahti, Erkki

    2015-01-01

    Cell-penetrating peptides (CPPs) have been widely used to deliver nanomaterials and other types of macromolecules into mammalian cells for therapeutic and diagnostic use. Cationic CPPs that bind to heparan sulfate (HS) proteoglycans on the cell surface induce potent endocytosis; however, the role of other surface receptors in this process is unclear. We describe the convergence of an HS-dependent pathway with the C-end rule (CendR) mechanism that enables peptide ligation with neuropilin-1 (NRP1), a cell surface receptor known to be involved in angiogenesis and vascular permeability. NRP1 binds peptides carrying a positive residue at the carboxyl terminus, a feature that is compatible with cationic CPPs, either intact or after proteolytic processing. We used CPP and CendR peptides, as well as HS- and NRP1-binding motifs from semaphorins, to explore the commonalities and differences of the HS and NRP1 pathways. We show that the CendR-NRP1 interaction determines the ability of CPPs to induce vascular permeability. We also show at the ultrastructural level, using a novel cell entry synchronization method, that both the HS and NRP1 pathways can initiate a macropinocytosis-like process and visualize these CPP-cargo complexes going through various endosomal compartments. Our results provide new insights into how CPPs exploit multiple surface receptor pathways for intracellular delivery. PMID:26601141

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

    PubMed

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

    2015-12-21

    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. PMID:26568525

  12. 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

  13. Massive glycosaminoglycan-dependent entry of Trp-containing cell-penetrating peptides induced by exogenous sphingomyelinase or cholesterol depletion.

    PubMed

    Bechara, Chérine; Pallerla, Manjula; Burlina, Fabienne; Illien, Françoise; Cribier, Sophie; Sagan, Sandrine

    2015-02-01

    Among non-invasive cell delivery strategies, cell-penetrating peptide (CPP) vectors represent interesting new tools. To get fundamental knowledge about the still debated internalisation mechanisms of these peptides, we modified the membrane content of cells, typically by hydrolysis of sphingomyelin or depletion of cholesterol from the membrane outer leaflet. We quantified and visualised the effect of these viable cell surface treatments on the internalisation efficiency of different CPPs, among which the most studied Tat, R9, penetratin and analogues, that all carry the N-terminal biotin-Gly4 tag cargo. Under these cell membrane treatments, only penetratin and R6W3 underwent a massive glycosaminoglycan (GAG)-dependent entry in cells. Internalisation of the other peptides was only slightly increased, similarly in the absence or the presence of GAGs for R9, and only in the presence of GAGs for Tat and R6L3. Ceramide formation (or cholesterol depletion) is known to lead to the reorganisation of membrane lipid domains into larger platforms, which can serve as a trap and cluster receptors. These results show that GAG clustering, enhanced by formation of ceramide, is efficiently exploited by penetratin and R6W3, which contains Trp residues in their sequence but not Tat, R9 and R6L3. Hence, these data shed new lights on the differences in the internalisation mechanism and pathway of these peptides that are widely used in delivery of cargo molecules. PMID:25112713

  14. Cell penetrating peptides released from thermosensitive nanoparticles suppress proinflammatory cytokine response by specifically targeting inflamed cartilage explants

    PubMed Central

    Panitch, Alyssa

    2014-01-01

    Cell penetrating anti-inflammatory peptide KAFAKLAARLYRKALARQLGVAA (KAFAK) has the ability to suppress pro-inflammatory cytokines TNF-α and IL-6 when released from degradable and non-degradable Poly(NIPAm-AMPS) nanoparticles. In vitro human macrophage model with THP1 human monocytes and ex vivo bovine knee cartilage tissue both showed a dose dependent suppression of pro-inflammatory cytokines when treated with KAFAK loaded poly(NIPAm-AMPS) nanoparticles. When bovine knee cartilage explants were treated with KAFAK loaded poly(NIPAm-AMPS) nanoparticles, rapid and highly selective targeting of only damaged tissue occurred. This study has demonstrated selective targeting and therapeutic efficacy of KAFAK when released from both degradable and non degradable poly(NIPAm-AMPS) nanoparticles in in vitro and ex vivo models. As a result, poly(NIPAm-AMPS) nanoparticles loaded with KAFAK could a very effective tool to treat osteoarthritis. PMID:23041412

  15. 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

  16. Real-time In Vivo Molecular Detection of Primary Tumors and Metastases with Ratiometric Activatable Cell-Penetrating Peptides

    PubMed Central

    Savariar, Elamprakash N.; Felsen, Csilla N.; Nashi, Nadia; Jiang, Tao; Ellies, Lesley G.; Steinbach, Paul; Tsien, Roger Y.; Nguyen, Quyen T.

    2013-01-01

    Management of metastatic disease is integral to cancer treatment. Evaluation of metastases often requires surgical removal of all anatomically susceptible lymph nodes for ex vivo pathologic examination. We report a family of novel ratiometric activatable cell-penetrating peptides, which contain Cy5 as far red fluorescent donor and Cy7 as near-infrared fluorescent acceptor. Cy5 is quenched in favor of Cy7 reemission until the intervening linker is cut by tumor-associated matrix metalloproteinases-2 and 9 (MMP2,9) or elastases. Such cleavage increases the Cy5:Cy7 emission ratio 40-fold and triggers tissue retention of the Cy5-containing fragment. This ratiometric increase provides an accelerated and quantifiable metric to identify primary tumors and metastases to liver and lymph nodes with increased sensitivity and specificity. This technique represents a significant advance over existing nonratiometric protease sensors and sentinel lymph node detection methods, which give no information about cancer invasion. PMID:23188503

  17. Cell-penetrating peptides as noninvasive transmembrane vectors for the development of novel multifunctional drug-delivery systems.

    PubMed

    Zhang, Dongdong; Wang, Jiaxi; Xu, Donggang

    2016-05-10

    Unique characteristics, such as nontoxicity and rapid cellular internalization, allow the cell-penetrating peptides (CPPs) to transport hydrophilic macromolecules into cells, thus, enabling them to execute biological functions. However, some CPPs have limitations due to nonspecificity and easy proteolysis. To overcome such defects, the CPP amino acid sequence can be modified, replaced, and reconstructed for optimization. CPPs can also be used in combination with other drug vectors, fused with their preponderances to create novel multifunctional drug-delivery systems that increase the stability during blood circulation, and also develop novel preparations capable of targeted delivery, along with sustainable and controllable release. Further improvements in CPP structure can facilitate the penetration of macromolecules into diverse biomembrane structures, such as the blood brain barrier, gastroenteric mucosa, and skin dermis. The ability of CPP to act as transmembrane vectors improves the clinical application of some biomolecules to treat central nervous system diseases, increase oral bioavailability, and develop percutaneous-delivery dosage form. PMID:26993425

  18. 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.

  19. Cationic Cell-Penetrating Peptide Binds to Planar Lipid Bilayers Containing Negatively Charged Lipids but does not Induce Conductive Pores

    PubMed Central

    Gurnev, Philip A.; Yang, Sung-Tae; Melikov, Kamran C.; Chernomordik, Leonid V.; Bezrukov, Sergey M.

    2013-01-01

    Using a cation-selective gramicidin A channel as a sensor of the membrane surface charge, we studied interactions of oligoarginine peptide R9C, a prototype cationic cell-penetrating peptide (CPP), with planar lipid membranes. We have found that R9C sorption to the membrane depends strongly on its lipid composition from virtually nonexistent for membranes made of uncharged lipids to very pronounced for membranes containing negatively charged lipids, with charge overcompensation at R9C concentrations exceeding 1 μM. The sorption was reversible as it was removed by addition of polyanionic dextran sulfate to the membrane bathing solution. No membrane poration activity of R9C (as would be manifested by increased bilayer conductance) was detected in the charged or neutral membranes, including those with asymmetric negative/neutral and negative/positive lipid leaflets. We conclude that interaction of R9C with planar lipid bilayers does not involve pore formation in all studied lipid combinations up to 20 μM peptide concentration. However, R9C induces leakage of negatively charged but not neutral liposomes in a process that involves lipid mixing between liposomes. Our findings suggest that direct traversing of CPPs through the uncharged outer leaflet of the plasma membrane bilayer is unlikely and that permeabilization necessarily involves both anionic lipids and CPP-dependent fusion between opposing membranes. PMID:23663836

  20. Heat shock protein-mediated cell penetration and cytosolic delivery of macromolecules by a telomerase-derived peptide vaccine.

    PubMed

    Lee, Seoung-Ae; Kim, Bo-Ram; Kim, Bu-Kyung; Kim, Dong-Won; Shon, Won-Jun; Lee, Na-Rae; Inn, Kyung-Soo; Kim, Bum-Joon

    2013-10-01

    A reverse-transcriptase-subunit of telomerase (hTERT) derived peptide, GV1001, has been developed as a vaccine against various cancers. Here, we report an unexpected function of GV1001 as a cell-penetrating peptide (CPP). GV1001 was delivered into a variety of cells including various cancer cell lines and primary blood cells. Moreover, the delivered GV1001 was predominantly located in the cytoplasm of the cells, while a significantly higher proportion of TAT peptide was localized in the nucleus. Macromolecules such as proteins, DNA and siRNA, which were linked to GV1001 by direct covalent conjugation or non-covalent complexation through poly-lysine, were successfully delivered into cells, indicating that GV1001 can be used as a carrier for macromolecules. Expression of the delivered DNA, and lowered expression of the target gene by the delivered siRNA, suggest the potential therapeutic use of GV1001. Pull-down analysis identified Heat Shock Protein 90 (HSP90) and 70 (HSP70) as GV1001 interacting proteins. Treatment of Anti-HSP90 and HSP70 antibodies lowered the internalization of GV1001, indicating that the interaction is critical for the efficient internalization of GV1001. Collectively, the results of this study suggest the pharmaceutical potential of GV1001, already proven safe in clinical trials, as a carrier for the delivery of macromolecular therapeutics into cells, in addition to its own anti-cancer activity. PMID:23827187

  1. 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. PMID:26094944

  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. PMID:20189781

  3. Relationships between Membrane Binding, Affinity and Cell Internalization Efficacy of a Cell-Penetrating Peptide: Penetratin as a Case Study

    PubMed Central

    Alves, Isabel D.; Bechara, Cherine; Walrant, Astrid; Zaltsman, Yefim; Jiao, Chen-Yu; Sagan, Sandrine

    2011-01-01

    Background Penetratin is a positively charged cell-penetrating peptide (CPP) that has the ability to bind negatively charged membrane components, such as glycosaminoglycans and anionic lipids. Whether this primary interaction of penetratin with these cell surface components implies that the peptide will be further internalized is not clear. Methodology Using mass spectrometry, the amount of internalized and membrane bound penetratin remaining after washings, were quantified in three different cell lines: wild type (WT), glycosaminoglycans- (GAGneg) and sialic acid-deficient (SAneg) cells. Additionally, the affinity and kinetics of the interaction of penetratin to membrane models composed of pure lipids and membrane fragments from the referred cell lines was investigated, as well as the thermodynamics of such interactions using plasmon resonance and calorimetry. Principal Findings Penetratin internalized with the same efficacy in the three cell lines at 1 µM, but was better internalized at 10 µM in SAneg>WT>GAGneg. The heat released by the interaction of penetratin with these cells followed the ranking order of internalization efficiency. Penetratin had an affinity of 10 nM for WT cells and µM for SAneg and GAGneg cells and model membrane of phospholipids. The remaining membrane-bound penetratin after cells washings was similar in WT and GAGneg cells, which suggested that these binding sites relied on membrane phospholipids. The interaction of penetratin with carbohydrates was more superficial and reversible while it was stronger with phospholipids, likely because the peptide can intercalate between the fatty acid chains. Conclusion/Significance These results show that accumulation and high-affinity binding of penetratin at the cell-surface do not reflect the internalization efficacy of the peptide. Altogether, these data further support translocation (membrane phospholipids interaction) as being the internalization pathway used by penetratin at low

  4. 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

  5. The acceleration of boron neutron capture therapy using multi-linked mercaptoundecahydrododecaborate (BSH) fused cell-penetrating peptide.

    PubMed

    Michiue, Hiroyuki; Sakurai, Yoshinori; Kondo, Natsuko; Kitamatsu, Mizuki; Bin, Feng; Nakajima, Kiichiro; Hirota, Yuki; Kawabata, Shinji; Nishiki, Tei-ichi; Ohmori, Iori; Tomizawa, Kazuhito; Miyatake, Shin-ichi; Ono, Koji; Matsui, Hideki

    2014-03-01

    New anti-cancer therapy with boron neutron capture therapy (BNCT) is based on the nuclear reaction of boron-10 with neutron irradiation. The median survival of BNCT patients with glioblastoma was almost twice as long as those receiving standard therapy in a Japanese BNCT clinical trial. In this clinical trial, two boron compounds, BPA (boronophenylalanine) and BSH (sodium borocaptate), were used for BNCT. BPA is taken up into cells through amino acid transporters that are expressed highly in almost all malignant cells, but BSH cannot pass through the cell membrane and remains outside the cell. We simulated the energy transfer against the nucleus at different locations of boron from outside the cell to the nuclear region with neutron irradiation and concluded that there was a marked difference between inside and outside the cell in boron localization. To overcome this disadvantage of BSH in BNCT, we used a cell-penetrating peptide system for transduction of BSH. CPP (cell-membrane penetrating peptide) is very common peptide domains that transduce many physiologically active substances into cells in vitro and in vivo. BSH-fused CPPs can penetrate the cell membrane and localize inside a cell. To increase the boron ratio in one BSH-peptide molecule, 8BSH fused to 11R with a dendritic lysine structure was synthesized and administrated to malignant glioma cells and a brain tumor mouse model. 8BSH-11R localized at the cell nucleus and showed a very high boron value in ICP results. With neutron irradiation, the 8BSH-11R administrated group showed a significant cancer killing effect compared to the 100 times higher concentration of BSH-administrated group. We concluded that BSH-fused CPPs were one of the most improved and potential boron compounds in the next-stage BNCT trial and 8BSH-11R may be applied in the clinical setting. PMID:24452095

  6. Endocytic Trafficking of Nanoparticles Delivered by Cell-penetrating Peptides Comprised of Nona-arginine and a Penetration Accelerating Sequence

    PubMed Central

    Liu, Betty R.; Lo, Shih-Yen; Liu, Chia-Chin; Chyan, Chia-Lin; Huang, Yue-Wern; Aronstam, Robert S.; Lee, Han-Jung

    2013-01-01

    Cell-penetrating peptides (CPPs) can traverse cellular membranes and deliver biologically active molecules into cells. In this study, we demonstrate that CPPs comprised of nona-arginine (R9) and a penetration accelerating peptide sequence (Pas) that facilitates escape from endocytic lysosomes, denoted as PR9, greatly enhance the delivery of noncovalently associated quantum dots (QDs) into human A549 cells. Mechanistic studies, intracellular trafficking analysis and a functional gene assay reveal that endocytosis is the main route for intracellular delivery of PR9/QD complexes. Endocytic trafficking of PR9/QD complexes was monitored using both confocal and transmission electron microscopy (TEM). Zeta-potential and size analyses indicate the importance of electrostatic forces in the interaction of PR9/QD complexes with plasma membranes. Circular dichroism (CD) spectroscopy reveals that the secondary structural elements of PR9 have similar conformations in aqueous buffer at pH 7 and 5. This study of nontoxic PR9 provides a basis for the design of optimized cargo delivery that allows escape from endocytic vesicles. PMID:23840594

  7. Cell penetrating peptides as a therapeutic strategy in chronic lymphocytic leukemia.

    PubMed

    Arrouss, Issam; Decaudin, Didier; Choquet, Sylvain; Azar, Nabih; Parizot, Christophe; Zini, Jean M; Nemati, Fariba; Rebollo, Angelita

    2015-01-01

    PP2A is a serine/threonine phosphatase critical to a number of physiological and developmental processes. In this manuscript, we show that a peptide, specifically blocking the caspase- 9/PP2A interaction, DPT-C9h, induces apoptosis in primary tumour B cells isolated from peripheral blood mononuclear cells or bone marrow of chronic lymphocytic leukemia (CLL) patients, but not on B cells obtained from healthy donors (HD). Moreover, in both CLL patients and HD, DPT-C9h does not induce apoptosis on T- and NKcells and monocytes. Our results strongly suggest that DPT-C9h peptide has tumour specificity and that caspase-9/PP2Ac interaction constitutes a novel therapeutic approach for the treatment in CLL patients. PMID:25687226

  8. 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

  9. 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

  10. Mechanism of antifungal activity of antimicrobial peptide APP, a cell-penetrating peptide derivative, against Candida albicans: intracellular DNA binding and cell cycle arrest.

    PubMed

    Li, Lirong; Sun, Jin; Xia, Shufang; Tian, Xu; Cheserek, Maureen Jepkorir; Le, Guowei

    2016-04-01

    We investigated the antifungal properties and anti-candidal mechanism of antimicrobial peptide APP. The minimum inhibitory concentration of APP was 8 μM against Candida albicans and Aspeogillus flavus, the concentration against Saccharomyces cerevisiae and Cryptococcus neoformans was 16 μM, while 32 μM inhibited Aspergilla niger and Trichopyton rubrum. APP caused slight depolarization (12.32 ± 0.87%) of the membrane potential of intact C. albicans cells when it exerted its anti-candidal activity and only caused 21.52 ± 0.48% C. albicans cell membrane damage. APP interacted with cell wall membrane, caused potassium efflux and nucleotide leakage. However, confocal fluorescence microscopy experiment and flow cytometry confirmed that FITC-labeled APP penetrated C. albicans cell membrane with 52.31 ± 1.88% cell-penetrating efficiency and accumulated in the cytoplasm. Then, APP interact with C. albicans genomic DNA and completely suppressed DNA migration above weight ratio (peptide/DNA) of 2, and significantly arrested cell cycles during the S-phase (S-phase cell population was 27.09 ± 0.73%, p < 0.05) after penetrating the cell membrane. Results indicated that APP kills C. albicans for efficient cell-penetrating efficiency, strong DNA-binding affinity and significant physiological changes inducing S-phase arrest in intracellular environment. PMID:26743655

  11. Early endosomal escape of a cyclic cell-penetrating peptide allows effective cytosolic cargo delivery.

    PubMed

    Qian, Ziqing; LaRochelle, Jonathan R; Jiang, Bisheng; Lian, Wenlong; Hard, Ryan L; Selner, Nicholas G; Luechapanichkul, Rinrada; Barrios, Amy M; Pei, Dehua

    2014-06-24

    Cyclic heptapeptide cyclo(FΦRRRRQ) (cFΦR4, where Φ is l-2-naphthylalanine) was recently found to be efficiently internalized by mammalian cells. In this study, its mechanism of internalization was investigated by perturbing various endocytic events through the introduction of pharmacologic agents and genetic mutations. The results show that cFΦR4 binds directly to membrane phospholipids, is internalized into human cancer cells through endocytosis, and escapes from early endosomes into the cytoplasm. Its cargo capacity was examined with a wide variety of molecules, including small-molecule dyes, linear and cyclic peptides of various charged states, and proteins. Depending on the nature of the cargos, they may be delivered by endocyclic (insertion of cargo into the cFΦR4 ring), exocyclic (attachment of cargo to the Gln side chain), or bicyclic approaches (fusion of cFΦR4 and cyclic cargo rings). The overall delivery efficiency (i.e., delivery of cargo into the cytoplasm and nucleus) of cFΦR4 was 4-12-fold higher than those of nonaarginine, HIV Tat-derived peptide, or penetratin. The higher delivery efficiency, coupled with superior serum stability, minimal toxicity, and synthetic accessibility, renders cFΦR4 a useful transporter for intracellular cargo delivery and a suitable system for investigating the mechanism of endosomal escape. PMID:24896852

  12. 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

  13. Design and Screening of a Glial Cell-Specific, Cell Penetrating Peptide for Therapeutic Applications in Multiple Sclerosis

    PubMed Central

    Heffernan, Corey; Sumer, Huseyin; Guillemin, Gilles J.; Manuelpillai, Ursula; Verma, Paul J.

    2012-01-01

    Multiple Sclerosis (MS) is an autoimmune, neurodegenerative disease of the central nervous system (CNS) characterized by demyelination through glial cell loss. Current and proposed therapeutic strategies to arrest demyelination and/or promote further remyelination include: (i) modulation of the host immune system; and/or (ii) transplantation of myelinating/stem or progenitor cells to the circulation or sites of injury. However, significant drawbacks are inherent with both approaches. Cell penetrating peptides (CPP) are short amino acid sequences with an intrinsic ability to translocate across plasma membranes, and theoretically represent an attractive vector for delivery of therapeutic peptides or nanoparticles to glia to promote cell survival or remyelination. The CPPs described to date are commonly non-selective in the cell types they transduce, limiting their therapeutic application in vivo. Here, we describe a theoretical framework for design of a novel CPP sequence that selectively transduces human glial cells (excluding non-glial cell types), and conduct preliminary screens of purified, recombinant CPPs with immature and matured human oligodendrocytes and astrocytes, and two non-glial cell types. A candidate peptide, termed TD2.2, consistently transduced glial cells, was significantly more effective at transducing immature oligodendrocytes than matured progeny, and was virtually incapable of transducing two non-glial cell types: (i) human neural cells and (ii) human dermal fibroblasts. Time-lapse confocal microscopy confirms trafficking of TD2.2 (fused to EGFP) to mature oligodendrocytes 3–6 hours after protein application in vitro. We propose selectivity of TD2.2 for glial cells represents a new therapeutic strategy for the treatment of glial-related disease, such as MS. PMID:23049807

  14. Structure Analysis and Conformational Transitions of the Cell Penetrating Peptide Transportan 10 in the Membrane-Bound State

    PubMed Central

    Strandberg, Erik; Verdurmen, Wouter P. R.; Bürck, Jochen; Ehni, Sebastian; Mykhailiuk, Pavel K.; Afonin, Sergii; Gerthsen, Dagmar; Komarov, Igor V.; Brock, Roland; Ulrich, Anne S.

    2014-01-01

    Structure analysis of the cell-penetrating peptide transportan 10 (TP10) revealed an exemplary range of different conformations in the membrane-bound state. The bipartite peptide (derived N-terminally from galanin and C-terminally from mastoparan) was found to exhibit prominent characteristics of (i) amphiphilic α-helices, (ii) intrinsically disordered peptides, as well as (iii) β-pleated amyloid fibrils, and these conformational states become interconverted as a function of concentration. We used a complementary approach of solid-state 19F-NMR and circular dichroism in oriented membrane samples to characterize the structural and dynamical behaviour of TP10 in its monomeric and aggregated forms. Nine different positions in the peptide were selectively substituted with either the L- or D-enantiomer of 3-(trifluoromethyl)-bicyclopent-[1.1.1]-1-ylglycine (CF3-Bpg) as a reporter group for 19F-NMR. Using the L-epimeric analogs, a comprehensive three-dimensional structure analysis was carried out in lipid bilayers at low peptide concentration, where TP10 is monomeric. While the N-terminal region is flexible and intrinsically unstructured within the plane of the lipid bilayer, the C-terminal α-helix is embedded in the membrane with an oblique tilt angle of ∼55° and in accordance with its amphiphilic profile. Incorporation of the sterically obstructive D-CF3-Bpg reporter group into the helical region leads to a local unfolding of the membrane-bound peptide. At high concentration, these helix-destabilizing C-terminal substitutions promote aggregation into immobile β-sheets, which resemble amyloid fibrils. On the other hand, the obstructive D-CF3-Bpg substitutions can be accommodated in the flexible N-terminus of TP10 where they do not promote aggregation at high concentration. The cross-talk between the two regions of TP10 thus exerts a delicate balance on its conformational switch, as the presence of the α-helix counteracts the tendency of the unfolded N

  15. Arginine-Rich Peptides Destabilize the Plasma Membrane, Consistent with a Pore Formation Translocation Mechanism of Cell-Penetrating Peptides

    PubMed Central

    Herce, H.D.; Garcia, A.E.; Litt, J.; Kane, R.S.; Martin, P.; Enrique, N.; Rebolledo, A.; Milesi, V.

    2009-01-01

    Abstract Recent molecular-dynamics simulations have suggested that the arginine-rich HIV Tat peptides translocate by destabilizing and inducing transient pores in phospholipid bilayers. In this pathway for peptide translocation, Arg residues play a fundamental role not only in the binding of the peptide to the surface of the membrane, but also in the destabilization and nucleation of transient pores across the bilayer. Here we present a molecular-dynamics simulation of a peptide composed of nine Args (Arg-9) that shows that this peptide follows the same translocation pathway previously found for the Tat peptide. We test experimentally the hypothesis that transient pores open by measuring ionic currents across phospholipid bilayers and cell membranes through the pores induced by Arg-9 peptides. We find that Arg-9 peptides, in the presence of an electrostatic potential gradient, induce ionic currents across planar phospholipid bilayers, as well as in cultured osteosarcoma cells and human smooth muscle cells. Our results suggest that the mechanism of action of Arg-9 peptides involves the creation of transient pores in lipid bilayers and cell membranes. PMID:19804722

  16. Arginine-rich peptides destabilize the plasma membrane, consistent with a pore formation translocation mechanism of cell-penetrating peptides.

    PubMed

    Herce, H D; Garcia, A E; Litt, J; Kane, R S; Martin, P; Enrique, N; Rebolledo, A; Milesi, V

    2009-10-01

    Recent molecular-dynamics simulations have suggested that the arginine-rich HIV Tat peptides translocate by destabilizing and inducing transient pores in phospholipid bilayers. In this pathway for peptide translocation, Arg residues play a fundamental role not only in the binding of the peptide to the surface of the membrane, but also in the destabilization and nucleation of transient pores across the bilayer. Here we present a molecular-dynamics simulation of a peptide composed of nine Args (Arg-9) that shows that this peptide follows the same translocation pathway previously found for the Tat peptide. We test experimentally the hypothesis that transient pores open by measuring ionic currents across phospholipid bilayers and cell membranes through the pores induced by Arg-9 peptides. We find that Arg-9 peptides, in the presence of an electrostatic potential gradient, induce ionic currents across planar phospholipid bilayers, as well as in cultured osteosarcoma cells and human smooth muscle cells. Our results suggest that the mechanism of action of Arg-9 peptides involves the creation of transient pores in lipid bilayers and cell membranes. PMID:19804722

  17. Integrating Display and Delivery Functionality with a Cell Penetrating Peptide Mimic as a Scaffold for Intracellular Multivalent Multitargeting.

    PubMed

    Bai, Yugang; Nguyen, Lien; Song, Ziyuan; Peng, Shaohong; Lee, JuYeon; Zheng, Nan; Kapoor, Iti; Hagler, Lauren D; Cai, Kaimin; Cheng, Jianjun; Chan, H Y Edwin; Zimmerman, Steven C

    2016-08-01

    The construction of a multivalent ligand is an effective way to increase affinity and selectivity toward biomolecular targets with multiple-ligand binding sites. Adopting this strategy, we used a known cell-penetrating peptide (CPP) mimic as a scaffold to develop a series of multivalent ligand constructs that bind to the expanded dCTG (CTG(exp)) and rCUG nucleotide repeats (CUG(exp)) known to cause myotonic dystrophy type I (DM1), an incurable neuromuscular disease. By assembling this polyvalent construct, the hydrophobic ligands are solubilized and delivered into cell nuclei, and their enhanced binding affinity leads to the inhibition of ribonuclear foci formation and a reversal of splicing defects, all at low concentrations. Some of the multivalent ligands are shown to inhibit selectively the in vitro transcription of (CTG·CAG)74, to reduce the concentration of the toxic CUG RNA in DM1 model cells, and to show phenotypic improvement in vivo in a Drosophila model of DM1. This strategy may be useful in drug design for other trinucleotide repeat disorders and more broadly for intracellular multivalent targeting. PMID:27355522

  18. Guanidine-Containing Methacrylamide (Co)polymers via aRAFT: Toward a Cell Penetrating Peptide Mimica

    PubMed Central

    Treat, Nicolas J.; Smith, DeeDee; Teng, Chengwen; Flores, Joel D.; Abel, Brooks A.; York, Adam W.; Huang, Faqing; McCormick, Charles L.

    2011-01-01

    We report the synthesis and controlled radical homo- and block copolymerization of 3-guanidinopropyl methacrylamide (GPMA) utilizing aqueous reversible addition-fragmentation chain transfer (aRAFT) polymerization. The resulting homopolymer and block copolymer with N-(2-hydroxypropyl) methacrylamide (HPMA) were prepared to mimic the behavior of cell penetrating peptides (CPPs) and poly(arginine) (> 6 units) which have been shown to cross cell membranes. The homopolymerization mediated by 4-cyano-4-(ethylsulfanylthiocarbonylsulfanyl)pentanoic acid (CEP) in aqueous buffer exhibited pseudo-first-order kinetics and linear growth of molecular weight with conversion. Retention of the “living” thiocarbonylthio ω-end-group was demonstrated through successful chain extension of the GPMA macroCTA yielding GPMA37-b-GPMA61 (Mw/Mn =1.05). Block copolymers of GPMA with the non-immunogenic, biocompatible HPMA were synthesized yielding HPMA271-b-GPMA13 (Mw/Mn = 1.15). Notably, intracellular uptake was confirmed by fluorescence microscopy, confocal laser scanning microscopy, and flow cytometry experiments after 2.5 h incubation with KB cells at 4 °C and at 37 °C utilizing FITC-labeled, GPMA-containing copolymers. The observed facility of cellular uptake and the structural control afforded by aRAFT polymerization suggest significant potential for these synthetic (co)polymers as drug delivery vehicles in targeted therapies. PMID:22639734

  19. 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

  20. Surgical molecular navigation with a Ratiometric Activatable Cell Penetrating Peptide improves intraoperative identification and resection of small salivary gland cancers

    PubMed Central

    Hussain, Timon; Savariar, Elamprakash N.; Diaz-Perez, Julio A.; Messer, Karen; Pu, Minya; Tsien, Roger Y.; Nguyen, Quyen T.

    2015-01-01

    Background We evaluated the use of intraoperative fluorescence guidance by enzymatically cleavable ratiometric activatable cell-penetrating peptide (RACPPPLGC(Me)AG) containing Cy5 as a fluorescent donor and Cy7 as a fluorescent acceptor for salivary gland cancer surgery in a mouse model. Methods Surgical resection of small parotid gland cancers in mice was performed with fluorescence guidance or white light (WL) imaging alone. Tumor identification accuracy, operating time and tumor free survival were compared. Results RACPP guidance aided tumor detection (positive histology in 90% (27/30) vs. 48% (15/31) for WL, p<0.001). A ~25% ratiometric signal increase as the threshold to distinguish between tumor and adjacent tissue, yielded >90% detection sensitivity and specificity. Operating time was reduced by 54% (p<0.001), tumor free survival was increased with RACPP guidance (p=0.025). Conclusions RACPP provides real-time intraoperative guidance leading to improved survival. Ratiometric signal thresholds can be set according to desired detection accuracy levels for future RACPP applications. PMID:25521629

  1. Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA.

    PubMed

    Ramakrishna, Suresh; Kwaku Dad, Abu-Bonsrah; Beloor, Jagadish; Gopalappa, Ramu; Lee, Sang-Kyung; Kim, Hyongbum

    2014-06-01

    RNA-guided endonucleases (RGENs) derived from the CRISPR/Cas system represent an efficient tool for genome editing. RGENs consist of two components: Cas9 protein and guide RNA. Plasmid-mediated delivery of these components into cells can result in uncontrolled integration of the plasmid sequence into the host genome, and unwanted immune responses and potential safety problems that can be caused by the bacterial sequences. Furthermore, this delivery method requires transfection tools. Here we show that simple treatment with cell-penetrating peptide (CPP)-conjugated recombinant Cas9 protein and CPP-complexed guide RNAs leads to endogenous gene disruptions in human cell lines. The Cas9 protein was conjugated to CPP via a thioether bond, whereas the guide RNA was complexed with CPP, forming condensed, positively charged nanoparticles. Simultaneous and sequential treatment of human cells, including embryonic stem cells, dermal fibroblasts, HEK293T cells, HeLa cells, and embryonic carcinoma cells, with the modified Cas9 and guide RNA, leads to efficient gene disruptions with reduced off-target mutations relative to plasmid transfections, resulting in the generation of clones containing RGEN-induced mutations. Our CPP-mediated RGEN delivery process provides a plasmid-free and additional transfection reagent-free method to use this tool with reduced off-target effects. We envision that our method will facilitate RGEN-directed genome editing. PMID:24696462

  2. A specific aptamer-cell penetrating peptides complex delivered siRNA efficiently and suppressed prostate tumor growth in vivo.

    PubMed

    Diao, Yanjun; Liu, Jiayun; Ma, Yueyun; Su, Mingquan; Zhang, Hongyi; Hao, Xiaoke

    2016-05-01

    Specific and efficient delivery of siRNA into intended tumor cells remains as a challenge, even though RNAi has been exploited as a new strategy for prostate cancer therapy. This work aims to address both specificity and efficiency of SURVIVIN-siRNA delivery by constructing a therapeutic complex using combinatorial strategies. A fusion protein STD was first expressed to serve as a backbone, consisting of streptavidin, a cell-penetrating peptide called Trans-Activator of Transcription (TAT) and a double-stranded RNA binding domain. A biotinylated Prostate Specific Membrane Antigen (PSMA) specific aptamer A10 and SURVIVIN-siRNA were then linked to STD protein to form the therapeutic complex. This complex could specifically targeted PSMA(+) tumor cells. Compared to lipofectamine and A10-siRNA chimera, it demonstrated higher efficiency in delivering siRNA into target cells by 19.2% and 59.9%, and increased apoptosis by 16.8% and 26.1% respectively. Upon systemic administration, this complex also showed significant efficacy in suppressing tumor growth in athymic mice (p <0.001). We conclude that this therapeutic complex could specifically and efficiently deliver SURVIVIN-siRNA to target cells and suppressed tumor growth in vivo, which indicates its potential to be used as a new strategy in prostate cancer therapy. PMID:26954374

  3. The next generation cell-penetrating peptide and carbon dot conjugated nano-liposome for transdermal delivery of curcumin.

    PubMed

    Patra, Santanu; Roy, Ekta; Madhuri, Rashmi; Sharma, Prashant K

    2016-03-01

    To overcome the problems associated with conventional liposomes in transdermal drug delivery like limited penetration ability and poor stability, in this article we report a new generation of cell penetrating peptide polyarginine containing nano-liposomes conjugated with carbon dots. The newly synthesized, cost-effective liposomic precursors were used for the fabrication of liposomes. The resulting liposomes have a bilayer structure like that of conventional liposomes with much smaller size, higher stability, and high penetration ability. The nano-liposomes show high stability at room temperature for three months without any change in size or encapsulation efficiency. The incorporation of carbon dots also opens up their application in fluorescence cell imaging studies, which is very well supported by the fluorescence microscopic analysis of the liposome skin penetration. The as-prepared nano-liposomes do not show any cytotoxicity for MCF-7 cells, even at high concentrations; however, when drug loaded liposomes are applied, they can kill the cancer cells with a high rate. The synthesized nano-liposomes have the potential to be used as an efficient, stable, biocompatible nanocarrier for transdermal drug delivery. PMID:26631310

  4. 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

  5. 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. PMID:25797209

  6. 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.

  7. Cell-penetrating peptides mediated protein cross-membrane delivery and its use in bacterial vector vaccine.

    PubMed

    Ma, Jimei; Xu, Jinmei; Guan, Lingyu; Hu, Tianjian; Liu, Qin; Xiao, Jingfan; Zhang, Yuanxing

    2014-07-01

    It is an attractive strategy to develop a recombinant bacterial vector vaccine by expressing exogenous protective antigen to induce the immune response, and the main concern is how to enhance the cellular internalization of antigen produced by bacterial vector. Cell-penetrating peptides (CPPs) are short cationic/amphipathic peptides which facilitate cellular uptake of various molecular cargoes and therefore have great potentials in vector vaccine design. In this work, eleven different CPPs were fused to the C-terminus of EGFP respectively, and the resultant EGFP-CPP fusion proteins were expressed and purified to assay their cross-membrane transport in macrophage J774 A.1 cells. Among the tested CPPs, TAT showed an excellent capability to deliver the cargo protein EGFP into cytoplasm. In order to establish an efficient antigen delivery system in Escherichia coli, the EGFP-TAT synthesis circuit was combined with an in vivo inducible lysis circuit PviuA-E in E. coli to form an integrated antigen delivery system, the resultant E. coli was proved to be able to lyse upon the induction of a mimic in vivo signal and thus release intracellular EGFP-TAT intensively, which were assumed to undergo a more efficient intracellular delivery by CPP to evoke protective immune responses. Based on the established antigen delivery system, the protective antigen gene flgD from an invasive intracellular fish pathogen Edwardsiella tarda EIB202, was applied to establish an E. coli recombinant vector vaccine. This E. coli vector vaccine presented superior immune protection (RPS = 63%) under the challenge with E. tarda EIB202, suggesting that the novel antigen delivery system had great potential in bacterial vector vaccine applications. PMID:24746937

  8. Enzyme-triggered delivery of chlorambucil from conjugates based on the cell-penetrating peptide BP16.

    PubMed

    Soler, Marta; González-Bártulos, Marta; Figueras, Eduard; Ribas, Xavi; Costas, Miquel; Massaguer, Anna; Planas, Marta; Feliu, Lidia

    2015-02-01

    The undecapeptide KKLFKKILKKL-NH2 (BP16) is a non-toxic cell-penetrating peptide (CPP) that is mainly internalized into cancer cells through a clathrin dependent endocytic mechanism and localizes in late endosomes. Moreover, this CPP is able to enhance the cellular uptake of chlorambucil (CLB) improving its cytotoxicity. In this work, we further explored the cell-penetrating properties of BP16 and those of its arginine analogue BP308. We investigated the influence on the cytotoxicity and on the cellular uptake of conjugating CLB at the N- or the C-terminal end of these undecapeptides. The effect of incorporating the cathepsin B-cleavable sequence Gly-Phe-Leu-Gly in CLB-BP16 and CLB-BP308 conjugates was also evaluated. The activity of CLB was significantly improved when conjugated at the N- or the C-terminus of BP16, or at the N-terminus of BP308. While CLB alone was not active (IC50 of 73.7 to >100 μM), the resulting conjugates displayed cytotoxic activity against CAPAN-1, MCF-7, PC-3, 1BR3G and SKMEL-28 cell lines with IC50 values ranging from 8.7 to 25.5 μM. These results were consistent with the internalization properties observed for the corresponding 5(6)-carboxyfluorescein-labeled conjugates. The presence of the tetrapeptide Gly-Phe-Leu-Gly at either the N- or the C-terminus of CLB-BP16 conjugates further increased the efficacy of CLB (IC50 of 3.6 to 16.2 μM), which could be attributed to its selective release in the lysosomal compartment. Enzymatic assays with cathepsin B showed the release of CLB-Gly-OH from these sequences within a short time. Therefore, the combination of BP16 with an enzymatic cleavable sequence can be used as a drug delivery system for the effective uptake and release of drugs in cancer cells. PMID:25474438

  9. 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

  10. 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. PMID:26181841

  11. 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

  12. 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

  13. 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. PMID:26216471

  14. Enhanced anti-tumor activity of trichosanthin after combination with a human-derived cell-penetrating peptide, and a possible mechanism of activity.

    PubMed

    Lu, Ye-Zhou; Li, Peng-Fei; Li, Yan-Zhong; Luo, Fan; Guo, Chao; Lin, Bin; Cao, Xue-Wei; Zhao, Jian; Wang, Fu-Jun

    2016-07-01

    Trichosanthin (TCS), a type I ribosome-inactivating protein (RIP-I) and renowned Chinese traditional medicine, displays a broad spectrum of biological and pharmacological properties. Particularly, its anti-tumor activity has received a great deal of attention. However, the cellular mechanism for TCS uptake varies with different tumor cell lines, leading to discrepancies in its reported ability to penetrate cells. In this study, HBD, a human derived cell-penetrating peptide (CPP), was used to improve the delivery of TCS into several types of tumor cells, including HeLa cells. Recombinant TCS (rTCS) with or without the fused HBD peptide was expressed in Escherichia coli cells and successfully purified by Ni-NTA affinity chromatography. The cellular uptake efficiency of FITC-labelled-rTCS-HBD was observed in HeLa cells and compared with the uptake efficiency of non-HBD conjugated rTCS under the same conditions using laser confocal microscopy. Moreover, the IC50 value of rTCS-HBD in the tested tumor cells was much lower than that of rTCS, indicating that HBD could efficiently deliver the rTCS into tumor cells. When compared with rTCS, rTCS-HBD induced higher rates of apoptosis in HeLa cells as analyzed by flow cytometry. Furthermore, the apoptotic events observed in HeLa cells incubated with HBD-fused rTCS included activation of Caspase-9, decrease in the Bcl-2/Bax ratio, and cleavage of PARP. These results strongly suggest the participation of mitochondria in apoptosis. This report illustrates one possible method for achieving the efficient transport of TCS into cells using a CPP as a vector, and increases the likelihood that TCS can be used in the clinic. PMID:27050721

  15. Effect of different intestinal conditions on the intermolecular interaction between insulin and cell-penetrating peptide penetratin and on its contribution to stimulation of permeation through intestinal epithelium.

    PubMed

    Kamei, Noriyasu; Aoyama, Yukina; Khafagy, El-Sayed; Henmi, Mao; Takeda-Morishita, Mariko

    2015-08-01

    Our recent studies have shown that the coadministration of cell-penetrating peptides (CPPs) is a potential strategy for oral delivery of peptide- and protein-based biopharmaceuticals. The intermolecular interaction between drug and CPP is an essential factor in the effective delivery of these drugs, but the characteristics of the interaction under the conditions of the intestinal lumen remain unknown. In this study, therefore, we examined the characteristics of binding of the amphipathic CPP penetratin to insulin and the efficiency of its enhancement of epithelial insulin transport at different pH and in simulated intestinal fluids (SIFs). The binding between insulin and penetratin was pH dependent and particularly decreased at pH 5.0. In addition, we clarified that the sodium taurocholate (NaTC) present in two types of SIF (fasted-state SIF [FaSSIF] and fed-state SIF [FeSSIF]) affected binding efficiency. However, the permeation of insulin through a Caco-2 cell monolayer was significantly facilitated by coincubation with l- or d-penetratin at various pH values. Moreover, the permeation-stimulating effect of l-penetratin was observed in FaSSIF containing NaTC and lecithin, but not in 3mM NaTC solution, suggesting that the presence of lecithin was the key factor in maintaining the ability of penetratin to enhance the intestinal absorption of biopharmaceuticals. This report describes the essential considerations for in vivo use and clinical application of a CPP-based oral delivery strategy. PMID:25960330

  16. 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.

  17. Antitumor activity of tripterine via cell-penetrating peptide-coated nanostructured lipid carriers in a prostate cancer model

    PubMed Central

    Yuan, Ling; Liu, Congyan; Chen, Yan; Zhang, Zhenhai; Zhou, Lei; Qu, Ding

    2013-01-01

    Background The purpose of this study was to evaluate the antitumor effect of cell-penetrating peptide-coated tripterine-loaded nanostructured lipid carriers (CT-NLC) on prostate tumor cells in vitro and in vivo. Methods CT-NLC were developed to improve the hydrophilicity of tripterine. The antiproliferative effects of CT-NLC, tripterine-loaded nanostructured lipid carriers (T-NLC), and free tripterine in a human prostatic carcinoma cell line (PC-3) and a mouse prostate carcinoma cell line (RM-1) were evaluated using an MTT assay. The advantage of CT-NLC over T-NLC and free tripterine with regard to antitumor activity in vivo was evaluated in a prostate tumor-bearing mouse model. The induced tumor necrosis factor-alpha and interleukin-6 cytokine content was investigated by enzyme-linked immunosorbent assay to determine the effect of CT-NLC, T-NLC, and free tripterine on immune responses. Histologic and TUNEL assays were carried out to investigate the mechanisms of tumor necrosis and apoptosis. Results CT-NLC, T-NLC, and free tripterine showed high antiproliferative activity in a dose-dependent manner, with an IC50 of 0.60, 0.81, and 1.02 μg/mL in the PC-3 cell line and 0.41, 0.54, and 0.89 μg/mL in the RM-1 cell line after 36 hours. In vivo, the tumor inhibition rates for cyclophosphamide, high-dose (4 mg/kg) and low-dose (2 mg/kg) tripterine, high-dose (4 mg/kg) and low-dose (2 mg/kg) T-NLC, high-dose (4 mg/kg) and low-dose (2 mg/kg) CT-NLC were 76.51%, 37.07%, 29.53%, 63.56%, 48.25%, 72.68%, and 54.50%, respectively, showing a dose-dependent pattern. The induced tumor necrosis factor-alpha and interleukin-6 cytokine content after treatment with CT-NLC and T-NLC was significantly higher than that of high-dose tripterine. Moreover, CT-NLC showed the expected advantage of inducing necrosis and apoptosis in prostate tumor cells. Conclusion CT-NLC noticeably enhanced antitumor activity in vitro and in vivo and showed dramatically improved cytotoxicity in normal cells

  18. 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

  19. 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

  20. Enhanced cellular internalization of CdTe quantum dots mediated by arginine- and tryptophan-rich cell-penetrating peptides as efficient carriers.

    PubMed

    Farkhani, Samad Mussa; Johari-Ahar, Mohammad; Zakeri-Milani, Parvin; Shahbazi Mojarrad, Javid; Valizadeh, Hadi

    2016-09-01

    Quantum dots (QDs), as a new class of fluorescent tags, have been widely used for biomedical applications. Despite their various advantages, QDs do not efficiently enter cells on their own, and aggregation often occurs following internalization. In the present study, we have designed three QD-cell-penetrating peptide (CPP) complexes to increase the uptake of QD into cells. The results demonstrated that R9 and R5W3R4 form relatively stable noncovalent complexes with QDs, considerably increasing the rate and efficiency of QD uptake by A549 cells. These data suggest that cationic CPPs could efficiently transfer QDs into cells in a non-toxic manner. PMID:25884240

  1. Co-operative Membrane Disruption Between Cell-penetrating Peptide and Cargo: Implications for the Therapeutic Use of the Bcl-2 Converter Peptide D-NuBCP-9-r8

    PubMed Central

    Watkins, Catherine L; Sayers, Edward J; Allender, Chris; Barrow, David; Fegan, Christopher; Brennan, Paul; Jones, Arwyn T

    2011-01-01

    Delivering apoptosis inducing peptides to cells is an emerging area in cancer and molecular therapeutics. Here, we have identified an alternative mechanism of action for the proapoptotic chimeric peptide D-NuBCP-9-r8. Integral to D-NuBCP-9-r8 is the Nur-77-derived D-isoform sequence fsrslhsll that targets Bcl-2, and the cell-penetrating peptide (CPP) octaarginine (r8) that is required for intracellular delivery. We find that the N-terminal phenylalanine of fsrslhsll acts in synergy with the cell-penetrating moiety to enhance peptide uptake at low nontoxic levels and cause rapid membrane blebbing and cell necrosis at higher (IC50) concentrations. These effects were not observed when a single phenylalanine–alanine mutation was introduced at the N-terminus of D-NuBCP-9-r8. Using primary samples from chronic lymphocytic leukemia (CLL) patients and cancer cell lines, we show that NuBCP-9-r8 induced toxicity, via membrane disruption, is independent of Bcl-2 expression. Overall, this study demonstrates a new mechanism of action for this peptide and cautions its use as a highly specific entity for targeting Bcl-2. For delivery of therapeutic peptides the work emphasizes that key amino acids in cargo, located several residues away from the cell-penetrating sequence, can significantly influence their cellular uptake and mode of action. PMID:21934653

  2. Interference with RUNX1/ETO Leukemogenic Function by Cell-Penetrating Peptides Targeting the NHR2 Oligomerization Domain

    PubMed Central

    Bartel, Yvonne; Grez, Manuel; Wichmann, Christian

    2013-01-01

    The leukemia-associated fusion protein RUNX1/ETO is generated by the chromosomal translocation t(8;21) which appears in about 12% of all de novo acute myeloid leukemias (AMLs). Essential for the oncogenic potential of RUNX1/ETO is the oligomerization of the chimeric fusion protein through the nervy homology region 2 (NHR2) within ETO. In previous studies, we have shown that the intracellular expression of peptides containing the NHR2 domain inhibits RUNX1/ETO oligomerization, thereby preventing cell proliferation and inducing differentiation of RUNX1/ETO transformed cells. Here, we show that introduction of a recombinant TAT-NHR2 fusion polypeptide into the RUNX1/ETO growth-dependent myeloid cell line Kasumi-1 results in decreased cell proliferation and increased numbers of apoptotic cells. This effect was highly specific and mediated by binding the TAT-NHR2 peptide to ETO sequences, as TAT-polypeptides containing the oligomerization domain of BCR did not affect cell proliferation or apoptosis in Kasumi-1 cells. Thus, the selective interference with NHR2-mediated oligomerization by peptides represents a challenging but promising strategy for the inhibition of the leukemogenic potential of RUNX1/ETO in t(8;21)-positive leukemia. PMID:23865046

  3. 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

  4. 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

  5. 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.

  6. A+-Helix of Protein C Inhibitor (PCI) Is a Cell-penetrating Peptide That Mediates Cell Membrane Permeation of PCI*

    PubMed Central

    Yang, Hanjiang; Wahlmüller, Felix Christof; Sarg, Bettina; Furtmüller, Margareta; Geiger, Margarethe

    2015-01-01

    Protein C inhibitor (PCI) is a serpin with broad protease reactivity. It binds glycosaminoglycans and certain phospholipids that can modulate its inhibitory activity. PCI can penetrate through cellular membranes via binding to phosphatidylethanolamine. The exact mechanism of PCI internalization and the intracellular role of the serpin are not well understood. Here we showed that testisin, a glycosylphosphatidylinositol-anchored serine protease, cleaved human PCI and mouse PCI (mPCI) at their reactive sites as well as at sites close to their N terminus. This cleavage was observed not only with testisin in solution but also with cell membrane-anchored testisin on U937 cells. The cleavage close to the N terminus released peptides rich in basic amino acids. Synthetic peptides corresponding to the released peptides of human PCI (His1–Arg11) and mPCI (Arg1–Ala18) functioned as cell-penetrating peptides. Because intact mPCI but not testisin-cleaved mPCI was internalized by Jurkat T cells, a truncated mPCI mimicking testisin-cleaved mPCI was created. The truncated mPCI lacking 18 amino acids at the N terminus was not taken up by Jurkat T cells. Therefore our model suggests that testisin or other proteases could regulate the internalization of PCI by removing its N terminus. This may represent one of the mechanisms regulating the intracellular functions of PCI. PMID:25488662

  7. Membrane-Bound Dynamic Structure of an Arginine-Rich Cell-Penetrating Peptide, the Protein Transduction Domain of HIV TAT, from Solid-State NMR

    PubMed Central

    Su, Yongchao; Waring, Alan J.; Ruchala, Piotr; Hong, Mei

    2010-01-01

    The protein transduction domain of HIV-1 TAT, TAT(48-60), is an efficient cell-penetrating peptide (CPP) that diffuses across the lipid membranes of cells despite eight cationic Arg and Lys residues. To understand its mechanism of membrane translocation against the free energy barrier, we have conducted solid-state NMR experiments to determine the site-specific conformation, dynamics, and lipid interaction of the TAT peptide in anionic lipid bilayers. We found that TAT(48-60) is a highly dynamic and nearly random-coil peptide in the lipid bilayer, and inserts into the membrane-water interface near the glycerol backbone region. Arg-phosphate salt bridge interaction was revealed by short guanidinium-phosphate distances and restricted dynamics of the guanidinium. Together with the observation of strong peptide-water cross peaks in 1H spin diffusion spectra, these results indicate that TAT binding to the membrane-water interface is stabilized not only by electrostatic attraction to the anionic lipids, but also by intermolecular hydrogen bonding with the lipid phosphates and water, which may take the role of intramolecular hydrogen bonds in canonical secondary structures. The random-coil structure of TAT and another CPP, penetratin, suggests that the lack of amphipathic structure is essential for rapid translocation of these Arg-rich CPPs across the lipid membrane without causing permanent damages to the membrane integrity. PMID:20550193

  8. 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

  9. Synthesis, cellular uptake and HIV-1 Tat-dependent trans-activation inhibition activity of oligonucleotide analogues disulphide-conjugated to cell-penetrating peptides

    PubMed Central

    Turner, John J.; Arzumanov, Andrey A.; Gait, Michael J.

    2005-01-01

    Oligonucleotides composed of 2′-O-methyl and locked nucleic acid residues complementary to HIV-1 trans-activation responsive element TAR block Tat-dependent trans-activation in a HeLa cell assay when delivered by cationic lipids. We describe an improved procedure for synthesis and purification under highly denaturing conditions of 5′-disulphide-linked conjugates of 3′-fluorescein labelled oligonucleotides with a range of cell-penetrating peptides and investigate their abilities to enter HeLa cells and block trans-activation. Free uptake of 12mer OMe/LNA oligonucleotide conjugates to Tat (48–58), Penetratin and R9F2 was observed in cytosolic compartments of HeLa cells. Uptake of the Tat conjugate was enhanced by N-terminal addition of four Lys or Arg residues or a second Tat peptide. None of the conjugates entered the nucleus or inhibited trans-activation when freely delivered, but inhibition was obtained in the presence of cationic lipids. Nuclear exclusion was seen for free delivery of Tat (48–58), Penetratin and R9 conjugates of 16mer phosphorothioate OMe oligonucleotide. Uptake into human fibroblast cytosolic compartments was seen for Tat, Penetratin, R9F2 and Transportan conjugates. Large enhancements of HeLa cell uptake into cytosolic compartments were seen when free Tat peptide was added to Tat conjugate of 12mer OMe/LNA oligonucleotide or Penetratin peptide to Penetratin conjugate of the same oligonucleotide. PMID:15640444

  10. Reversible Sheet–Turn Conformational Change of a Cell-Penetrating Peptide in Lipid Bilayers Studied by Solid-State NMR

    PubMed Central

    Su, Yongchao; Mani, Rajeswari; Doherty, Tim; Waring, Alan J.

    2014-01-01

    The membrane-bound conformation of a cell-penetrating peptide, penetratin, is investigated using solid-state NMR spectroscopy. The 13C chemical shifts of 13C, 15N-labeled residues in the peptide indicate a reversible conformational change from β-sheet at low temperature to coil-like at high temperature. This conformational change occurs for all residues examined between positions 3 and 13, at peptide/lipid molar ratios of 1:15 and 1:30, in membranes with 25–50% anionic lipids, and in both saturated DMPC/DMPG (1,2-dimyristoyl-sn-glycero-3-phosphatidylchloline/1,2-dimyristoyl-sn-glycero-3-phosphatidylglycerol) membranes and unsaturated POPC/POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol) membranes. Thus, it is an intrinsic property of penetratin. The coil state of the peptide has C– H order parameters of 0.23–0.52 for Cα and Cβ sites, indicating that the peptide backbone is unstructured. Moreover, chemical shift anisotropy lineshapes are uniaxially averaged, suggesting that the peptide backbone undergoes uniaxial rotation around the bilayer normal. These observations suggest that the dynamic state of penetratin at high temperature is a structured turn instead of an isotropic random coil. The thermodynamic parameters of this sheet–turn transition are extracted and compared to other membrane peptides reported to exhibit conformational changes. We suggest that the function of this turn conformation may be to reduce hydrophobic interactions with the lipid chains and facilitate penetratin translocation across the bilayer without causing permanent membrane damage. PMID:18656895

  11. 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.

  12. Parallel synthesis of cell-penetrating peptide conjugates of PMO toward exon skipping enhancement in Duchenne muscular dystrophy.

    PubMed

    O'Donovan, Liz; Okamoto, Itaru; Arzumanov, Andrey A; Williams, Donna L; Deuss, Peter; Gait, Michael J

    2015-02-01

    We describe two new methods of parallel chemical synthesis of libraries of peptide conjugates of phosphorodiamidate morpholino oligonucleotide (PMO) cargoes on a scale suitable for cell screening prior to in vivo analysis for therapeutic development. The methods represent an extension of the SELection of PEPtide CONjugates (SELPEPCON) approach previously developed for parallel peptide-peptide nucleic acid (PNA) synthesis. However, these new methods allow for the utilization of commercial PMO as cargo with both C- and N-termini unfunctionalized. The synthetic methods involve conjugation in solution phase, followed by rapid purification via biotin-streptavidin immobilization and subsequent reductive release into solution, avoiding the need for painstaking high-performance liquid chromatography purifications. The synthesis methods were applied for screening of PMO conjugates of a 16-member library of variants of a 10-residue ApoE peptide, which was suggested for blood-brain barrier crossing. In this work the conjugate library was tested in an exon skipping assay using skeletal mouse mdx cells, a model of Duchene's muscular dystrophy where higher activity peptide-PMO conjugates were identified compared with the starting peptide-PMO. The results demonstrate the power of the parallel synthesis methods for increasing the speed of optimization of peptide sequences in conjugates of PMO for therapeutic screening. PMID:25412073

  13. Parallel Synthesis of Cell-Penetrating Peptide Conjugates of PMO Toward Exon Skipping Enhancement in Duchenne Muscular Dystrophy

    PubMed Central

    O'Donovan, Liz; Okamoto, Itaru; Arzumanov, Andrey A.; Williams, Donna L.; Deuss, Peter

    2015-01-01

    We describe two new methods of parallel chemical synthesis of libraries of peptide conjugates of phosphorodiamidate morpholino oligonucleotide (PMO) cargoes on a scale suitable for cell screening prior to in vivo analysis for therapeutic development. The methods represent an extension of the SELection of PEPtide CONjugates (SELPEPCON) approach previously developed for parallel peptide-peptide nucleic acid (PNA) synthesis. However, these new methods allow for the utilization of commercial PMO as cargo with both C- and N-termini unfunctionalized. The synthetic methods involve conjugation in solution phase, followed by rapid purification via biotin-streptavidin immobilization and subsequent reductive release into solution, avoiding the need for painstaking high-performance liquid chromatography purifications. The synthesis methods were applied for screening of PMO conjugates of a 16-member library of variants of a 10-residue ApoE peptide, which was suggested for blood-brain barrier crossing. In this work the conjugate library was tested in an exon skipping assay using skeletal mouse mdx cells, a model of Duchene's muscular dystrophy where higher activity peptide-PMO conjugates were identified compared with the starting peptide-PMO. The results demonstrate the power of the parallel synthesis methods for increasing the speed of optimization of peptide sequences in conjugates of PMO for therapeutic screening. PMID:25412073

  14. 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

  15. 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

  16. Synthesis of Glycopolymer Containing Cell-Penetrating Peptides as Inducers of Recombinant Protein Expression under the Control of Lactose Operator/Repressor Systems.

    PubMed

    Katagiri, Kei; Takasu, Akinori; Higuchi, Masahiro

    2016-05-01

    We recently reported on newly synthesized S-galactosyl oligo(Arg) conjugates to overcome the serious problem of the passage through the E. coli cell membrane. Following in vivo expression of green fluorescent protein (GFP) induced by each of the S-galactosyl (Arg)n constructs (n = 5, 6, 8) at the T5 promoter in E. coli for 18 h, we visually observed that the cultures fluoresced green light when excited with UV light. The fluorescence intensities for these cultures were greater than that found for a control culture, indicating that the peptides had induced GFP expression. In order to accomplish higher expression efficiency, we investigated the cluster effect and structural fine-tuning of new poly(2-oxazoline) containing CysArgArg as the cell-penetrating peptide (CPP) and S-galactosides when acting as inducers of recombinant protein expression under the control of lac operator/repressor systems in this article. Quantitative fluorescence intensities (calculated per molecule) also supported the observations that the cell-penetrating glyco poly(2-oxazoline)s were better inducers of GFP expression than glyco poly(2-oxazoline) containing no CPP or isopropyl β-d-thiogalactoside. Because the level of GFP expression was directly related to the number of sugar residues in each glyco poly(2-oxazoline), we propose that a cluster effect of the S-galactosides attached to the cell-penetrating poly(2-oxazoline) is responsible for how well the galactosides inhibited the lac repressor to activate the protein expression under the control of the lac operator/repressor system. A similar tendency was observed when the T7 promoter was placed upstream of the gene for an artificial extracellular matrix protein and glyco poly(2-oxazoline)s-CPP conjugates were used as inducers. To assess how the glyco poly(2-oxazoline) penetrate the cell membrane, we labeled the glyco poly(2-oxazoline) using 1-amino pyrene and directly observed the penetration process. Furthermore, we could visualize protein

  17. 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

  18. Efficient entry of cell-penetrating peptide nona-arginine into adherent cells involves a transient increase in intracellular calcium

    PubMed Central

    Melikov, Kamran; Hara, Ann; Yamoah, Kwabena; Zaitseva, Elena; Zaitsev, Eugene; Chernomordik, Leonid V.

    2015-01-01

    Understanding the mechanism of entry of cationic peptides such as nona-arginine (R9) into cells remains an important challenge to their use as efficient drug-delivery vehicles. At nanomolar to low micromolar R9 concentrations and at physiological temperature, peptide entry involves endocytosis. In contrast, at a concentration ≥10 μM, R9 induces a very effective non-endocytic entry pathway specific for cationic peptides. We found that a similar entry pathway is induced at 1–2 μM concentrations of R9 if peptide application is accompanied by a rapid temperature drop to 15°C. Both at physiological and at sub-physiological temperatures, this entry mechanism was inhibited by depletion of the intracellular ATP pool. Intriguingly, we found that R9 at 10–20 μM and 37°C induces repetitive spikes in intracellular Ca2+ concentration. This Ca2+ signalling correlated with the efficiency of the peptide entry. Pre-loading cells with the Ca2+ chelator BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid) inhibited both Ca2+ spikes and peptide entry, suggesting that an increase in intracellular Ca2+ precedes and is required for peptide entry. One of the hallmarks of Ca2+ signalling is a transient cell-surface exposure of phosphatidylserine (PS), a lipid normally residing only in the inner leaflet of the plasma membrane. Blocking the accessible PS with the PS-binding domain of lactadherin strongly inhibited non-endocytic R9 entry, suggesting the importance of PS externalization in this process. To conclude, we uncovered a novel mechanistic link between calcium signalling and entry of cationic peptides. This finding will enhance our understanding of the properties of plasma membrane and guide development of future drug-delivery vehicles. PMID:26272944

  19. The Antimicrobial Domains of Wheat Puroindolines Are Cell-Penetrating Peptides with Possible Intracellular Mechanisms of Action

    PubMed Central

    Alfred, Rebecca L.; Palombo, Enzo A.; Panozzo, Joseph F.; Bhave, Mrinal

    2013-01-01

    The puroindoline proteins (PINA and PINB) of wheat display lipid-binding properties which affect the grain texture, a critical parameter for wheat quality. Interestingly, the same proteins also display antibacterial and antifungal properties, attributed mainly to their Tryptophan-rich domain (TRD). Synthetic peptides based on this domain also display selectivity towards bacterial and fungal cells and do not cause haemolysis of mammalian cells. However, the mechanisms of these activities are unclear, thus limiting our understanding of the in vivo roles of PINs and development of novel applications. This study investigated the mechanisms of antimicrobial activities of synthetic peptides based on the TRD of the PINA and PINB proteins. Calcein dye leakage tests and transmission electron microscopy showed that the peptides PuroA, Pina-M and Pina-W→F selectively permeabilised the large unilamellar vesicles (LUVs) made with negatively charged phospholipids mimicking bacterial membranes, but were ineffective against LUVs made with zwitterionic phospholipids mimicking eukaryotic membranes. Propidium iodide fluorescence tests of yeast (Saccharomyces cerevisiae) cells showed the peptides were able to cause loss of membrane integrity, PuroA and Pina-M being more efficient. Scanning electron micrographs of PINA-based peptide treated yeast cells showed the formation of pits or pores in cell membranes and release of cellular contents. Gel retardation assays indicated the peptides were able to bind to DNA in vitro, and the induction of filamental growth of E. coli cells indicated in vivo inhibition of DNA synthesis. Together, the results strongly suggest that the PIN-based peptides exert their antimicrobial effects by pore formation in the cell membrane, likely by a carpet-like mechanism, followed by intracellular mechanisms of activity. PMID:24098387

  20. Roles of Arginine and Lysine Residues in the Translocation of a Cell-Penetrating Peptide from 13C, 31P and 19F Solid-State NMR

    PubMed Central

    Su, Yongchao; Doherty, Tim; Waring, Alan J.; Ruchala, Piotr; Hong, Mei

    2009-01-01

    Cell-penetrating peptides (CPPs) are small cationic peptides that cross the cell membrane while carrying macromolecular cargoes. We use solid-state NMR to investigate the structure and lipid interaction of two cationic residues, Arg10 and Lys13, in the CPP penetratin. 13C chemical shifts indicate that Arg10 adopts a rigid β-strand conformation in the liquid-crystalline state of anionic lipid membranes. This behavior contrasts with all other residues observed so far in this peptide, which adopt a dynamic β-turn conformation with coil-like chemical shifts at physiological temperature. Low-temperature 13C-31P distances between the peptide and the lipid phosphates indicate that both the Arg10 guanidinium Cζ and the Lys13 Cε lie in close proximity to the lipid 31P (4.0 - 4.2 Å), proving the existence of charge-charge interaction for both Arg10 and Lys13 in the gel-phase membrane. However, since lysine substitution in CPPs are known to reduce their translocation ability, we propose that low temperature stabilizes both lysine and arginine interactions with the phosphates, whereas at high temperature the lysine-phosphate interaction is much weaker than the arginine-phosphate interaction. This is supported by the unusually high rigidity of the Arg10 sidechain and its β-strand conformation at high temperature. The latter is proposed to be important for ion pair formation by allowing close approach of the lipid headgroups to guanidinium sidechains. 19F and 13C spin diffusion experiments indicate that penetratin is oligomerized into β-sheets in gel-phase membranes. These solid-state NMR data indicate that guanidinium-phosphate interactions exist in penetratin, and guanidinium groups play a stronger structural role than ammonium groups in the lipid-assisted translocation of CPPs across liquid-crystalline cell membranes. PMID:19364134

  1. 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

  2. Visualization and Quantitative Assessment of the Brain Distribution of Insulin through Nose-to-Brain Delivery Based on the Cell-Penetrating Peptide Noncovalent Strategy.

    PubMed

    Kamei, Noriyasu; Shingaki, Tomotaka; Kanayama, Yousuke; Tanaka, Misa; Zochi, Riyo; Hasegawa, Koki; Watanabe, Yasuyoshi; Takeda-Morishita, Mariko

    2016-03-01

    Our recent work suggested that intranasal coadministration with the cell-penetrating peptide (CPP) penetratin increased the brain distribution of the peptide drug insulin. The present study aimed to distinctly certify the ability of penetratin to facilitate the nose-to-brain delivery of insulin by quantitatively evaluating the distribution characteristics in brain using radioactive (64)Cu-NODAGA-insulin. Autoradiography and analysis using a gamma counter of brain areas demonstrated that the accumulation of radioactivity was greatest in the olfactory bulb, the anterior part of the brain closest to the administration site, at 15 min after intranasal administration of (64)Cu-NODAGA-insulin with l- or d-penetratin. The brain accumulation of (64)Cu-NODAGA-insulin with penetratin was confirmed by ELISA using unlabeled insulin in which intact insulin was delivered to the brain after intranasal coadministration with l- or d-penetratin. By contrast, quantification of cerebrospinal fluid (CSF) samples showed increased insulin concentration in only the anterior portion of the CSF at 15 min after intranasal coadministration with l-penetratin. This study gives the first concrete proof that penetratin can accelerate the direct transport of insulin from the nasal cavity to the brain parenchyma. Further optimization of intranasal administration with CPP may increase the efficacy of delivery of biopharmaceuticals to the brain while reducing the risk of systemic drug exposure. PMID:26795701

  3. 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

  4. 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

  5. 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

  6. Cell-penetrating conjugates of coproporphyrins with oligoarginine peptides: rational design and application for sensing intracellular O2.

    PubMed

    Dmitriev, Ruslan I; Ropiak, Honorata M; Ponomarev, Gelii V; Yashunsky, Dmitri V; Papkovsky, Dmitri B

    2011-12-21

    A panel of phosphorescent oligoarginine conjugates of tetracarboxylic Pt(II)-coproporphyrin I dye (PtCP), monosubstituted with long peptides or tetra-substituted with short peptides and having different linkers and peripheral groups, is described. Their photophysical properties, cell loading efficiency, and mechanisms of transport into the cell were investigated and compared. The conjugates were seen to rely on endocytotic mechanisms of cell entry, which are different from that of the unconjugated oligoarginine peptide, and show diverse patterns of intracellular distribution. On the basis of this study, the tetra-substituted PtCP conjugate displaying whole cell distribution was selected for the sensing of intracellular O(2). This probe has been tested in biological experiments on a fluorescence plate reader, including the monitoring of in situ oxygenation of respiring cells and their responses to metabolic stimulation. Similar conjugates of the phosphorescent Pd(II)-coprorphyrin and fluorescent coproporphyrin-ketone were also synthesized and assessed for the sensing of low levels intracellular O(2) and ratiometric pH-sensing, respectively. The results produced and the structure-activity relationships determined can facilitate the rational design of new bioconjugates of porphyrin dyes tailored to specific applications. PMID:22035070

  7. The cell-penetrating peptide, Pep-1, has activity against intracellular chlamydial growth but not extracellular forms of Chlamydia trachomatis

    PubMed Central

    Park, Narae; Yamanaka, Kinrin; Tran, Dat; Chandrangsu, Pete; Akers, Johnny C.; de Leon, Jessica C.; Morrissette, Naomi S.; Selsted, Michael E.; Tan, Ming

    2009-01-01

    Objectives In the course of studies to identify novel treatment strategies against the pathogenic bacterium, Chlamydia, we tested the carrier peptide, Pep-1, for activity against an intracellular infection. Methods Using a cell culture model of Chlamydia trachomatis infection, the effect of Pep-1 was measured by incubating the peptide with extracellular chlamydiae prior to infection, or by adding Pep-1 to the medium at varying times after infection, and assaying for inhibition of inclusion formation. Results Pep-1 had a concentration-dependent effect on chlamydial growth with 100% inhibition of inclusion formation at 8 mg/L peptide. There was a window of susceptibility during the chlamydial developmental cycle with a maximal effect when treatment was begun within 12 h of infection. Pep-1 treatment caused a severe reduction in the production of infectious progeny even when started later, when the effect on inclusion formation was minimal. Furthermore, electron micrographs showed a paucity of progeny elementary bodies (EBs) in the inclusion. In contrast, pre-incubation of EBs with Pep-1 prior to infection did not affect inclusion formation. Taken together, these findings indicate that the antichlamydial effect was specific for the intracellular stage of chlamydial infection. By comparison, Pep-1 had no antimicrobial activity against Escherichia coli and Staphylococcus aureus or the obligate intracellular parasite, Toxoplasma gondii. Conclusions Pep-1 has antichlamydial activity by preventing intracellular chlamydial growth and replication but has no effect on extracellular chlamydiae. PMID:18957395

  8. Cell-penetrating peptide-siRNA conjugate loaded YSA-modified nanobubbles for ultrasound triggered siRNA delivery.

    PubMed

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

    2015-12-01

    Due to the absence of effective in vivo delivery systems, the employment of small interference RNA (siRNA) in the clinic has been hindered. In this paper, a new siRNA targeting system for EphA2-positive tumors was developed, based on ultrasound-sensitive nanobubbles (NBs) and cell-permeable peptides (CPPs). Here, a CPP-siRNA conjugate (CPP-siRNA) was entrapped in an ephrin mimetic peptide (YSA peptide)-modified NB (CPP-siRNA/YSA-NB) and the penetration of the CPP-siRNA was temporally masked; local ultrasound stimulation triggered the release of CPP-siRNA from the NBs and activated its penetration. Subsequent research demonstrated that the CPP-siRNA/YSA-NBs had particle sizes of approximately 200 nm and a siRNA entrapment efficiency of more than 85%. The in vitro release results showed that over 90% of the encapsulated CPP-siRNA released from the NBs in the presence of ultrasound, while less than 1.5% of that (30 min) released without ultrasound. Cell experiments showed a the higher CPP-siRNA cellular uptake of CPP-siRNA/YSA-NB among the various formulations in human breast adenocarcinoma cells (MCF-7, EphA2 positive cells). Additionally, after systemic administration in mice, CPP-siRNA/YSA-NB accumulated in the tumor, augmented c-Myc silencing and delayed tumor progression. In conclusion, the application of CPP-siRNA/YSA-NB with ultrasound may provide a strategy for the selective and efficient delivery of siRNA. PMID:26492155

  9. 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. PMID:26176270

  10. Modified nanoparticles with cell-penetrating peptide and amphipathic chitosan derivative for enhanced oral colon absorption of insulin: preparation and evaluation.

    PubMed

    Guo, Feng; Zhang, Mengyuan; Gao, Yuan; Zhu, Siqi; Chen, Shuangxi; Liu, Wenyu; Zhong, Haijun; Liu, Jianping

    2016-07-01

    Colon is an ideal absorptive site for oral protein and peptide drug (insulin), and yet it poses multiple barriers against the drug absorption, such as the barriers against the drug diffusion from colon lumen toward the absorptive mucosa and permeation across colon epithelium. In this study, modified nanoparticles (Tat-CS-NPs) with cell-penetrating peptide (Tat) and amphipathic chitosan derivative (a-CS) were used as carriers to improve colonic absorption of the drug. With a-CS as emulsifier and poly(lactic-co-glycolic acid) as matrix material, Tat-CS-NPs were prepared and evaluated in vitro/vivo. Using Caco-2 cell monolayers to imitate the colonic epithelial cells, it was found that the cellular uptake amount and transcellular transportation performance of Tat-CS-NPs were much enhanced compared with those of CS-NPs and PVA-NPs. The efficacy evaluation on diabetic rat models demonstrated that the hypoglycemic effect of Tat-CS-NPs loaded with insulin was 6.89 times higher than that of PVA-NPs, and 1.79 times higher than that of CS-NPs. By gavage of [(99m)Tc] isotope labeled Tat-CS-NPs in mini-pigs and single-photon emission computed tomography (SPECT) on pigs' gastrointestinal tract, it was further proved that the nanoparticles could reach colon and produce pharmacological effect. In conclusion, Tat-CS-NPs as vehicles for colon-specific drug delivery may be an efficient approach to improve oral bioavailability of protein and peptide drugs. PMID:26181840

  11. 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

  12. Attachment of cell-binding ligands to arginine-rich cell-penetrating peptides enables cytosolic translocation of complexed siRNA.

    PubMed

    Zeller, Skye; Choi, Chang Seon; Uchil, Pradeep D; Ban, Hong-Seok; Siefert, Alyssa; Fahmy, Tarek M; Mothes, Walther; Lee, Sang-Kyung; Kumar, Priti

    2015-01-22

    Cell-penetrating peptides (CPPs), such as nona-arginine (9R), poorly translocate siRNA into cells. Our studies demonstrate that attaching 9R to ligands that bind cell surface receptors quantitatively increases siRNA uptake and importantly, allows functional delivery of complexed siRNA. The mechanism involved accumulation of ligand-9R:siRNA microparticles on the cell membrane, which induced transient membrane inversion at the site of ligand-9R binding and rapid siRNA translocation into the cytoplasm. siRNA release also occurred late after endocytosis when the ligand was attached to the L isoform of 9R, but not the protease-resistant 9DR, prolonging mRNA knockdown. This critically depended on endosomal proteolytic activity, implying that partial CPP degradation is required for endosome-to-cytosol translocation. The data demonstrate that ligand attachment renders simple polycationic CPPs effective for siRNA delivery by restoring their intrinsic property of translocation. PMID:25544044

  13. Targeting the EGFR/PCNA Signaling Suppresses Tumor Growth of Triple-Negative Breast Cancer Cells with Cell-Penetrating PCNA Peptides

    PubMed Central

    Yu, Yung-Luen; Chou, Ruey-Hwang; Liang, Jia-Hong; Chang, Wei-Jung; Su, Kuo-Jung; Tseng, Yen-Ju; Huang, Wei-Chien; Wang, Shao-Chun; Hung, Mien-Chie

    2013-01-01

    Tyrosine 211 (Y211) phosphorylation of proliferation cell nuclear antigen (PCNA) coincides with pronounced cancer cell proliferation and correlates with poor survival of breast cancer patients. In epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI)-resistant cells, both nuclear EGFR (nEGFR) expression and PCNA Y211 phosphorylation are increased. Moreover, the resistance to EGFR TKI is a major clinical problem in treating EGFR-overexpressing triple-negative breast cancer (TNBC). Thus, effective treatment to combat resistance is urgently needed. Here, we show that treatment of cell-penetrating PCNA peptide (CPPP) inhibits growth and induces apoptosis of human TNBC cells. The Y211F CPPP specifically targets EGFR and competes directly for PCNA tyrosine Y211 phosphorylation and prevents nEGFR from binding PCNA in vivo; it also suppresses tumor growth by sensitizing EGFR TKI resistant cells, which have enhanced nEGFR function and abrogated classical EGFR membrane signaling. Furthermore, we identify an active motif of CPPP, RFLNFF (RF6 CPPP), which is necessary and sufficient to inhibit TKI-resistant TNBC cell growth of orthotopic implanted tumor in mice. Finally, the activity of its synthetic retro-inverted derivative, D-RF6 CPPP, on an equimolar basis, is more potent than RF6 CPPP. Our study reveals a drug candidate with translational potential for the future development of safe and effective therapeutic for EGFR TKI resistance in TNBC. PMID:23593472

  14. Attachment of cell-binding ligands to arginine-rich cell penetrating peptides enables cytosolic translocation of complexed siRNA

    PubMed Central

    Zeller, Skye; Choi, Changseon; Uchil, Pradeep D.; Ban, Hongseok; Siefert, Alyssa; Fahmy, Tarek M.; Mothes, Walther; Lee, Sang Kyung; Kumar, Priti

    2014-01-01

    SUMMARY Cell penetrating peptides (CPPs) like nona-arginine (9R) poorly translocate siRNA into cells. Our studies demonstrate that attaching 9R to ligands that bind cell-surface receptors quantitatively increases siRNA uptake and importantly, allows functional delivery of complexed siRNA. The mechanism involved accumulation of ligand-9R:siRNA microparticles on the cell membrane, which induced transient membrane inversion at the site of ligand-9R binding and rapid siRNA translocation into the cytoplasm. siRNA release also occurred late after endocytosis when the ligand was attached to the L isoform of 9R, but not the protease-resistant 9DR, prolonging mRNA knockdown. This critically depended on endosomal proteolytic activity implying partial CPP degradation is required for endosome to cytosol translocation. The data demonstrate that ligand attachment renders simple polycationic CPPs effective for siRNA delivery by restoring their intrinsic property of translocation. PMID:25544044

  15. 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

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

    PubMed

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

    2016-01-19

    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

  17. 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

  18. Efficient siRNA Delivery Using Novel Cell-Penetrating Peptide-siRNA Conjugate-Loaded Nanobubbles and Ultrasound.

    PubMed

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

    2016-06-01

    Because of the absence of tolerable and effective carriers for in vivo delivery, the applications of small interfering RNA (siRNA) in the clinic for therapeutic purposes have been limited. In this study, development of a novel siRNA delivery system based on ultrasound-sensitive nanobubbles (NBs, nano-sized echogenic liposomes) and cell-permeable peptides (CPPs) is described. A CPP-siRNA conjugate was entrapped in an NB, (CPP-siRNA)-NB, and the penetration of CPP-siRNA was temporally masked; local ultrasound stimulation triggered the release of CPP-siRNA from the NBs and activated its penetration. Subsequent research revealed that the (CPP-siRNA)-NBs had a mean particle size of 201 ± 2.05 nm and a siRNA entrapment efficiency >85%. In vitro release results indicated that >90% of the encapsulated CPP-siRNA was released from NBs in the presence of ultrasound, whereas <1.5% (30 min) was released in the absence of ultrasound. Cell experiments indicated higher cellular CPP-siRNA uptake of (CPP-siRNA)-NBs with ultrasound among the various formulations in human breast adenocarcinoma cells (HT-1080). Additionally, after systemic administration in mice, (CPP-siRNA)-NBs accumulated in the tumor, augmented c-myc silencing and delayed tumor progression. In conclusion, the application of (CPP-siRNA)-NBs with ultrasound may constitute an approach to selective targeted delivery of siRNA. PMID:27012462

  19. In vivo proof of concept of oral insulin delivery based on a co-administration strategy with the cell-penetrating peptide penetratin.

    PubMed

    Nielsen, Ebbe Juel Bech; Yoshida, Shinya; Kamei, Noriyasu; Iwamae, Ruisha; Khafagy, El-Sayed; Olsen, Jørgen; Rahbek, Ulrik Lytt; Pedersen, Betty Lomstein; Takayama, Kozo; Takeda-Morishita, Mariko

    2014-09-10

    Oral delivery of insulin is blocked by low intestinal absorption caused by the poor permeability of insulin across cellular membranes and the susceptibility to enzymatic degradation in the gastrointestinal tract. Cell-penetrating peptides (CPPs) have been investigated for a number of years as oral absorption enhancers for hydrophilic macromolecules. Penetratin, a cationic and amphipathic CPP, effectively enhances insulin absorption and we were able to alleviate the enzymatic barrier by using the enzymatic resistant D-form of penetratin. In this study, mice were dosed orally with a physical mixture of insulin and penetratin. Blood glucose concentrations were measured and a pharmacological availability (PA) of 18.2% was achieved in mice dosed with insulin and D-penetratin. Following the promising data, we investigated the degradation parameters of insulin and penetratin in rat intestinal fluid. As expected, L-penetratin was degraded rapidly whereas D-penetratin had a halflife of 67±7min in 10-fold diluted gastrointestinal fluid. Insulin degradation was slowed by the presence of penetratin in intestinal fluid. The half-life of insulin increased from 24.9±4.5min to 55.6±14min and 90.5±11.8min in the presence of L- and D-penetratin respectively. In conclusion, both Land D-penetratin acted as oral absorption enhancers at select CPP concentrations for insulin and the current study is the first solid evidence of pharmacological activity of oral insulin delivery systems based on non-covalent intermolecular interactions with penetratin. PMID:24973720

  20. Cell-penetrating peptides do not cross mitochondrial membranes even when conjugated to a lipophilic cation: evidence against direct passage through phospholipid bilayers

    PubMed Central

    2004-01-01

    CPPs (cell-penetrating peptides) facilitate the cellular uptake of covalently attached oligonucleotides, proteins and other macromolecules, but the mechanism of their uptake is disputed. Two models are proposed: direct movement through the phospholipid bilayer and endocytic uptake. Mitochondria are a good model system to distinguish between these possibilities, since they have no vesicular transport systems. Furthermore, CPP-mediated delivery of macromolecules to the mitochondrial matrix would be a significant breakthrough in the study of mitochondrial function and dysfunction, and could also lead to new therapies for diseases caused by mitochondrial damage. Therefore we investigated whether two CPPs, penetratin and Tat, could act as mitochondrial delivery vectors. We also determined whether conjugation of the lipophilic cation TPP (triphenylphosphonium) to penetratin or Tat facilitated their uptake into mitochondria, since TPP leads to uptake of attached molecules into mitochondria driven by the membrane potential. Neither penetratin nor Tat, nor their TPP conjugates, are internalized by isolated mitochondria, indicating that these CPPs cannot cross mitochondrial phospholipid bilayers. Tat and TPP–Tat are taken up by cells, but they accumulate in endosomes and do not reach mitochondria. We conclude that CPPs cannot cross mitochondrial phospholipid bilayers, and therefore cannot deliver macromolecules directly to mitochondria. Our findings shed light on the mechanism of uptake of CPPs by cells. The lack of direct movement of CPPs through mitochondrial phospholipid bilayers, along with the observed endosomal accumulation of Tat and TPP–Tat in cells, makes it unlikely that CPPs enter cells by direct membrane passage, and instead favours cellular uptake via an endocytic pathway. PMID:15270716

  1. A cell-penetrating peptide based on the interaction between c-Src and connexin43 reverses glioma stem cell phenotype

    PubMed Central

    Gangoso, E; Thirant, C; Chneiweiss, H; Medina, J M; Tabernero, A

    2014-01-01

    Connexin43 (Cx43), the main gap junction channel-forming protein in astrocytes, is downregulated in malignant gliomas. These tumors are composed of a heterogeneous population of cells that include many with stem-cell-like properties, called glioma stem cells (GSCs), which are highly tumorigenic and lack Cx43 expression. Interestingly, restoring Cx43 reverses GSC phenotype and consequently reduces their tumorigenicity. In this study, we investigated the mechanism by which Cx43 exerts its antitumorigenic effects on GSCs. We have focused on the tyrosine kinase c-Src, which interacts with the intracellular carboxy tail of Cx43. We found that Cx43 regulates c-Src activity and proliferation in human GSCs expanded in adherent culture. Thus, restoring Cx43 in GSCs inhibited c-Src activity, which in turn promoted the downregulation of the inhibitor of differentiation Id1. Id1 sustains stem cell phenotype as it controls the expression of Sox2, responsible for stem cell self-renewal, and promotes cadherin switching, which has been associated to epithelial–mesenchymal transition. Our results show that both the ectopic expression of Cx43 and the inhibition of c-Src reduced Id1, Sox2 expression and promoted the switch from N- to E-cadherin, suggesting that Cx43, by inhibiting c-Src, downregulates Id1 with the subsequent changes in stem cell phenotype. On the basis of this mechanism, we found that a cell-penetrating peptide, containing the region of Cx43 that interacts with c-Src, mimics the effect of Cx43 on GSC phenotype, confirming the relevance of the interaction between Cx43 and c-Src in the regulation of the malignant phenotype and pinpointing this interaction as a promising therapeutic target. PMID:24457967

  2. Cell-penetrating peptide and antibiotic combination therapy: a potential alternative to combat drug resistance in methicillin-resistant Staphylococcus aureus.

    PubMed

    Randhawa, Harmandeep Kaur; Gautam, Ankur; Sharma, Minakshi; Bhatia, Rakesh; Varshney, Grish C; Raghava, Gajendra Pal Singh; Nandanwar, Hemraj

    2016-05-01

    The diverse pattern of resistance by methicillin-resistant Staphylococcus aureus (MRSA) is the major obstacle in the treatment of its infections. The key reason of resistance is the poor membrane permeability of drug molecules. Over the last decade, cell-penetrating peptides (CPPs) have emerged as efficient drug delivery vehicles and have been exploited to improve the intracellular delivery of numerous therapeutic molecules in preclinical studies. Therefore, to overcome the drug resistance, we have investigated for the first time the effects of two CPPs (P3 and P8) in combination with four antibiotics (viz. oxacillin, erythromycin, norfloxacin, and vancomycin) against MRSA strains. We found that both CPPs internalized into the MRSA efficiently at very low concentration (<10 μM) which was non-toxic to bacteria as well as mammalian cells and showed no significant hemolytic activity. However, the combinations of CPPs (≤10 μM) and antibiotics showed high toxicity against MRSA as compared to antibiotics alone. The significant finding is that P3 and P8 could lower the MICs against oxacillin, norfloxacin, and vancomycin to susceptible levels (generally <1 μg/mL) for almost all five clinical isolates. Further, the bacterial cell death was confirmed by scanning electron microscopy as well as propidium iodide uptake assay. Simultaneously, time-kill kinetics revealed the increased uptake of antibiotics. In summary, CPPs assist to restore the effectiveness of antibiotics at much lower concentration, eliminate the antibiotic toxicity, and represent the CPP-antibiotic combination therapy as a potential novel weapon to combat MRSA infections. PMID:26837216

  3. Selective Intracellular Delivery of Recombinant Arginine Deiminase (ADI) Using pH-Sensitive Cell Penetrating Peptides To Overcome ADI Resistance in Hypoxic Breast Cancer Cells.

    PubMed

    Yeh, Tzyy-Harn; Chen, Yun-Ru; Chen, Szu-Ying; Shen, Wei-Chiang; Ann, David K; Zaro, Jennica L; Shen, Li-Jiuan

    2016-01-01

    Arginine depletion strategies, such as pegylated recombinant arginine deiminase (ADI-PEG20), offer a promising anticancer treatment. Many tumor cells have suppressed expression of a key enzyme, argininosuccinate synthetase 1 (ASS1), which converts citrulline to arginine. These tumor cells become arginine auxotrophic, as they can no longer synthesize endogenous arginine intracellularly from citrulline, and are therefore sensitive to arginine depletion therapy. However, since ADI-PEG20 only depletes extracellular arginine due to low internalization, ASS1-expressing cells are not susceptible to treatment since they can synthesize arginine intracellularly. Recent studies have found that several factors influence ASS1 expression. In this study, we evaluated the effect of hypoxia, frequently encountered in many solid tumors, on ASS1 expression and its relationship to ADI-resistance in human MDA-MB-231 breast cancer cells. It was found that MDA-MB-231 cells developed ADI resistance in hypoxic conditions with increased ASS1 expression. To restore ADI sensitivity as well as achieve tumor-selective delivery under hypoxia, we constructed a pH-sensitive cell penetrating peptide (CPP)-based delivery system to carry ADI inside cells to deplete both intra- and extracellular arginine. The delivery system was designed to activate the CPP-mediated internalization only at the mildly acidic pH (6.5-7) associated with the microenvironment of hypoxic tumors, thus achieving better selectivity toward tumor cells. The pH sensitivity of the CPP HBHAc was controlled by recombinant fusion to a histidine-glutamine (HE) oligopeptide, generating HBHAc-HE-ADI. The tumor distribution of HBHAc-HE-ADI was comparable to ADI-PEG20 in a mouse xenograft model of human breast cancer cells in vivo. In addition, HBHAc-HE-ADI showed increased in vitro cellular uptake in cells incubated in a mildly acidic pH (hypoxic conditions) compared to normal pH (normoxic conditions), which correlated with p

  4. 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

  5. Tumor Detection at 3 Tesla with an Activatable Cell Penetrating Peptide Dendrimer (ACPPD-Gd), a T1 Magnetic Resonance (MR) Molecular Imaging Agent

    PubMed Central

    Malone, Christopher D.; Olson, Emilia S.; Mattrey, Robert F.; Jiang, Tao; Tsien, Roger Y.; Nguyen, Quyen T.

    2015-01-01

    Purpose The ability to detect small malignant lesions with magnetic resonance imaging (MRI) is limited by inadequate accumulations of Gd with standard chelate agents. To date, no T1-targeted agents have proven superiority to Gd chelates in their ability to detect small tumors at clinically relevant field strengths. Activatable cell-penetrating peptides and their Gd-loaded dendrimeric form (ACPPD-Gd) have been shown to selectively accumulate in tumors. In this study we compared the performance of ACPPD-Gd vs. untargeted Gd chelates to detect small tumors in rodent models using a clinical 3T-MR system. Materials and Methods This study was approved by the Institutional-Animal Care-and-Use Committee. 2 of 4 inguinal breast fat pads of 16 albino-C57BL/6 mice were inoculated with tumor Py8119 cells and the other 2 with saline at random. MRI at 3T was performed at 4, 9, and 14 days after inoculation on 8 mice 24-hours after injection of 0.036mmol Gd/kg (ACPPD-Gd), and before and 2–3 minutes after 0.1 mmol/kg gadobutrol on the other 8 mice. T1-weighted (T1w) tumor signal normalized to muscle, was compared among the non-contrast, gadobutrol, and ACPPD-Gd groups using ANOVA. Experienced and trainee readers blinded to experimental conditions assessed for the presence of tumor in each of the 4 breast regions. Receiver operator characteristic (ROC) curves and area-under-curve (AUC) values were constructed and analyzed. Results Tumors ≥1mm3 were iso-intense to muscle without contrast on T1w sequences. They enhanced diffusely and homogeneously by 57±20% (p<0.001) 24 hours after ACPPD-Gd and by 25±13% (p<0.001) immediately after gadobutrol. The nearly 2-fold difference was similar for small tumors (1-5mm3) (45±19% vs. 19±18%, p = 0.03). ACPPD-Gd tended to improve tumor detection by an experienced reader (AUC 0.98 vs 0.91) and significantly more for a trainee (0.93 vs. 0.82, p = 0.02) compared to gadobutrol. This improvement was more pronounced when obvious tumors (>5mm3

  6. 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

  7. Delivery of siRNA Complexed with Palmitoylated α-Peptide/β-Peptoid Cell-Penetrating Peptidomimetics: Membrane Interaction and Structural Characterization of a Lipid-Based Nanocarrier System.

    PubMed

    Jing, Xiaona; Foged, Camilla; Martin-Bertelsen, Birte; Yaghmur, Anan; Knapp, Kolja M; Malmsten, Martin; Franzyk, Henrik; Nielsen, Hanne M

    2016-06-01

    Proteolytically stable α-peptide/β-peptoid peptidomimetics constitute promising cell-penetrating carrier candidates exhibiting superior cellular uptake as compared to commonly used cell-penetrating peptides (CPPs). The aim of the present study was to explore the potential of these peptidomimetics for delivery of small interfering RNA (siRNA) to the cytosol by incorporation of a palmitoylated peptidomimetic construct into a cationic lipid-based nanocarrier system. The optimal construct was selected on the basis of the effect of palmitoylation and the influence of the length of the peptidomimetic on the interaction with model membranes and the cellular uptake. Palmitoylation enhanced the peptidomimetic adsorption to supported lipid bilayers as studied by ellipsometry. However, both palmitoylation and increased peptidomimetic chain length were found to be beneficial in the cellular uptake studies using fluorophore-labeled analogues. Thus, the longer palmitoylated peptidomimetic was chosen for further formulation of siRNA in a dioleoylphosphatidylethanolamine/cholesteryl hemisuccinate (DOPE/CHEMS) nanocarrier system, and the resulting nanoparticles were found to mediate efficient gene silencing in vitro. Cryo-transmission electron microscopy (cryo-TEM) revealed multilamellar, onion-like spherical vesicles, and small-angle X-ray scattering (SAXS) analysis confirmed that the majority of the lipids in the nanocarriers were organized in lamellar structures, yet coexisted with a hexagonal phase, which is important for efficient nanocarrier-mediated endosomal escape of siRNA ensuring cytosolic delivery. The present work is a proof-of-concept for the use of α-peptides/β-peptoid peptidomimetics in an efficient delivery system that may be more generally exploited for the intracellular delivery of biomacromolecular drugs. PMID:26654841

  8. 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

  9. 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

  10. Dual Myostatin and Dystrophin Exon Skipping by Morpholino Nucleic Acid Oligomers Conjugated to a Cell-penetrating Peptide Is a Promising Therapeutic Strategy for the Treatment of Duchenne Muscular Dystrophy

    PubMed Central

    Malerba, Alberto; Kang, Jagjeet K; McClorey, Graham; Saleh, Amer F; Popplewell, Linda; Gait, Michael J; Wood, Matthew JA; Dickson, George

    2012-01-01

    The knockdown of myostatin, a negative regulator of skeletal muscle mass may have important implications in disease conditions accompanied by muscle mass loss like cancer, HIV/AIDS, sarcopenia, muscle atrophy, and Duchenne muscular dystrophy (DMD). In DMD patients, where major muscle loss has occurred due to a lack of dystrophin, the therapeutic restoration of dystrophin expression alone in older patients may not be sufficient to restore the functionality of the muscles. We recently demonstrated that phosphorodiamidate morpholino oligomers (PMOs) can be used to re-direct myostatin splicing and promote the expression of an out-of-frame transcript so reducing the amount of the synthesized myostatin protein. Furthermore, the systemic administration of the same PMO conjugated to an octaguanidine moiety (Vivo-PMO) led to a significant increase in the mass of soleus muscle of treated mice. Here, we have further optimized the use of Vivo-PMO in normal mice and also tested the efficacy of the same PMO conjugated to an arginine-rich cell-penetrating peptide (B-PMO). Similar experiments conducted in mdx dystrophic mice showed that B-PMO targeting myostatin is able to significantly increase the tibialis anterior (TA) muscle weight and when coadministered with a B-PMO targeting the dystrophin exon 23, it does not have a detrimental interaction. This study confirms that myostatin knockdown by exon skipping is a potential therapeutic strategy to counteract muscle wasting conditions and dual myostatin and dystrophin skipping has potential as a therapy for DMD. PMID:23250360

  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. 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

  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. Decreased outer membrane permeability protects mycobacteria from killing by ubiquitin-derived peptides.

    PubMed

    Purdy, Georgiana E; Niederweis, Michael; Russell, David G

    2009-09-01

    Ubiquitin-derived peptides are bactericidal in vitro and contribute to the mycobactericidal activity of the lysosome. To further define interactions of ubiquitin-derived peptides with mycobacteria, we screened for mutants with increased resistance to the bactericidal activity of the synthetic ubiquitin-derived peptide Ub2. The four Ub2-resistant Mycobacterium smegmatis mutants were also resistant to the bactericidal action of other antimicrobial peptides and macrophages. Two mutants were in the mspA gene encoding the main M. smegmatis porin. Using a translocation-deficient MspA point mutant, we showed that susceptibility of M. smegmatis to Ub2 was independent of MspA channel activity. Instead, the M. smegmatis Ub2-resistant mutants shared a common phenotype of decreased cell wall permeability compared with wild-type bacteria. Expression of mspA rendered Mycobacterium tuberculosis CDC1551 more susceptible both to ubiquitin-derived peptides in vitro and to lysosomal killing in macrophages. Finally, biochemical assays designed to assess membrane integrity indicated that Ub2 treatment impairs membrane function of M. smegmatis and M. tuberculosis cells. The M. smegmatis Ub2-resistant mutants were more resistant than wild-type M. smegmatis to this damage. We conclude that Ub2 targets mycobacterial membranes and that reduced membrane permeability provides mycobacteria intrinsic resistance against antimicrobial compounds including bactericidal ubiquitin-derived peptides. PMID:19682257

  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. Human neutrophil peptide-1 decreases during ageing in selected Mexican population.

    PubMed

    Rivas-Santiago, Bruno; Castañeda-Delgado, Julio E; de Haro-Acosta, Jeny; Torres-Juarez, Flor; Frausto-Lujan, Isabel; Marin-Luevano, Paulina; González-Amaro, Roberto; Enciso-Moreno, Jose A

    2016-04-01

    Antimicrobial peptide innate immunity plays a central role in the susceptibility to infectious diseases, as has been described extensively in different settings. However, the role that these molecules play in the immunity mediated by polymorphonuclear phagocytes as part of the innate immunity of ageing individuals has not been described. In the present study, we addressed the question whether antimicrobial activity in polymorphonuclear cells from elderly individuals was altered in comparison with young adults. We compared phagocytosis index, bacterial killing efficiency, myeloperoxidase activity and cathelicidin expression. Results showed that there were no statistical differences among groups. However, human neutrophil peptide-1 (HNP-1) was decreased in the elderly individuals group. Results suggest that the decreased HNP-1 production in the polymorphonuclear phagocytes form elderly individuals might have an important participation in the increased susceptibility to infectious diseases. PMID:26323500

  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. Atrial natriuretic peptide decreases blood volume in intact and anephric rats

    SciTech Connect

    Trippodo, N.C.; Chien, Y.W.; Pegram, B.L.; Cole, F.E.; MacPhee, A.A.; Kardon, M.B.

    1986-03-05

    Atrial natriuretic peptide (ANP) reportedly lowers atrial pressure and increases hematocrit, suggesting venodilation and/or decreased blood volume (BV). To examine these possibilities, rat ANP (99-126) was administered to Inactinanesthetized rats (313 +/- 9 g, +/- SE) at 0.5 ..mu..g/kg/min for 30 minutes. Urine flow increased by 0.05 ml/min (p < 0.001) during the last 15 minutes of infusion. Mean arterial pressure (MAP) and thoracic central venous pressure (CVP) decreased (p < 0.001) by 12 and 0.5 mmHg, respectively; hematocrit increased by 4.1 units (p < 0.001) and BV (/sup 51/Cr-RBC) decreased by 3.4 ml/kg (p < 0.001). Mean circulatory filling pressure, measured by inflating an intracardiac balloon to briefly stop the circulation, did not change. Distribution of BV between the thoracic and spanchnic organs (whole-animal freezing in liquid nitrogen) was not measurably altered. The results suggest that the decrease in CVP was related more to decreased BV than to venodilation. To investigate possible mechanisms for the decreased BV, the same dose of ANP was administered to anephric rats. MAP decreased by 8 mmHg (p < 0.001); hematocrit increased by 2.4 units (p < 0.001) and BV decreased by 1.7 ml/kg (p < 0.05). The results indicate that short-term administration of ANP decreases blood volume by causing intravascular fluid to shift into the interstitium as well as by inducing diuresis.

  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. PMID:27237727

  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. 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

  4. Culture on electrospun polyurethane scaffolds decreases atrial natriuretic peptide expression by cardiomyocytes in vitro.

    PubMed

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

    2008-12-01

    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 shows 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

  5. Decreased susceptibility of Staphylococcus aureus small-colony variants toward human antimicrobial peptides.

    PubMed

    Gläser, Regine; Becker, Karsten; von Eiff, Christof; Meyer-Hoffert, Ulf; Harder, Jürgen

    2014-09-01

    Staphylococcus aureus is a frequent resident of human nose and skin in many individuals, but it is also able to cause a variety of serious infections including those of the skin and soft tissue. There is increasing evidence that particularly persistent, relapsing, and difficult-to-treat infections caused by S. aureus are associated with the formation of the small-colony variant (SCV) phenotype. The aim of this study was to investigate the hypothesis that (i) skin-derived antimicrobial peptides (AMPs) exhibit a reduced activity against SCVs and (ii) that switching into the SCV phenotype may endow S. aureus with a decreased susceptibility toward the killing activity of human stratum corneum. Here, we show that clinically derived S. aureus SCVs are less susceptible to the bactericidal activity of different human skin-derived AMPs as compared with their isogenic corresponding wild-type strains. Similarly, a S. aureus hemB mutant displaying the SCV phenotype was less susceptible to the antimicrobial activity of AMPs than its hemB-complemented mutant. These findings were accompanied by a higher resistance of SCVs to the killing activity of human stratum corneum. Switching into the SCV phenotype may help S. aureus to subvert cutaneous innate defense, thus contributing to the establishment and persistence of infection. PMID:24717245

  6. A cell-penetrating foldamer with a bioreducible linkage for intracellular delivery of DNA.

    PubMed

    Douat, Céline; Aisenbrey, Christopher; Antunes, Stéphanie; Decossas, Marion; Lambert, Olivier; Bechinger, Burkhard; Kichler, Antoine; Guichard, Gilles

    2015-09-14

    Despite significant advances in foldamer chemistry, tailored delivery systems based on foldamer architectures, which provide a high level of control over secondary structure, are curiously rare among non-viral technologies for transporting nucleic acids into cells. A potent pH-responsive, bioreducible cell-penetrating foldamer (CPF) was developed through covalent dimerization of a short (8-mer) amphipathic oligourea sequence bearing histidine-type units. This CPF exhibits a high capacity to assemble with pDNA and mediates efficient delivery of nucleic acids into the cell. Furthermore, it does not adversely affect cellular viability and was shown to compare favorably with a cognate peptide transfection agent based on His-rich sequences. PMID:26246005

  7. 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

  8. 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

  9. DECREASED S-NITROSATION OF PEPTIDE THIOLS IN THE MEMBRANE INTERIOR

    PubMed Central

    Zhang, Hao; Andrekopoulos, Christopher; Xu, Yingkai; Joseph, Joy; Hogg, Neil; Feix, Jimmy; Kalyanaraman, B.

    2009-01-01

    It has been proposed that autoxidation of nitric oxide (·NO) stimulates S-nitrosation of thiols located in the hydrophobic milieu. We tested whether thiols located in hydrophobic membranes undergo enhanced S-nitrosation in the presence of ·NO/O2. Transmembrane cysteinyl peptides, C4 (AcNH-KKACALA(LA)6KK-CONH2); C8 (AcNH-KKALALACALA(LA)3KK-CONH2), were incorporated into dilauroylphosphatidylcholine (DLPC) bilayers; their location in the membrane was determined by EPR spin labeling. The peptides, C8 and C4, and GSH (300 μM) were treated with a ·NO donor, DEA-NONOate, and nitrosothiol formation determined under different O2 levels. Surprisingly, the more hydrophobic cysteinyl peptide, C8, did not yield any S-nitrosated product compared to GSH in the aqueous phase or C4 peptide in the liposomes in the presence of ·NO/O2. This data suggests that thiols located deeply in the hydrophobic core of the membrane may be less likely to undergo S-nitrosation in the presence of ·NO/O2. PMID:19573593

  10. 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.

  11. A Cell-penetrating Helical Polymer For siRNA Delivery to Mammalian Cells

    PubMed Central

    Gabrielson, Nathan P; Lu, Hua; Yin, Lichen; Kim, Kyung Hoon; Cheng, Jianjun

    2012-01-01

    Cell-penetrating peptides (CPPs) are routinely used for intracellular delivery of a variety of cargo, including drugs, genes, and short interfering RNA (siRNA). Most CPPs are active only upon exposure to acidic environments inside of late endosomes, thereby facilitating the endosomal escape of internalized vectors. Here, we describe the generation of a synthetic polypeptide—PVBLGn-8—that is able to adopt a helical structure independent of pH. Like other CPPs, the helical structure of PVBLGn-8 allows the polypeptide to destabilize membranes. However, since the helix is stable at all physiologically relevant pH values between pH 2 and pH 7.4, the membrane permeation properties of PVBLGn-8 are irreversible. Given its pH-insensitive activity, our results suggest that PVBLGn-8 is able to facilitate efficient siRNA delivery by causing pore formation in the cell membranes through which either free or complexed siRNA is able to diffuse. This nonspecific form of entry into the cell cytosol may prove useful when trying to deliver siRNA to cells which have proven to be difficult to transfect. PMID:22643866

  12. 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

  13. CRMP-2 peptide mediated decrease of high and low voltage-activated calcium channels, attenuation of nociceptor excitability, and anti-nociception in a model of AIDS therapy-induced painful peripheral neuropathy

    PubMed Central

    2012-01-01

    Background The ubiquity of protein-protein interactions in biological signaling offers ample opportunities for therapeutic intervention. We previously identified a peptide, designated CBD3, that suppressed inflammatory and neuropathic behavioral hypersensitivity in rodents by inhibiting the ability of collapsin response mediator protein 2 (CRMP-2) to bind to N-type voltage-activated calcium channels (CaV2.2) [Brittain et al. Nature Medicine 17:822–829 (2011)]. Results and discussion Here, we utilized SPOTScan analysis to identify an optimized variation of the CBD3 peptide (CBD3A6K) that bound with greater affinity to Ca2+ channels. Molecular dynamics simulations demonstrated that the CBD3A6K peptide was more stable and less prone to the unfolding observed with the parent CBD3 peptide. This mutant peptide, conjugated to the cell penetrating motif of the HIV transduction domain protein TAT, exhibited greater anti-nociception in a rodent model of AIDS therapy-induced peripheral neuropathy when compared to the parent TAT-CBD3 peptide. Remarkably, intraperitoneal administration of TAT-CBD3A6K produced none of the minor side effects (i.e. tail kinking, body contortion) observed with the parent peptide. Interestingly, excitability of dissociated small diameter sensory neurons isolated from rats was also reduced by TAT-CBD3A6K peptide suggesting that suppression of excitability may be due to inhibition of T- and R-type Ca2+ channels. TAT-CBD3A6K had no effect on depolarization-evoked calcitonin gene related peptide (CGRP) release compared to vehicle control. Conclusions Collectively, these results establish TAT-CBD3A6K as a peptide therapeutic with greater efficacy in an AIDS therapy-induced model of peripheral neuropathy than its parent peptide, TAT-CBD3. Structural modifications of the CBD3 scaffold peptide may result in peptides with selectivity against a particular subset of voltage-gated calcium channels resulting in a multipharmacology of action on the target. PMID

  14. Comparative study on the interaction of cell-penetrating polycationic polymers with lipid membranes.

    PubMed

    Takechi, Yuki; Tanaka, Hirokazu; Kitayama, Hiroki; Yoshii, Haruka; Tanaka, Masafumi; Saito, Hiroyuki

    2012-01-01

    Cell-penetrating peptides are arginine- and lysine-rich cationic peptides that can readily enter cells not only by themselves but also carrying other macromolecular cargos. In fact, we have reported that polycationic polymer such as poly-l-lysine (PLL) and poly-l-arginine (PLA) translocate through negatively charged phospholipid liposome membranes. In this work, we made a comparative study of the interaction of PLL or PLA with lipid membranes consisting of negatively charged phospholipids to understand the role of basic amino acid residue (i.e. arginine and lysine) in the membrane-penetrating activity of polypeptides. PLA and PLL translocated into giant unilamellar vesicle composed of soybean phospholipids. ζ-potential and turbidity measurements demonstrated the electrostatic binding of PLL and PLA to large unilamellar vesicle (LUV). Fluorescence studies using membrane probes revealed that the binding of PLA and PLL to LUV affects the hydration and packing of the membrane interface region, in which the membrane insertion of PLA appeared to be greater than PLL. Differential scanning calorimetry showed that the enthalpy of the gel to liquid-crystalline phase transition for dipalmitoyl phosphatidylglycerol vesicle was greatly reduced by binding of PLL and PLA, in which the reduction is much larger in PLA than in PLL. Circular dichroism measurements in 2,2,2-trifluoroethanol/water mixture or in the presence of LUV indicated that the propensity of PLA to form α-helical structure is greater than PLL. Consistently, attenuated total reflection-Fourier transform infrared spectroscopy revealed that there is greater α-helical structure in PLA bound to LUV compared to PLL, which has much less ordered structure. Furthermore, isothermal titration calorimetry measurements demonstrated that the contribution of enthalpy to the energetics of binding to LUV is two-fold larger in PLA than in PLL. These results suggest that the stronger interaction of arginine residue with

  15. EGFP-Based Protein Nanoparticles with Cell-Penetrating Peptide for Efficient siRNA Delivery.

    PubMed

    Guan, Xingang; Hu, Xiuli; Cui, Fengchao; Li, Yunqi; Jing, Xiabing; Xie, Zhigang

    2015-11-01

    Development of an innovative nucleic acid nanocarriers still represents a challenge. In this study, we develop a protein nanoparticle (H6-TatEGFP) and examine its siRNA condensing activity. Gel retardation assay show that protein nanoparticle can condense siRNA into stable nanoparticle/siRNA complexes. UsingCy3-labelled siRNA, we also evaluate siRNA transport characteristic of protein nanoparticles in tumor cells, the results indicate that H6-TatEGFP nanoparticle may be a potential nanocarrier for siRNA in tumor cells. PMID:26109167

  16. 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

  17. Cell-Penetrating, Guanidinium-Rich Oligophosphoesters: Effective and Versatile Molecular Transporters for Drug and Probe Delivery.

    PubMed

    McKinlay, Colin J; Waymouth, Robert M; Wender, Paul A

    2016-03-16

    The design, synthesis, and biological evaluation of a new family of highly effective cell-penetrating molecular transporters, guanidinium-rich oligophosphoesters, are described. These unique transporters are synthesized in two steps, irrespective of oligomer length, by the organocatalytic ring-opening polymerization (OROP) of 5-membered cyclic phospholane monomers followed by oligomer deprotection. Varying the initiating alcohol results in a wide variety of cargo attachment strategies for releasable or nonreleasable transporter applications. Initiation of oligomerization with a fluorescent probe produces, upon deprotection, a transporter-probe conjugate that is shown to readily enter multiple cell lines in a dose-dependent manner. These new transporters are superior in cell uptake to previously studied guanidinium-rich oligocarbonates and oligoarginines, showing over 2-fold higher uptake than the former and 6-fold higher uptake than the latter. Initiation with a protected thiol gives, upon deprotection, thiol-terminated transporters which can be thiol-click conjugated to a variety of probes, drugs and other cargos as exemplified by the conjugation and delivery of the model probe fluorescein-maleimide and the medicinal agent paclitaxel (PTX) into cells. Of particular significance given that drug resistance is a major cause of chemotherapy failure, the PTX-transporter conjugate, designed to evade Pgp export and release free PTX after cell entry, shows efficacy against PTX-resistant ovarian cancer cells. Collectively this study introduces a new and highly effective class of guanidinium-rich cell-penetrating transporters and methodology for their single-step conjugation to drugs and probes, and demonstrates that the resulting drug/probe-conjugates readily enter cells, outperforming previously reported guanidinium-rich oligocarbonates and peptide transporters. PMID:26900771

  18. Human Cytomegalovirus variant peptides adapt by decreasing their total coordination upon binding to a T cell receptor

    PubMed Central

    Antipas, Georgios S.E.; Germenis, Anastasios E.

    2015-01-01

    The tertiary structure of the native Cytomegalovirus peptide (NLV) presented by HLA-A2 and bound to the RA14 T cell receptor was used as a reference for the calculation of atomic coordination differences of both the NLV as well as of a number of singly substituted NLV variants in the absence of TCR. Among the pMHC complexes, the native peptide was found to exhibit the highest total coordination difference in respect to the reference structure, suggesting that it experienced the widest structural adaptation upon recognition by the TCR. In addition, the peptide on the isolated NLV-MHC complex was over-coordinated as compared to the rest of the variants. Moreover, the trend was found to account for a set of measured dissociation constants and critical concentrations for target-cell lysis for all variants in complexation with RA14: functionally, all variant peptides were established to be either weak agonists or null peptides, while, at the same time, our current study established that they were also under-coordinated in respect to NLV. It could, thus, be argued that the most ‘efficient’ structural adaptation upon pMHC recognition by the TCR requires of the peptide to undergo the widest under-coordination possible. The main structural characteristic which differentiated the NLV in respect to the variants was a the presence of 16 oxygen atoms (waters) in the former׳s second coordination shell which accounted for over-coordination of roughly 100% and 30% in the O–O and C–O partials respectively. In fact, in the absence of second shell oxygens, the NLV peptide was decidedly under-coordinated in respect to all of the variants, as also suggested by the C–C partial. PMID:26958591

  19. Increasing peptide identifications and decreasing search times for ETD spectra by pre-processing and calculation of parent precursor charge

    PubMed Central

    2012-01-01

    Background Electron Transfer Dissociation [ETD] can dissociate multiply charged precursor polypeptides, providing extensive peptide backbone cleavage. ETD spectra contain charge reduced precursor peaks, usually of high intensity, and whose pattern is dependent on its parent precursor charge. These charge reduced precursor peaks and associated neutral loss peaks should be removed before these spectra are searched for peptide identifications. ETD spectra can also contain ion-types other than c and z˙. Modifying search strategies to accommodate these ion-types may aid in increased peptide identifications. Additionally, if the precursor mass is measured using a lower resolution instrument such as a linear ion trap, the charge of the precursor is often not known, reducing sensitivity and increasing search times. We implemented algorithms to remove these precursor peaks, accommodate new ion-types in noise filtering routine in OMSSA and to estimate any unknown precursor charge, using Linear Discriminant Analysis [LDA]. Results Spectral pre-processing to remove precursor peaks and their associated neutral losses prior to protein sequence library searches resulted in a 9.8% increase in peptide identifications at a 1% False Discovery Rate [FDR] compared to previous OMSSA filter. Modifications to the OMSSA noise filter to accommodate various ion-types resulted in a further 4.2% increase in peptide identifications at 1% FDR. Moreover, ETD spectra when searched with charge states obtained from the precursor charge determination algorithm is shown to be up to 3.5 times faster than the general range search method, with a minor 3.8% increase in sensitivity. Conclusion Overall, there is an 18.8% increase in peptide identifications at 1% FDR by incorporating the new precursor filter, noise filter and by using the charge determination algorithm, when compared to previous versions of OMSSA. PMID:22321509

  20. Cell-Penetrating Poly(disulfide) Assisted Intracellular Delivery of Mesoporous Silica Nanoparticles for Inhibition of miR-21 Function and Detection of Subsequent Therapeutic Effects.

    PubMed

    Yu, Changmin; Qian, Linghui; Ge, Jingyan; Fu, Jiaqi; Yuan, Peiyan; Yao, Samantha C L; Yao, Shao Q

    2016-08-01

    The design of drug delivery systems capable of minimal endolysosomal trapping, controlled drug release, and real-time monitoring of drug effect is highly desirable for personalized medicine. Herein, by using mesoporous silica nanoparticles (MSNs) coated with cell-penetrating poly(disulfide)s and a fluorogenic apoptosis-detecting peptide (DEVD-AAN), we have developed a platform that could be uptaken rapidly by mammalian cells via endocytosis-independent pathways. Subsequent loading of these MSNs with small molecule inhibitors and antisense oligonucleotides resulted in intracellular release of these drugs, leading to combination inhibition of endogenous miR-21 activities which was immediately detectable by the MSN surface-coated peptide using two-photon fluorescence microscopy. PMID:27325284

  1. 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

  2. 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.)

  3. Astrocytes Protect Neurons from Aβ1-42 Peptide-Induced Neurotoxicity Increasing TFAM and PGC-1 and Decreasing PPAR-γ and SIRT-1

    PubMed Central

    Aguirre-Rueda, Diana; Guerra-Ojeda, Sol; Aldasoro, Martin; Iradi, Antonio; Obrador, Elena; Ortega, Angel; Mauricio, M. Dolores; Vila, Jose Mª; Valles, Soraya L.

    2015-01-01

    One of the earliest neuropathological events in Alzheimer's disease is accumulation of astrocytes at sites of Aβ1-42 depositions. Our results indicate that Aβ1-42 toxic peptide increases lipid peroxidation, apoptosis and cell death in neurons but not in astrocytes in primary culture. Aβ1-42-induced deleterious neuronal effects are not present when neurons and astrocytes are mixed cultured. Stimulation of astrocytes with toxic Aβ1-42 peptide increased p-65 and decreased IκB resulting in inflammatory process. In astrocytes Aβ1-42 decreases protein expressions of sirtuin 1 (SIRT-1) and peroxisome proliferator-activated receptor γ (PPAR-γ) and over-expresses peroxisome proliferator-activated receptor γ coactivator 1 (PGC-1) and mitochondrial transcription factor A (TFAM), protecting mitochondria against Aβ1-42-induced damage and promoting mitochondrial biogenesis. In summary our data suggest that astrocytes may have a key role in protecting neurons, increasing neural viability and mitochondrial biogenesis, acquiring better oxidative stress protection and perhaps modulating inflammatory processes against Aβ1-42 toxic peptide. This might be a sign of a complex epigenetic process in Alzheimer's disease development. PMID:25552918

  4. Discovery of the cell-penetrating function of A2 domain derived from LTA subunit of Escherichia coli heat-labile enterotoxin.

    PubMed

    Liu, Di; Guo, Hua; Zheng, Wenyun; Zhang, Na; Wang, Tianwen; Wang, Ping; Ma, Xingyuan

    2016-06-01

    Heat-labile enterotoxin (LT) is a protein toxin produced by enterotoxigenic Escherichia coli (ETEC). As a bacterial toxin, LT holotoxin can enter intestinal epithelial cells and cause diarrhea. In addition, LT is also a powerful mucosal adjuvant capable of enhancing the strong immune responses to co-administered antigens. However, the LT immunological mechanism is still not clear in some aspects, especially with the respect to how the LTA subunit functions alone. Here, we discovered that the A2 domain of LTA could carry a fluorescent protein into cells, whose function is similar to a cell-penetrating peptide. The transmembrane-transporting ability of the A2 domain is non-specific in its cell-penetrating function, which was shown through testing with different cell types. Moreover, the LTA2 fusion protein penetrated a fluorescently labeled cell membrane that identified LTA2 internalization through membrane transport pathways, and showed it finally localized in the endoplasmic reticulum. Furthermore, low-temperature stress and pharmacological agent treatments showed that the LTA2 internalization route is a temperature-dependent process involving the clathrin-mediated endocytosis and the macropinocytosis pathways. These results could explain the internalization of the LTA subunit alone without the LTB pentamer, contributing to a better understanding of LTA working as a mucosal adjuvant; they also suggest that the A2 domain could be used as a novel transport vehicle for research and treatment of disease. PMID:26960316

  5. Cyclisation increases the stability of the sea anemone peptide APETx2 but decreases its activity at acid-sensing ion channel 3.

    PubMed

    Jensen, Jonas E; Mobli, Mehdi; Brust, Andreas; Alewood, Paul F; King, Glenn F; Rash, Lachlan D

    2012-07-01

    APETx2 is a peptide isolated from the sea anemone Anthopleura elegantissima. It is the most potent and selective inhibitor of acid-sensing ion channel 3 (ASIC3) and it is currently in preclinical studies as a novel analgesic for the treatment of chronic inflammatory pain. As a peptide it faces many challenges in the drug development process, including the potential lack of stability often associated with therapeutic peptides. In this study we determined the susceptibility of wild-type APETx2 to trypsin and pepsin and tested the applicability of backbone cyclisation as a strategy to improve its resistance to enzymatic degradation. Cyclisation with either a six-, seven- or eight-residue linker vastly improved the protease resistance of APETx2 but substantially decreased its potency against ASIC3. This suggests that either the N- or C-terminus of APETx2 is involved in its interaction with the channel, which we confirmed by making N- and C-terminal truncations. Truncation of either terminus, but especially the N-terminus, has detrimental effects on the ability of APETx2 to inhibit ASIC3. The current work indicates that cyclisation is unlikely to be a suitable strategy for stabilising APETx2, unless linkers can be engineered that do not interfere with binding to ASIC3. PMID:22851922

  6. Cyclisation Increases the Stability of the Sea Anemone Peptide APETx2 but Decreases Its Activity at Acid-Sensing Ion Channel 3

    PubMed Central

    Jensen, Jonas E.; Mobli, Mehdi; Brust, Andreas; Alewood, Paul F.; King, Glenn F.; Rash, Lachlan D.

    2012-01-01

    APETx2 is a peptide isolated from the sea anemone Anthopleura elegantissima. It is the most potent and selective inhibitor of acid-sensing ion channel 3 (ASIC3) and it is currently in preclinical studies as a novel analgesic for the treatment of chronic inflammatory pain. As a peptide it faces many challenges in the drug development process, including the potential lack of stability often associated with therapeutic peptides. In this study we determined the susceptibility of wild-type APETx2 to trypsin and pepsin and tested the applicability of backbone cyclisation as a strategy to improve its resistance to enzymatic degradation. Cyclisation with either a six-, seven- or eight-residue linker vastly improved the protease resistance of APETx2 but substantially decreased its potency against ASIC3. This suggests that either the N- or C-terminus of APETx2 is involved in its interaction with the channel, which we confirmed by making N- and C-terminal truncations. Truncation of either terminus, but especially the N-terminus, has detrimental effects on the ability of APETx2 to inhibit ASIC3. The current work indicates that cyclisation is unlikely to be a suitable strategy for stabilising APETx2, unless linkers can be engineered that do not interfere with binding to ASIC3. PMID:22851922

  7. Glucagon-Like Peptide-1 Receptor Activation in the Ventral Tegmental Area Decreases the Reinforcing Efficacy of Cocaine.

    PubMed

    Schmidt, Heath D; Mietlicki-Baase, Elizabeth G; Ige, Kelsey Y; Maurer, John J; Reiner, David J; Zimmer, Derek J; Van Nest, Duncan S; Guercio, Leonardo A; Wimmer, Mathieu E; Olivos, Diana R; De Jonghe, Bart C; Hayes, Matthew R

    2016-06-01

    Cocaine addiction continues to be a significant public health problem for which there are currently no effective FDA-approved treatments. Thus, there is a clear need to identify and develop novel pharmacotherapies for cocaine addiction. Recent evidence indicates that activation of glucagon-like peptide-1 (GLP-1) receptors in the ventral tegmental area (VTA) reduces intake of highly palatable food. As the neural circuits and neurobiological mechanisms underlying drug taking overlap to some degree with those regulating food intake, these findings suggest that activation of central GLP-1 receptors may also attenuate cocaine taking. Here, we show that intra-VTA administration of the GLP-1 receptor agonist exendin-4 (0.05 μg) significantly reduced cocaine, but not sucrose, self-administration in rats. We also demonstrate that cocaine taking is associated with elevated plasma corticosterone levels and that systemic infusion of cocaine activates GLP-1-expressing neurons in the nucleus tractus solitarius (NTS), a hindbrain nucleus that projects monosynaptically to the VTA. To determine the potential mechanisms by which cocaine activates NTS GLP-1-expressing neurons, we microinjected corticosterone (0.5 μg) directly into the hindbrain fourth ventricle. Intraventricular corticosterone attenuated cocaine self-administration and this effect was blocked in animals pretreated with the GLP-1 receptor antagonist exendin-(9-39) (10 μg) in the VTA. Finally, AAV-shRNA-mediated knockdown of VTA GLP-1 receptors was sufficient to augment cocaine self-administration. Taken together, these findings indicate that increased activation of NTS GLP-1-expressing neurons by corticosterone may represent a homeostatic response to cocaine taking, thereby reducing the reinforcing efficacy of cocaine. Therefore, central GLP-1 receptors may represent a novel target for cocaine addiction pharmacotherapies. PMID:26675243

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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

  13. The Neuropilin-1 Inhibitor, ATWLPPR Peptide, Prevents Experimental Diabetes-Induced Retinal Injury by Preserving Vascular Integrity and Decreasing Oxidative Stress

    PubMed Central

    Wang, Jun; Wang, Shuaiwei; Li, Mengling; Wu, Dongdong; Liu, Fang; Yang, Ruisheng; Ji, Shaoping; Ji, Ailing; Li, Yanzhang

    2015-01-01

    Neuropilin-1 (NRP-1) is a transmembrane glycoprotein. As a VEGF co-receptor, NRP1 significantly enhances VEGFR2 signaling and promotes vascular permeability and migration. The purpose of this study was to evaluate the effects of an NRP-1 inhibitor, ATWLPPR peptide, on the early stages of diabetic retinopathy. Eight-week-old male C57BL/6 mice were divided into three groups: a Normal group, a Diabetes (DB) ATWLPPR treatment group and a DB saline group. Electroretinography (ERG), fundus fluorescence angiography (FFA) and leukostasis were examined to evaluate the retinal injury induced by diabetes at the end of the fifth week after STZ injection. Occludin expression and extravasation of albumin were measured to determine the extent of vascular injury. The oxidative stress level and the levels of inflammation-associated proteins were also assayed. The results indicated that treatment with ATWLPPR prevents the abnormal condition of ERG (amplitudes of b-wave decreased and implicit time increased) and vascular injury (occludin degradation and increase in extravasated albumin). These effects were associated with a reduction in the oxidase stress level and the expression of VEGF, GFAP, and ICAM-1. We conclude that ATWLPPR, an NRP-1 inhibitor, may reduce the early retinal damage induced by diabetes by preserving vascular integrity and decreasing the oxidative stress level. Blockade of NRP-1 may be a new therapeutic strategy for the early stages of DR. PMID:26554379

  14. 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

  15. 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

  16. 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

  17. The peptide carrier Pep-1 forms biologically efficient nanoparticle complexes

    SciTech Connect

    Munoz-Morris, Maria A.; Heitz, Frederic; Divita, Gilles . E-mail: gilles.divita@crbm.cnrs.fr; Morris, May C.

    2007-04-20

    Cell-penetrating peptides (CPPs) constitute a family of peptides whose unique characteristic is their ability to insert into and cross biological membranes. Cell-penetrating peptide carriers of the Pep family are amphipathic peptides which have been shown to deliver peptides and proteins into a wide variety of cells through formation of non-covalent complexes with their cargo. In this study, we have investigated the morphological features of different Pep-1/cargo complexes by scanning electron microscopy and light scattering measurements. We provide first-time evidence that biologically efficient complexes of Pep-1/p27Kip tumour suppressor physically exist in the form of discrete nanoparticles. Moreover, we have characterized the relationship between the Pep-1/cargo ratio, the size and homogeneity of the nanoparticles formed, and their efficiency in delivering the cargo into cells, and report that particle size and homogeneity is both directly dependent on the ratio of Pep-1/cargo formulations, and responsible for their biological efficiency.

  18. Sub-lethal levels of amyloid β-peptide oligomers decrease non-transferrin-bound iron uptake and do not potentiate iron toxicity in primary hippocampal neurons.

    PubMed

    SanMartín, C D; Paula-Lima, A C; Hidalgo, C; Núñez, M T

    2012-08-01

    Two major lesions are pathological hallmarks in Alzheimer's disease (AD): the presence of neurofibrillary tangles formed by intracellular aggregates of the hyperphosphorylated form of the cytoskeletal tau protein, and of senile plaques composed of extracellular aggregates of amyloid beta (Aβ) peptide. Current hypotheses regard soluble amyloid beta oligomers (AβOs) as pathological causative agents in AD. These aggregates cause significant calcium deregulation and mediate neurotoxicity by disrupting synaptic activity. Additionally, the presence of high concentrations of metal ions such as copper, zinc, aluminum and iron in neurofibrillary tangles and senile plaques, plus the fact that they accelerate the rate of formation of Aβ fibrils and AβOs in vitro, suggests that accumulation of these metals in the brain is relevant to AD pathology. A common cellular response to AβOs and transition metals such as copper and iron is the generation of oxidative stress, with the ensuing damage to cellular components. Using hippocampal neurons in primary culture, we report here the effects of treatment with AβOs on the (+)IRE and (-)IRE mRNA levels of the divalent metal transporter DMT1. We found that non-lethal AβOs concentrations decreased DMT1 (-)IRE without affecting DMT1 (+)IRE mRNA levels, and inhibited non-transferrin bound iron uptake. In addition, since both iron and AβOs induce oxidative damage, we studied whether their neurotoxic effects are synergistic. In the range of concentrations and times used in this study, AβOs did not potentiate iron-induced cell death while iron chelation did not decrease AβOs-induced cell death. The lack of synergism between iron and AβOs suggests that these two neurotoxic agents converge in a common target, which initiates signaling processes that promote neurodegeneration. PMID:22526560

  19. A novel apolipoprotein C-II mimetic peptide that activates lipoprotein lipase and decreases serum triglycerides in apolipoprotein E-knockout mice.

    PubMed

    Amar, Marcelo J A; Sakurai, Toshihiro; Sakurai-Ikuta, Akiko; Sviridov, Denis; Freeman, Lita; Ahsan, Lusana; Remaley, Alan T

    2015-02-01

    Apolipoprotein A-I (apoA-I) mimetic peptides are currently being developed as possible new agents for the treatment of cardiovascular disease based on their ability to promote cholesterol efflux and their other beneficial antiatherogenic properties. Many of these peptides, however, have been reported to cause transient hypertriglyceridemia due to inhibition of lipolysis by lipoprotein lipase (LPL). We describe a novel bihelical amphipathic peptide (C-II-a) that contains an amphipathic helix (18A) for binding to lipoproteins and stimulating cholesterol efflux as well as a motif based on the last helix of apolipoprotein C-II (apoC-II) that activates lipolysis by LPL. The C-II-a peptide promoted cholesterol efflux from ATP-binding cassette transporter ABCA1-transfected BHK cells similar to apoA-I mimetic peptides. Furthermore, it was shown in vitro to be comparable to the full-length apoC-II protein in activating lipolysis by LPL. When added to serum from a patient with apoC-II deficiency, it restored normal levels of LPL-induced lipolysis and also enhanced lipolysis in serum from patients with type IV and V hypertriglyceridemia. Intravenous injection of C-II-a (30 mg/kg) in apolipoprotein E-knockout mice resulted in a significant reduction of plasma cholesterol and triglycerides of 38 ± 6% and 85 ± 7%, respectively, at 4 hours. When coinjected with the 5A peptide (60 mg/kg), the C-II-a (30 mg/kg) peptide was found to completely block the hypertriglyceridemic effect of the 5A peptide in C57Bl/6 mice. In summary, C-II-a is a novel peptide based on apoC-II, which promotes cholesterol efflux and lipolysis and may therefore be useful for the treatment of apoC-II deficiency and other forms of hypertriglyceridemia. PMID:25395590

  20. A Novel Apolipoprotein C-II Mimetic Peptide That Activates Lipoprotein Lipase and Decreases Serum Triglycerides in Apolipoprotein E–Knockout Mice

    PubMed Central

    Sakurai, Toshihiro; Sakurai-Ikuta, Akiko; Sviridov, Denis; Freeman, Lita; Ahsan, Lusana; Remaley, Alan T.

    2015-01-01

    Apolipoprotein A-I (apoA-I) mimetic peptides are currently being developed as possible new agents for the treatment of cardiovascular disease based on their ability to promote cholesterol efflux and their other beneficial antiatherogenic properties. Many of these peptides, however, have been reported to cause transient hypertriglyceridemia due to inhibition of lipolysis by lipoprotein lipase (LPL). We describe a novel bihelical amphipathic peptide (C-II-a) that contains an amphipathic helix (18A) for binding to lipoproteins and stimulating cholesterol efflux as well as a motif based on the last helix of apolipoprotein C-II (apoC-II) that activates lipolysis by LPL. The C-II-a peptide promoted cholesterol efflux from ATP-binding cassette transporter ABCA1-transfected BHK cells similar to apoA-I mimetic peptides. Furthermore, it was shown in vitro to be comparable to the full-length apoC-II protein in activating lipolysis by LPL. When added to serum from a patient with apoC-II deficiency, it restored normal levels of LPL-induced lipolysis and also enhanced lipolysis in serum from patients with type IV and V hypertriglyceridemia. Intravenous injection of C-II-a (30 mg/kg) in apolipoprotein E–knockout mice resulted in a significant reduction of plasma cholesterol and triglycerides of 38 ± 6% and 85 ± 7%, respectively, at 4 hours. When coinjected with the 5A peptide (60 mg/kg), the C-II-a (30 mg/kg) peptide was found to completely block the hypertriglyceridemic effect of the 5A peptide in C57Bl/6 mice. In summary, C-II-a is a novel peptide based on apoC-II, which promotes cholesterol efflux and lipolysis and may therefore be useful for the treatment of apoC-II deficiency and other forms of hypertriglyceridemia. PMID:25395590

  1. Memantine treatment decreases levels of secreted Alzheimer's amyloid precursor protein (APP) and amyloid beta (A beta) peptide in the human neuroblastoma cells.

    PubMed

    Ray, Balmiki; Banerjee, Pradeep K; Greig, Nigel H; Lahiri, Debomoy K

    2010-02-01

    Memantine, an uncompetitive NMDA receptor antagonist, is a FDA-approved drug used for the treatment of moderate-to-severe Alzheimer's disease (AD). Several studies have documented protective roles of memantine against amyloid beta (A beta) peptide-mediated damage to neurons in both in vitro and in vivo models. Memantine is also effective in reducing amyloid burden in the brain of APP transgenic mice. However, the exact mechanism by which memantine provides protection against A beta-mediated neurodegenerative cascade, including APP metabolism, remains to be elucidated. Herein, we investigated the effect of memantine on levels of the secreted form of A beta precursor protein (APP), secreted A beta and cell viability markers under short/acute conditions. We treated neuronal SK-N-SH cells with 10 microM memantine and measured levels of secreted total APP (sAPP), APP alpha isoform and A beta((1-40)) in a time dependent manner for up to 24h. Memantine significantly decreased the levels of the secreted form of sAPP, sAPP alpha and A beta((1-40)) compared to vehicle treated cells. This change started as early as 8h and continued for up to 24h of drug treatment. Unlike sAPP, a slight non-significant increase in total intracellular APP level was observed in 24-h treated memantine cells. Taken together, these results suggest a role for memantine in the transport or trafficking of APP molecules away from the site of their proteolytic cleavage by the secretase enzymes. Such a novel property of memantine warrants further study to define its therapeutic utility. PMID:19948208

  2. 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

  3. 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.

  4. 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

  5. Targeting Protease-Activated Receptor-1 with Cell-Penetrating Pepducins in Lung Cancer

    PubMed Central

    Cisowski, Jaroslaw; O'Callaghan, Katie; Kuliopulos, Athan; Yang, John; Nguyen, Nga; Deng, Qing; Yang, Eric; Fogel, Michael; Tressel, Sarah; Foley, Caitlin; Agarwal, Anika; Hunt, Stephen W.; McMurry, Tom; Brinckerhoff, Larry; Covic, Lidija

    2011-01-01

    Protease-activated receptors (PARs) are G-protein–coupled receptors that are activated by proteolytic cleavage and generation of a tethered ligand. High PAR1 expression has been documented in a variety of invasive cancers of epithelial origin. In the present study, we investigated the contribution of the four PAR family members to motility of lung carcinomas and primary tumor samples from patients. We found that of the four PARs, only PAR1 expression was highly increased in the lung cancer cell lines. Primary lung cancer cells isolated from patient lung tumors migrated at a 10- to 40-fold higher rate than epithelial cells isolated from nonmalignant lung tissue. Cell-penetrating pepducin inhibitors were generated against the first (i1) and third (i3) intracellular loops of PAR1 and tested for their ability to inhibit PAR1-driven migration and extracellular regulated kinase (ERK)1/2 activity. The PAR1 pepducins showed significant inhibition of cell migration in both primary and established cell lines similar to silencing of PAR1 expression with short hairpin RNA (shRNA). Unlike i1 pepducins, the i3 loop pepducins were effective inhibitors of PAR1-mediated ERK activation and tumor growth. Comparable in efficacy with Bevacizumab, monotherapy with the PAR1 i3 loop pepducin P1pal-7 provided significant 75% inhibition of lung tumor growth in nude mice. We identify the PAR1–ERK1/2 pathway as a feasible target for therapy in lung cancer. PMID:21703428

  6. TOE1 is an inhibitor of HIV-1 replication with cell-penetrating capability

    PubMed Central

    Sperandio, Sabina; Barat, Corinne; Cabrita, Miguel A.; Gargaun, Ana; Berezovski, Maxim V.; Tremblay, Michel J.; de Belle, Ian

    2015-01-01

    Target of Egr1 (TOE1) is a nuclear protein localized primarily in nucleoli and Cajal bodies that was identified as a downstream target of the immediate early gene Egr1. TOE1 displays a functional deadenylation domain and has been shown to participate in spliceosome assembly. We report here that TOE1 can function as an inhibitor of HIV-1 replication and show evidence that supports a direct interaction of TOE1 with the viral specific transactivator response element as part of the inhibitory mechanism. In addition, we show that TOE1 can be secreted by activated CD8+ T lymphocytes and can be cleaved by the serine protease granzyme B, one of the main components of cytotoxic granules. Both full-length and cleaved TOE1 can spontaneously cross the plasma membrane and penetrate cells in culture, retaining HIV-1 inhibitory activity. Antiviral potency of TOE1 and its cell-penetrating capability have been identified to lie within a 35-amino-acid region containing the nuclear localization sequence. PMID:26056259

  7. Manipulation of Cell Cycle and Chromatin Configuration by Means of Cell-Penetrating Geminin

    PubMed Central

    Yasunaga, Shin’ichiro; Kurogi, Toshiaki; Santo, Mimoko; Masuhiro, Yoshikazu; Hanazawa, Shigemasa; Ohtsubo, Motoaki; Naka, Kazuhito; Takihara, Yoshihiro

    2016-01-01

    Geminin regulates chromatin remodeling and DNA replication licensing which play an important role in regulating cellular proliferation and differentiation. Transcription of the Geminin gene is regulated via an E2F-responsive region, while the protein is being closely regulated by the ubiquitin-proteasome system. Our objective was to directly transduce Geminin protein into cells. Recombinant cell-penetrating Geminin (CP-Geminin) was generated by fusing Geminin with a membrane translocating motif from FGF4 and was efficiently incorporated into NIH 3T3 cells and mouse embryonic fibroblasts. The withdrawal study indicated that incorporated CP-Geminin was quickly reduced after removal from medium. We confirmed CP-Geminin was imported into the nucleus after incorporation and also that the incorporated CP-Geminin directly interacted with Cdt1 or Brahma/Brg1 as the same manner as Geminin. We further demonstrated that incorporated CP-Geminin suppressed S-phase progression of the cell cycle and reduced nuclease accessibility in the chromatin, probably through suppression of chromatin remodeling, indicating that CP-Geminin constitutes a novel tool for controlling chromatin configuration and the cell cycle. Since Geminin has been shown to be involved in regulation of stem cells and cancer cells, CP-Geminin is expected to be useful for elucidating the role of Geminin in stem cells and cancer cells, and for manipulating their activity. PMID:27195810

  8. Peptide-mediated delivery: an overview of pathways for efficient internalization.

    PubMed

    Pae, Janely; Pooga, Margus

    2014-11-01

    Poor cellular delivery and low bioavailability of novel potent therapeutic molecules continue to remain the bottleneck of modern cancer and gene therapy. Cell-penetrating peptides have provided immense opportunities for the intracellular delivery of bioactive cargos and have led to the first exciting successes in experimental therapy of muscular dystrophies. This review focuses on the mechanisms by which cell-penetrating peptides gain access to the cell interior and deliver cargos. Recent advances in augmenting delivery efficacy and facilitation of endosomal escape of cargo are presented, and the cell-penetrating peptide-mediated delivery of two of the most popular classes of cargo molecules, oligonucleotides and proteins, is analyzed. The arsenal of tools for oligonucleotide delivery has dramatically expanded in the last decade enabling harnessing of cell-surface receptors for targeted delivery. PMID:25491671

  9. 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…

  10. Soluble expression and stability enhancement of transcription factors using 30Kc19 cell-penetrating protein.

    PubMed

    Ryu, Jina; Park, Hee Ho; Park, Ju Hyun; Lee, Hong Jai; Rhee, Won Jong; Park, Tai Hyun

    2016-04-01

    Transcription factors have been studied as an important drug candidate. Ever since the successful generation of induced pluripotent stem cells (iPSCs), there has been tremendous interest in reprogramming transcription factors. Because of the safety risks involved in a virus-based approach, many researchers have been trying to deliver transcription factors using nonintegrating materials. Thus, delivery of transcription factors produced as recombinant proteins in E. coli was proposed as an alternative method. However, the low level of soluble expression and instability of such recombinant proteins are potential barriers. We engineered a Bombyx mori 30Kc19 protein as a fusion partner for transcription factors to overcome those problems. We have previously reported that 30Kc19 protein can be produced as a soluble form in E. coli and has a cell-penetrating property and a protein-stabilizing effect. Transcription factors fused with 30Kc19 (Oct4-30Kc19, Sox2-30Kc19, c-Myc-30Kc19, L-Myc-30Kc19, and Klf4-30Kc19) were produced as recombinant proteins. Interestingly, Oct4 and L-Myc were expressed as a soluble form by conjugating with 30Kc19 protein, whereas Oct4 alone and L-Myc alone aggregated. The 30Kc19 protein also enhanced the stability of transcription factors both in vitro and in cells. In addition, 30Kc19-conjugated transcription factors showed rapid delivery into cells and transcriptional activity significantly increased. Overall, 30Kc19 protein conjugation simultaneously enhanced soluble expression, stability, and transcriptional activity of transcription factors. We propose that the conjugation with 30Kc19 protein is a novel approach to solve the technical bottleneck of gene regulation using transcription factors. PMID:26668030

  11. TAT-BH4 and TAT-Bcl-xL peptides protect against sepsis-induced lymphocyte apoptosis in vivo.

    PubMed

    Hotchkiss, Richard S; McConnell, Kevin W; Bullok, Kristin; Davis, Christopher G; Chang, Katherine C; Schwulst, Steven J; Dunne, Jeffrey C; Dietz, Gunnar P H; Bähr, Mathias; McDunn, Jonathan E; Karl, Irene E; Wagner, Tracey H; Cobb, J Perren; Coopersmith, Craig M; Piwnica-Worms, David

    2006-05-01

    Apoptosis is a key pathogenic mechanism in sepsis that induces extensive death of lymphocytes and dendritic cells, thereby contributing to the immunosuppression that characterizes the septic disorder. Numerous animal studies indicate that prevention of apoptosis in sepsis improves survival and may represent a potential therapy for this highly lethal disorder. Recently, novel cell-penetrating peptide constructs such as HIV-1 TAT basic domain and related peptides have been developed to deliver bioactive cargoes and peptides into cells. In the present study, we investigated the effects of sepsis-induced apoptosis in Bcl-x(L) transgenic mice and in wild-type mice treated with an antiapoptotic TAT-Bcl-x(L) fusion protein and TAT-BH4 peptide. Lymphocytes from Bcl-x(L) transgenic mice were resistant to sepsis-induced apoptosis, and these mice had a approximately 3-fold improvement in survival. TAT-Bcl-x(L) and TAT-BH4 prevented Escherichia coli-induced human lymphocyte apoptosis ex vivo and markedly decreased lymphocyte apoptosis in an in vivo mouse model of sepsis. In conclusion, TAT-conjugated antiapoptotic Bcl-2-like peptides may offer a novel therapy to prevent apoptosis in sepsis and improve survival. PMID:16622015

  12. 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

  13. 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

  14. Decrease in Formalin-Inactivated Respiratory Syncytial Virus (FI-RSV) Enhanced Disease with RSV G Glycoprotein Peptide Immunization in BALB/c Mice

    PubMed Central

    Rey, Gertrud U.; Miao, Congrong; Caidi, Hayat; Trivedi, Suvang U.; Harcourt, Jennifer L.; Tripp, Ralph A.; Anderson, Larry J.; Haynes, Lia M.

    2013-01-01

    Respiratory syncytial virus (RSV) is a high priority target for vaccine development. One concern in RSV vaccine development is that a non-live virus vaccine would predispose for enhanced disease similar to that seen with the formalin inactivated RSV (FI-RSV) vaccine. Since a mAb specific to RSV G protein can reduce pulmonary inflammation and eosinophilia seen after RSV infection of FI-RSV vaccinated mice, we hypothesized that RSV G peptides that induce antibodies with similar reactivity may limit enhanced disease after subunit or other non-live RSV vaccines. In support of this hypothesis, we show that FI-RSV vaccinated mice administered RSV G peptide vaccines had a significant reduction in enhanced disease after RSV challenge. These data support the importance of RSV G during infection to RSV disease pathogenesis and suggest that use of appropriately designed G peptide vaccines to reduce the risk of enhanced disease with non-live RSV vaccines merits further study. PMID:24376637

  15. Enhanced cellular uptake of short polyarginine peptides through fatty acylation and cyclization.

    PubMed

    Oh, Donghoon; Nasrolahi Shirazi, Amir; Northup, Kevin; Sullivan, Brian; Tiwari, Rakesh Kumar; Bisoffi, Marco; Parang, Keykavous

    2014-08-01

    Many of the reported arginine-rich cell-penetrating peptides (CPPs) for the enhanced delivery of drugs are linear peptides composed of more than seven arginine residues to retain the cell penetration properties. Herein, we synthesized a class of nine polyarginine peptides containing 5 and 6 arginines, namely, R5 and R6. We further explored the effect of acylation with long chain fatty acids (i.e., octanoic acid, dodecanoic acid, and hexadecanoic acid) and cyclization on the cell penetrating properties of the peptides. The fluorescence-labeled acylated cyclic peptide dodecanoyl-[R5] and linear peptide dodecanoyl-(R5) showed approximately 13.7- and 10.2-fold higher cellular uptake than that of control 5,6-carboxyfluorescein, respectively. The mechanism of the peptide internalization into cells was found to be energy-dependent endocytosis. Dodecanoyl-[R5] and dodecanoyl-[R6] enhanced the intracellular uptake of a fluorescence-labeled cell-impermeable negatively charged phosphopeptide (F'-GpYEEI) in human ovarian cancer cells (SK-OV-3) by 3.4-fold and 5.5-fold, respectively, as shown by flow cytometry. The cellular uptake of F'-GpYEEI in the presence of hexadecanoyl-[R5] was 9.3- and 6.0-fold higher than that in the presence of octanoyl-[R5] and dodecanoyl-[R5], respectively. Dodecanoyl-[R5] enhanced the cellular uptake of the phosphopeptide by 1.4-2.5-fold higher than the corresponding linear peptide dodecanoyl-(R5) and those of representative CPPs, such as hepta-arginine (CR7) and TAT peptide. These results showed that a combination of acylation by long chain fatty acids and cyclization on short arginine-containing peptides can improve their cell-penetrating property, possibly through efficient interaction of rigid positively charged R and hydrophobic dodecanoyl moiety with the corresponding residues in the cell membrane phospholipids. PMID:24978295

  16. The frog skin host-defense peptide CPF-SE1 improves glucose tolerance, insulin sensitivity and islet function and decreases plasma lipids in high-fat fed mice.

    PubMed

    Srinivasan, Dinesh; Ojo, Opeolu O; Owolabi, Bosede O; Conlon, J Michael; Flatt, Peter R; Abdel-Wahab, Yasser H A

    2015-10-01

    The frog skin host-defense peptide CPF-SE1 has previously been shown to stimulate the in vitro release of insulin from clonal BRIN-BD11 β-cells. In this study, the in vivo effects of the peptide were investigated in male NIH Swiss mice maintained on a high-fat diet to induce obesity and insulin resistance. Insulin-secretory responses of islets isolated from treated and untreated mice and changes in islet morphology were also examined. Total body fat, plasma glucagon, triglyceride and cholesterol concentrations were measured at the end of the treatment period. Acute intraperitoneal administration of CPF-SE1 (75 nmol body weight) to high-fat fed mice together with glucose (18 mmol/kg body weight) improved glucose tolerance and insulin responses compared to high-fat fed controls. Long term administration of CPF-SE1 (twice-daily administration of 75 nmol/kg body weight for 28 days) did not affect body weight or energy intake but decreased circulating glucose and increased insulin concentrations. Insulin sensitivity and insulin-secretory responses of islets to secretagogues were also significantly improved at 28 days in peptide-treated mice. In addition, significant decreases in plasma glucagon concentrations, pancreatic insulin and glucagon content, islet and beta cell area, body fat and plasma triglyceride levels were observed in CPF-SE1 treated with mice. In conclusion, CPF-SE1 improves beta cell function, insulin sensitivity and glycaemic control whilst reducing total body fat and circulating triglyceride levels. The peptide shows potential for development into an agent for treatment of patients with metabolic syndrome and type 2 diabetes. PMID:26123844

  17. Non-Metabolic Membrane Tubulation and Permeability Induced by Bioactive Peptides

    PubMed Central

    Lamazière, Antonin; Burlina, Fabienne; Wolf, Claude; Chassaing, Gérard; Trugnan, Germain; Ayala-Sanmartin, Jesus

    2007-01-01

    Background Basic cell-penetrating peptides are potential vectors for therapeutic molecules and display antimicrobial activity. The peptide-membrane contact is the first step of the sequential processes leading to peptide internalization and cell activity. However, the molecular mechanisms involved in peptide-membrane interaction are not well understood and are frequently controversial. Herein, we compared the membrane activities of six basic peptides with different size, charge density and amphipaticity: Two cell-penetrating peptides (penetratin and R9), three amphipathic peptides and the neuromodulator substance P. Methodology/Principal Findings Experiments of X ray diffraction, video-microscopy of giant vesicles, fluorescence spectroscopy, turbidimetry and calcein leakage from large vesicles are reported. Permeability and toxicity experiments were performed on cultured cells. The peptides showed differences in bilayer thickness perturbations, vesicles aggregation and local bending properties which form lipidic tubular structures. These structures invade the vesicle lumen in the absence of exogenous energy. Conclusions/Significance We showed that the degree of membrane permeabilization with amphipathic peptides is dependent on both peptide size and hydrophobic nature of the residues. We propose a model for peptide-induced membrane perturbations that explains the differences in peptide membrane activities and suggests the existence of a facilitated “physical endocytosis,” which represents a new pathway for peptide cellular internalization. PMID:17299584

  18. 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

  19. A neuroligin-1-derived peptide stimulates phosphorylation of the NMDA receptor NR1 subunit and rescues MK-801-induced decrease in long-term potentiation and memory impairment.

    PubMed

    Korshunova, Irina; Gjørlund, Michelle D; Owczarek, Sylwia; Petersen, Anders V; Perrier, Jean-François; Gøtzsche, Casper René; Berezin, Vladimir

    2015-03-01

    Neuroligins (NLs) are postsynaptic adhesion molecules, interacting with presynaptic neurexins (NXs), which determine the differential formation of excitatory (glutamatergic, NL1) and inhibitory (GABAergic, NL2) synapses. We have previously demonstrated that treatment with a NL2-derived peptide, neurolide-2, reduces sociability and increase animal aggression. We hypothesized that interfering with NL1 function at the excitatory synapses might regulate synaptic plasticity and learning, and counteract memory deficits induced by N-methyl-d-aspartate (NMDA) receptor inhibition. First, neuronal NMDA receptor phosphorylation after treatment with NL1 or a mimetic peptide, neurolide-1, was quantified by immunoblotting. Subsequently, we investigated effects of neurolide-1 on long-term potentiation (LTP) induction in hippocampal slices compromised by NMDA receptor inhibitor MK-801. Finally, we investigated neurolide-1 effects on short- and long-term social and spatial memory in social recognition, Morris water-maze, and Y-maze tests. We found that subcutaneous neurolide-1 administration, restored hippocampal LTP compromised by NMDA receptor inhibitor MK-801. It counteracted MK-801-induced memory deficit in the water-maze and Y-maze tests after long-term treatment (24 h and 1-2 h before the test), but not after short-term exposure (1-2 h). Long-term exposure to neurolide-1 also facilitated social recognition memory. In addition, neurolide-1-induced phosphorylation of the NMDA receptor NR1 subunit on a site important for synaptic trafficking, potentially favoring synaptic receptor retention. Our findings emphasize the role of NL1-NMDA receptor interaction in cognition, and identify neurolide-1, as a valuable pharmacological tool to examine the in vivo role of postsynaptic NL1 in cognitive behavior in physiological and pathological conditions. PMID:26038702

  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. PMID:25143246

  1. Use of a novel cytotoxic HEXIM1 peptide in the directed breast cancer therapy

    PubMed Central

    Neo, Shu Hui; Lew, Qiao Jing; Koh, Ser Mei; Zheng, Lu; Bi, Xuezhi; Chao, Sheng-Hao

    2016-01-01

    Hexamethylene bisacetamide-inducible protein 1 (HEXIM1) is best known as the inhibitor of positive transcription elongation factor b (P-TEFb) and is recently identified as a novel positive regulator of p53. We previously showed the basic region (BR) of HEXIM1 mediates the binding of HEXIM1 to a nucleolar protein, nucleophosmin (NPM), and can be ubiquitinated by human double minute 2 protein. Here we identify a cytotoxic peptide derived from the BR of HEXIM1. When fused with a cell-penetrating peptide, the HEXIM1 BR peptide triggers rapid cytotoxic effect independent of p53. Similarly, when the BR peptide is linked with a breast cancer cell targeting peptide, LTV, the LTV-BR fusion peptide exhibits specific killing of breast cancer cells, which is not observed with the commonly used cytotoxic peptide, KLA. Importantly, the BR peptide fails to enter cells by itself and does not induce any cytotoxic effects when it is not guided by any cell-penetrating or cancer targeting peptides. We showed that HEXIM1 BR peptide depolarizes mitochondrial membrane potential in a p53-dependent manner and its cell-killing activity is not suppressed by caspase inhibition. Furthermore, we observed an accumulation of the internalized BR peptide in the nucleoli of treated cells and an altered localization of NPM. These results illustrate a novel mechanism which the BR peptide induces cell death and can potentially be used as a novel therapeutic strategy against breast cancer. PMID:26734838

  2. 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

  3. Decreased glycation and structural protection properties of γ-glutamyl-S-allyl-cysteine peptide isolated from fresh garlic scales (Allium sativum L.).

    PubMed

    Tan, Dehong; Zhang, Yao; Chen, Lulu; Liu, Ling; Zhang, Xuan; Wu, Zhaoxia; Bai, Bing; Ji, Shujuan

    2015-01-01

    The antiglycative effect of γ-glutamyl-S-allyl-cysteine (GSAC) peptide isolated from fresh garlic scales was investigated in the bovine serum albumin (BSA)/glucose system. GSAC inhibited the increase of fluorescence intensity at about 440 nm in a concentration-dependent manner and reduced reacted free lysine side chains by 10.9%, 24.7% and 37.7%, as the GSAC concentrations increased from 0.1 to 2.5 mg mL(-1). Glycation-specific decline in BSA α-helix content (from 61.3% to 55.6%) and increase in β-sheet (from 2.1% to 5.4%) were prevented by GSAC (2.5 mg mL(-1)) in vitro, implying its stabilisation effect. GSAC treatment (2.5 mg mL(-1)) suppressed protein crosslinking to form polymers. Additionally, GSAC (10, 40, and 160 μg mL(-1)) showed radical-scavenging and metal-chelating capacities. In conclusion, GSAC has an antiglycative effect, which may involve its radical-scavenging and metal-chelating capacities. PMID:25631559

  4. Structure-Function Analysis of the Glioma Targeting NFL-TBS.40-63 Peptide Corresponding to the Tubulin-Binding Site on the Light Neurofilament Subunit

    PubMed Central

    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. PMID:23152907

  5. Vitamin B12 Conjugation of Peptide-YY3–36 Decreases Food Intake Compared to Native Peptide-YY3–36 Upon Subcutaneous Administration in Male Rats

    PubMed Central

    Henry, Kelly E.; Elfers, Clinton T.; Burke, Rachael M.; Chepurny, Oleg G.; Holz, George G.; Blevins, James E.

    2015-01-01

    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-PYY3–36 conjugate was tested against native PYY3–36, 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-PYY3–36 conjugate (EC50 26.5 nM) compared with native PYY3–36 (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-PYY3–36 or PYY3–36 injection relative to saline or B12-PYYC36. Food intake was reduced during a 5-day treatment for both B12-PYY3–36- (24%, P = .001) and PYY3–36-(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-PYY3–36 treatment (−26%, −29%, −27%) compared with the PYY3–36 treatment (−3%, −21%, −16%), and B12-PYY3–36 generated a significantly longer inhibition of food intake vs PYY3–36 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-PYY3–36 conjugate compared with the native PYY3–36. PMID:25658456

  6. 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). PMID:25658456

  7. 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

  8. Decreased calcitonin gene-related peptide expression in the dorsal root ganglia of TNF-deficient mice in a monoiodoacetate-induced knee osteoarthritis model

    PubMed Central

    Taniguchi, Aya; Ishikawa, Tetsuhiro; Miyagi, Masayuki; Kamoda, Hiroto; Sakuma, Yoshihiro; Oikawa, Yasuhiro; Kubota, Go; Inage, Kazuhide; Sainoh, Takeshi; Nakamura, Junichi; Aoki, Yasuchika; Toyone, Tomoaki; Inoue, Gen; Suzuki, Miyako; Yamauchi, Kazuyo; Suzuki, Takane; Takahashi, Kazuhisa; Ohtori, Seiji; Orita, Sumihisa

    2015-01-01

    Background: The detailed mechanisms of knee osteoarthritis (OA) pain have not been clarified, but involvement of inflammatory cytokines such as tumor necrosis factor-alpha (TNF) has been suggested. The present study aimed to investigate the more detailed neurological involvement of TNF in joint pain using a TNF-knockout mouse OA model. Methods: The right knees of twelve-week-old C57BL/6J wild and TNF-deficient knockout (TNF-ko) mice (n=15, each group) were given a single intra-articular injection of 10 µg monoiodoacetate in 10 mL sterile saline. The left knees were only punctured as the control. Evaluations were performed immediately after the injection (baseline) and at 7, 14, and 28 days after the injection with a subsequent intra-articular injection of neurotracer into both knees. The animals were evaluated for immunofluorescence of the lumbar dorsal root ganglia (DRG) innervating the knee joints. The injected knees were observed macroscopically and mouse pain-related behaviors were scored. Results: Macroscopic observation showed similar knee OA development in both wild and TNF-ko mice. Calcitonin gene-related peptide (CGRP, a neuropeptide identified as a inflammatory pain-related biomarker) was significantly increased in DRG neurons innervating OA-induced knee joints with significantly less CGRP expression in TNF-ko animals. Pain-related behavior scoring showed a significant increase in pain in OA-induced joints, but there was no significant difference in pain observed between the wild and TNF-ko mice. Conclusions: The result of the present study indicates the possible association of TNF-alpha in OA pain but not OA development. PMID:26722492

  9. 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

  10. 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

  11. 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

  12. Antimicrobial peptides in 2014.

    PubMed

    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

  13. 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

  14. 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

  15. 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

  16. 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

  17. A Dual-Ligand Liposomal System Composed of a Cell-Penetrating Peptide and a Mitochondrial RNA Aptamer Synergistically Facilitates Cellular Uptake and Mitochondrial Targeting.

    PubMed

    Yamada, Yuma; Furukawa, Ryo; Harashima, Hideyoshi

    2016-05-01

    It has been reported that the use of mitochondrial RNA aptamers including RNase P (RP) results in the selective mitochondrial delivery of endogenous and exogenous RNAs. The issue of whether these aptamers would be useful ligands for the mitochondrial targeting of a nanoparticle has not been demonstrated to date because nanocarriers modified with these RNA aptamers are insufficiently internalized by cells. We report here on the development of a dual-ligand liposomal system composed of octaarginine (R8), a device that enhances cellular uptake, and an RP aptamer for mitochondrial targeting to permit a nanocarrier to be efficiently delivered to mitochondria. Surprisingly, the cellular uptake of the R8-modified nanocarrier was facilitated by modification with an RP aptamer. The optimal composition of a nanocarrier needed for efficient cellular uptake and mitochondrial targeting was determined. In a confocal laser scanning microscopy analysis, the dual-ligand-modified nanocarrier was found to result in effective mitochondrial targeting through an ATP-dependent pathway and was much more effective than a single-ligand R8-modified nanocarrier. This is the first report of the regulation of intracellular trafficking by a mitochondrial RNA aptamer-modified nanocarrier system. PMID:27056631

  18. A common landscape for membrane-active peptides

    PubMed Central

    Last, Nicholas B; Schlamadinger, Diana E; Miranker, Andrew D

    2013-01-01

    Three families of membrane-active peptides are commonly found in nature and are classified according to their initial apparent activity. Antimicrobial peptides are ancient components of the innate immune system and typically act by disruption of microbial membranes leading to cell death. Amyloid peptides contribute to the pathology of diverse diseases from Alzheimer's to type II diabetes. Preamyloid states of these peptides can act as toxins by binding to and permeabilizing cellular membranes. Cell-penetrating peptides are natural or engineered short sequences that can spontaneously translocate across a membrane. Despite these differences in classification, many similarities in sequence, structure, and activity suggest that peptides from all three classes act through a small, common set of physical principles. Namely, these peptides alter the Brownian properties of phospholipid bilayers, enhancing the sampling of intrinsic fluctuations that include membrane defects. A complete energy landscape for such systems can be described by the innate membrane properties, differential partition, and the associated kinetics of peptides dividing between surface and defect regions of the bilayer. The goal of this review is to argue that the activities of these membrane-active families of peptides simply represent different facets of what is a shared energy landscape. PMID:23649542

  19. Functional recovery of human cells harbouring the mitochondrial DNA mutation MERRF A8344G via peptide-mediated mitochondrial delivery.

    PubMed

    Chang, Jui-Chih; Liu, Ko-Hung; Li, Yu-Chi; Kou, Shou-Jen; Wei, Yau-Huei; Chuang, Chieh-Sen; Hsieh, Mingli; Liu, Chin-San

    2013-01-01

    We explored the feasibility of mitochondrial therapy using the cell-penetrating peptide Pep-1 to transfer mitochondrial DNA (mtDNA) between cells and rescue a cybrid cell model of the mitochondrial disease myoclonic epilepsy with ragged-red fibres (MERRF) syndrome. Pep-1-conjugated wild-type mitochondria isolated from parent cybrid cells incorporating a mitochondria-specific tag were used as donors for mitochondrial delivery into MERRF cybrid cells (MitoB2) and mtDNA-depleted Rho-zero cells (Mitoρ°). Forty-eight hours later, translocation of Pep-1-labelled mitochondria into the mitochondrial regions of MitoB2 and Mitoρ° host cells was observed (delivery efficiencies of 77.48 and 82.96%, respectively). These internalized mitochondria were maintained for at least 15 days in both cell types and were accompanied by mitochondrial function recovery and cell survival by preventing mitochondria-dependent cell death. Mitochondrial homeostasis analyses showed that peptide-mediated mitochondrial delivery (PMD) also increased mitochondrial biogenesis in both cell types, but through distinct regulatory pathways involving mitochondrial dynamics. Dramatic decreases in mitofusin-2 (MFN2) and dynamin-related protein 1/fission 1 were observed in MitoB2 cells, while Mitoρ° cells showed a significant increase in optic atrophy 1 and MFN2. These findings suggest that PMD can be used as a potential therapeutic intervention for mitochondrial disorders. PMID:23006856

  20. 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.

  1. Discovery of a Direct Ras Inhibitor by Screening a Combinatorial Library of Cell-Permeable Bicyclic Peptides

    PubMed Central

    2015-01-01

    Cyclic peptides have great potential as therapeutic agents and research tools. However, their applications against intracellular targets have been limited, because cyclic peptides are generally impermeable to the cell membrane. It was previously shown that fusion of cyclic peptides with a cyclic cell-penetrating peptide resulted in cell-permeable bicyclic peptides that are proteolytically stable and biologically active in cellular assays. In this work, we tested the generality of the bicyclic approach by synthesizing a combinatorial library of 5.7 × 106 bicyclic peptides featuring a degenerate sequence in the first ring and an invariant cell-penetrating peptide in the second ring. Screening of the library against oncoprotein K-Ras G12V followed by hit optimization produced a moderately potent and cell-permeable K-Ras inhibitor, which physically blocks the Ras-effector interactions in vitro, inhibits the signaling events downstream of Ras in cancer cells, and induces apoptosis of the cancer cells. Our approach should be generally applicable to developing cell-permeable bicyclic peptide inhibitors against other intracellular proteins. PMID:26645887

  2. 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

  3. 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

  4. Recent Developments in Peptide-Based Nucleic Acid Delivery

    PubMed Central

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

    2008-01-01

    Despite the fact that non-viral nucleic acid delivery systems are generally considered to be less efficient than viral vectors, they have gained much interest in recent years due to their superior safety profile compared to their viral counterpart. Among these synthetic vectors are cationic polymers, branched dendrimers, cationic liposomes and cell-penetrating peptides (CPPs). The latter represent an assortment of fairly unrelated sequences essentially characterised by a high content of basic amino acids and a length of 10–30 residues. CPPs are capable of mediating the cellular uptake of hydrophilic macromolecules like peptides and nucleic acids (e.g. siRNAs, aptamers and antisense-oligonucleotides), which are internalised by cells at a very low rate when applied alone. Up to now, numerous sequences have been reported to show cell-penetrating properties and many of them have been used to successfully transport a variety of different cargos into mammalian cells. In recent years, it has become apparent that endocytosis is a major route of internalisation even though the mechanisms underlying the cellular translocation of CPPs are poorly understood and still subject to controversial discussions. In this review, we will summarise the latest developments in peptide-based cellular delivery of nucleic acid cargos. We will discuss different mechanisms of entry, the intracellular fate of the cargo, correlation studies of uptake versus biological activity of the cargo as well as technical problems and pitfalls. PMID:19325804

  5. Recent developments in peptide-based nucleic acid delivery.

    PubMed

    Veldhoen, Sandra; Laufer, Sandra D; Restle, Tobias

    2008-06-01

    Despite the fact that non-viral nucleic acid delivery systems are generally considered to be less efficient than viral vectors, they have gained much interest in recent years due to their superior safety profile compared to their viral counterpart. Among these synthetic vectors are cationic polymers, branched dendrimers, cationic liposomes and cell-penetrating peptides (CPPs). The latter represent an assortment of fairly unrelated sequences essentially characterised by a high content of basic amino acids and a length of 10-30 residues. CPPs are capable of mediating the cellular uptake of hydrophilic macromolecules like peptides and nucleic acids (e.g. siRNAs, aptamers and antisense-oligonucleotides), which are internalised by cells at a very low rate when applied alone. Up to now, numerous sequences have been reported to show cell-penetrating properties and many of them have been used to successfully transport a variety of different cargos into mammalian cells. In recent years, it has become apparent that endocytosis is a major route of internalisation even though the mechanisms underlying the cellular translocation of CPPs are poorly understood and still subject to controversial discussions. In this review, we will summarise the latest developments in peptide-based cellular delivery of nucleic acid cargos. We will discuss different mechanisms of entry, the intracellular fate of the cargo, correlation studies of uptake versus biological activity of the cargo as well as technical problems and pitfalls. PMID:19325804

  6. 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

  7. Alanine scan and (2)H NMR analysis of the membrane-active peptide BP100 point to a distinct carpet mechanism of action.

    PubMed

    Zamora-Carreras, Héctor; Strandberg, Erik; Mühlhäuser, Philipp; Bürck, Jochen; Wadhwani, Parvesh; Jiménez, M Ángeles; Bruix, Marta; Ulrich, Anne S

    2016-06-01

    The short membrane-active peptide BP100 [KKLFKKILKYL-NH2] is known as an effective antimicrobial and cell penetrating agent. For a functional alanine scan each of the 11 amino acids was replaced with deuterated Ala-d3, one at a time. MIC assays showed that a substitution of Lys did not affect the antimicrobial activity, but it decreased when a hydrophobic residue was replaced. In most cases, a reduction in hydrophobicity led to a decrease in hemolysis, and some peptide analogues had an improved therapeutic index. Circular dichroism showed that BP100 folds as an amphiphilic α-helix in a bilayer. Its alignment was determined from (2)H NMR in oriented membranes of different composition. The azimuthal rotation angle was the same under all conditions, but the average helix tilt angle and the dynamical behavior of the peptide varied in a systematic manner. In POPC/POPG bilayers, with a negative spontaneous curvature, the peptide was found to lie flat on the bilayer surface, and with little wobble. In DMPC/DMPG, with a positive spontaneous curvature, BP100 at higher concentrations became tilted obliquely into the membrane, with the uncharged C-terminus inserted more deeply into the lipid bilayer, experiencing significant fluctuations in tilt angle. In DMPC/DMPG/lyso-MPC, with a pronounced positive spontaneous curvature, the helix tilted even further and became even more mobile. The 11-mer BP100 is obviously too short to form transmembrane pores. We conclude that BP100 operates via a carpet mechanism, whereby the C-terminus gets inserted into the hydrophobic core of the bilayer, which leads to membrane perturbation and induces transient permeability. PMID:26975251

  8. 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.

  9. 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

  10. Peptides Displayed as High Density Brush Polymers Resist Proteolysis and Retain Bioactivity

    PubMed Central

    2015-01-01

    We describe a strategy for rendering peptides resistant to proteolysis by formulating them as high-density brush polymers. The utility of this approach is demonstrated by polymerizing well-established cell-penetrating peptides (CPPs) and showing that the resulting polymers are not only resistant to proteolysis but also maintain their ability to enter cells. The scope of this design concept is explored by studying the proteolytic resistance of brush polymers composed of peptides that are substrates for either thrombin or a metalloprotease. Finally, we demonstrate that the proteolytic susceptibility of peptide brush polymers can be tuned by adjusting the density of the polymer brush and offer in silico models to rationalize this finding. We contend that this strategy offers a plausible method of preparing peptides for in vivo use, where rapid digestion by proteases has traditionally restricted their utility. PMID:25314576

  11. IKK NBD peptide inhibits LPS induced pulmonary inflammation and alters sphingolipid metabolism in a murine model.

    PubMed

    von Bismarck, Philipp; Winoto-Morbach, Supandi; Herzberg, Mona; Uhlig, Ulrike; Schütze, Stefan; Lucius, Ralph; Krause, Martin F

    2012-06-01

    Airway epithelial NF-κB is a key regulator of host defence in bacterial infections and has recently evolved as a target for therapeutical approaches. Evidence is accumulating that ceramide, generated by acid sphingomyelinase (aSMase), and sphingosine-1-phosphate (S1-P) are important mediators in host defence as well as in pathologic processes of acute lung injury. Little is known about the regulatory mechanisms of pulmonary sphingolipid metabolism in bacterial infections of the lung. The objective of this study was to evaluate the influence of NF-κB on sphingolipid metabolism in Pseudomonas aeruginosa LPS-induced pulmonary inflammation. In a murine acute lung injury model with intranasal Pseudomonas aeruginosa LPS we investigated TNF-α, KC (murine IL-8), IL-6, MCP-1 and neutrophilic infiltration next to aSMase activity and ceramide and S1-P lung tissue concentrations. Airway epithelial NF-κB was inhibited by topically applied IKK NBD, a cell penetrating NEMO binding peptide. This treatment resulted in significantly reduced inflammation and suppression of aSMase activity along with decreased ceramide and S1-P tissue concentrations down to levels observed in healthy animals. In conclusion our results confirm that changes in sphingolipid metabolim due to Pseudomonas aeruginosa LPS inhalation are regulated by NF-κB translocation. This confirms the critical role of airway epithelial NF-κB pathway for the inflammatory response to bacterial pathogens and underlines the impact of sphingolipids in inflammatory host defence mechanisms. PMID:22469869

  12. 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

  13. 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. PMID:26447818

  14. 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

  15. 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

  16. 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 Central

    2015-01-01

    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 99mTc-labeled Arg-X-Asp-conjugated and X-Ala-Asp-conjugated α-melanocyte stimulating hormone (α-MSH) peptides. RSD-β-Ala-(Arg11)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-(Arg11)CCMSH (2), RVD-β-Ala-(Arg11)CCMSH (3), RAD-β-Ala-(Arg11)CCMSH (4), NAD-β-Ala-(Arg11)CCMSH (5), and EAD-β-Ala-(Arg11)CCMSH (6) peptides were synthesized and evaluated for their melanocortin 1 (MC1) receptor binding affinities in B16/F1 melanoma cells. The biodistribution of their 99mTc-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 99mTc-peptides. 99mTc-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 99mTc-peptides at 2 h postinjection. The B16/F1 melanoma lesions could be clearly visualized by single photon emission computed tomography (SPECT)/CT using 99mTc-4 as an imaging probe. PMID:25290883

  17. 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

  18. Peptides Used in the Delivery of Small Noncoding RNA

    PubMed Central

    2015-01-01

    RNA interference (RNAi) is an endogenous process in which small noncoding RNAs, including small interfering RNAs (siRNAs) and microRNAs (miRNAs), post-transcriptionally regulate gene expressions. In general, siRNA and miRNA/miRNA mimics are similar in nature and activity except their origin and specificity. Although both siRNAs and miRNAs have been extensively studied as novel therapeutics for a wide range of diseases, the large molecular weight, anionic surface charges, instability in blood circulation, and intracellular trafficking to the RISC after cellular uptake have hindered the translation of these RNAs from bench to clinic. As a result, a great variety of delivery systems have been investigated for safe and effective delivery of small noncoding RNAs. Among these systems, peptides, especially cationic peptides, have emerged as a promising type of carrier due to their inherent ability to condense negatively charged RNAs, ease of synthesis, controllable size, and tunable structure. In this review, we will focus on three major types of cationic peptides, including poly(l-lysine) (PLL), protamine, and cell penetrating peptides (CPP), as well as peptide targeting ligands that have been extensively used in RNA delivery. The delivery strategies, applications, and limitations of these cationic peptides in siRNA/miRNA delivery will be discussed. PMID:25157701

  19. Dysferlin-peptides reallocate mutated dysferlin thereby restoring function.

    PubMed

    Schoewel, Verena; Marg, Andreas; Kunz, Severine; Overkamp, Tim; Carrazedo, Romy Siegert; Zacharias, Ute; Daniel, Peter T; Spuler, Simone

    2012-01-01

    Mutations in the dysferlin gene cause the most frequent adult-onset limb girdle muscular dystrophy, LGMD2B. There is no therapy. Dysferlin is a membrane protein comprised of seven, beta-sheet enriched, C2 domains and is involved in Ca(2+)dependent sarcolemmal repair after minute wounding. On the protein level, point mutations in DYSF lead to misfolding, aggregation within the endoplasmic reticulum, and amyloidogenesis. We aimed to restore functionality by relocating mutant dysferlin. Therefore, we designed short peptides derived from dysferlin itself and labeled them to the cell penetrating peptide TAT. By tracking fluorescently labeled short peptides we show that these dysferlin-peptides localize in the endoplasmic reticulum. There, they are capable of reducing unfolded protein response stress. We demonstrate that the mutant dysferlin regains function in membrane repair in primary human myotubes derived from patients' myoblasts by the laser wounding assay and a novel technique to investigate membrane repair: the interventional atomic force microscopy. Mutant dysferlin abuts to the sarcolemma after peptide treatment. The peptide-mediated approach has not been taken before in the field of muscular dystrophies. Our results could redirect treatment efforts for this condition. PMID:23185377

  20. Membrane-Active Peptides and the Clustering of Anionic Lipids

    PubMed Central

    Wadhwani, P.; Epand, R.F.; Heidenreich, N.; Bürck, J.; Ulrich, A.S.; Epand, R.M.

    2012-01-01

    There is some overlap in the biological activities of cell-penetrating peptides (CPPs) and antimicrobial peptides (AMPs). We compared nine AMPs, seven CPPs, and a fusion peptide with regard to their ability to cluster anionic lipids in a mixture mimicking the cytoplasmic membrane of Gram-negative bacteria, as measured by differential scanning calorimetry. We also studied their bacteriostatic effect on several bacterial strains, and examined their conformational changes upon membrane binding using circular dichroism. A remarkable correlation was found between the net positive charge of the peptides and their capacity to induce anionic lipid clustering, which was independent of their secondary structure. Among the peptides studied, six AMPs and four CPPs were found to have strong anionic lipid clustering activity. These peptides also had bacteriostatic activity against several strains (particularly Gram-negative Escherichia coli) that are sensitive to lipid clustering agents. AMPs and CPPs that did not cluster anionic lipids were not toxic to E. coli. As shown previously for several types of AMPs, anionic lipid clustering likely contributes to the mechanism of antibacterial action of highly cationic CPPs. The same mechanism could explain the escape of CPPs from intracellular endosomes that are enriched with anionic lipids. PMID:22853904

  1. 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.

  2. 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

  3. 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.

  4. 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.

  5. Combination therapy of anti-cancer bioactive peptide with Cisplatin decreases chemotherapy dosing and toxicity to improve the quality of life in xenograft nude mice bearing human gastric cancer

    PubMed Central

    2014-01-01

    Background A great challenge of cancer chemotherapy is to eliminate cancer cells and concurrently maintain the quality of life (QOL) for cancer patients. Previously, we identified a novel anti-cancer bioactive peptide (ACBP), a peptide induced in goat spleen or liver following immunization with human gastric cancer protein extract. ACBP alone exhibited anti-tumor activity without measurable side effects. Thus, we hypothesize that ACBP and combined chemotherapy could improve the efficacy of treatment and lead to a better QOL. Results In this study, ACBP was isolated and purified from immunized goat liver, and designated as ACBP-L. The anti-tumor activity was investigated in a previously untested human gastric cancer MGC-803 cell line and tumor model. ACBP-L inhibited cell proliferation in vitro in a dose and time dependent manner, titrated by MTT assay. The effect of ACBP-L on cell morphology was observed through light and scanning electron microscopy. In vivo ACBP-L alone significantly inhibited MGC-803 tumor growth in a xenograft nude mouse model without measurable side effects. Treatment with the full dosage of Cisplatin alone (5 mg/kg every 5 days) strongly suppressed tumor growth. However, the QOL in these mice had been significantly affected when measured by food intakes and body weight. The combinatory regiment of ACBP-L with a fewer doses of Cisplatin (5 mg/kg every 10 days) resulted in a similar anti-tumor activity with improved QOL. 18F-FDG PET/CT scan was used to examine the biological activity in tumors of live animals and indicated the consistent treatment effects. The tumor tissues were harvested after treatment, and ACBP-L and Cisplatin treatment suppressed Bcl-2, and induced Bax, Caspase 3, and Caspase 8 molecules as detected by RT-PCR and immunohistochemistry. The combinatory regiment induced stronger Bax and Caspase 8 protein expression. Conclusion Our current finding in this gastric cancer xenograft animal model demonstrated that ACBP-L could

  6. 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

  7. 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

  8. 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

  9. 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

  10. Guanidinium-rich, glycerol-derived oligocarbonates: a new class of cell-penetrating molecular transporters that complex, deliver, and release siRNA.

    PubMed

    Wender, Paul A; Huttner, Melanie A; Staveness, Daryl; Vargas, Jessica R; Xu, Adele F

    2015-03-01

    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

  11. Targeting a Metalloprotease-PAR1 Signaling System with Cell-Penetrating Pepducins Inhibits Angiogenesis, Ascites, and Progression of Ovarian Cancer

    PubMed Central

    Agarwal, Anika; Covic, Lidija; Sevigny, Leila M; Kaneider, Nicole C; Lazarides, Katherine; Azabdaftari, Gissou; Sharifi, Sheida; Kuliopulos, Athan

    2009-01-01

    Gene chip and proteomic analyses of tumors and stromal tissue has led to the identification of dozens of candidate tumor and host components potentially involved in tumor-stromal interactions, angiogenesis, and progression of invasive disease. In particular, matrix metalloproteases have emerged as important biomarkers and prognostic factors for invasive and metastatic cancers. From an initial screen of benign versus malignant patient fluids, we delineated a metalloprotease cascade comprising MMP-14, MMP-9 and MMP-1 that culminates in activation of PAR1—a G protein-coupled protease-activated receptor upregulated in diverse cancers. In xenograft models of advanced peritoneal ovarian cancer, PAR1-dependent angiogenesis, ascites formation and metastasis was effectively inhibited by intraperitoneal administration of cell-penetrating pepducins based on the intracellular loops of PAR1. These data provide an in vivo proof-of-concept that targeting the metalloprotease-PAR1 signaling system may be a novel therapeutic approach in the treatment of ovarian cancer. PMID:18790755

  12. Targeting a metalloprotease-PAR1 signaling system with cell-penetrating pepducins inhibits angiogenesis, ascites, and progression of ovarian cancer.

    PubMed

    Agarwal, Anika; Covic, Lidija; Sevigny, Leila M; Kaneider, Nicole C; Lazarides, Katherine; Azabdaftari, Gissou; Sharifi, Sheida; Kuliopulos, Athan

    2008-09-01

    Gene chip and proteomic analyses of tumors and stromal tissue has led to the identification of dozens of candidate tumor and host components potentially involved in tumor-stromal interactions, angiogenesis, and progression of invasive disease. In particular, matrix metalloproteases (MMP) have emerged as important biomarkers and prognostic factors for invasive and metastatic cancers. From an initial screen of benign versus malignant patient fluids, we delineated a metalloprotease cascade comprising MMP-14, MMP-9, and MMP-1 that culminates in activation of PAR1, a G protein-coupled protease-activated receptor up-regulated in diverse cancers. In xenograft models of advanced peritoneal ovarian cancer, PAR1-dependent angiogenesis, ascites formation, and metastasis were effectively inhibited by i.p. administration of cell-penetrating pepducins based on the intracellular loops of PAR1. These data provide an in vivo proof-of-concept that targeting the metalloprotease-PAR1 signaling system may be a novel therapeutic approach in the treatment of ovarian cancer. PMID:18790755

  13. Systematic screening of the cellular uptake of designed alpha-helix peptides.

    PubMed

    Usui, Kenji; Kikuchi, Takuya; Mie, Masayasu; Kobatake, Eiry; Mihara, Hisakazu

    2013-05-01

    The cellular penetration (CP) activity of functional molecules has attracted significant attention as one of the most promising new approaches for drug delivery. In particular, cell-penetrating peptides (CPPs) have been studied extensively in cellular engineering. Because there have been few large-scale systematic studies to identify peptide sequences with optimal CP activity or that are suitable for further applications in cell engineering, such as cell-specific penetration and cell-selective culture, we screened and compared the cellular uptake (CU) activity of 54 systematically designed α-helical peptides in HeLa cells. Furthermore, the CU activity of 24 designed peptides was examined in four cell lines using a cell fingerprinting technique and statistical approaches. The CU activities in various cells depended on amino acid residues of peptide sequences as well as charge, α-helical content and hydrophobicity of the peptides. Notably, the mutation of a single residue significantly altered the CU ability of a peptide, highlighting the variability of cell uptake mechanisms. Moreover, these results demonstrated the feasibility of cell-selective culture by conducting cell-selective permeation and death in cultures containing two cell types. These studies may lead to further peptide library design and screening for new classes of CPPs with useful functions. PMID:23498920

  14. Poly-arginine and arginine-rich peptides are neuroprotective in stroke models.

    PubMed

    Meloni, Bruno P; Brookes, Laura M; Clark, Vince W; Cross, Jane L; Edwards, Adam B; Anderton, Ryan S; Hopkins, Richard M; Hoffmann, Katrin; Knuckey, Neville W

    2015-06-01

    Using cortical neuronal cultures and glutamic acid excitotoxicity and oxygen-glucose deprivation (OGD) stroke models, we demonstrated that poly-arginine and arginine-rich cell-penetrating peptides (CPPs), are highly neuroprotective, with efficacy increasing with increasing arginine content, have the capacity to reduce glutamic acid-induced neuronal calcium influx and require heparan sulfate preotoglycan-mediated endocytosis to induce a neuroprotective effect. Furthermore, neuroprotection could be induced with immediate peptide treatment or treatment up to 2 to 4 hours before glutamic acid excitotoxicity or OGD, and with poly-arginine-9 (R9) when administered intravenously after stroke onset in a rat model. In contrast, the JNKI-1 peptide when fused to the (non-arginine) kFGF CPP, which does not rely on endocytosis for uptake, was not neuroprotective in the glutamic acid model; the kFGF peptide was also ineffective. Similarly, positively charged poly-lysine-10 (K10) and R9 fused to the negatively charged poly-glutamic acid-9 (E9) peptide (R9/E9) displayed minimal neuroprotection after excitotoxicity. These results indicate that peptide positive charge and arginine residues are critical for neuroprotection, and have led us to hypothesize that peptide-induced endocytic internalization of ion channels is a potential mechanism of action. The findings also question the mode of action of different neuroprotective peptides fused to arginine-rich CPPs. PMID:25669902

  15. Poly-arginine and arginine-rich peptides are neuroprotective in stroke models

    PubMed Central

    Meloni, Bruno P; Brookes, Laura M; Clark, Vince W; Cross, Jane L; Edwards, Adam B; Anderton, Ryan S; Hopkins, Richard M; Hoffmann, Katrin; Knuckey, Neville W

    2015-01-01

    Using cortical neuronal cultures and glutamic acid excitotoxicity and oxygen-glucose deprivation (OGD) stroke models, we demonstrated that poly-arginine and arginine-rich cell-penetrating peptides (CPPs), are highly neuroprotective, with efficacy increasing with increasing arginine content, have the capacity to reduce glutamic acid-induced neuronal calcium influx and require heparan sulfate preotoglycan-mediated endocytosis to induce a neuroprotective effect. Furthermore, neuroprotection could be induced with immediate peptide treatment or treatment up to 2 to 4 hours before glutamic acid excitotoxicity or OGD, and with poly-arginine-9 (R9) when administered intravenously after stroke onset in a rat model. In contrast, the JNKI-1 peptide when fused to the (non-arginine) kFGF CPP, which does not rely on endocytosis for uptake, was not neuroprotective in the glutamic acid model; the kFGF peptide was also ineffective. Similarly, positively charged poly-lysine-10 (K10) and R9 fused to the negatively charged poly-glutamic acid-9 (E9) peptide (R9/E9) displayed minimal neuroprotection after excitotoxicity. These results indicate that peptide positive charge and arginine residues are critical for neuroprotection, and have led us to hypothesize that peptide-induced endocytic internalization of ion channels is a potential mechanism of action. The findings also question the mode of action of different neuroprotective peptides fused to arginine-rich CPPs. PMID:25669902

  16. 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

  17. Cationic Membrane Peptides: Atomic-Level Insight of Structure-Activity Relationships from Solid-State NMR

    PubMed Central

    Su, Yongchao; Li, Shenhui; Hong, Mei

    2012-01-01

    Many membrane-active peptides, such as cationic cell-penetrating peptides (CPPs) and antimicrobial peptides (AMPs), conduct their biological functions by interacting with the cell membrane. The interactions of charged residues with lipids and water facilitate membrane insertion, translocation or disruption of these highly hydrophobic species. In this mini-review we will summarize high-resolution structural and dynamic findings towards the understanding of the structure-activity relationship of lipid membrane-bound CPPs and AMPs, as examples of the current development of solid-state NMR (SSNMR) techniques for studying membrane peptides. We will present the most recent atomic-resolution structure of the guanidinium-phosphate complex, as constrained from experimentally measured site-specific distances. These SSNMR results will be valuable specifically for understanding the intracellular translocation pathway of CPPs and antimicrobial mechanism of AMPs, and more generally broaden our insight into how cationic macromolecules interact with and cross the lipid membrane. PMID:23108593

  18. Fluorescence Correlation Spectroscopy Reveals Highly Efficient Cytosolic Delivery of Certain Penta-Arg Proteins and Stapled Peptides

    PubMed Central

    Steinauer, Angela; Rhoades, Elizabeth; Schepartz, Alanna

    2015-01-01

    We used fluorescence correlation spectroscopy (FCS) to accurately and precisely determine the relative efficiencies with which three families of “cell-penetrating peptides” traffic to the cytosol of mammalian cells. We find that certain molecules containing a “penta-arg” motif reach the cytosol, intact, with efficiencies greater than 50%. This value is at least 10-fold higher than that observed for the widely studied cationic sequence derived from HIV Tat or polyarginine Arg8, and equals that of hydrocarbon-stapled peptides that are active in cells and animals. Moreover, we show that the efficiency with which stapled peptides reach the cytosol, as determined by FCS, correlates directly with their efficacy in cell-based assays. We expect that these findings and the associated technology will aid the design of peptides, proteins, and peptide mimetics that predictably and efficiently reach the interior of mammalian cells. PMID:25679876

  19. 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

  20. 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

  1. Structural studies on the forward and reverse binding modes of peptides to the chaperone DnaK.

    PubMed

    Zahn, Michael; Berthold, Nicole; Kieslich, Björn; Knappe, Daniel; Hoffmann, Ralf; Sträter, Norbert

    2013-07-24

    Hsp70 chaperones have been implicated in assisting protein folding of newly synthesized polypeptide chains, refolding of misfolded proteins, and protein trafficking. For these functions, the chaperones need to exhibit a significant promiscuity in binding to different sequences of hydrophobic peptide stretches. To characterize the structural basis of sequence specificity and flexibility of the Escherichia coli Hsp70 chaperone DnaK, we have analyzed crystal structures of the substrate binding domain of the protein in complex with artificially designed peptides as well as small proline-rich antimicrobial peptides. The latter peptides from mammals and insects were identified to target DnaK after cell penetration. Interestingly, the complex crystal structures reveal two different peptide binding modes. The peptides can bind either in a forward or in a reverse direction to the conventional substrate binding cleft of DnaK in an extended conformation. Superposition of the two binding modes shows a remarkable similarity in the side chain orientations and hydrogen bonding pattern despite the reversed peptide orientation. The DnaK chaperone has evolved to bind peptides in both orientations in the substrate binding cleft with comparable energy without rearrangements of the protein. Optimal hydrophobic interactions with binding pockets -2 to 0 appear to be the main determinant for the orientation and sequence position of peptide binding. PMID:23562829

  2. Peptide arrays for screening cancer specific peptides.

    PubMed

    Ahmed, Sahar; Mathews, Anu Stella; Byeon, Nara; Lavasanifar, Afsaneh; Kaur, Kamaljit

    2010-09-15

    In this paper, we describe a novel method to screen peptides for specific recognition by cancer cells. Seventy peptides were synthesized on a cellulose membrane in an array format, and a direct method to study the peptide-whole cell interaction was developed. The relative binding affinity of the cells for different peptides with respect to a lead 12-mer p160 peptide, identified by phage display, was evaluated using the CyQUANT fluorescence of the bound cells. Screening allowed identification of at least five new peptides that displayed higher affinity (up to 3-fold) for MDA-MB-435 and MCF-7 human cancer cells compared to the p160 peptide. These peptides showed very little binding to the control (noncancerous) human umbilical vein endothelial cells (HUVECs). Three of these peptides were synthesized separately and labeled with fluorescein isothiocyanate (FITC) to study their uptake and interaction with the cancer and control cells using confocal laser scanning microscopy and flow cytometry. The results confirmed the high and specific affinity of an 11-mer peptide 11 (RGDPAYQGRFL) and a 10-mer peptide 18 (WXEAAYQRFL) for the cancer cells versus HUVECs. Peptide 11 binds different receptors on target cancer cells as its sequence contains multiple recognition motifs, whereas peptide 18 binds mainly to the putative p160 receptor. The peptide array-whole cell binding assay reported here is a complementary method to phage display for further screening and optimization of cancer targeting peptides for cancer therapy and diagnosis. PMID:20799711

  3. Antibacterial activity of novel cationic peptides against clinical isolates of multi-drug resistant Staphylococcus pseudintermedius from infected dogs.

    PubMed

    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

  4. 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

  5. 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 ...

  6. Construction of surfactant-like tetra-tail amphiphilic peptide with RGD ligand for encapsulation of porphyrin for photodynamic therapy.

    PubMed

    Chen, Jing-Xiao; Wang, Hui-Yuan; Li, Cao; Han, Kai; Zhang, Xian-Zheng; Zhuo, Ren-Xi

    2011-02-01

    A surfactant-like tetra-tail amphiphilic peptide, [(C(18))(2)K](2)KR(8)GRGDS was designed and synthesized for targeted drug delivery. The resulting peptide-amphiphile, consisting of four hydrophobic aliphatic tails and a hydrophilic peptide head group, was able to self-assemble into nanosized micelles in aqueous medium at low concentration. Ibuprofen and doxorubicin (DOX) was loaded into peptide micelles as model hydrophobic drugs respectively, and the sustained release behavior was observed. Due to the incorporation of targeted arginine-glycine-aspartic acid (RGD) sequences and cell penetrating peptide (CPP) residue octaarginine (R(8)), the micelles could be recognized specifically by cancer cells, as well as transport through the cell membrane efficiently. The observation of laser-scanning confocal microscopy confirmed effective cellular uptaking of porphyrin-loaded peptide micelles. Furthermore, the porphyrin-loaded micelles exhibited low dark toxicity and high phototoxicity against cancer cells, indicating the powerful potential for effective photodynamic therapy. Combined with the low cytotoxicity of the peptide against both HeLa and 293T cell lines, the surfactant-like peptide developed in this study may be promising in clinical application for targeted drug delivery. PMID:21084116

  7. Transient Focal Membrane Deformation Induced by Arginine-rich Peptides Leads to Their Direct Penetration into Cells

    PubMed Central

    Hirose, Hisaaki; Takeuchi, Toshihide; Osakada, Hiroko; Pujals, Sílvia; Katayama, Sayaka; Nakase, Ikuhiko; Kobayashi, Shouhei; Haraguchi, Tokuko; Futaki, Shiroh

    2012-01-01

    Endocytosis has been implicated in the cellular uptake of arginine-rich, cell-penetrating peptides (CPPs). However, accumulating evidence suggests that certain conditions allow the direct, non-endocytic penetration of arginine-rich peptides through the plasma membrane. We previously showed that Alexa Fluor 488-labeled dodeca-arginine (R12-Alexa488) directly enters cells at specific sites on the plasma membrane and subsequently diffuses throughout cells. In this study, we found that the peptide influx was accompanied by the formation of unique, “particle-like” multivesicular structures on the plasma membrane, together with topical inversion of the plasma membrane. Importantly, the conjugation of dodeca-arginine (R12) to Alexa Fluor 488 or a peptide tag derived from hemagglutinin (HAtag) significantly accelerated particle formation, suggesting that the chemical properties of the attached molecules (cargo molecules) may contribute to translocation of the R12 peptide. Coincubation with R12-HAtag allowed the membrane-impermeable R4-Alexa488 to permeate cells. These results suggest that R12 peptides attached to hydrophobic cargo molecules stimulate dynamic morphological alterations in the plasma membrane, and that these structural changes allow the peptides to permeate the plasma membrane. These findings may provide a novel mode of cell permeabilization by arginine-rich peptides as a means of drug delivery. PMID:22334015

  8. 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

  9. Uptake and cellular distribution of nucleolar targeting peptides (NrTPs) in different cell types.

    PubMed

    Rodrigues, Margarida; Andreu, David; Santos, Nuno C

    2015-03-01

    Nucleolar targeting peptides (NrTPs) are a family of cell penetrating peptides (CPPs) derived from crotamine, a rattlesnake venom toxin. They were named NrTPs for their remarkable nucleolus-homing properties and have been studied for their potential as drug delivery vehicles. Live cell microscopy experiments were conducted to monitor NrTP uptake and distribution in different cell types, including primary cells (PBMCs and erythrocytes) and different immortalized cell lines (HeLa, BHK21, BV-173, and MOLT-4). Uptake dependence on cell type (primary vs. immortalized, suspension vs. adherent, cancer vs. healthy cells), peptide concentration and cell viability were evaluated. To gain further insight on the internalization mechanism, uptake kinetics was also monitored. Results showed the uptake and distribution pattern as strongly dependent on peptide sequence, peptide concentration and membrane constituents. Under similar conditions, NrTP6 is more internalized than NrTP1, NrTP2 and NrTP5. Additionally, while internalization of NrTP7 and NrTP8 may cause cytotoxicity, NrTP6 is noncytotoxic. Higher peptide concentrations can be correlated to nucleolar targeting, although even at low concentrations a residual number of cells reveal positive nucleolar labeling. NrTPs were successfully internalized into all cell types tested except erythrocytes. PMID:25620660

  10. The role of spontaneous lipid curvature in the interaction of interfacially active peptides with membranes.

    PubMed

    Koller, Daniel; Lohner, Karl

    2014-09-01

    Research on antimicrobial peptides is in part driven by urgent medical needs such as the steady increase in pathogens being resistant to antibiotics. Despite the wealth of information compelling structure-function relationships are still scarce and thus the interfacial activity model has been proposed to bridge this gap. This model also applies to other interfacially active (membrane active) peptides such as cytolytic, cell penetrating or antitumor peptides. One parameter that is strongly linked to interfacial activity is the spontaneous lipid curvature, which is experimentally directly accessible. We discuss different parameters such as H-bonding, electrostatic repulsion, changes in monolayer surface area and lateral pressure that affect induction of membrane curvature, but also vice versa how membrane curvature triggers peptide response. In addition, the impact of membrane lipid composition on the formation of curved membrane structures and its relevance for diverse mode of action of interfacially active peptides and in turn biological activity are described. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova. PMID:24853655

  11. Structure and dynamics of cationic membrane peptides and proteins: Insights from solid-state NMR

    PubMed Central

    Hong, Mei; Su, Yongchao

    2011-01-01

    Many membrane peptides and protein domains contain functionally important cationic Arg and Lys residues, whose insertion into the hydrophobic interior of the lipid bilayer encounters significant energy barriers. To understand how these cationic molecules overcome the free energy barrier to insert into the lipid membrane, we have used solid-state NMR spectroscopy to determine the membrane-bound topology of these peptides. A versatile array of solid-state NMR experiments now readily yields the conformation, dynamics, orientation, depth of insertion, and site-specific protein–lipid interactions of these molecules. We summarize key findings of several Arg-rich membrane peptides, including β-sheet antimicrobial peptides, unstructured cell-penetrating peptides, and the voltage-sensing helix of voltage-gated potassium channels. Our results indicate the central role of guanidinium-phosphate and guanidinium-water interactions in dictating the structural topology of these cationic molecules in the lipid membrane, which in turn account for the mechanisms of this functionally diverse class of membrane peptides. PMID:21344534

  12. 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. PMID:25966787

  13. 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

  14. 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

  15. 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

    2015-06-01

    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.

  16. 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.

  17. Molecular structure and pronounced conformational flexibility of doxorubicin in free and conjugated state within a drug-peptide compound.

    PubMed

    Tsoneva, Yana; Jonker, Hendrik R A; Wagner, Manfred; Tadjer, Alia; Lelle, Marco; Peneva, Kalina; Ivanova, Anela

    2015-02-19

    The search for targeted drug delivery systems requires the design of drug-carrier complexes, which could both reach the malignant cells and preserve the therapeutic substance activity. A promising strategy aimed at enhancing the uptake and reducing the systemic toxicity is to bind covalently the drug to a cell-penetrating peptide. To understand the structure-activity relationship in such preparations, the chemotherapeutic drug doxorubicin was investigated by unrestrained molecular dynamics simulations, supported by NMR, which yielded its molecular geometry in aqueous environment. Furthermore, the structure and dynamics of a conjugate of the drug with a cell-penetrating peptide was obtained from molecular dynamics simulations in aqueous solution. The geometries of the unbound compounds were characterized at different temperatures, as well as the extent to which they change after covalent binding and whether/how they influence each other in the drug-peptide conjugate. The main structural fragments that affect the conformational ensemble of every molecule were found. The results show that the transitions between different substructures of the three compounds require a modest amount of energy. At increased temperature, either more conformations become populated as a result of the thermal fluctuations or the relative shares of the various conformers equalize at the nanosecond scale. These frequent structural interconversions suggest expressed conformational freedom of the molecules. Conjugation into the drug-peptide compound partially immobilizes the molecules of the parent compounds. Nevertheless, flexibility still exists, as well as an effective intra- and intermolecular hydrogen bonding that stabilizes the structures. We observe compact packing of the drug within the peptide that is also based on stacking interactions. All this outlines the drug-peptide conjugate as a prospective building block of a more complex drug-carrier system. PMID:25603129

  18. Cell Penetrant Inhibitors of the KDM4 and KDM5 Families of Histone Lysine Demethylases. 2. Pyrido[3,4-d]pyrimidin-4(3H)-one Derivatives.

    PubMed

    Westaway, Susan M; Preston, Alex G S; Barker, Michael D; Brown, Fiona; Brown, Jack A; Campbell, Matthew; Chung, Chun-wa; Drewes, Gerard; Eagle, Robert; Garton, Neil; Gordon, Laurie; Haslam, Carl; Hayhow, Thomas G; Humphreys, Philip G; Joberty, Gerard; Katso, Roy; Kruidenier, Laurens; Leveridge, Melanie; Pemberton, Michelle; Rioja, Inma; Seal, Gail A; Shipley, Tracy; Singh, Onkar; Suckling, Colin J; Taylor, Joanna; Thomas, Pamela; Wilson, David M; Lee, Kevin; Prinjha, Rab K

    2016-02-25

    Following the discovery of cell penetrant pyridine-4-carboxylate inhibitors of the KDM4 (JMJD2) and KDM5 (JARID1) families of histone lysine demethylases (e.g., 1), further optimization led to the identification of non-carboxylate inhibitors derived from pyrido[3,4-d]pyrimidin-4(3H)-one. A number of exemplars such as compound 41 possess interesting activity profiles in KDM4C and KDM5C biochemical and target-specific, cellular mechanistic assays. PMID:26771203

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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. PMID:24341748

  7. 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

  8. Peptide conjugation of 2'-O-methyl phosphorothioate antisense oligonucleotides enhances cardiac uptake and exon skipping in mdx mice.

    PubMed

    Jirka, Silvana M G; Heemskerk, Hans; Tanganyika-de Winter, Christa L; Muilwijk, Daan; Pang, Kar Him; de Visser, Peter C; Janson, Anneke; Karnaoukh, Tatyana G; Vermue, Rick; 't Hoen, Peter A C; van Deutekom, Judith C T; Aguilera, Begoña; Aartsma-Rus, Annemieke

    2014-02-01

    Antisense oligonucleotide (AON)-mediated exon skipping is a promising therapeutic approach for Duchenne muscular dystrophy that is currently being tested in various clinical trials. This approach is based on restoring the open reading frame of dystrophin transcripts resulting in shorter but partially functional dystrophin proteins as found in patients with Becker muscular dystrophy. After systemic administration, a large proportion of AONs ends up in the liver and kidneys. Therefore, enhancing AON uptake by skeletal and cardiac muscle would improve the AONs' therapeutic effect. For phosphorodiamidate morpholino oligomer, AONs use nonspecific positively charged cell penetrating peptides to enhance efficacy. However, this is challenging for negatively charged 2'-O-methyl phosphorothioate oligomer. Therefore, we screened a 7-mer phage display peptide library to identify muscle and heart homing peptides in vivo in the mdx mouse model and found a promising candidate peptide capable of binding muscle cells in vitro and in vivo. Upon systemic administration in dystrophic mdx mice, conjugation of a 2'-O-methyl phosphorothioate AON to this peptide indeed improved uptake in skeletal and cardiac muscle, and resulted in higher exon skipping levels with a significant difference in heart and diaphragm. Based on these results, peptide conjugation represents an interesting strategy to enhance the therapeutic effect of exon skipping with 2'-O-methyl phosphorothioate AONs for Duchenne muscular dystrophy. PMID:24320790

  9. 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 ...

  10. 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 ...

  11. 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

  12. [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

  13. 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.

  14. Effective photoinactivation of Gram-positive and Gram-negative bacterial strains using an HIV-1 Tat peptide-porphyrin conjugate.

    PubMed

    Bourré, Ludovic; Giuntini, Francesca; Eggleston, Ian M; Mosse, Charles A; Macrobert, Alexander J; Wilson, Michael

    2010-12-01

    Given that cell-penetrating peptides (CPP) are cationic and often amphipathic, similar to membrane-active antimicrobial peptides, it may be possible to use CPP conjugation to improve the delivery of photosensitisers for antimicrobial photodynamic therapy (antimicrobial PDT). We investigated the possibility of using a Tat peptide to deliver the photosensitiser, tetrakis(phenyl)porphyrin (TPP) and kill bacteria. The Tat peptide is a positively-charged mammalian cell-penetrating peptide with potent antimicrobial activity but no haemolytic activity. Fluorescence spectroscopy revealed that the bioconjugate can bind to and/or be incorporated into all bacterial species tested. All species were susceptible to the Tat-porphyrin, with the bactericidal effect being dependent on both the concentration and the light dose. Using the highest light dose, treatment with the Tat-porphyrin achieved reductions of 6.6 log(10) and 6.37 log(10) in the viable counts of Staphylococcus aureus and Streptococcus pyogenes, and reductions of 5.74 log(10) and 6.6 log(10) in the viable counts of Pseudomonas aeruginosa and Escherichia coli. Moreover, the Tat moiety appears to confer antimicrobial properties to the conjugate, particularly for the Gram positive strains, based on the observation of dark toxicity using 1 μM of Tat-porphyrin. Finally, the conjugate induced membrane destabilization by synergistic action of the peptide and PDT, resulting in carboxyfluorescein leakage from bacterial membrane-mimicking liposomes. These findings demonstrate that the use of CPP to deliver a photosensitiser is an effective way of improving the uptake and the treatment efficacy of antimicrobial PDT. PMID:20931134

  15. A Novel Intracellular Peptide Derived from G1/S Cyclin D2 Induces Cell Death*

    PubMed Central

    de Araujo, Christiane B.; Russo, Lilian C.; Castro, Leandro M.; Forti, Fábio L.; do Monte, Elisabete R.; Rioli, Vanessa; Gozzo, Fabio C.; Colquhoun, Alison; Ferro, Emer S.

    2014-01-01

    Intracellular peptides are constantly produced by the ubiquitin-proteasome system, and many are probably functional. Here, the peptide WELVVLGKL (pep5) from G1/S-specific cyclin D2 showed a 2-fold increase during the S phase of HeLa cell cycle. pep5 (25–100 μm) induced cell death in several tumor cells only when it was fused to a cell-penetrating peptide (pep5-cpp), suggesting its intracellular function. In vivo, pep5-cpp reduced the volume of the rat C6 glioblastoma by almost 50%. The tryptophan at the N terminus of pep5 is essential for its cell death activity, and N terminus acetylation reduced the potency of pep5-cpp. WELVVL is the minimal active sequence of pep5, whereas Leu-Ala substitutions totally abolished pep5 cell death activity. Findings from the initial characterization of the cell death/signaling mechanism of pep5 include caspase 3/7 and 9 activation, inhibition of Akt2 phosphorylation, activation of p38α and -γ, and inhibition of proteasome activity. Further pharmacological analyses suggest that pep5 can trigger cell death by distinctive pathways, which can be blocked by IM-54 or a combination of necrostatin-1 and q-VD-OPh. These data further support the biological and pharmacological potential of intracellular peptides. PMID:24764300

  16. Synthetic Multivalent Antifungal Peptides Effective against Fungi

    PubMed Central

    Li, Jianguo; Nandhakumar, Muruganantham; Aung, Thet Tun; Goh, Eunice; Chang, Jamie Ya Ting; Saraswathi, Padhmanaban; Tang, Charles; Safie, Siti Radiah Binte; Lin, Lim Yih; Riezman, Howard; Lei, Zhou; Verma, Chandra S.; Beuerman, Roger W.

    2014-01-01

    Taking advantage of the cluster effect observed in multivalent peptides, this work describes antifungal activity and possible mechanism of action of tetravalent peptide (B4010) which carries 4 copies of the sequence RGRKVVRR through a branched lysine core. B4010 displayed better antifungal properties than natamycin and amphotericin B. The peptide retained significant activity in the presence of monovalent/divalent cations, trypsin and serum and tear fluid. Moreover, B4010 is non-haemolytic and non-toxic to mice by intraperitoneal (200 mg/kg) or intravenous (100 mg/kg) routes. S. cerevisiae mutant strains with altered membrane sterol structures and composition showed hyper senstivity to B4010. The peptide had no affinity for cell wall polysaccharides and caused rapid dissipation of membrane potential and release of vital ions and ATP when treated with C. albicans. We demonstrate that additives which alter the membrane potential or membrane rigidity protect C. albicans from B4010-induced lethality. Calcein release assay and molecular dynamics simulations showed that the peptide preferentially binds to mixed bilayer containing ergosterol over phophotidylcholine-cholesterol bilayers. The studies further suggested that the first arginine is important for mediating peptide-bilayer interactions. Replacing the first arginine led to a 2–4 fold decrease in antifungal activities and reduced membrane disruption properties. The combined in silico and in vitro approach should facilitate rational design of new tetravalent antifungal peptides. PMID:24498363

  17. [Plant signaling peptides. Cysteine-rich peptides].

    PubMed

    Ostrowski, Maciej; Kowalczyk, Stanisław

    2015-01-01

    Recent bioinformatic and genetic analyses of several model plant genomes have revealed the existence of a highly abundant group of signaling peptides that are defined as cysteine-rich peptides (CRPs). CRPs are usually in size between 50 and 90 amino acid residues, they are positively charged, and they contain 4-16 cysteine residues that are important for the correct conformational folding. Despite the structural differences among CRP classes, members from each class have striking similarities in their molecular properties and function. The present review presents the recent progress in research on signaling peptides from several families including: EPF/EPFL, SP11/SCR, PrsS, RALF, LURE, and some other peptides belonging to CRP group. There is convincing evidence indicating multiple roles for these CRPs as signaling molecules during the plant life cycle, ranging from stomata development and patterning, self-incompatibility, pollen tube growth and guidance, reproductive processes, and nodule formation. PMID:26281357

  18. 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

  19. 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

  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. Synthetic therapeutic peptides: science and market.

    PubMed

    Vlieghe, Patrick; Lisowski, Vincent; Martinez, Jean; Khrestchatisky, Michel

    2010-01-01

    The decreasing number of approved drugs produced by the pharmaceutical industry, which has been accompanied by increasing expenses for R&D, demands alternative approaches to increase pharmaceutical R&D productivity. This situation has contributed to a revival of interest in peptides as potential drug candidates. New synthetic strategies for limiting metabolism and alternative routes of administration have emerged in recent years and resulted in a large number of peptide-based drugs that are now being marketed. This review reports on the unexpected and considerable number of peptides that are currently available as drugs and the chemical strategies that were used to bring them into the market. As demonstrated here, peptide-based drug discovery could be a serious option for addressing new therapeutic challenges. PMID:19879957

  2. Peptide fibrils with altered stability, activity, and cell selectivity

    PubMed Central

    Chen, Long; Liang, Jun F.

    2014-01-01

    Peptides have some unique and superior features compared to proteins. However, the use of peptides as therapeutics is hampered by their low stability and cell selectivity. In this study, a new lytic peptide (CL-1, FLGALFRALSRLL) was constructed. Under the physiological condition, peptide CL-1 self-assembled into dynamically stable aggregates with fibrils-like structures. Aggregated CL-1 demonstrated dramatically altered activity and stability in comparison with single molecule CL-1 and other lytic peptides: when incubated with co-cultured bacteria and tissue cells, CL-1 aggregates killed bacteria selectively but spared co-cultured human cells; CL-1 aggregates kept intact in human serum for more than five hours. Peptide-cell interaction studies performed on lipid monolayers and live human tissue cells revealed that in comparison with monomeric CL-1, aggregated CL-1 had decreased cell affinity and membrane insertion capability on tissue cells. A dynamic process involving aggregate dissociation and rearrangement seemed to be an essential step for membrane bound CL-1 aggregates to realize its cytotoxicity to tissue cells. Our study suggests that peptide aggregation could be as important as the charge and secondary structure of a peptide in affecting peptide-cell interactions. Controlling peptide self-assembly represents a new way to increase the stability and cell selectivity of bioactive peptides for wide biomedical applications. PMID:23713839

  3. Peptide-micelle hybrids containing fasudil for targeted delivery to the pulmonary arteries and arterioles to treat pulmonary arterial hypertension.

    PubMed

    Gupta, Nilesh; Ibrahim, Hany M; Ahsan, Fakhrul

    2014-11-01

    This study investigates the respirability and efficacy of peptide-micelle hybrid nanoparticles as carriers for inhalational therapy of pulmonary arterial hypertension (PAH). CARSKNKDC (CAR), a cell-penetrating and lung-homing peptide, conjugated polyethylene glycol-distearoyl-phosphoethanolamine micelles containing fasudil, an investigational anti-PAH drug, were prepared by solvent evaporation method and characterized for various physicochemical properties. The pharmacokinetics and pharmacological efficacy of hybrid particles containing fasudil were evaluated in healthy rats and monocrotaline-induced PAH rats. CAR micelles containing fasudil had an entrapment efficiency of approximately 58%, showed controlled release of the drug, and were monodispersed with an average size of approximately 14 nm. Nuclear magnetic resonance scan confirmed the drug's presence in the core of peptide-micelle hybrid particles. Compared with plain micelles, CAR peptide increased the cellular uptake by approximately 1.7-fold and extended the drug half-life by approximately fivefold. The formulations were more prone to accumulate in the pulmonary vasculature than in the peripheral blood, which is evident from the ratio of the extent of reduction of pulmonary and systemic arterial pressures. On the whole, this study demonstrates that peptide-polymer hybrid micelles can serve as inhalational carriers for PAH therapy. PMID:25266507

  4. Statistical Analysis of Peptide-Induced Graded and All-or-None Fluxes in Giant Vesicles

    PubMed Central

    Wheaten, Sterling A.; Lakshmanan, Aruna; Almeida, Paulo F.

    2013-01-01

    Antimicrobial, cytolytic, and cell-penetrating peptides induce pores or perturbations in phospholipid membranes that result in fluxes of dyes into or out of lipid vesicles. Here we examine the fluxes induced by four of these membrane-active peptides in giant unilamellar vesicles. The type of flux is determined from the modality of the distributions of vesicles as a function of their dye content using the statistical Hartigan dip test. Graded and all-or-none fluxes correspond to unimodal and bimodal distributions, respectively. To understand how these distributions arise, we perform Monte Carlo simulations of peptide-induced dye flux into vesicles using a very simple model. The modality of the distributions depends on the rate constants of pore opening and closing, and dye flux. If the rate constants of pore opening and closing are both much smaller than that of dye flux through the pore, all-or-none influx occurs. However, if one of them, especially the rate constant for pore opening, increases significantly relative to the flux rate constant, the process becomes graded. In the experiments, we find that the flux type is the same in giant and large vesicles, for all peptides except one. But this one exception indicates that the flux type cannot be used to unambiguously predict the mechanism of membrane permeabilization by the peptides. PMID:23870264

  5. 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. PMID:23391494

  6. In Vitro Efficient Transfection by CM18-Tat11 Hybrid Peptide: A New Tool for Gene-Delivery Applications

    PubMed Central

    Salomone, Fabrizio; Cardarelli, Francesco; Signore, Giovanni; Boccardi, Claudia; Beltram, Fabio

    2013-01-01

    Cell penetrating peptides (CPPs) are actively researched as non-viral molecular carriers for the controlled delivery of nucleic acids into cells, but widespread application is severely hampered by their trapping into endosomes. Here we show that the recently introduced endosomolytic CM18-Tat11 hybrid peptide (KWKLFKKIGAVLKVLTTG-YGRKKRRQRRR, residues 1-7 of Cecropin-A, 2-12 of Melittin, and 47-57 of HIV-1 Tat protein) can be exploited to obtain a self-assembled peptide-DNA vector which maintains the CM18-Tat11 ability to enter cells and destabilize vesicular membranes, concomitantly yielding high DNA transfection efficiency with no detectable cytotoxic effects. Different peptide-DNA stoichiometric ratios were tested to optimize vector size, charge, and stability characteristics. The transfection efficiency of selected candidates is quantitatively investigated by the luciferase-reporter assay. Vector intracellular trafficking is monitored in real time and in live cells by confocal microscopy. In particular, fluorescence resonant energy transfer (FRET) between suitably-labeled peptide and DNA modules was exploited to monitor complex disassembly during endocytosis, and this process is correlated to transfection timing and efficiency. We argue that these results can open the way to the rational design and application of CM18-Tat11–based systems for gene-delivery purposes. PMID:23922923

  7. Antimicrobial Peptides in Human Sepsis.

    PubMed

    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. PMID

  8. 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

  9. 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

  10. MODELING OF THE ENDOSOMOLYTIC ACTIVITY OF HA2-TAT PEPTIDES WITH RED BLOOD CELLS AND GHOSTS†

    PubMed Central

    Lee, Ya-Jung; Johnson, Gregory; Pellois, Jean-Philippe

    2011-01-01

    HA2-TAT is a peptide-based delivery agent that combines the pH-sensitive HA2 fusion peptide from Influenza and the cell-penetrating peptide TAT from HIV. This chimeric peptide is engineered to induce the cellular uptake of macromolecules into endosomes via the TAT moiety and to respond to the acidifying lumen of endosomes to cause membrane leakage and release of macromolecules into cells via the HA2 moiety. The question of how HA2 and TAT affect the properties of one another remains, however, unanswered and the behavior of the peptide inside endosomes is mostly uncharacterized. To address these issues, the binding and membrane leakage activity of a glutamic acid-enriched analogue E5-TAT was assessed with red blood cells and giant unilamellar vesicles as membrane models for endosomes. Hemolysis and microscopy assays reveal that E5-TAT binds to membranes in a pH-dependent manner and causes membrane leakage by inducing the formation of pores through which macromolecules can escape. The TAT moiety contributes to this activity by causing a shift in the pH response of E5 and by binding to negatively charged phospholipids. On the other hand, TAT binding to glycosaminoglycans reduces the lytic activity of E5-TAT. Addition of TAT to the C-terminus of E5 can therefore either increase or inhibit the activity of E5 depending on the cellular components present at the membrane. Taken together, these results suggest a model for the endosomolytic activity of the peptide and provide the basis for the molecular design of future delivery agents. PMID:20704453

  11. Critical Evaluation and Compilation of Physicochemical Determinants and Membrane Interactions of MMGP1 Antifungal Peptide.

    PubMed

    Pushpanathan, Muthuirulan; Pooja, Sharma; Gunasekaran, Paramasamy; Rajendhran, Jeyaprakash

    2016-05-01

    A growing issue of pathogen resistance to antibiotics has fostered the development of innovative approaches for novel drug development. Here, we report the physicochemical and biological properties of an antifungal peptide, MMGP1, based on computational analysis. Computation of physicochemical properties has revealed that the natural biological activities of MMGP1 are coordinated by its intrinsic properties such as net positive charge (+5.04), amphipathicity, high hydrophobicity, low hydrophobic moment, and higher isoelectric point (11.915). Prediction of aggregation hot spots in MMGP1 had revealed the presence of potentially aggregation-prone segments that can nucleate in vivo aggregation (on the membrane), whereas no aggregating regions were predicted for in vitro aggregation (in solutions) of MMGP1. This ability of MMGP1 to form oligomeric aggregates on membrane further substantiates its direct-cell penetrating potency. Monte Carlo simulation of the interactions of MMGP1 in the aqueous phase and different membrane environments revealed that increasing the proportion of acidic lipids on membrane had led to increase in the peptide helicity. Furthermore, the peptide adopts energetically favorable transmembrane configuration, by inserting peptide loop and helix termini into the membrane containing >60% of anionic lipids. The charged lipid-based insertion of MMGP1 into membrane might be responsible for the selectivity of peptide toward fungal cells. Additionally, MMGP1 possessed DNA-binding property. Computational docking has identified DNA-binding residues (TRP3, SER4, MET7, ARG8, PHE10, ALA11, GLY20, THR21, ARG22, MET23, TRP34, and LYS36) in MMGP1 crucial for its DNA-binding property. Furthermore, computational mutation analysis revealed that aromatic amino acids are crucial for in vivo aggregation, membrane insertion, and DNA-binding property of MMGP1. These data provide new insight into the molecular determinants of MMGP1 antifungal activity and also serves as

  12. 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

  13. Antihypertensive peptides from curd

    PubMed Central

    Dabarera, Melani Chathurika; Athiththan, Lohini V.; Perera, Rasika P.

    2015-01-01

    Introduction: Curd (Dadhi) peptides reduce hypertension by inhibiting angiotensin converting enzyme (ACE) and serum cholesterol. Peptides vary with bacterial species and milk type used during fermentation. Aim: To isolate and assay the antihypertensive peptides, before and after digestion, in two commercially available curd brands in Sri Lanka. Materials and Methods: Whey (Dadhi Mastu) separated by high-speed centrifugation was isolated using reverse-phase-high- performance liquid chromatography (HPLC). Eluted fractions were analyzed for ACE inhibitory activity using modified Cushman and Cheung method. Curd samples were subjected to enzymatic digestion with pepsin, trypsin, and carboxypeptidase-A at their optimum pH and temperature. Peptides isolated using reverse-phase-HPLC was assayed for ACE inhibitory activity. Results: Whey peptides of both brands gave similar patterns (seven major and five minor peaks) in HPLC elution profile. Smaller peptides concentration was higher in brand 1 and penta-octapeptides in brand 2. Pentapeptide had the highest ACE inhibitory activity (brand 2–90% and brand 1–73%). After digestion, di and tri peptides with similar inhibitory patterns were obtained in both which were higher than before digestion. Thirteen fractions were obtained, where nine fractions showed more than 70% inhibition in both brands with 96% ACE inhibition for a di-peptide. Conclusion: Curd has ACE inhibitory peptides and activity increases after digestion. PMID:27011726

  14. 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

  15. Polycyclic peptide therapeutics.

    PubMed

    Baeriswyl, Vanessa; Heinis, Christian

    2013-03-01

    Owing to their excellent binding properties, high stability, and low off-target toxicity, polycyclic peptides are an attractive molecule format for the development of therapeutics. Currently, only a handful of polycyclic peptides are used in the clinic; examples include the antibiotic vancomycin, the anticancer drugs actinomycin D and romidepsin, and the analgesic agent ziconotide. All clinically used polycyclic peptide drugs are derived from natural sources, such as soil bacteria in the case of vancomycin, actinomycin D and romidepsin, or the venom of a fish-hunting coil snail in the case of ziconotide. Unfortunately, nature provides peptide macrocyclic ligands for only a small fraction of therapeutic targets. For the generation of ligands of targets of choice, researchers have inserted artificial binding sites into natural polycyclic peptide scaffolds, such as cystine knot proteins, using rational design or directed evolution approaches. More recently, large combinatorial libraries of genetically encoded bicyclic peptides have been generated de novo and screened by phage display. In this Minireview, the properties of existing polycyclic peptide drugs are discussed and related to their interesting molecular architectures. Furthermore, technologies that allow the development of unnatural polycyclic peptide ligands are discussed. Recent application of these technologies has generated promising results, suggesting that polycyclic peptide therapeutics could potentially be developed for a broad range of diseases. PMID:23355488

  16. Peptide folding simulations.

    PubMed

    Gnanakaran, S; Nymeyer, Hugh; Portman, John; Sanbonmatsu, Kevin Y; García, Angel E

    2003-04-01

    Developments in the design of small peptides that mimic proteins in complexity, recent advances in nanosecond time-resolved spectroscopy methods to study peptides and the development of modern, highly parallel simulation algorithms have come together to give us a detailed picture of peptide folding dynamics. Two newly implemented simulation techniques, parallel replica dynamics and replica exchange molecular dynamics, can now describe directly from simulations the kinetics and thermodynamics of peptide formation, respectively. Given these developments, the simulation community now has the tools to verify and validate simulation protocols and models (forcefields). PMID:12727509

  17. Strategies for identifying synthetic peptides to act as inhibitors of NADPH oxidases, or "all that you did and did not want to know about Nox inhibitory peptides".

    PubMed

    Dahan, Iris; Pick, Edgar

    2012-07-01

    Phagocytes utilize reactive oxygen species (ROS) to kill pathogenic microorganisms. The source of ROS is an enzymatic complex (the NADPH oxidase), comprising a membrane-associated heterodimer (flavocytochrome b (558)), consisting of subunits Nox2 and p22(phox), and four cytosolic components (p47(phox), p67(phox), p40(phox), and Rac). The primordial ROS (superoxide) is generated by the reduction of molecular oxygen by NADPH via redox centers located on Nox2. This process is activated by the translocation of the cytosolic components to the membrane and their assembly with Nox2. Membrane translocation is preceded by interactions among cytosolic components. A number of proteins structurally and functionally related to Nox2 have been discovered in many cells (the Nox family) and these have pleiotropic functions related to the production of ROS. An intense search is underway to design therapeutic means to modulate Nox-dependent overproduction of ROS, associated with diseases. Among drug candidates, a central position is held by synthetic peptides reflecting domains in oxidase components involved in NADPH oxidase assembly. Peptides, corresponding to domains in Nox2, p22(phox), p47(phox), and Rac, found to be oxidase activation inhibitory in vitro, are reviewed. Usually, peptides are inhibitory only when added preceding assembly of the complex. Although competition with intact components seems most likely, less obvious mechanisms are, sometimes, at work. The use of peptides as inhibitory drugs in vivo requires the development of methods to assure cell penetration, resistance to degradation, and avoidance of toxicity, and modest successes have been achieved. The greatest challenge remains the discovery of peptide inhibitors acting specifically on individual Nox isoforms. PMID:22562603

  18. Rationally Designed Interfacial Peptides Are Efficient In Vitro Inhibitors of HIV-1 Capsid Assembly with Antiviral Activity

    PubMed Central

    Bocanegra, Rebeca; Nevot, María; Doménech, Rosa; López, Inmaculada; Abián, Olga; Rodríguez-Huete, Alicia; Cavasotto, Claudio N.; Velázquez-Campoy, Adrián; Gómez, Javier; Martínez, Miguel Ángel; Neira, José Luis; Mateu, Mauricio G.

    2011-01-01

    Virus capsid assembly constitutes an attractive target for the development of antiviral therapies; a few experimental inhibitors of this process for HIV-1 and other viruses have been identified by screening compounds or by selection from chemical libraries. As a different, novel approach we have undertaken the rational design of peptides that could act as competitive assembly inhibitors by mimicking capsid structural elements involved in intersubunit interfaces. Several discrete interfaces involved in formation of the mature HIV-1 capsid through polymerization of the capsid protein CA were targeted. We had previously designed a peptide, CAC1, that represents CA helix 9 (a major part of the dimerization interface) and binds the CA C-terminal domain in solution. Here we have mapped the binding site of CAC1, and shown that it substantially overlaps with the CA dimerization interface. We have also rationally modified CAC1 to increase its solubility and CA-binding affinity, and designed four additional peptides that represent CA helical segments involved in other CA interfaces. We found that peptides CAC1, its derivative CAC1M, and H8 (representing CA helix 8) were able to efficiently inhibit the in vitro assembly of the mature HIV-1 capsid. Cocktails of several peptides, including CAC1 or CAC1M plus H8 or CAI (a previously discovered inhibitor of CA polymerization), or CAC1M+H8+CAI, also abolished capsid assembly, even when every peptide was used at lower, sub-inhibitory doses. To provide a preliminary proof that these designed capsid assembly inhibitors could eventually serve as lead compounds for development of anti-HIV-1 agents, they were transported into cultured cells using a cell-penetrating peptide, and tested for antiviral activity. Peptide cocktails that drastically inhibited capsid assembly in vitro were also able to efficiently inhibit HIV-1 infection ex vivo. This study validates a novel, entirely rational approach for the design of capsid assembly

  19. α-Helix peptides designed from EBV-gH protein display higher antigenicity and induction of monocyte apoptosis than the native peptide

    PubMed Central

    Melo-Cardenas, Johanna; Guevara, Tatiana; Echeverria, Ignacia; Rodriguez, Isabel C.; Vanegas, Magnolia; Amzel, Mario; Patarroyo, Manuel E.

    2010-01-01

    We tested the hypothesis that stabilizing α-helix of Epstein–Barr virus gH-derived peptide 11438 used for binding human cells will increase its biological activity. Non-stable α-helix of peptide 11438 was unfolded in an entropy-driven process, despite the opposing effect of the enthalpy factor. Adding and/or changing amino acids in peptide 11438 allowed the designing of peptides 33207, 33208 and 33210; peptides 33208 and 33210 displayed higher helical content due to a decreased unfolding entropy change as was determined by AGADIR, molecular dynamics and circular dichroism analysis. Peptides 33207, 33208 and 33210 inhibited EBV invasion of peripheral blood mononuclear cells and displayed epitopes more similar to native protein than peptide 11438; these peptides could be useful for detecting antibodies induced by native gH protein since they displayed high reactivity with anti-EBV antibodies. Anti-peptide 33207 antibodies showed higher reactivity with EBV than anti-peptide 11438 antibodies being useful for inducing antibodies against EBV. Anti-peptide 33210 antibodies inhibit EBV invasion of epithelial cells better than anti-peptide 11438 antibodies. Peptide 33210 bound to normal T lymphocytes and Raji cells stronger than peptide 11438 and also induced apoptosis of monocytes and Raji cells but not of normal T cells in a similar way to EBV-gH. Peptide 33210 inhibited the monocytes’ development toward dendritic cells better than EBV and peptide 11438. In conclusion, stabilizing the α-helix in peptides 33208 and 33210 designed from peptide 11438 increased the antigenicity and the ability of the antibodies induced by peptides of inhibiting EBV invasion of host cells. PMID:20473772

  20. 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 produced by the body and insulin injected ...

  1. Laminin-111-derived peptides and cancer

    PubMed Central

    Kikkawa, Yamato; Hozumi, Kentaro; Katagiri, Fumihiko; Nomizu, Motoyoshi; Kleinman, Hynda K.; Koblinski, Jennifer E.

    2013-01-01

    Laminin-111 is a large trimeric basement membrane glycoprotein with many active sites. In particular, four peptides active in tumor malignancy studies have been identified in laminin-111 using a systematic peptide screening method followed by various assays. Two of the peptides (IKVAV and AG73) are found on the α1 chain, one (YIGSR) of the β1 chain and one (C16) on the γ1 chain. The four peptides have distinct activities and receptors. Since three of the peptides (IKVAV, AG73 and C16) strongly promote tumor growth, this may explain the potent effects laminin-111 has on malignant cells. The peptide, YIGSR, decreases tumor growth and experimental metastasis via a 32/67 kD receptor while IKVAV increases tumor growth, angiogenesis and protease activity via integrin receptors. AG73 increases tumor growth and metastases via syndecan receptors. C16 increases tumor growth and angiogenesis via integrins. Identification of such sites on laminin-111 will have use in defining strategies to develop therapeutics for cancer. PMID:23263633

  2. 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.

  3. 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

  4. 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

  5. Peptide nanoparticle delivery of charge-neutral splice-switching morpholino oligonucleotides.

    PubMed

    Järver, Peter; Zaghloul, Eman M; Arzumanov, Andrey A; Saleh, Amer F; McClorey, Graham; Hammond, Suzan M; Hällbrink, Mattias; Langel, Ülo; Smith, C I Edvard; Wood, Matthew J A; Gait, Michael J; El Andaloussi, Samir

    2015-04-01

    Oligonucleotide analogs have provided novel therapeutics targeting various disorders. However, their poor cellular uptake remains a major obstacle for their clinical development. Negatively charged oligonucleotides, such as 2'-O-Methyl RNA and locked nucleic acids have in recent years been delivered successfully into cells through complex formation with cationic polymers, peptides, liposomes, or similar nanoparticle delivery systems. However, due to the lack of electrostatic interactions, this promising delivery method has been unsuccessful to date using charge-neutral oligonucleotide analogs. We show here that lipid-functionalized cell-penetrating peptides can be efficiently exploited for cellular transfection of the charge-neutral oligonucleotide analog phosphorodiamidate morpholino. The lipopeptides form complexes with splice-switching phosphorodiamidate morpholino oligonucleotide and can be delivered into clinically relevant cell lines that are otherwise difficult to transfect while retaining biological activity. To our knowledge, this is the first study to show delivery through complex formation of biologically active charge-neutral oligonucleotides by cationic peptides. PMID:25594433

  6. 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.

  7. 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.

  8. An anti-apoptotic peptide improves survival in lethal total body irradiation

    PubMed Central

    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.

    2009-01-01

    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 μM) 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. PMID:19303399

  9. 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

  10. 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...

  11. Bovine hemoglobin: an attractive source of antibacterial peptides.

    PubMed

    Nedjar-Arroume, Naïma; Dubois-Delval, Véronique; Adje, Estelle Yaba; Traisnel, Jonathan; Krier, François; Mary, Patrice; Kouach, Mostafa; Briand, Gilbert; Guillochon, Didier

    2008-06-01

    A peptic hemoglobin hydrolysate was fractioned by a semi-preparative reversed-phase HPLC and some fractions have an antibacterial activity against four bacteria strains: Micrococcus luteus A270, Listeria innocua, Escherichia coli and Salmonella enteritidis. These fractions were analyzed by ESI/MS and ESI/MS/MS, in order to characterize the peptides in these fractions. Each fraction contains at least three peptides and some fractions contain five peptides. All these fractions were purified several times by HPLC to obtain pure peptides. Thirty antibacterial peptides were identified. From the isolated antibacterial peptides, 24 peptides were derived from the alpha chains of hemoglobin and 6 peptides were derived from the beta chains of hemoglobin. The lowest concentration of these peptides (minimum inhibitory concentration (MIC)) necessary to completely inhibit the growth of four bacteria strain was determined. The cell population of all of the tested bacteria species decreased by at least 97% after a 24-h incubation with any of the peptides at the minimum inhibitory concentration. PMID:18342399

  12. 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

  13. Does clozapine decrease smoking?

    PubMed

    de Leon, Jose; Diaz, Francisco J; Josiassen, Richard C; Cooper, Thomas B; Simpson, George M

    2005-06-01

    McEvoy et al.'s study in 1999, which used cotinine levels but had limited power, suggested that clozapine treatment may be associated with a mild smoking decrease (particularly when plasma clozapine levels are > 150 ng/ml). Some naturalistic studies also suggest that clozapine treatment may be associated with a mild smoking decrease. The present study included 38 schizophrenic daily smokers from a double-blind clozapine trial. Five analyses were tested for significant decreases in plasma cotinine levels from a haloperidol baseline to: (1) the end of clozapine trials regarding clozapine doses (100, 300 or 600 mg/day), (2) the end of the clozapine trial where the highest plasma clozapine level was achieved, (3) the end of the clozapine trial where a clozapine level in the 150-450 ng/ml range was achieved, (4) the end of the first clozapine trial regardless of clozapine dose, and (5) the end of the last clozapine trial in the study. The first and straightforward analysis by dose showed no clozapine effects on smoking. The second and the third analyses (an attempt to mimic the design by McEvoy et al. [McEvoy, J.P., Freudenreich, O., Wilson, W.H., 1999. Smoking and therapeutic response to clozapine in patients with schizophrenia. Biol. Psychiat. 46, 125-129.]) also indicated that there was not a significant effect of clozapine on smoking. The fourth and five analyses were also negative. None of the five analyses in our clozapine trial demonstrated that clozapine had major effects on smoking. This study cannot rule out that in some subjects, clozapine treatment may be associated with a small decrease in smoking. New prospective longitudinal studies using repeated cotinine and clozapine levels are needed to explore whether clozapine may reduce smoking in some patients. PMID:15951089

  14. Functional evaluation of fluorescein-labeled derivatives of a peptide inhibitor of the EGF receptor dimerization.

    PubMed

    Toyama, Kei; Mizuguchi, Takaaki; Nomura, Wataru; Tamamura, Hirokazu

    2016-08-15

    A cyclic decapeptide (1, ), which acts on the extracellular region of the EGF receptor, preventing it from dimerizing, has been developed. Peptide 2, which was labeled with fluorescein at the N-terminus of peptide 1, was synthesized based on structure-activity relationship studies. Peptide 2 essentially retained the inhibitory activity of peptide 1 against the receptor autophosphorylation. Confocal microscopy studies revealed that in carcinoma cells, the fluorescence of peptide 2 was localized inside some vesicles. Treatment of intact cells by peptide 1 in combination with peptide 2 decreased the fluorescence intensity significantly compared to treatment with only peptide 2. These results indicate that peptide 2 competes with peptide 1 for binding to the cellular surface. Six derivatives of peptide 2, in which constituent amino acids, with the exception of two cysteines and proline were randomized, were synthesized and used to treat the cells. Peptides 6 and 9 showed the highest fluorescence intensity in cells. From the results of the EGF receptor autophosphorylation assay, these two derivatives were proven to have higher inhibitory activity than peptide 2, which would therefore be a useful delivery peptide and fluorescent probe to find new inhibitors against the EGF receptor. Peptides 6 and 9 are promising leads for EGF receptor inhibitors. PMID:27283787

  15. 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.

  16. Effect of β-sheet propensity on peptide aggregation

    NASA Astrophysics Data System (ADS)

    Bellesia, Giovanni; Shea, Joan-Emma

    2009-04-01

    The effect of β-sheet propensity on the structural features of peptide aggregates was investigated using an off-lattice coarse-grained peptide model. A phase diagram as a function of temperature and β-sheet propensity reveals a diverse family of supramolecular assemblies. Highly rigid peptides (peptides with high β-sheet propensity) are seen to assemble predominantly into fibrillar structures. Increasing the flexibility of the peptide (reducing β-sheet propensity) leads to a variety of structures, including fibrils, β-barrel structures, and amorphous aggregates. Nonfibrillar entities have been suggested as primary causative agents in amyloid diseases and our simulations indicate that mutations that decrease β-sheet propensity will decrease fibril formation and favor the formation of such toxic oligomers. Parallels between β-sheet aggregates and nematic liquid crystals are discussed.

  17. 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

  18. A Huntingtin-based peptide inhibitor of caspase-6 provides protection from mutant Huntingtin-induced motor and behavioral deficits

    PubMed Central

    Aharony, Israel; Ehrnhoefer, Dagmar E.; Shruster, Adi; Qiu, Xiaofan; Franciosi, Sonia; Hayden, Michael R.; Offen, Daniel

    2015-01-01

    Over the past decade, increasing evidence has implied a significant connection between caspase-6 activity and the pathogenesis of Huntington's disease (HD). Consequently, inhibiting caspase-6 activity was suggested as a promising therapeutic strategy to reduce mutant Huntingtin toxicity, and to provide protection from mutant Huntingtin-induced motor and behavioral deficits. Here, we describe a novel caspase-6 inhibitor peptide based on the huntingtin caspase-6 cleavage site, fused with a cell-penetrating sequence. The peptide reduces mutant Huntingtin proteolysis by caspase-6, and protects cells from mutant Huntingtin toxicity. Continuous subcutaneous administration of the peptide protected pre-symptomatic BACHD mice from motor deficits and behavioral abnormalities. Moreover, administration of the peptide in an advanced disease state resulted in the partial recovery of motor performance, and an alleviation of depression-related behavior and cognitive deficits. Our findings reveal the potential of substrate-based caspase inhibition as a therapeutic strategy, and present a promising agent for the treatment of HD. PMID:25616965

  19. 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

  20. 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

  1. Developing a Dissociative Nanocontainer for Peptide Drug Delivery.

    PubMed

    Kelly, Patrick; Anand, Prachi; Uvaydov, Alexander; Chakravartula, Srinivas; Sherpa, Chhime; Pires, Elena; O'Neil, Alison; Douglas, Trevor; Holford, Mandë

    2015-10-01

    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

  2. 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

  3. 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

  4. Synthetic antimicrobial peptide design.

    PubMed

    Powell, W A; Catranis, C M; Maynard, C A

    1995-01-01

    To guide the design of potential plant pathogen-resistance genes, synthetic variants of naturally occurring antimicrobial gene products were evaluated. Five 20-amino acid (ESF1, ESF4, ESF5, ESF6, ESF13), one 18-amino acid (ESF12), and one 17-amino acid (ESF17) amphipathic peptide sequences were designed, synthesized, and tested with in vitro bioassays. Positive charges on the hydrophilic side of the peptide were shown to be essential for antifungal activity, yet the number of positive charges could be varied with little or no change in activity. The size could be reduced to 18 amino acids, but at 17 amino acids a significant reduction in activity was observed. ESF1, 5, 6, and 12 peptides were inhibitory to the germination of conidia from Cryphonectria parasitica, Fusarium oxysporum f. sp. lycopersici, and Septoria musiva but did not inhibit the germination of pollen from Castanea mollissima and Salix lucida. ESF12 also had no effect on the germination of Malus sylvestris and Lycopersicon esculentum pollen, but inhibited the growth of the bacteria Agrobacterium tumefaciens, Erwinia amylovora, and Pseudomonas syringae. The minimal inhibitory concentrations of the active ESF peptides were similar to those of the naturally occurring control peptides, magainin II and cecropin B. The significant differential in sensitivity between the microbes and plant cells indicated that the active ESF peptides are potentially useful models for designing plant pathogen-resistance genes. PMID:7579625

  5. Antimitotic peptides and depsipeptides.

    PubMed

    Hamel, Ernest; Covell, David G

    2002-01-01

    Tubulin is the target for an ever increasing number of unusual peptides and depsipeptides that were originally isolated from a wide variety of organisms. Since tubulin is the major component of cellular microtubules, which maintain cell shape in interphase and form the mitotic spindle, most of these compounds are highly toxic to mammalian cells. These peptides and depsipeptides disrupt cellular microtubules and prevent formation of a functional spindle, resulting in the accumulation of cultured cells in the G2/M phase of the cell cycle through specific inhibition of mitosis. At the biochemical level, the compounds all inhibit the assembly of tubulin into polymer and, in the cases where it has been studied, strongly suppress microtubule dynamics at low concentrations. In most cases the peptides and depsipeptides inhibit the binding of vinblastine and vincristine to tubulin in a noncompetitive manner, inhibit tubulin-dependent GTP hydrolysis, and interfere with nucleotide turnover at the exchangeable GTP site on beta-tubulin. Most of the peptides and depsipeptides induce tubulin to form oligomers of aberrant morphology, including tubulin rings that vary in diameter depending on the (depsi) peptide under study. The purpose of this review is to give an overview of the cellular, biochemical, in vivo, and SAR aspects of this group of compounds. We also summarize initial efforts by computer modeling to decipher a pharmacophore among the diverse structures of these peptides and depsipeptides. PMID:12678750

  6. 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

  7. Development of JNK2-Selective Peptide Inhibitors that Inhibit Breast Cancer Cell Migration

    PubMed Central

    Kaoud, Tamer S.; Mitra, Shreya; Lee, Sunbae; Taliaferro, Juliana; Cantrell, Michael; Linse, Klaus D.; Van Den Berg, Carla L.; Dalby, Kevin N.

    2012-01-01

    Despite their lack of selectivity towards c-Jun N-terminal kinase (JNK) isoforms, peptides derived from the JIP (JNK Interacting Protein) scaffolds linked to the cell-penetrating peptide TAT are widely used to investigate JNK-mediated signaling events. To engineer an isoform-selective peptide inhibitor, several JIP-based peptide sequences were designed and tested. A JIP sequence connected through a flexible linker to either the N-terminus of an inverted TAT sequence (JIP10-Δ-TATi), or to a poly-arginine sequence (JIP10-Δ-R9) enabled the potent inhibition of JNK2 (IC50~90 nM) and exhibited 10-fold selectivity for JNK2 over JNK1 and JNK3. Examination of both peptides in HEK293 cells revealed a potent ability to inhibit the induction of both JNK activation and c-Jun phosphorylation in cells treated with anisomycin. Notably, Western blot analysis indicates that only a fraction of total JNK must be activated to elicit robust c-Jun phosphorylation. To examine the potential of each peptide to selectively modulate JNK2 signaling in vivo, their ability to inhibit the migration of Polyoma Middle-T Antigen Mammary Tumor (PyVMT) cells was assessed. PyVMTjnk2-/- cells exhibit a lower migration potential compared to PyVMTjnk2+/+ cells, and this migration potential is restored through the over-expression of GFP-JNK2α. Both JIP10-Δ-TATi and JIP10-Δ-R9 inhibit the migration of PyVMTjnk2+/+ cells and PyVMTjnk2-/- cells expressing GFP-JNK2α. However, neither peptide inhibits the migration of PyVMTjnk2-/- cells. A control form of JIP10-Δ-TATi containing a single leucine to arginine mutation lacks ability to inhibit JNK2 in vitro cell-free and cell-based assays and does not inhibit the migration of PyVMTjnk2+/+ cells. Together, these data suggest that JIP10-Δ-TATi and JIP10-Δ-R9 inhibit the migration of PyVMT cells through the selective inhibition of JNK2. Finally, the mechanism of inhibition of a D-retro-inverso JIP peptide, previously reported to inhibit JNK, was examined

  8. Anti-Legionella activity of staphylococcal hemolytic peptides.

    PubMed

    Marchand, A; Verdon, J; Lacombe, C; Crapart, S; Héchard, Y; Berjeaud, J M

    2011-05-01

    A collection of various Staphylococci was screened for their anti-Legionella activity. Nine of the tested strains were found to secrete anti-Legionella compounds. The culture supernatants of the strains, described in the literature to produce hemolytic peptides, were successfully submitted to a two step purification process. All the purified compounds, except one, corresponded to previously described hemolytic peptides and were not known for their anti-Legionella activity. By comparison of the minimal inhibitory concentrations, minimal permeabilization concentrations, decrease in the number of cultivable bacteria, hemolytic activity and selectivity, the purified peptides could be separated in two groups. First group, with warnericin RK as a leader, corresponds to the more hemolytic and bactericidal peptides. The peptides of the second group, represented by the PSMα from Staphylococcus epidermidis, appeared bacteriostatic and poorly hemolytic. PMID:21291938

  9. Dramatic rigidification of a peptide-decorated lamellar phase

    NASA Astrophysics Data System (ADS)

    Tsapis, N.; Urbach, W.; Ober, R.

    2001-04-01

    We have performed small-angle x-ray scattering on a lamellar (Lα) phase made of a nonionic surfactant (C12E4), decane, and water, after the insertion of a triblock peptide. The hydrophilic part of the peptide is rigid and organized in an α helix in the presence of membranes. Surface tension measurements and spectrofluorometry show that the peptide lies on the membrane surface. The Caillé parameter η and the smectic compressibility modulus B¯ decrease with peptide concentration, whereas the membrane bending rigidity κ increases threefold for mole ratio of peptide to surfactant as low as 5.2×10-4. The published models for rigid inclusions in membranes cannot account for this dramatic rigidification. However, experimental results are well fitted by a Heuristic renormalization of the membrane thickness.

  10. Peptides of the constant region of antibodies display fungicidal activity.

    PubMed

    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

  11. Electromembrane extraction of peptides.

    PubMed

    Balchen, Marte; Reubsaet, Léon; Pedersen-Bjergaard, Stig

    2008-06-20

    Rapid extraction of eight different peptides using electromembrane extraction (EME) was demonstrated for the first time. During an extraction time of 5 min, the model peptides migrated from a 500 microL aqueous acidic sample solution, through a thin supported liquid membrane (SLM) of an organic liquid sustained in the pores in the wall of a porous hollow fiber, and into a 25 microL aqueous acidic acceptor solution present inside the lumen of the hollow fiber. The driving force of the extraction was a 50 V potential sustained across the SLM, with the positive electrode in the sample and the negative electrode in the acceptor solution. The nature and the composition of the SLM were highly important for the EME process, and a mixture of 1-octanol and 15% di(2-ethylhexyl) phosphate was found to work properly. Using 1mM HCl as background electrolyte in the sample and 100 mM HCl in the acceptor solution, and agitation at 1050 rpm, enrichment up to 11 times was achieved. Recoveries were found to be dependent on the structure of the peptide, indicating that the polarity and the number of ionized groups were important parameters affecting the extraction efficiency. The experimental findings suggested that electromembrane extraction of peptides is possible and may be a valuable tool for future extraction of peptides. PMID:18479691

  12. 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

  13. 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

  14. Natriuretic peptide-guided management in heart failure.

    PubMed

    Chioncel, Ovidiu; Collins, Sean P; Greene, Stephen J; Ambrosy, Andrew P; Vaduganathan, Muthiah; Macarie, Cezar; Butler, Javed; Gheorghiade, Mihai

    2016-08-01

    Heart failure is a clinical syndrome that manifests from various cardiac and noncardiac abnormalities. Accordingly, rapid and readily accessible methods for diagnosis and risk stratification are invaluable for providing clinical care, deciding allocation of scare resources, and designing selection criteria for clinical trials. Natriuretic peptides represent one of the most important diagnostic and prognostic tools available for the care of heart failure patients. Natriuretic peptide testing has the distinct advantage of objectivity, reproducibility, and widespread availability.The concept of tailoring heart failure management to achieve a target value of natriuretic peptides has been tested in various clinical trials and may be considered as an effective method for longitudinal biomonitoring and guiding escalation of heart failure therapies with overall favorable results.Although heart failure trials support efficacy and safety of natriuretic peptide-guided therapy as compared with usual care, the relationship between natriuretic peptide trajectory and clinical benefit has not been uniform across the trials, and certain subgroups have not shown robust benefit. Furthermore, the precise natriuretic peptide value ranges and time intervals of testing are still under investigation. If natriuretic peptides fail to decrease following intensification of therapy, further work is needed to clarify the optimal pharmacologic approach. Despite decreasing natriuretic peptide levels, some patients may present with other high-risk features (e.g. elevated troponin). A multimarker panel investigating multiple pathological processes will likely be an optimal alternative, but this will require prospective validation.Future research will be needed to clarify the type and magnitude of the target natriuretic peptide therapeutic response, as well as the duration of natriuretic peptide-guided therapy in heart failure patients. PMID:27110656

  15. Sustained Dystrophin Expression Induced by Peptide-conjugated Morpholino Oligomers in the Muscles of mdx Mice

    PubMed Central

    Jearawiriyapaisarn, Natee; Moulton, Hong M; Buckley, Brian; Roberts, Jennifer; Sazani, Peter; Fucha