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Sample records for hoxd10 gene delivery

  1. HoxD10 gene delivery using adenovirus/adeno-associate hybrid virus inhibits the proliferation and tumorigenicity of GH4 pituitary lactotrope tumor cells

    SciTech Connect

    Cho, Mi Ae; Yashar, Parham; Kim, Suk Kyoung; Noh, Taewoong; Gillam, Mary P.; Lee, Eun Jig Jameson, J. Larry

    2008-07-04

    Prolactinoma is one of the most common types of pituitary adenoma. It has been reported that a variety of growth factors and cytokines regulating cell growth and angiogenesis play an important role in the growth of prolactinoma. HoxD10 has been shown to impair endothelial cell migration, block angiogenesis, and maintain a differentiated phenotype of cells. We investigated whether HoxD10 gene delivery could inhibit the growth of prolactinoma. Rat GH4 lactotrope tumor cells were infected with adenovirus/adeno-associated virus (Ad/AAV) hybrid vectors carrying the mouse HoxD10 gene (Hyb-HoxD10) or the {beta}-galactosidase gene (Hyb-Gal). Hyb-HoxD10 expression inhibited GH4 cell proliferation in vitro. The expression of FGF-2 and cyclin D2 was inhibited in GH4 cells infected with Hyb-HoxD10. GH4 cells transduced with Hyb-HoxD10 did not form tumors in nude mice. These results indicate that the delivery of HoxD10 could potentially inhibit the growth of PRL-secreting tumors. This approach may be a useful tool for targeted therapy of prolactinoma and other neoplasms.

  2. miR-10b promotes invasion by targeting HOXD10 in colorectal cancer

    PubMed Central

    WANG, YUNFENG; LI, ZHEN; ZHAO, XUHONG; ZUO, XIAOMING; PENG, ZHIHAI

    2016-01-01

    Studies have shown that homeobox D10 (HOXD10) is the target gene of microRNA-10b (miR-10b) and is closely associated with the inhibition of cell migration and invasion. Ras homolog family member C (RhoC) has been reported to promote tumor metastasis in various types of cancer. The effect of miR-10b on colorectal cancer (CRC) metastasis and the associated molecular mechanisms remain elusive. The present study aimed to investigate whether miR-10b could promote invasion by targeting HOXD10 in CRC by exploring the association between miR-10b and HOXD10 expression in CRC patients. The findings revealed that miR-10b levels were elevated in the CRC specimens and significantly correlated with advanced clinical stage and lymph node metastasis. In addition, HOXD10 was a direct target of miR-10b, and the increased expression of RhoC and downregulation of HOXD10 correlated with the increased expression level of miR-10b. HOXD10 protein level was also markedly attenuated in lymph node metastasis-positive tumor tissues compared with lymph node metastasis-free tumor tissues. These findings suggest that miR-10b may stimulate the upregulation of RhoC through targeting HOXD10, thus promoting the invasion and migration in CRC tumor. PMID:27347170

  3. Interaction between Tbx1 and Hoxd10 and connection with TGFβ-BMP signal pathway during kidney development.

    PubMed

    Fu, Yu; Li, Fei; Zhao, Diana Yue; Zhang, Jing-Shu; Lv, Yuan; Li-Ling, Jesse

    2014-02-15

    Renal malformations are commonly found among patients carrying a 22q11 deletion which renders loss of Tbx1 gene, an important transcriptional factor implicated in a number of developmental processes. Smad1 is known to interact with Tbx1, but the exact mechanism remains unknown. In this study, we have measured the expression of Tbx1 in both murine and human tissues using RT-PCR, and analyzed its protein product and protein-protein interactions with Western blotting and immunoprecipitation assays. Precipitated proteins were verified with mass spectrometry. As discovered, Tbx1 binds with Hoxd10. Tbx1 and Hoxd10 genes also have similar expression profiles during murine kidney development. Based on homology between mouse and human, we hypothesized that such interaction also exists in human. Through a RNA interference experiment using a human embryonic kidney HEK293 cell line, we demonstrated that TBX1 can alter TGF-β/BMP, an important signaling pathway, through interacting with HOXD10. Above findings may shed light on the mechanism of TBX1 mutations leading to renal malformations found in patients carrying a 22q11 deletion. PMID:22842189

  4. Programming neural Hoxd10: in vivo evidence that early node-associated signals predominate over paraxial mesoderm signals at posterior spinal levels.

    PubMed

    Omelchenko, Natalia; Lance-Jones, Cynthia

    2003-09-01

    Studies of the programming of Hox patterns at anterior spinal levels suggest that these events are accomplished through an integration of Hensen's node-derived and paraxial mesoderm signaling. We have used in vivo tissue manipulation in the avian embryo to examine the respective roles of node- derived and other local signals in the programming of a Hox pattern at posterior spinal levels. Hoxd10 is highly expressed in the lumbosacral (LS) spinal cord and adjacent paraxial mesoderm. At stages of LS neural tube formation (stages 12-14), the tailbud contains the remnants of Hensen's node and the primitive streak. Hoxd10 expression was analyzed after transposition of LS neural segments with and without the tailbud, after isolation of normally positioned LS segments from the stage 13 tailbud, and after axial displacement of posterior paraxial mesoderm. Data suggest that inductive signals from the tailbud are primarily responsible for the programming of Hoxd10 at neural plate and the earliest neural tube stages. After these stages, the LS neural tube appears to differ from more anterior neural segments in its lack of dependence on Hox-inductive signals from local tissues, including paraxial mesoderm. Our data also suggest that a graded system of repressive signals for posterior Hox genes is present at cervical and thoracic levels and likely to originate from paraxial mesoderm.

  5. Homeobox D10 Gene, a Candidate Tumor Suppressor, Is Downregulated through Promoter Hypermethylation and Associated with Gastric Carcinogenesis

    PubMed Central

    Wang, Liangjing; Chen, Shujie; Xue, Meng; Zhong, Jing; Wang, Xian; Gan, Lihong; Lam, Emily KY; Liu, Xin; Zhang, Jianbin; Zhou, Tianhua; Yu, Jun; Jin, Hongchuan; Si, Jianmin

    2012-01-01

    Homeobox D10 (HoxD10 ) gene plays a critical role in cell differentiation and morphogenesis during development. However, the function of HoxD10 in tumor progression remains largely unknown. We demonstrate that the expression of HoxD10 is commonly downregulated in gastric cancer tissues (n = 33) and cell lines (n = 8) relative to normal stomach tissues. Functionally, reexpression of HoxD10 results in significant inhibition of cell survival, induction of cell apoptosis, and impairment of cell migration and invasion. Moreover, ectopic expression of HoxD10 suppresses gastric tumor growth in a mouse xenograft model. To identify target candidates of HoxD10, we performed cDNA microarray and showed that HoxD10 regulates multiple downstream genes including IGFBP3. Reintroduction of HoxD10 transcriptionally upregulates IGFBP3, activates caspase 3 and caspase 8, and subsequently induces cell apoptosis. Methylation specific PCR revealed that HoxD10 promoter DNA was hypermethylated in gastric cancer cell lines. Additionally, 5-aza demethylation treatment could transiently reactivate the expression of HoxD10 in gastric cancer cells. HoxD10 promoter methylation frequently was detected in gastric cancer tissues obtained from endoscopic biopsies (85.7%, 24/28) and surgically resected samples (82.6%, 57/69). Intestinal metaplasia tissues showed a 60% methylation rate (18/30), but no detectable methylation in normal stomach tissues (0%, 0/10). Taken together, our results suggest that HoxD10 functions as a candidate tumor suppressor in gastric cancer, which is inactivated through promoter hypermethylation. PMID:22160393

  6. Electroporation-mediated gene delivery.

    PubMed

    Young, Jennifer L; Dean, David A

    2015-01-01

    Electroporation has been used extensively to transfer DNA to bacteria, yeast, and mammalian cells in culture for the past 30 years. Over this time, numerous advances have been made, from using fields to facilitate cell fusion, delivery of chemotherapeutic drugs to cells and tissues, and most importantly, gene and drug delivery in living tissues from rodents to man. Electroporation uses electrical fields to transiently destabilize the membrane allowing the entry of normally impermeable macromolecules into the cytoplasm. Surprisingly, at the appropriate field strengths, the application of these fields to tissues results in little, if any, damage or trauma. Indeed, electroporation has even been used successfully in human trials for gene delivery for the treatment of tumors and for vaccine development. Electroporation can lead to between 100 and 1000-fold increases in gene delivery and expression and can also increase both the distribution of cells taking up and expressing the DNA as well as the absolute amount of gene product per cell (likely due to increased delivery of plasmids into each cell). Effective electroporation depends on electric field parameters, electrode design, the tissues and cells being targeted, and the plasmids that are being transferred themselves. Most importantly, there is no single combination of these variables that leads to greatest efficacy in every situation; optimization is required in every new setting. Electroporation-mediated in vivo gene delivery has proven highly effective in vaccine production, transgene expression, enzyme replacement, and control of a variety of cancers. Almost any tissue can be targeted with electroporation, including muscle, skin, heart, liver, lung, and vasculature. This chapter will provide an overview of the theory of electroporation for the delivery of DNA both in individual cells and in tissues and its application for in vivo gene delivery in a number of animal models. PMID:25620008

  7. Electroporation-mediated gene delivery.

    PubMed

    Young, Jennifer L; Dean, David A

    2015-01-01

    Electroporation has been used extensively to transfer DNA to bacteria, yeast, and mammalian cells in culture for the past 30 years. Over this time, numerous advances have been made, from using fields to facilitate cell fusion, delivery of chemotherapeutic drugs to cells and tissues, and most importantly, gene and drug delivery in living tissues from rodents to man. Electroporation uses electrical fields to transiently destabilize the membrane allowing the entry of normally impermeable macromolecules into the cytoplasm. Surprisingly, at the appropriate field strengths, the application of these fields to tissues results in little, if any, damage or trauma. Indeed, electroporation has even been used successfully in human trials for gene delivery for the treatment of tumors and for vaccine development. Electroporation can lead to between 100 and 1000-fold increases in gene delivery and expression and can also increase both the distribution of cells taking up and expressing the DNA as well as the absolute amount of gene product per cell (likely due to increased delivery of plasmids into each cell). Effective electroporation depends on electric field parameters, electrode design, the tissues and cells being targeted, and the plasmids that are being transferred themselves. Most importantly, there is no single combination of these variables that leads to greatest efficacy in every situation; optimization is required in every new setting. Electroporation-mediated in vivo gene delivery has proven highly effective in vaccine production, transgene expression, enzyme replacement, and control of a variety of cancers. Almost any tissue can be targeted with electroporation, including muscle, skin, heart, liver, lung, and vasculature. This chapter will provide an overview of the theory of electroporation for the delivery of DNA both in individual cells and in tissues and its application for in vivo gene delivery in a number of animal models.

  8. Nonviral Vectors for Gene Delivery

    NASA Astrophysics Data System (ADS)

    Baoum, Abdulgader Ahmed

    2011-12-01

    The development of nonviral vectors for safe and efficient gene delivery has been gaining considerable attention recently. An ideal nonviral vector must protect the gene against degradation by nuclease in the extracellular matrix, internalize the plasma membrane, escape from the endosomal compartment, unpackage the gene at some point and have no detrimental effects. In comparison to viruses, nonviral vectors are relatively easy to synthesize, less immunogenic, low in cost, and have no limitation in the size of a gene that can be delivered. Significant progress has been made in the basic science and applications of various nonviral gene delivery vectors; however, the majority of nonviral approaches are still inefficient and often toxic. To this end, two nonviral gene delivery systems using either biodegradable poly(D,L-lactide- co-glycolide) (PLG) nanoparticles or cell penetrating peptide (CPP) complexes have been designed and studied using A549 human lung epithelial cells. PLG nanoparticles were optimized for gene delivery by varying particle surface chemistry using different coating materials that adsorb to the particle surface during formation. A variety of cationic coating materials were studied and compared to more conventional surfactants used for PLG nanoparticle fabrication. Nanoparticles (˜200 nm) efficiently encapsulated plasmids encoding for luciferase (80-90%) and slowly released the same for two weeks. After a delay, moderate levels of gene expression appeared at day 5 for certain positively charged PLG particles and gene expression was maintained for at least two weeks. In contrast, gene expression mediated by polyethyleneimine (PEI) ended at day 5. PLG particles were also significantly less cytotoxic than PEI suggesting the use of these vehicles for localized, sustained gene delivery to the pulmonary epithelium. On the other hand, a more simple method to synthesize 50-200 nm complexes capable of high transfection efficiency or high gene knockdown was

  9. Composite Nanoparticles for Gene Delivery

    PubMed Central

    Wang, Yuhua; Huang, Leaf

    2016-01-01

    Nanoparticle-mediated gene and siRNA delivery has been an appealing area to gene therapists when they attempt to treat the diseases by manipulating the genetic information in the target cells. However, the advances in materials science could not keep up with the demand for multifunctional nanomaterials to achieve desired delivery efficiency. Researchers have thus taken an alternative approach to incorporate various materials into single composite nanoparticle using different fabrication methods. This approach allows nanoparticles to possess defined nanostructures as well as multiple functionalities to overcome the critical extracellular and intracellular barriers to successful gene delivery. This chapter will highlight the advances of fabrication methods that have the most potential to translate nanoparticles from bench to bedside. Furthermore, a major class of composite nanoparticle–lipid-based composite nanoparticles will be classified based on the components and reviewed in details. PMID:25409605

  10. Nanoparticle-Mediated Gene Delivery

    NASA Astrophysics Data System (ADS)

    Jin, Sha; Leach, John C.; Ye, Kaiming

    Nonviral gene delivery has been gaining considerable attention recently. Although the efficacy of DNA transfection, which is a major concern, is low in nonviral vector-mediated gene transfer compared with viral ones, nonviral vectors are relatively easy to prepare, less immunogenic and oncogenic, and have no potential of virus recombination and no limitation on the size of a transferred gene. The ability to incorporate genetic materials such as plasmid DNA, RNA, and siRNA into functionalized nanoparticles with little toxicity demonstrates a new era in pharmacotherapy for delivering genes selectively to tissues and cells. In this chapter, we highlight the basic concepts and applications of nonviral gene delivery using super paramagnetic iron oxide nanoparticles and functionalized silica nanoparticles. The experimental protocols related to these topics are described in the chapter.

  11. Gene doping: gene delivery for olympic victory.

    PubMed

    Gould, David

    2013-08-01

    With one recently recommended gene therapy in Europe and a number of other gene therapy treatments now proving effective in clinical trials it is feasible that the same technologies will soon be adopted in the world of sport by unscrupulous athletes and their trainers in so called 'gene doping'. In this article an overview of the successful gene therapy clinical trials is provided and the potential targets for gene doping are highlighted. Depending on whether a doping gene product is secreted from the engineered cells or is retained locally to, or inside engineered cells will, to some extent, determine the likelihood of detection. It is clear that effective gene delivery technologies now exist and it is important that detection and prevention plans are in place. PMID:23082866

  12. Gene doping: gene delivery for olympic victory

    PubMed Central

    Gould, David

    2013-01-01

    With one recently recommended gene therapy in Europe and a number of other gene therapy treatments now proving effective in clinical trials it is feasible that the same technologies will soon be adopted in the world of sport by unscrupulous athletes and their trainers in so called ‘gene doping’. In this article an overview of the successful gene therapy clinical trials is provided and the potential targets for gene doping are highlighted. Depending on whether a doping gene product is secreted from the engineered cells or is retained locally to, or inside engineered cells will, to some extent, determine the likelihood of detection. It is clear that effective gene delivery technologies now exist and it is important that detection and prevention plans are in place. PMID:23082866

  13. Gene doping: gene delivery for olympic victory.

    PubMed

    Gould, David

    2013-08-01

    With one recently recommended gene therapy in Europe and a number of other gene therapy treatments now proving effective in clinical trials it is feasible that the same technologies will soon be adopted in the world of sport by unscrupulous athletes and their trainers in so called 'gene doping'. In this article an overview of the successful gene therapy clinical trials is provided and the potential targets for gene doping are highlighted. Depending on whether a doping gene product is secreted from the engineered cells or is retained locally to, or inside engineered cells will, to some extent, determine the likelihood of detection. It is clear that effective gene delivery technologies now exist and it is important that detection and prevention plans are in place.

  14. Cationic Bolaamphiphiles for Gene Delivery

    NASA Astrophysics Data System (ADS)

    Tan, Amelia Li Min; Lim, Alisa Xue Ling; Zhu, Yiting; Yang, Yi Yan; Khan, Majad

    2014-05-01

    Advances in medical research have shed light on the genetic cause of many human diseases. Gene therapy is a promising approach which can be used to deliver therapeutic genes to treat genetic diseases at its most fundamental level. In general, nonviral vectors are preferred due to reduced risk of immune response, but they are also commonly associated with low transfection efficiency and high cytotoxicity. In contrast to viral vectors, nonviral vectors do not have a natural mechanism to overcome extra- and intracellular barriers when delivering the therapeutic gene into cell. Hence, its design has been increasingly complex to meet challenges faced in targeting of, penetration of and expression in a specific host cell in achieving more satisfactory transfection efficiency. Flexibility in design of the vector is desirable, to enable a careful and controlled manipulation of its properties and functions. This can be met by the use of bolaamphiphile, a special class of lipid. Unlike conventional lipids, bolaamphiphiles can form asymmetric complexes with the therapeutic gene. The advantage of having an asymmetric complex lies in the different purposes served by the interior and exterior of the complex. More effective gene encapsulation within the interior of the complex can be achieved without triggering greater aggregation of serum proteins with the exterior, potentially overcoming one of the great hurdles faced by conventional single-head cationic lipids. In this review, we will look into the physiochemical considerations as well as the biological aspects of a bolaamphiphile-based gene delivery system.

  15. Gene delivery by functional inorganic nanocarriers.

    PubMed

    Loh, Xian Jun; Lee, Tung-Chun

    2012-08-01

    Gene delivery into cells to elicit cellular response has received a great attention recently. Viruses, lipids, peptides, cationic polymers and certain inorganic nanomaterials have been reported as gene delivery vectors. In this review, we focus on the recent literature on gene delivery using inorganic nanoparticles. This emerging field of study is concisely summarized and illustrated by selected examples and recent patents. New approaches and directions towards the practical use of multifunctional nanocarriers are highlighted.

  16. Viral and nonviral delivery systems for gene delivery.

    PubMed

    Nayerossadat, Nouri; Maedeh, Talebi; Ali, Palizban Abas

    2012-01-01

    Gene therapy is the process of introducing foreign genomic materials into host cells to elicit a therapeutic benefit. Although initially the main focus of gene therapy was on special genetic disorders, now diverse diseases with different patterns of inheritance and acquired diseases are targets of gene therapy. There are 2 major categories of gene therapy, including germline gene therapy and somatic gene therapy. Although germline gene therapy may have great potential, because it is currently ethically forbidden, it cannot be used; however, to date human gene therapy has been limited to somatic cells. Although numerous viral and nonviral gene delivery systems have been developed in the last 3 decades, no delivery system has been designed that can be applied in gene therapy of all kinds of cell types in vitro and in vivo with no limitation and side effects. In this review we explain about the history of gene therapy, all types of gene delivery systems for germline (nuclei, egg cells, embryonic stem cells, pronuclear, microinjection, sperm cells) and somatic cells by viral [retroviral, adenoviral, adeno association, helper-dependent adenoviral systems, hybrid adenoviral systems, herpes simplex, pox virus, lentivirus, Epstein-Barr virus)] and nonviral systems (physical: Naked DNA, DNA bombardant, electroporation, hydrodynamic, ultrasound, magnetofection) and (chemical: Cationic lipids, different cationic polymers, lipid polymers). In addition to the above-mentioned, advantages, disadvantages, and practical use of each system are discussed. PMID:23210086

  17. Race, genes and preterm delivery.

    PubMed Central

    Fiscella, Kevin

    2005-01-01

    High rates of preterm delivery (PTD) among African Americans are the leading cause of excess infant mortality among African Americans. Failure to fully explain racial disparity in PTD has led to speculation that genetic factors might contribute to this disparity. Current evidence suggests that genetic factors contribute to PTD, but this does not imply that genetic factors contribute to racial disparity in PTD. Environmental factors clearly contribute to PTD. Many of these factors acting over a women's life prior to pregnancy disproportionately affect African Americans and contribute significantly to racial disparity in PTD. Thus, inferring genetic contribution to racial disparity in PTD by attempting to control for environmental factors measured at a single point in time is flawed. There is emerging evidence of gene-environment interactions for PTD, some of which disproportionately affect African Americans. There is also evidence of racial differences in the prevalence of polymorphisms potentially related to PTD. However, to date there is no direct evidence that these differences contribute significantly to racial disparity in PTD. Given the complexity of polygenic conditions such as PTD, the possibility of any single gene contributing substantially to racial disparity in PTD seems remote. PMID:16334498

  18. Cyclodextrins in non-viral gene delivery.

    PubMed

    Lai, Wing-Fu

    2014-01-01

    Cyclodextrins (CDs) are naturally occurring cyclic oligosaccharides. They consist of (α-1,4)-linked glucose units, and possess a basket-shaped topology with an "inner-outer" amphiphilic character. Over the years, substantial efforts have been undertaken to investigate the possible use of CDs in drug delivery and controlled drug release, yet the potential of CDs in gene delivery has received comparatively less discussion in the literature. In this article, we will first discuss the properties of CDs for gene delivery, followed by a synopsis of the use of CDs in development and modification of non-viral gene carriers. Finally, areas that are noteworthy in CD-based gene delivery will be highlighted for future research. Due to the application prospects of CDs, it is anticipated that CDs will continue to emerge as an important tool for vector development, and will play significant roles in facilitating non-viral gene delivery in the forthcoming decades. PMID:24103652

  19. Diseases originate and terminate by genes: unraveling nonviral gene delivery.

    PubMed

    Swami, Rajan; Singh, Indu; Khan, Wahid; Ramakrishna, Sistla

    2013-12-01

    The world is driving in to the era of transformation of chemical therapeutic molecules to biological genetic material therapeutics, and that is where the biological drugs especially "genes" come into existence. These genes worked as "magical bullets" to specifically silence faulty genes responsible for progression of diseases. Viral gene delivery research is far ahead of nonviral gene delivery technique. However, with more advancement in polymer science, new ways are opening for better and efficient nonviral gene delivery. But efficient delivery method is always considered as a bottleneck for gene delivery as success of which will decide the fate of gene in cells. During the past decade, it became evident that extracellular as well as intracellular barriers compromise the transfection efficiency of nonviral vectors. The challenge for gene therapy research is to pinpoint the rate-limiting steps in this complex process and implement strategies to overcome the biological physiochemical and metabolic barriers encountered during targeting. The synergy between studies that investigate the mechanism of breaking in and breaking out of nonviral gene delivery carrier through various extracellular and intracellular barriers with desired characteristics will enable the rational design of vehicles and revolutionize the treatment of various diseases.

  20. Local drug and gene delivery through microbubbles.

    PubMed

    Unger, E C; Hersh, E; Vannan, M; Matsunaga, T O; McCreery, T

    2001-01-01

    Ultrasound contrast agents (microbubbles) lower the threshold for cavitation by ultrasound energy. Ultrasound microbubbles may be used as cavitation nuclei for drug and gene delivery. By tailoring the physical properties of microbubbles and coating materials, drugs and genetic drugs can be incorporated into ultrasound contrast agents. As the microbubbles enter the region of insonation, the microbubbles cavitate, locally releasing the therapeutic agents. Cavitation also causes a local shockwave that improves cellular uptake of the therapeutic agent. As a result of the human genome project and continuing advances in molecular biology, many therapeutic genes have been discovered. In the cardiovascular system, gene therapy has the potential to improve myocardial vascularization and ameliorate congestive heart failure. For successful development of clinical gene therapy, however, effective gene delivery vectors are needed. Ultrasound contrast agents can be used to develop new, more effective vectors for gene delivery. Transthoracic ultrasound can be focused on the heart so that an intravenous injection of gene-bearing microbubbles will deliver genes relatively selectively to the myocardium. Using this technique, we have produced high levels of transgene expression in the insonated region of the myocardium. This new technology, using microbubbles and ultrasound for drug and gene delivery, merits further study and development.

  1. Endovascular Gene Delivery from a Stent Platform: Gene- Eluting Stents

    PubMed Central

    Fishbein, Ilia; Chorny, Michael; Adamo, Richard F; Forbes, Scott P; Corrales, Ricardo A; Alferiev, Ivan S; Levy, Robert J

    2015-01-01

    A synergistic impact of research in the fields of post-angioplasty restenosis, drug-eluting stents and vascular gene therapy over the past 15 years has shaped the concept of gene-eluting stents. Gene-eluting stents hold promise of overcoming some biological and technical problems inherent to drug-eluting stent technology. As the field of gene-eluting stents matures it becomes evident that all three main design modules of a gene-eluting stent: a therapeutic transgene, a vector and a delivery system are equally important for accomplishing sustained inhibition of neointimal formation in arteries treated with gene delivery stents. This review summarizes prior work on stent-based gene delivery and discusses the main optimization strategies required to move the field of gene-eluting stents to clinical translation. PMID:26225356

  2. Computer-assisted hydrodynamic gene delivery.

    PubMed

    Suda, Takeshi; Suda, Kieko; Liu, Dexi

    2008-06-01

    The recently developed hydrodynamic delivery method makes it possible to deliver DNA and RNA into parenchyma cells by intravascular injection of nucleic acid-containing solution. While this procedure is effective in rodents, it is difficult to perform in large animals, because manual control while delivering the injection cannot be sufficiently reliable for achieving a just-right hydrodynamic pressure in targeted tissue. In order to overcome this problem, we have developed a computer-controlled injection device that uses real-time intravascular pressure as a regulator. Using the new injection device, and mouse liver as the model organ, we demonstrated continuous injection at a single pressure and different pressures, and also serial (repeated) injections at intervals of 250 ms, by programming the computer according to the need. When assessed by reporter plasmids, the computer-controlled injection device exhibits gene delivery efficiency similar to that of conventional hydrodynamic injection. The device is also effective in gene delivery to kidney and muscle cells in rats, with plasmids or adenoviral vectors as gene carriers. Successful gene delivery to liver and kidney was also demonstrated in pigs, with the computer-controlled injection being combined with image-guided catheterization. These results represent a significant advance in in vivo gene delivery research, with potential for use in gene therapy in humans.

  3. Polymeric Gene Delivery for Diabetic Treatment

    PubMed Central

    2011-01-01

    Several polymers were used to delivery genes to diabetic animals. Polyaminobutyl glycolic acid was utilized to deliver IL-10 plasmid DNA to prevent autoimmune insulitis of non-obese diabetic (NOD) mouse. Polyethylene glycol grafted polylysine was combined with antisense glutamic acid decarboxylase (GAD) MRNA to represent GAD autoantigene expression. GLP1 and TSTA (SP-EX4) were delivered by bioreducible polymer to stop diabetic progression. Fas siRNA delivery was carried out to treat diabetic NOD mice animal. PMID:21977450

  4. Magnetic nanoparticles for gene and drug delivery

    PubMed Central

    McBain, Stuart C; Yiu, Humphrey HP; Dobson, Jon

    2008-01-01

    Investigations of magnetic micro- and nanoparticles for targeted drug delivery began over 30 years ago. Since that time, major progress has been made in particle design and synthesis techniques, however, very few clinical trials have taken place. Here we review advances in magnetic nanoparticle design, in vitro and animal experiments with magnetic nanoparticle-based drug and gene delivery, and clinical trials of drug targeting. PMID:18686777

  5. Gene therapy prospects--intranasal delivery of therapeutic genes.

    PubMed

    Podolska, Karolina; Stachurska, Anna; Hajdukiewicz, Karolina; Małecki, Maciej

    2012-01-01

    Gene therapy is recognized to be a novel method for the treatment of various disorders. Gene therapy strategies involve gene manipulation on broad biological processes responsible for the spreading of diseases. Cancer, monogenic diseases, vascular and infectious diseases are the main targets of gene therapy. In order to obtain valuable experimental and clinical results, sufficient gene transfer methods are required. Therapeutic genes can be administered into target tissues via gene carriers commonly defined as vectors. The retroviral, adenoviral and adeno-associated virus based vectors are most frequently used in the clinic. So far, gene preparations may be administered directly into target organs or by intravenous, intramuscular, intratumor or intranasal injections. It is common knowledge that the number of gene therapy clinical trials has rapidly increased. However, some limitations such as transfection efficiency and stable and long-term gene expression are still not resolved. Consequently, great effort is focused on the evaluation of new strategies of gene delivery. There are many expectations associated with intranasal delivery of gene preparations for the treatment of diseases. Intranasal delivery of therapeutic genes is regarded as one of the most promising forms of pulmonary gene therapy research. Gene therapy based on inhalation of gene preparations offers an alternative way for the treatment of patients suffering from such lung diseases as cystic fibrosis, alpha-1-antitrypsin defect, or cancer. Experimental and first clinical trials based on plasmid vectors or recombinant viruses have revealed that gene preparations can effectively deliver therapeutic or marker genes to the cells of the respiratory tract. The noninvasive intranasal delivery of gene preparations or conventional drugs seems to be very encouraging, although basic scientific research still has to continue.

  6. Electrostatic Surface Modifications to Improve Gene Delivery

    PubMed Central

    Shmueli, Ron B.; Anderson, Daniel G.

    2010-01-01

    Importance of the field Gene therapy has the potential to treat a wide variety of diseases including genetic diseases and cancer. Areas covered in this review This review introduces biomaterials used for gene delivery and then focuses on the use of electrostatic surface modifications to improve gene delivery materials. These modifications have been used to stabilize therapeutics in vivo, add cell-specific targeting ligands, and promote controlled release. Coatings of nanoparticles and microparticles as well as non-particulate surface coatings are covered in this review. Electrostatic principles are crucial for the development of multilayer delivery structures fabricated by the layer-by-layer method. What the reader will gain The reader will gain knowledge about the composition of biomaterials used for surface modifications and how these coatings and multilayers can be utilized to improve spatial control and efficiency of delivery. Examples are shown for the delivery of nucleic acids, including DNA and siRNA, to in vitro and in vivo systems. Take home message The versatile and powerful approach of electrostatic coatings and multilayers will lead to the development of enhanced gene therapies. PMID:20201712

  7. Gene delivery using ultrasound contrast agents.

    PubMed

    Unger, E C; Hersh, E; Vannan, M; McCreery, T

    2001-05-01

    With the human genome product and continuing advances in molecular biology many therapeutic genes have been discovered. In the cardiovascular system, gene therapy has the potential to improve myocardial vascularization and ameliorate congestive heart failure. For successful development of clinical gene therapy, however, effective gene delivery vectors are needed. Ultrasound contrast agents can be used to develop new, more effective vectors for gene delivery. Ultrasound contrast agents lower the threshold for cavitation by ultrasound energy. Using physical properties of microbubbles and coating materials, genetic drugs have been incorporated into ultrasound contrast agents. Gene-bearing microbubbles can be injected IV and ultrasound energy applied to the target region. As the microbubbles enter the region of insonation, the microbubbles cavitate, locally releasing DNA. Cavitation also likely causes a local shockwave that improves cellular uptake of DNA. With transthoracic ultrasound, using commercially available diagnostic ultrasound system and an IV injection of gene-bearing microbubbles, high levels of transgene expression are observed in the insonated region of the myocardium. This new technology using microbubbles and ultrasound for gene delivery merits further study and development.

  8. Recent Trends of Polymer Mediated Liposomal Gene Delivery System

    PubMed Central

    Lee, Sang-Soo; George Priya Doss, C.; Yagihara, Shin; Kim, Do-Young

    2014-01-01

    Advancement in the gene delivery system have resulted in clinical successes in gene therapy for patients with several genetic diseases, such as immunodeficiency diseases, X-linked adrenoleukodystrophy (X-ALD) blindness, thalassemia, and many more. Among various delivery systems, liposomal mediated gene delivery route is offering great promises for gene therapy. This review is an attempt to depict a portrait about the polymer based liposomal gene delivery systems and their future applications. Herein, we have discussed in detail the characteristics of liposome, importance of polymer for liposome formulation, gene delivery, and future direction of liposome based gene delivery as a whole. PMID:25250340

  9. "Programmed packaging" for gene delivery.

    PubMed

    Hyodo, M; Sakurai, Y; Akita, H; Harashima, H

    2014-11-10

    We report on the development of a multifunctional envelope-type nano device (MEND) based on our packaging concept "Programmed packaging" to control not only intracellular trafficking but also the biodistribution of encapsulated compounds such as nucleic acids/proteins/peptides. Our strategy for achieving this is based on molecular mechanisms of cell biology such as endocytosis, vesicular trafficking, etc. In this review, we summarize the concept of programmed packaging and discuss some of our recent successful examples of using MENDs. Systematic evolution of ligands by exponential enrichment (SELEX) was applied as a new methodology for identifying a new ligand toward cell or mitochondria. The delivery of siRNA to tumors and the tumor vasculature was achieved using pH sensitive lipid (YSK05), which was newly designed and optimized under in vivo conditions. The efficient delivery of pDNA to immune cells such as dendritic cells has also been developed using the KALA ligand, which can be a breakthrough technology for DNA vaccine. Finally, ss-cleavable and pH-activated lipid-like surfactant (ssPalm) which is a lipid like material with pH-activatable and SS-cleavable properties is also introduced as a proof of our concept. PMID:24780263

  10. Novel Polymeric Nanoparticles for Pulmonary Gene Delivery

    NASA Astrophysics Data System (ADS)

    Fields, Rachel Jennifer

    The lung is an important target for gene and drug therapy of many diseases such as chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), tubuerculosis (TB) and lung cancer. In fact, the pulmonary route has been employed as a means of delivering drugs for centuries, dating back 4000 years to India where inhaled vapors were used for medicinal purpose. Currently, pulmonary administration of small, hydrophobic drugs leads to rapid local and systemic absorption. However, delivery of large biomacromolecules, such as therapeutic genes, has not yet been accomplished. Here, I test the hypothesis that a rationally engineered nanoparticle (NP) vector can improve delivery of large biomacromolecules. . In this dissertation I tested this hypothesis using a hybrid NP delivery system consisting of a blend of poly(lactic-co-glycolic acid) (PLGA) and a poly(beta-amino ester) (PBAE), a cationic polymer that is particularly useful for delivery of nucleic acids.. PBAE/PLGA nanoparticles (15% PBAE) loaded with plasmid DNA were surface modified with cell-penetrating peptides (CPPs) via a PEGylated phospholipid linker. This optimized NP formulation was able to induce substantial intracellular uptake and transfect lung epithelial cells in vitro while imparting minimal cellular toxicity. In order to determine the most effective method to deliver these NPs to the lung I used fluorescently labeled particles to study the biodistribution of particles after administration to the lung of mice via various administration routes. I determined that the intranasal route was most effective. I further investigated this route and determined that an average of 37.1 +/- 15.1 % of lung cells had NP association after 4hrs. I also investigated the association of particles with different lung cell types like macrophages and alveolar epithelial cells and determined that our best particle formulations associated with approximately 80% of both of these cell types. To demonstrate the ability of the

  11. Mesocopic Structure of Nonviral Gene Delivery Vectors

    NASA Astrophysics Data System (ADS)

    van Zanten, John; Hanes, Justin; Lai, Eva; Har-El, Yah-El

    2003-03-01

    In general, little is known about nonviral gene delivery vector formation kinetics and the interplay between vector structure, especially mesoscopic structure, and transfection efficiency. DNA complexation with various condensing agents is a self-assembly process driven primarily by electrostatic interactions and counterion release. DNA complexation kinetics influence two physical parameters that directly affect gene delivery and expression efficiency: DNA complex geometric size and molar mass (density). In this study we demonstrate the utility of time resolved multiangle laser light scattering (TR-MALLS) for probing DNA complexation kinetics, determining DNA complex size and density in real time and monitoring nonviral vector stability in liquid formulations and serum. The condensing agents considered are poly-L-lysine, polyethylenimine and various cationic liposome formulations.

  12. Application of Magnetic Nanoparticles to Gene Delivery

    PubMed Central

    Kami, Daisuke; Takeda, Shogo; Itakura, Yoko; Gojo, Satoshi; Watanabe, Masatoshi; Toyoda, Masashi

    2011-01-01

    Nanoparticle technology is being incorporated into many areas of molecular science and biomedicine. Because nanoparticles are small enough to enter almost all areas of the body, including the circulatory system and cells, they have been and continue to be exploited for basic biomedical research as well as clinical diagnostic and therapeutic applications. For example, nanoparticles hold great promise for enabling gene therapy to reach its full potential by facilitating targeted delivery of DNA into tissues and cells. Substantial progress has been made in binding DNA to nanoparticles and controlling the behavior of these complexes. In this article, we review research on binding DNAs to nanoparticles as well as our latest study on non-viral gene delivery using polyethylenimine-coated magnetic nanoparticles. PMID:21747701

  13. Polymers for gene delivery across length scales

    NASA Astrophysics Data System (ADS)

    Putnam, David

    2006-06-01

    A number of human diseases stem from defective genes. One approach to treating such diseases is to replace, or override, the defective genes with normal genes, an approach called 'gene therapy'. However, the introduction of correctly functioning DNA into cells is a non-trivial matter, and cells must be coaxed to internalize, and then use, the DNA in the desired manner. A number of polymer-based synthetic systems, or 'vectors', have been developed to entice cells to use exogenous DNA. These systems work across the nano, micro and macro length scales, and have been under continuous development for two decades, with varying degrees of success. The design criteria for the construction of more-effective delivery vectors at each length scale are continually evolving. This review focuses on the most recent developments in polymer-based vector design at each length scale.

  14. An intestinal Trojan horse for gene delivery

    NASA Astrophysics Data System (ADS)

    Peng, Haisheng; Wang, Chao; Xu, Xiaoyang; Yu, Chenxu; Wang, Qun

    2015-02-01

    The intestinal epithelium forms an essential element of the mucosal barrier and plays a critical role in the pathophysiological response to different enteric disorders and diseases. As a major enteric dysfunction of the intestinal tract, inflammatory bowel disease is a genetic disease which results from the inappropriate and exaggerated mucosal immune response to the normal constituents in the mucosal microbiota environment. An intestine targeted drug delivery system has unique advantages in the treatment of inflammatory bowel disease. As a new concept in drug delivery, the Trojan horse system with the synergy of nanotechnology and host cells can achieve better therapeutic efficacy in specific diseases. Here, we demonstrated the feasibility of encapsulating DNA-functionalized gold nanoparticles into primary isolated intestinal stem cells to form an intestinal Trojan horse for gene regulation therapy of inflammatory bowel disease. This proof-of-concept intestinal Trojan horse will have a wide variety of applications in the diagnosis and therapy of enteric disorders and diseases.

  15. Synthetic virology: engineering viruses for gene delivery.

    PubMed

    Guenther, Caitlin M; Kuypers, Brianna E; Lam, Michael T; Robinson, Tawana M; Zhao, Julia; Suh, Junghae

    2014-01-01

    The success of gene therapy relies heavily on the performance of vectors that can effectively deliver transgenes to desired cell populations. As viruses have evolved to deliver genetic material into cells, a prolific area of research has emerged over the last several decades to leverage the innate properties of viruses as well as to engineer new features into them. Specifically, the field of synthetic virology aims to capitalize on knowledge accrued from fundamental virology research in order to design functionally enhanced gene delivery vectors. The enhanced viral vectors, or 'bionic' viruses, feature engineered components, or 'parts', that are natural (intrinsic to viruses or from other organisms) and synthetic (such as man-made polymers or inorganic nanoparticles). Various design strategies--rational, combinatorial, and pseudo-rational--have been pursued to create the hybrid viruses. The gene delivery vectors of the future will likely criss-cross the boundaries between natural and synthetic domains to harness the unique strengths afforded by the various functional parts that can be grafted onto virus capsids. Such research endeavors will further expand and enable enhanced control over the functional capacity of these nanoscale devices for biomedicine.

  16. Synthetic Virology: Engineering Viruses for Gene Delivery

    PubMed Central

    Guenther, Caitlin M.; Kuypers, Brianna E.; Lam, Michael T.; Robinson, Tawana M.; Zhao, Julia; Suh, Junghae

    2014-01-01

    The success of gene therapy relies heavily on the performance of vectors that can effectively deliver transgenes to desired cell populations. As viruses have evolved to deliver genetic material into cells, a prolific area of research has emerged over the last several decades to leverage the innate properties of viruses as well as to engineer new features into them. Specifically, the field of synthetic virology aims to capitalize on knowledge accrued from fundamental virology research in order to design functionally enhanced gene delivery vectors. The enhanced viral vectors, or “bionic” viruses, feature engineered components, or “parts”, that are natural (intrinsic to viruses or from other organisms) and synthetic (such as man-made polymers or inorganic nanoparticles). Various design strategies – rational, combinatorial, and pseudo-rational – have been pursued to create the hybrid viruses. The gene delivery vectors of the future will likely criss-cross the boundaries between natural and synthetic domains to harness the unique strengths afforded by the various functional parts that can be grafted onto virus capsids. Such research endeavours will further expand and enable enhanced control over the functional capacity of these nanoscale devices for biomedicine. PMID:25195922

  17. Tissue-Specific Gene Delivery via Nanoparticle Coating

    PubMed Central

    Harris, Todd J.; Green, Jordan J.; Fung, Peter W.; Langer, Robert; Anderson, Daniel G.; Bhatia, Sangeeta N.

    2009-01-01

    The use of biomaterials for gene delivery can potentially avoid many of the safety concerns with viral gene delivery. However, the efficacy of polymeric gene delivery methods is low, particularly in vivo. One significant concern is that the interior and exterior composition of polymeric gene delivery nanoparticles are often coupled, with a single polymer backbone governing all functions from biophysical properties of the polymer/DNA particle to DNA condensation and release. In this work we develop electrostatically adsorbed poly(glutamic acid)-based peptide coatings to alter the exterior composition of a core gene delivery particle and thereby affect tissue-specificity of gene delivery function in vivo. We find that with all coating formulations tested, the coatings reduce potential toxicity associated with uncoated cationic gene delivery nanoparticles following systemic injection. Particles coated with a low 2.5:1 peptide:DNA weight ratio (w/w) form large 2 micron sized particles in the presence of serum that can facilitate specific gene delivery to the liver. The same particles coated at a higher 20:1 w/w form small 200 nm particles in the presence of serum that can facilitate specific gene delivery to the spleen and bone marrow. Thus, variations in nanoparticle peptide coating density can alter the tissue-specificity of gene delivery in vivo. PMID:19850333

  18. [Developments in gene delivery vectors for ocular gene therapy].

    PubMed

    Khabou, Hanen; Dalkara, Deniz

    2015-05-01

    Gene therapy is quickly becoming a reality applicable in the clinic for inherited retinal diseases. Its remarkable success in safety and efficacy, in clinical trials for Leber's congenital amaurosis (LCA) type II generated significant interest and opened up possibilities for a new era of retinal gene therapies. Success in these clinical trials was mainly due to the favorable characteristics of the retina as a target organ. The eye offers several advantages as it is readily accessible and has some degree of immune privilege making it suitable for application of viral vectors. The viral vectors most frequently used for retinal gene delivery are lentivirus, adenovirus and adeno-associated virus (AAV). Here we will discuss the use of these viral vectors in retinal gene delivery with a strong focus on favorable properties of AAV. Thanks to its small size, AAV diffuses well in the inter-neural matrix making it suitable for applications in neural retina. Building on this initial clinical success with LCA II, we have now many opportunities to extend this proof-of-concept to other retinal diseases using AAV as a vector. This article will discuss what are some of the most imminent cellular targets for such therapies and the AAV toolkit that has been built to target these cells successfully. We will also discuss some of the challenges that we face in translating AAV-based gene therapies to the clinic.

  19. Targeting of Synthetic Gene Delivery Systems

    PubMed Central

    2003-01-01

    Safe, efficient, and specific delivery of therapeutic genes remains an important bottleneck for the development of gene therapy. Synthetic, nonviral systems have a unique pharmaceutical profile with potential advantages for certain applications. Targeting of the synthetic vector improves the specificity of gene medicines through a modulation of the carriers' biodistribution, thus creating a dose differential between healthy tissue and the target site. The biodistribution of current carrier systems is being influenced to a large extent by intrinsic physicochemical characteristics, such as charge and size. Consequently, such nonspecific interactions can interfere with specific targeting, for example, by ligands. Therefore, a carrier complex should ideally be inert, that is, free from intrinsic properties that would bias its distribution away from the target site. Strategies such as coating of DNA carrier complexes with hydrophilic polymers have been used to mask some of these intrinsic targeting effects and avoid nonspecific interactions. Preexisting endogenous ligand-receptor interactions have frequently been used for targeting to certain cell types or tumours. Recently exogenous ligands have been derived from microorganisms or, like antibodies or phage-derived peptides, developed de novo. In animal models, such synthetic vectors have targeted remote sites such as a tumour. Furthermore, the therapeutic proof of the concept has been demonstrated for fitting combinations of synthetic vectors and therapeutic gene. PMID:12721518

  20. Lipopolyplexes as nanomedicines for therapeutic gene delivery.

    PubMed

    García, Leire; Urbiola, Koldo; Düzgüneş, Nejat; Tros de Ilarduya, Conchita

    2012-01-01

    We describe an efficient, nonviral gene transfer system that employs polyethylenimine (PEI 800, 25, 22 kDa), and 1,2-dioleoyl-3-(trimethylammonium) propane (DOTAP) and cholesterol (Chol) as lipids (lipopolyplex), at three different lipid/DNA molar ratios (2/1, 5/1, and 17/1), employing five different formulation strategies. PEIs of 800, 25, and 22 kDa are highly effective in condensing plasmid DNA, leading to a complete condensation at N/P⁺/⁻ ratios above 4. Increasing the molar ratio lipid/DNA in the complex results in higher positive values of the zeta potential, while the particle size increases in some protocols, but not in others. PEI of molecular weight 800 kDa used in the formulation of lipopolyplexes results in bigger particles compared to that obtained with the smaller PEI species. Transfection activity is measured using pCMVLuc expressing luciferase is maximal by using strategies 3 and 4 and an N/P molar ratio of 17/1. These complexes have a high efficiency of gene delivery to liver cancer cells, even in the presence of a high serum concentration. Complexes formed with linear PEI are more effective than lipopolyplexes containing branched PEI. The ternary complexes are much more efficient than conventional lipoplexes (cationic lipid and DNA) and polyplexes (cationic polymer and DNA). The same behavior is observed for complexes prepared with the therapeutic gene pCMVIL-12 expressing interleukin-12.

  1. Cyclodextrin-based gene delivery systems.

    PubMed

    Ortiz Mellet, Carmen; García Fernández, José M; Benito, Juan M

    2011-03-01

    Cyclodextrin (CD) history has been largely dominated by their unique ability to form inclusion complexes with guests fitting in their hydrophobic cavity. Chemical funcionalization was soon recognized as a powerful mean for improving CD applications in a wide range of fields, including drug delivery, sensing or enzyme mimicking. However, 100 years after their discovery, CDs are still perceived as novel nanoobjects of undeveloped potential. This critical review provides an overview of different strategies to promote interactions between CD conjugates and genetic material by fully exploiting the inside-outside/upper-lower face anisotropy of the CD nanometric platform. Covalent modification, self-assembling and supramolecular ligation can be put forward with the ultimate goal to build artificial viruses for programmed and efficient gene therapy (222 references).

  2. Microneedles As a Delivery System for Gene Therapy

    PubMed Central

    Chen, Wei; Li, Hui; Shi, De; Liu, Zhenguo; Yuan, Weien

    2016-01-01

    Gene delivery systems can be divided to two major types: vector-based (either viral vector or non-viral vector) and physical delivery technologies. Many physical carriers, such as electroporation, gene gun, ultrasound start to be proved to have the potential to enable gene therapy. A relatively new physical delivery technology for gene delivery consists of microneedles (MNs), which has been studied in many fields and for many molecule types and indications. Microneedles can penetrate the stratum corneum, which is the main barrier for drug delivery through the skin with ease of administration and without significant pain. Many different kinds of MNs, such as metal MNs, coated MNs, dissolving MNs have turned out to be promising in gene delivery. In this review, we discussed the potential as well as the challenges of utilizing MNs to deliver nucleic acids for gene therapy. We also proposed that a combination of MNs and other gene delivery approaches may lead to a better delivery system for gene therapy. PMID:27303298

  3. Production and clinical development of nanoparticles for gene delivery

    PubMed Central

    Chen, Jie; Guo, Zhaopei; Tian, Huayu; Chen, Xuesi

    2016-01-01

    Gene therapy is a promising strategy for specific treatment of numerous gene-associated human diseases by intentionally altering the gene expression in pathological cells. A successful clinical application of gene-based therapy depends on an efficient gene delivery system. Many efforts have been attempted to improve the safety and efficiency of gene-based therapies. Nanoparticles have been proved to be the most promising vehicles for clinical gene therapy due to their tunable size, shape, surface, and biological behaviors. In this review, the clinical development of nanoparticles for gene delivery will be particularly highlighted. Several promising candidates, which are closest to clinical applications, will be briefly reviewed. Then, the recent developments of nanoparticles for clinical gene therapy will be identified and summarized. Finally, the development of nanoparticles for clinical gene delivery in future will be prospected. PMID:27088105

  4. Recent progress in nanomaterials for gene delivery applications.

    PubMed

    Keles, Erhan; Song, Yang; Du, Dan; Dong, Wen-Ji; Lin, Yuehe

    2016-08-16

    Nanotechnology-based gene delivery is the division of nanomedicine concerned with the synthesis, characterization, and functionalization of nanomaterials to be used in targeted-gene delivery applications. Nanomaterial-based gene delivery systems hold great promise for curing fatal inherited and acquired diseases, including neurological disorders, cancer, cardiovascular diseases, and acquired immunodeficiency syndrome (AIDS). However, their use in clinical applications is still controversial. To date, the Food and Drug Administration (FDA) has not approved any gene delivery system because of the unknown long-term toxicity and the low gene transfection efficiency of nanomaterials in vivo. Compared to viral vectors, nonviral gene delivery vectors are characterized by a low preexisting immunogenicity, which is important for preventing a severe immune response. In addition, nonviral vectors provide higher loading capacity and ease of fabrication. For these reasons, this review article focuses on applications of nonviral gene delivery systems, including those based on lipids, polymers, graphene, and other inorganic nanoparticles, and discusses recent advances in nanomaterials for gene therapy. Methods of synthesizing these nanomaterials are briefly described from a materials science perspective. Also, challenges, critical issues, and concerns about the in vivo applications of nanomaterial-based gene delivery systems are discussed. It should be noted that this article is not a comprehensive review of the literature. PMID:27480033

  5. Recent progress in nanomaterials for gene delivery applications.

    PubMed

    Keles, Erhan; Song, Yang; Du, Dan; Dong, Wen-Ji; Lin, Yuehe

    2016-08-16

    Nanotechnology-based gene delivery is the division of nanomedicine concerned with the synthesis, characterization, and functionalization of nanomaterials to be used in targeted-gene delivery applications. Nanomaterial-based gene delivery systems hold great promise for curing fatal inherited and acquired diseases, including neurological disorders, cancer, cardiovascular diseases, and acquired immunodeficiency syndrome (AIDS). However, their use in clinical applications is still controversial. To date, the Food and Drug Administration (FDA) has not approved any gene delivery system because of the unknown long-term toxicity and the low gene transfection efficiency of nanomaterials in vivo. Compared to viral vectors, nonviral gene delivery vectors are characterized by a low preexisting immunogenicity, which is important for preventing a severe immune response. In addition, nonviral vectors provide higher loading capacity and ease of fabrication. For these reasons, this review article focuses on applications of nonviral gene delivery systems, including those based on lipids, polymers, graphene, and other inorganic nanoparticles, and discusses recent advances in nanomaterials for gene therapy. Methods of synthesizing these nanomaterials are briefly described from a materials science perspective. Also, challenges, critical issues, and concerns about the in vivo applications of nanomaterial-based gene delivery systems are discussed. It should be noted that this article is not a comprehensive review of the literature.

  6. Retinal gene delivery by rAAV and DNA electroporation

    PubMed Central

    Venkatesh, Aditya; Ma, Shan; Langellotto, Fernanda; Gao, Guangping; Punzo, Claudio

    2013-01-01

    Ocular gene therapy is a fast growing area of research. The eye is an ideal organ for gene therapy since it is immune privileged, easily accessible, and direct viral delivery results primarily in local infection. Because the eye is not a vital organ, mutations in eye specific genes tend to be more common. To date, over 40 eye specific genes have been identified which harbor mutations that lead to blindness. Gene therapy with recombinant Adeno Associated Virus (rAAV) holds the promise to treat patients with such mutations. However, proof-of-concept and safety evaluation for gene therapy remains to be established for most of these diseases. This unit describes the in vivo delivery of genes to the mouse eye by rAAV-mediated gene transfer and plasmid DNA electroporation. Advantages and limitations of these methods are discussed, and detailed protocols for gene delivery, required materials, as well as subsequent tissue processing methods are described. PMID:23408132

  7. Getting the most from gene delivery by repeated DNA transfections

    NASA Astrophysics Data System (ADS)

    Montani, Maura; Marchini, Cristina; Badillo Pazmay, Gretta Veronica; Andreani, Cristina; Bartolacci, Caterina; Amici, Augusto; Pozzi, Daniela; Caracciolo, Giulio

    2015-06-01

    Intracellular delivery of reporter genes causes cells to be luminescent or fluorescent, this condition being of tremendous relevance in applied physics research. Potential applications range from the study of spatial distribution and dynamics of plasma membrane and cytosolic proteins up to the rational design of nanocarriers for gene therapy. Since efficiency of gene delivery is the main limit in most biophysical studies, versatile methods that can maximize gene expression are urgently needed. Here, we describe a robust methodology based on repeated gene delivery in mammalian cells. We find this procedure to be much more efficient than the more traditional route of gene delivery making it possible to get high-quality data without affecting cell viability. Implications for biophysical investigations are discussed.

  8. Investigation of a thiolated polymer in gene delivery

    NASA Astrophysics Data System (ADS)

    Bacalocostantis, Irene

    Thiol-containing bioreducible polymers show significant potential as delivery vectors in gene therapy, a rapidly growing field which seeks to treat genetic-based disorders by delivering functional synthetic genes to diseased cells. Studies have shown that thiolated polymers exhibit improved biodegradability and prolonged in vivo circulation times over non-thiolated polymers. However, the extent to which thiol concentrations impact the carrier's delivery potential has not been well explored. The aim of this dissertation is to investigate how relative concentrations of free thiols and disulfide crosslinks impact a polymeric carriers delivery performance with respect to DNA packaging, complex stability, cargo protection, gene release, internalization efficiency and cytotoxicity. To accomplish this goal, several fluorescent polymers containing varying concentrations of thiol groups were synthesized by conjugating thiol-pendant chains onto the primary amines of cationic poly(allylamine). In vitro delivery assays and characterization techniques were employed to assess the effect of thiols in gene delivery.

  9. Recent progresses in gene delivery-based bone tissue engineering.

    PubMed

    Lu, Chia-Hsin; Chang, Yu-Han; Lin, Shih-Yeh; Li, Kuei-Chang; Hu, Yu-Chen

    2013-12-01

    Gene therapy has converged with bone engineering over the past decade, by which a variety of therapeutic genes have been delivered to stimulate bone repair. These genes can be administered via in vivo or ex vivo approach using either viral or nonviral vectors. This article reviews the fundamental aspects and recent progresses in the gene therapy-based bone engineering, with emphasis on the new genes, viral vectors and gene delivery approaches.

  10. Recent progresses in gene delivery-based bone tissue engineering.

    PubMed

    Lu, Chia-Hsin; Chang, Yu-Han; Lin, Shih-Yeh; Li, Kuei-Chang; Hu, Yu-Chen

    2013-12-01

    Gene therapy has converged with bone engineering over the past decade, by which a variety of therapeutic genes have been delivered to stimulate bone repair. These genes can be administered via in vivo or ex vivo approach using either viral or nonviral vectors. This article reviews the fundamental aspects and recent progresses in the gene therapy-based bone engineering, with emphasis on the new genes, viral vectors and gene delivery approaches. PMID:23994567

  11. In Vivo Gene Delivery by Nonviral Vectors: Overcoming Hurdles?

    PubMed Central

    Zhang, Yuan; Satterlee, Andrew; Huang, Leaf

    2012-01-01

    The promise of cancer gene therapeutics is hampered by difficulties in the in vivo delivery to the targeted tumor cells, and systemic delivery remains to be the biggest challenge to be overcome. Here, we concentrate on systemic in vivo gene delivery for cancer therapy using nonviral vectors. In this review, we summarize the existing delivery barriers together with the requirements and strategies to overcome these problems. We will also introduce the current progress in the design of nonviral vectors, and briefly discuss their safety issues. PMID:22525514

  12. Rapid endosomal escape of prickly nanodiamonds: implications for gene delivery

    NASA Astrophysics Data System (ADS)

    Chu, Zhiqin; Miu, Kaikei; Lung, Pingsai; Zhang, Silu; Zhao, Saisai; Chang, Huan-Cheng; Lin, Ge; Li, Quan

    2015-06-01

    The prickly nanodiamonds easily entered cells via endocytosis followed by unique intracellular translocation characteristics—quick endosomal escape followed by stable residence in cytoplasm. Endosomal membrane rupturing is identified as the major route of nanodiamonds’ escaping the vesicle confinement and to the cytoplasm. Little cytotoxicity is observed to associate with the nanodiamonds’ cytosolic release. Such features enable its application for gene delivery, which requires both effective cellular uptake and cytosolic release of the gene. Taking green fluorescent protein gene as an example, we demonstrate the successful cytosolic delivery and expression of such a gene using the prickly nanodiamonds as carrier.

  13. Rapid endosomal escape of prickly nanodiamonds: implications for gene delivery.

    PubMed

    Chu, Zhiqin; Miu, Kaikei; Lung, Pingsai; Zhang, Silu; Zhao, Saisai; Chang, Huan-Cheng; Lin, Ge; Li, Quan

    2015-01-01

    The prickly nanodiamonds easily entered cells via endocytosis followed by unique intracellular translocation characteristics—quick endosomal escape followed by stable residence in cytoplasm. Endosomal membrane rupturing is identified as the major route of nanodiamonds' escaping the vesicle confinement and to the cytoplasm. Little cytotoxicity is observed to associate with the nanodiamonds' cytosolic release. Such features enable its application for gene delivery, which requires both effective cellular uptake and cytosolic release of the gene. Taking green fluorescent protein gene as an example, we demonstrate the successful cytosolic delivery and expression of such a gene using the prickly nanodiamonds as carrier. PMID:26123532

  14. Rapid endosomal escape of prickly nanodiamonds: implications for gene delivery

    PubMed Central

    Chu, Zhiqin; Miu, Kaikei; Lung, Pingsai; Zhang, Silu; Zhao, Saisai; Chang, Huan-Cheng; Lin, Ge; Li, Quan

    2015-01-01

    The prickly nanodiamonds easily entered cells via endocytosis followed by unique intracellular translocation characteristics—quick endosomal escape followed by stable residence in cytoplasm. Endosomal membrane rupturing is identified as the major route of nanodiamonds’ escaping the vesicle confinement and to the cytoplasm. Little cytotoxicity is observed to associate with the nanodiamonds’ cytosolic release. Such features enable its application for gene delivery, which requires both effective cellular uptake and cytosolic release of the gene. Taking green fluorescent protein gene as an example, we demonstrate the successful cytosolic delivery and expression of such a gene using the prickly nanodiamonds as carrier. PMID:26123532

  15. Theranostic agents for intracellular gene delivery with spatiotemporal imaging

    PubMed Central

    Knipe, Jennifer M.; Peters, Jonathan T.; Peppas, Nicholas A.

    2013-01-01

    Gene therapy is the modification of gene expression to treat a disease. However, efficient intracellular delivery and monitoring of gene therapeutic agents is an ongoing challenge. Use of theranostic agents with suitable targeted, controlled delivery and imaging modalities has the potential to greatly advance gene therapy. Inorganic nanoparticles including magnetic nanoparticles, gold nanoparticles, and quantum dots have been shown to be effective theranostic agents for the delivery and spatiotemporal tracking of oligonucleotides in vitro and even a few cases in vivo. Major concerns remain to be addressed including cytotoxicity, particularly of quantum dots; effective dosage of nanoparticles for optimal theranostic effect; development of real-time in vivo imaging; and further improvement of gene therapy efficacy. PMID:23606894

  16. Lentivectors for regulated and reversible cutaneous gene delivery.

    PubMed

    Siprashvili, Zurab; Khavari, Paul A

    2004-01-01

    Systemic administration of therapeutic proteins has value in treating a wide variety of disorders, including erythropoietin (Epo)-responsive anemias. Recombinant proteins, however, are costly and require repeated injections, while gene delivery approaches have suffered from inefficiency and difficulties with regulation. The skin effectively delivers polypeptides to the circulation, and improved approaches would support sustainable, topically regulated protein expression after a single vector injection. Toward this goal, we generated lentivectors in which both gene delivery and persistence in skin are regulated by administration of distinct steroid ligands. Following a single injection of regulated lentivector into human skin regenerated on immunodeficient mice, topical glucocorticoid ligands regulated Epo levels and hematocrit over time. Abrogation of gene delivery was achieved by both glucocorticoid cessation and proviral excision via a 4-hydroxytamoxifen-inducible Cre recombinase. These findings establish an approach to durable, topically controlled systemic delivery of therapeutic proteins from human skin tissue.

  17. Biodegradable polymer-metal complexes for gene and drug delivery.

    PubMed

    Hosseinkhani, Hossein; Hosseinkhani, Mohsen

    2009-01-01

    The delivery of genes and drugs into cells has increasingly attracted attention for the generation of genetically engineered cells. Successful drug delivery will have enormous academic, clinical, and practical impacts on gene therapy, cell and molecular biology, pharmaceutical and food industries, and bio-production. The major aim of gene therapy is to deliver genetic materials into cells effectively, genetically modifying and repairing cell functions with the possibility of inducing therapeutic healing of disease. The genetic material includes DNA, RNA, antisense, decoy DNA, and ribozymes. The aim is that the appropriate transfection would allow diseased cells to return to a healthy condition. The genetic manipulation is often manifested in the mechanisms of intracellular actions of genes and proteins, and may play an important role in making clear the key genes associated with various diseases. Based on fundamental and scientific knowledge, the delivery technology of genetic material should be applicable to producing various proteins of pharmaceutical value (e.g. cytokines, growth factors, and antibodies) and also to producing seeds resistant to harmful insects and cold weather damage. This implies that the cells might be enhanced to produce valuable pharmaceutical and food products. For each approach, it is important, for successful gene expression, to select an appropriate gene to be delivered as well as to develop the gene delivery technology to enhance transfection efficiency. This review will provide an overview of the enhanced gene expression of plasmid DNA complexed with new non-viral gene delivery vehicles by biodegradable biopolymer-metal complex, introducing our recent research data to emphasize the technical feasibility of biopolymer-metal complexes in gene therapy and biotechnology.

  18. Cationic Polymer Based Gene Delivery: Uptake and Intracellular Trafficking

    NASA Astrophysics Data System (ADS)

    Ho, Yoonkhei; Too, Heng-Phon

    2014-04-01

    To date, low transfection efficiency remains the major drawback of polymer based gene delivery. Many cell types including stem cells, fibroblast and neurons are known to be poorly transfected with polymer based gene carriers and the high toxicity severely restrict their utility in gene delivery. Continual efforts are made to identify cellular barriers to efficient transfection as these carriers have low immunogenicity, ease of manufacturing and scalability. Here, we summarize the current status of understanding on uptake mechanism of polymer-DNA complexes (polyplexes), their endosomal escape, cytosolic transport and nuclear entry of pDNA.

  19. Characterisation of gene delivery using liposomal bubbles and ultrasound

    NASA Astrophysics Data System (ADS)

    Koshima, Risa; Suzuki, Ryo; Oda, Yusuke; Hirata, Keiichi; Nomura, Tetsuya; Negishi, Yoichi; Utoguchi, Naoki; Kudo, Nobuki; Maruyama, Kazuo

    2011-09-01

    The combination of nano/microbubbles and ultrasound is a novel technique for a non-viral gene deliver. We have previously developed novel ultrasound sensitive liposomes (Bubble liposomes) which contain the ultrasound imaging gas perfluoropropane. In this study, Bubble liposomes were compared with cationic lipid (CL)-DNA complexes as potential gene delivery carriers into tumors in vivo. The delivery of genes by bubble liposomes depended on the intensity of the applied ultrasound. The transfection efficiency plateaued at 0.7 W/cm2 ultrasound intensity. Bubble liposomes efficiently transferred genes into cultured cells even when the cells were exposed to ultrasound for only 1 s. In addition, bubble liposomes were able to introduce the luciferase gene more effectively than CL-DNA complexes into mouse ascites tumor cells. We conclude that the combination of Bubble liposomes and ultrasound is a good method for gene transfer in vivo.

  20. Bioengineered Silk Protein-Based Gene Delivery Systems

    PubMed Central

    Numata, Keiji; Subramanian, Balajikarthick; Currie, Heather A.; Kaplan, David L.

    2009-01-01

    Silk proteins self-assemble into mechanically robust material structures that are also biodegradable and non-cytotoxic, suggesting utility for gene delivery. Since silk proteins can also be tailored in terms of chemistry, molecular weight and other design features via genetic engineering, further control of this system for gene delivery can be considered. In the present study, silk-based block copolymers were bioengineered with poly(l-lysine) domains for gene delivery. Ionic complexes of these silk-polylysine based block copolymers with plasmid DNA (pDNA) were prepared for gene delivery to human embryonic kidney (HEK) cells. The material systems were characterized by agarose gel electrophoresis, atomic force microscopy, and dynamic light scattering. The polymers self-assembled in solution and complexed plasmid DNA through ionic interactions. The pDNA complexes with 30-lysine residues prepared at a polymer/nucleotide ratio of 10 and with a solution diameter of 380 nm, showed the highest efficiency for transfection. The pDNA complexes were also immobilized on silk films and demonstrated direct cell transfection from these surfaces. The results demonstrate the potential of bioengineered silk proteins as a new family of highly tailored gene delivery systems. PMID:19577803

  1. Engineered nonviral nanocarriers for intracellular gene delivery applications.

    PubMed

    Ojea-Jiménez, Isaac; Tort, Olivia; Lorenzo, Julia; Puntes, Victor F

    2012-10-01

    The efficient delivery of nucleic acids into mammalian cells is a central aspect of cell biology and of medical applications, including cancer therapy and tissue engineering. Non-viral chemical methods have been received with great interest for transfecting cells. However, further development of nanocarriers that are biocompatible, efficient and suitable for clinical applications is still required. In this paper, the different material platforms for gene delivery are comparatively addressed, and the mechanisms of interaction with biological systems are discussed carefully.

  2. Engineered nonviral nanocarriers for intracellular gene delivery applications.

    PubMed

    Ojea-Jiménez, Isaac; Tort, Olivia; Lorenzo, Julia; Puntes, Victor F

    2012-10-01

    The efficient delivery of nucleic acids into mammalian cells is a central aspect of cell biology and of medical applications, including cancer therapy and tissue engineering. Non-viral chemical methods have been received with great interest for transfecting cells. However, further development of nanocarriers that are biocompatible, efficient and suitable for clinical applications is still required. In this paper, the different material platforms for gene delivery are comparatively addressed, and the mechanisms of interaction with biological systems are discussed carefully. PMID:22972254

  3. Smart Polymeric Nanoparticles for Cancer Gene Delivery

    PubMed Central

    2015-01-01

    The massive amount of human genetic information already available has accelerated the identification of target genes, making gene and nucleic acid therapy the next generation of medicine. Nanoparticle (NP)-based anticancer gene therapy treatment has received significant interest in this evolving field. Recent advances in vector technology have improved gene transfection efficiencies of nonviral vectors to a level similar to viruses. This review serves as an introduction to surface modifications of NPs based on polymeric structural improvements and target moieties. A discussion regarding the future perspective of multifunctional NPs in cancer therapy is also included. PMID:25531409

  4. Au nanoinjectors for electrotriggered gene delivery into the cell nucleus.

    PubMed

    Kang, Mijeong; Kim, Bongsoo

    2015-01-01

    Intracellular delivery of exogenous materials is an essential technique required for many fundamental biological researches and medical treatments. As our understanding of cell structure and function has been improved and diverse therapeutic agents with a subcellular site of action have been continuously developed, there is a demand to enhance the performance of delivering devices. Ideal intracellular delivery devices should convey various kinds of exogenous materials without deteriorating cell viability regardless of cell type and, furthermore, precisely control the location and the timing of delivery as well as the amount of delivered materials for advanced researches.In this chapter the development of a new intracellular delivery device, a nanoinjector made of a Au (gold) nanowire (a Au nanoinjector) is described in which delivery is triggered by external application of an electric pulse. As a model study, a gene was delivered directly into the nucleus of a neuroblastoma cell, and successful delivery without cell damage was confirmed by the expression of the delivered gene. The insertion of a Au nanoinjector directly into a cell can be generally applied to any kind of cell, and a high degree of surface modification of Au allows attachment of diverse materials such as proteins, small molecules, or nanoparticles as well as genes on Au nanoinjectors. This expands their applicability, and it is expected that they will provide important information on the effects of delivered exogenous materials and consequently contribute to the development of related therapeutic or clinical technologies.

  5. Hyperbranched polysiloxysilane nanoparticles for nonviral gene delivery vectors and nanoprobes

    NASA Astrophysics Data System (ADS)

    Kim, Won Jin; Bonoiu, Adela C.; Lee, Kwang-Sup; Hayakawa, Teruaki; Xia, Cheng; Kakimoto, Masa-aki; Pudavar, Haridas E.; Prasad, Paras N.

    2009-08-01

    We report an approach to produce predefined surface charge tunable gene delivery vectors using siloxysilsane-based polymer for gene delivery studies. To obtain nonviral vectors, new series of hyperbranched polysiloxysilane (HBPS) were synthesized, and the end groups in polymer structures have modified with hydrophilic molecules; in other words, carboxylic acid and quaternary ammonium groups were employed into terminal structures to give the amphiphilicity. The novelty of these amphiphilic HBPS polymers lies in the fact that nanoparticles with different zeta potential (surface charge density) can be easily tailored and functionalized. These polymeric nanoparticles which containing various chemical groups on the surface indicated altered surface charge distributions (from -40 to +64mV). Finally, the use of these nanoparticles as efficient gene delivery vectors was demonstrated by means of in vitro transfection study using β- galactosidase plasmid and pEGFP-N1 plasmid, and the most efficient combination was obtained using HBPS-CN30:70.

  6. Micelles and Nanoparticles for Ultrasonic Drug and Gene Delivery

    PubMed Central

    Husseini, Ghaleb A.; Pitt, William G.

    2008-01-01

    Drug delivery research employing micelles and nanoparticles has expanded in recent years. Of particular interest is the use of these nanovehicles that deliver high concentrations of cytotoxic drugs to diseased tissues selectively, thus reducing the agent’s side effects on the rest of the body. Ultrasound, traditionally used in diagnostic medicine, is finding a place in drug delivery in connection with these nanoparticles. In addition to their non-invasive nature and the fact that they can be focused on targeted tissues, acoustic waves have been credited with releasing pharmacological agents from nanocarriers, as well as rendering cell membranes more permeable. In this article, we summarize new technologies that combine the use of nanoparticles with acoustic power both in drug and gene delivery. Ultrasonic drug delivery from micelles usually employs polyether block copolymers, and has been found effective in vivo for treating tumors. Ultrasound releases drug from micelles, most probably via shear stress and shock waves from collapse of cavitation bubbles. Liquid emulsions and solid nanoparticles are used with ultrasound to deliver genes in vitro and in vivo. The small packaging allows nanoparticles to extravasate into tumor tissues. Ultrasonic drug and gene delivery from nano-carriers has tremendous potential because of the wide variety of drugs and genes that could be delivered to targeted tissues by fairly non-invasive means. PMID:18486269

  7. Surface modification of nonviral nanocarriers for enhanced gene delivery.

    PubMed

    Fortier, Charles; Durocher, Yves; De Crescenzo, Gregory

    2014-01-01

    Biomedical nanotechnology has given a new lease of life to gene therapy with the ever-developing and ever-diversifying nonviral gene delivery nanocarriers. These are designed to pass a series of barriers in order to bring their nucleic acid cargo to the right subcellular location of particular cells. For a given application, each barrier has its dedicated strategy, which translates into a physicochemical, biological and temporal identity of the nanocarrier surface. Different strategies have thus been explored to implement adequate surface identities on nanocarriers over time for systemic delivery. In that context, this review will mainly focus on organic nanocarriers, for which these strategies will be described and discussed. PMID:24354815

  8. Chitosan for gene delivery and orthopedic tissue engineering applications.

    PubMed

    Raftery, Rosanne; O'Brien, Fergal J; Cryan, Sally-Ann

    2013-01-01

    Gene therapy involves the introduction of foreign genetic material into cells in order exert a therapeutic effect. The application of gene therapy to the field of orthopaedic tissue engineering is extremely promising as the controlled release of therapeutic proteins such as bone morphogenetic proteins have been shown to stimulate bone repair. However, there are a number of drawbacks associated with viral and synthetic non-viral gene delivery approaches. One natural polymer which has generated interest as a gene delivery vector is chitosan. Chitosan is biodegradable, biocompatible and non-toxic. Much of the appeal of chitosan is due to the presence of primary amine groups in its repeating units which become protonated in acidic conditions. This property makes it a promising candidate for non-viral gene delivery. Chitosan-based vectors have been shown to transfect a number of cell types including human embryonic kidney cells (HEK293) and human cervical cancer cells (HeLa). Aside from its use in gene delivery, chitosan possesses a range of properties that show promise in tissue engineering applications; it is biodegradable, biocompatible, has anti-bacterial activity, and, its cationic nature allows for electrostatic interaction with glycosaminoglycans and other proteoglycans. It can be used to make nano- and microparticles, sponges, gels, membranes and porous scaffolds. Chitosan has also been shown to enhance mineral deposition during osteogenic differentiation of MSCs in vitro. The purpose of this review is to critically discuss the use of chitosan as a gene delivery vector with emphasis on its application in orthopedic tissue engineering. PMID:23676471

  9. Chitosan for gene delivery and orthopedic tissue engineering applications.

    PubMed

    Raftery, Rosanne; O'Brien, Fergal J; Cryan, Sally-Ann

    2013-05-15

    Gene therapy involves the introduction of foreign genetic material into cells in order exert a therapeutic effect. The application of gene therapy to the field of orthopaedic tissue engineering is extremely promising as the controlled release of therapeutic proteins such as bone morphogenetic proteins have been shown to stimulate bone repair. However, there are a number of drawbacks associated with viral and synthetic non-viral gene delivery approaches. One natural polymer which has generated interest as a gene delivery vector is chitosan. Chitosan is biodegradable, biocompatible and non-toxic. Much of the appeal of chitosan is due to the presence of primary amine groups in its repeating units which become protonated in acidic conditions. This property makes it a promising candidate for non-viral gene delivery. Chitosan-based vectors have been shown to transfect a number of cell types including human embryonic kidney cells (HEK293) and human cervical cancer cells (HeLa). Aside from its use in gene delivery, chitosan possesses a range of properties that show promise in tissue engineering applications; it is biodegradable, biocompatible, has anti-bacterial activity, and, its cationic nature allows for electrostatic interaction with glycosaminoglycans and other proteoglycans. It can be used to make nano- and microparticles, sponges, gels, membranes and porous scaffolds. Chitosan has also been shown to enhance mineral deposition during osteogenic differentiation of MSCs in vitro. The purpose of this review is to critically discuss the use of chitosan as a gene delivery vector with emphasis on its application in orthopedic tissue engineering.

  10. Chlorotoxin labeled magnetic nanovectors for targeted gene delivery to glioma.

    PubMed

    Kievit, Forrest M; Veiseh, Omid; Fang, Chen; Bhattarai, Narayan; Lee, Donghoon; Ellenbogen, Richard G; Zhang, Miqin

    2010-08-24

    Glioma accounts for 80% of brain tumors and currently remains one of the most lethal forms of cancers. Gene therapy could potentially improve the dismal prognosis of patients with glioma, but this treatment modality has not yet reached the bedside from the laboratory due to the lack of safe and effective gene delivery vehicles. In this study we investigate targeted gene delivery to C6 glioma cells in a xenograft mouse model using chlorotoxin (CTX) labeled nanoparticles. The developed nanovector consists of an iron oxide nanoparticle core, coated with a copolymer of chitosan, polyethylene glycol (PEG), and polyethylenimine (PEI). Green fluorescent protein (GFP) encoding DNA was bound to these nanoparticles, and CTX was then attached using a short PEG linker. Nanoparticles without CTX were also prepared as a control. Mice bearing C6 xenograft tumors were injected intravenously with the DNA-bound nanoparticles. Nanoparticle accumulation in the tumor site was monitored using magnetic resonance imaging and analyzed by histology, and GFP gene expression was monitored through Xenogen IVIS fluorescence imaging and confocal fluorescence microscopy. Interestingly, the CTX did not affect the accumulation of nanoparticles at the tumor site but specifically enhanced their uptake into cancer cells as evidenced by higher gene expression. These results indicate that this targeted gene delivery system may potentially improve treatment outcome of gene therapy for glioma and other deadly cancers.

  11. Practical delivery of genes to the marketplace.

    PubMed

    Fischhoff, David A; Cline, Molly N

    2009-01-01

    Although new technologies in genomics are powerful tools for discovering genes and gaining insight into their function, discovery of a gene itself does not ensure its practical application. Commercialization of transgenic crop plants has now taken place for more than a decade. Plant biotechnology, which can be seen as an extension of traditional plant breeding for crop improvement, offers one way to boost food, feed, fiber, and fuel production and has provided significant environmental and economic benefits. Like plant breeding, biotechnology introduces new traits with specific benefits into plants, and does so in a selective, precise, and controlled manner. Several steps are necessary before commercializing a crop with a biotechnology trait, including not only gene discovery and product development but also regulatory clearance, stewardship evaluation, and stakeholder dialogue. Examples will be drawn from the work at Monsanto on the development and commercialization of glyphosate-tolerant soybeans, which is representative of the first wave of agronomic traits. PMID:19347653

  12. Practical delivery of genes to the marketplace.

    PubMed

    Fischhoff, David A; Cline, Molly N

    2009-01-01

    Although new technologies in genomics are powerful tools for discovering genes and gaining insight into their function, discovery of a gene itself does not ensure its practical application. Commercialization of transgenic crop plants has now taken place for more than a decade. Plant biotechnology, which can be seen as an extension of traditional plant breeding for crop improvement, offers one way to boost food, feed, fiber, and fuel production and has provided significant environmental and economic benefits. Like plant breeding, biotechnology introduces new traits with specific benefits into plants, and does so in a selective, precise, and controlled manner. Several steps are necessary before commercializing a crop with a biotechnology trait, including not only gene discovery and product development but also regulatory clearance, stewardship evaluation, and stakeholder dialogue. Examples will be drawn from the work at Monsanto on the development and commercialization of glyphosate-tolerant soybeans, which is representative of the first wave of agronomic traits.

  13. Strategies on the nuclear-targeted delivery of genes

    PubMed Central

    Yao, Jing; Fan, Ying; Li, Yuanke; Huang, Leaf

    2016-01-01

    To improve the nuclear-targeted delivery of non-viral vectors, extensive effort has been carried out on the development of smart vectors which could overcome multiple barriers. The nuclear envelope presents a major barrier to transgene delivery. Viruses are capable of crossing the nuclear envelope to efficiently deliver their genome into the nucleus through the specialized protein components. However, non-viral vectors are preferred over viral ones because of the safety concerns associated with the latter. Non-viral delivery systems have been designed to include various types of components to enable nuclear translocation at the periphery of the nucleus. This review summarizes the progress of research regarding nuclear transport mechanisms. “Smart” non-viral vectors that have been modified by peptides and other small molecules are able to facilitate the nuclear translocation and enhance the efficacy of gene expression. The resulting technology may also enhance delivery of other macromolecules to the nucleus. PMID:23964565

  14. Gene delivery to the heart by magnetic nanobeads

    NASA Astrophysics Data System (ADS)

    Li, Wenzhong; Nesselmann, Catharina; Zhou, Zhaohui; Ong, Lee-Lee; Öri, Ferenc; Tang, Guping; Kaminski, Alexander; Lützow, Karola; Lendlein, Andreas; Liebold, Andreas; Stamm, Christof; Wang, John; Steinhoff, Gustav; Ma, Nan

    2007-04-01

    Gene delivery with non-viral gene vectors to the cardiovascular system suffers from low transfection efficiency. In this study, magnetic fields were investigated to assist cardiovascular gene delivery via magnetic nanobeads both in vitro and in vivo. The magnetic field was provided with a 1120 mT Nd-Fe-B permanent magnet while complexes of poly-ethyleneimine (PEI) and various DNA plasmids were conjugated with magnetic nanobeads (MNB) using a Sulfo-NHS-LC-Biotin linker. In vitro results showed that transfection in two cell lines was 30-80-fold higher in magnetically conjugated MNB/PEI/DNA complexes than transfection from PEI/DNA complexes alone. Similarly, in vivo results using mouse models showed 72 h after injection observable gene expression in the heart with conjugated MNB/PEI/DNA complexes, but barely with PEI/DNA complexes alone.

  15. The magnetofection method: using magnetic force to enhance gene delivery.

    PubMed

    Plank, Christian; Schillinger, Ulrike; Scherer, Franz; Bergemann, Christian; Rémy, Jean-Serge; Krötz, Florian; Anton, Martina; Lausier, Jim; Rosenecker, Joseph

    2003-05-01

    In order to enhance and target gene delivery we have previously established a novel method, termed magnetofection, which uses magnetic force acting on gene vectors that are associated with magnetic particles. Here we review the benefits, the mechanism and the potential of the method with regard to overcoming physical limitations to gene delivery. Magnetic particle chemistry and physics are discussed, followed by a detailed presentation of vector formulation and optimization work. While magnetofection does not necessarily improve the overall performance of any given standard gene transfer method in vitro, its major potential lies in the extraordinarily rapid and efficient transfection at low vector doses and the possibility of remotely controlled vector targeting in vivo.

  16. Physical non-viral gene delivery methods for tissue engineering.

    PubMed

    Mellott, Adam J; Forrest, M Laird; Detamore, Michael S

    2013-03-01

    The integration of gene therapy into tissue engineering to control differentiation and direct tissue formation is not a new concept; however, successful delivery of nucleic acids into primary cells, progenitor cells, and stem cells has proven exceptionally challenging. Viral vectors are generally highly effective at delivering nucleic acids to a variety of cell populations, both dividing and non-dividing, yet these viral vectors are marred by significant safety concerns. Non-viral vectors are preferred for gene therapy, despite lower transfection efficiencies, and possess many customizable attributes that are desirable for tissue engineering applications. However, there is no single non-viral gene delivery strategy that "fits-all" cell types and tissues. Thus, there is a compelling opportunity to examine different non-viral vectors, especially physical vectors, and compare their relative degrees of success. This review examines the advantages and disadvantages of physical non-viral methods (i.e., microinjection, ballistic gene delivery, electroporation, sonoporation, laser irradiation, magnetofection, and electric field-induced molecular vibration), with particular attention given to electroporation because of its versatility, with further special emphasis on Nucleofection™. In addition, attributes of cellular character that can be used to improve differentiation strategies are examined for tissue engineering applications. Ultimately, electroporation exhibits a high transfection efficiency in many cell types, which is highly desirable for tissue engineering applications, but electroporation and other physical non-viral gene delivery methods are still limited by poor cell viability. Overcoming the challenge of poor cell viability in highly efficient physical non-viral techniques is the key to using gene delivery to enhance tissue engineering applications.

  17. Bioengineered Silk Gene Delivery System for Nuclear Targeting

    PubMed Central

    Yigit, Sezin; Tokareva, Olena; Varone, Antonio; Georgakoudi, Irene

    2015-01-01

    Gene delivery research has gained momentum with the use of lipophilic vectors that mimic viral systems to increase transfection efficiency. However, maintaining cell viability with these systems remains a major challenge. Therefore biocompatible and nontoxic biopolymers that are designed by combining non-immunological viral mimicking components with suitable carriers have been explored to address these limitations. In the present study recombinant DNA technology was used to design a multi-functional gene delivery system for nuclear targeting, while also supporting cell viability. Spider dragline silk recombinant proteins were modified with DNA condensing units and the proton sponge endosomal escape pathway was utilized for enhanced delivery. Short-term transfection efficiency in a COS-7 cell line (adherent kidney cells isolated from African green monkey) was enhanced compared to lipofectamine and polyethyleneimine (PEI), as was cell viability with these recombinant bio-polyplexes. Endosomal escape and consequent nuclear targeting were shown with fluorescence microscopy. PMID:24889658

  18. Novel Approaches for Retinal Drug and Gene Delivery

    PubMed Central

    Kim, Stephen J.

    2014-01-01

    The ARVO 2014 minisymposium on “Novel Approaches for Retinal Drug and Gene Delivery” was held on May 6, 2014 in Orlando, FL. The main intent of the symposium was to review recent advances in retinal drug and gene delivery with specific emphasis on novel approaches that address current limitations and have the potential to translate into clinical practice. The symposium was sponsored by Translational Vision Science and Technology. PMID:25346872

  19. Gene Delivery Strategies to Promote Spinal Cord Repair

    PubMed Central

    Walthers, Christopher M; Seidlits, Stephanie K

    2015-01-01

    Gene therapies hold great promise for the treatment of many neurodegenerative disorders and traumatic injuries in the central nervous system. However, development of effective methods to deliver such therapies in a controlled manner to the spinal cord is a necessity for their translation to the clinic. Although essential progress has been made to improve efficiency of transgene delivery and reduce the immunogenicity of genetic vectors, there is still much work to be done to achieve clinical strategies capable of reversing neurodegeneration and mediating tissue regeneration. In particular, strategies to achieve localized, robust expression of therapeutic transgenes by target cell types, at controlled levels over defined time periods, will be necessary to fully regenerate functional spinal cord tissues. This review summarizes the progress over the last decade toward the development of effective gene therapies in the spinal cord, including identification of appropriate target genes, improvements to design of genetic vectors, advances in delivery methods, and strategies for delivery of multiple transgenes with synergistic actions. The potential of biomaterials to mediate gene delivery while simultaneously providing inductive scaffolding to facilitate tissue regeneration is also discussed. PMID:25922572

  20. Thymine, adenine and lipoamino acid based gene delivery systems.

    PubMed

    Skwarczynski, Mariusz; Ziora, Zyta M; Coles, Daniel J; Lin, I-Chun; Toth, Istvan

    2010-05-14

    A novel class of thymine, adenine and lipoamino acid based non-viral carriers for gene delivery has been developed. Their ability to bind to DNA by hydrogen bonding was confirmed by NMR diffusion, isothermal titration calorimetry and transmission electron microscopy experiments.

  1. Upconversion luminescence tracking of gene delivery via multifunctional nanocapsules.

    PubMed

    Bai, Xilin; Xu, Suying; Liu, Jiali; Wang, Leyu

    2016-04-01

    The real-time fluorescence tracking of gene delivery is very important as it helps to figure out how a vector enters a cell and also to follow its fate within the cell interior. Lanthanide-doped upconversion nanoparticles (UCNPs) have shown great potential in biomedical applications in virtue of their unique optical and biological properties. Herein, we report a simple and versatile strategy to fabricate a multifunctional nanocapsule for effective gene delivery and real-time luminescence tracking. The hydrophobic UCNPs were modified by positively charged amphiphilic polymer together with polyethylene glycol-poly(lactic-co-glycolic acid) (PEG-PLGA) polymer, affording biocompatible nanocapsules with high gene loading capacity and good stability. Red UC luminescence of UCNPs are able to track the delivery of nanocapsules in cells without background fluorescence interference, in the meantime, the green fluorescence of green fluorescence protein (GFP) expressed by the pDNA could subtly monitor the gene transfection efficacy. The results demonstrated that our nanocapsule has ideal biocompatibility, satisfactory gene loading capacity and great bioimaging ability, which is promising for imaging guided cell therapy and gene engineering. PMID:26838389

  2. Aerosol delivery of programmed cell death protein 4 using polysorbitol-based gene delivery system for lung cancer therapy.

    PubMed

    Kim, You-Kyoung; Xing, Lei; Chen, Bao-An; Xu, Fengguo; Jiang, Hu-Lin; Zhang, Can

    2014-11-01

    The development of a safe and effective gene delivery system is the most challenging obstacle to the broad application of gene therapy in the clinic. In this study, we report the development of a polysorbitol-based gene delivery system as an alternative gene carrier for lung cancer therapy. The copolymer was prepared by a Michael addition reaction between sorbitol diacrylate (SD) and spermine (SPE); the SD-SPE copolymer effectively condenses with DNA on the nanoscale and protects it from nucleases. SD-SPE/DNA complexes showed excellent transfection with low toxicity both in vitro and in vivo, and aerosol delivery of SD-SPE complexes with programmed cell death protein 4 DNA significantly suppressed lung tumorigenesis in K-ras(LA1) lung cancer model mice. These results demonstrate that SD-SPE has great potential as a gene delivery system based on its excellent biocompatibility and high gene delivery efficiency for lung cancer gene therapy. PMID:24983766

  3. Delivery of gene-expressing fragments using quantum dot

    NASA Astrophysics Data System (ADS)

    Hoshino, Akiyoshi; Manabe, Noriyoshi; Hanada, Sanshiro; Fujioka, Kouki; Yasuhara, Masato; Kondo, Akihiko; Yamamoto, Kenji

    2009-02-01

    Gene therapy is an attractive approach to supplement a deficient gene function. Although there has been some success with specific gene delivery using various methods including viral vectors and liposomes, most of these methods have a limited efficiency or also carry a risk for oncogenesis. Fluorescent nanoparticles, such as nanocrystal quantum dots (QDs), have potential to be applied to molecular biology and bioimaging, since some nanocrystals emit higher and longer lasting fluorescence than conventional organic probes do. We herein report that quantum dots (QDs) conjugated with nuclear localizing signal peptides (NLSP) successfully introduced the gene-fragments with promoter elements, which promoted the expression of the enhanced green fluorescent protein (eGFP) gene in mammalian cells. The expression of eGFP protein was observed when the QD/geneconstruct was added to the culture media. The gene-expression efficiency varied depending on multiple factors around QDs, such as 1) the reading direction of gene fragments, 2) the quantity of gene fragments attached on the surface of QD-constructs, 3) the surface electronic charges varied according to the structure of QD/gene-constructs, and 4) the particle size of QD/gene complex varied according to the structure and amounts of gene fragments. Using this QD/geneconstruct system, eGFP protein could be detected 28 days after the gene-introduction whereas the fluorescence of QDs was disappeared. This system therefore provides another method for the intracellular delivery of gene-fragments without using either viral vectors or specific liposomes. These results suggest that inappropriate treatment and disposal of QDs may still have risks to the environmental pollution including human health under certain conditions. Here we propose the further research for the immune and physiological responses in not only immune cells but also other cells, in order to clear the effect of all other nanoscale products as well as nanocrystal

  4. Gene Delivery Potential of Biofunctional Carbonate Apatite Nanoparticles in Lungs

    PubMed Central

    Alhaji, Suleiman Yusuf; Chowdhury, Ezharul Houque; Rosli, Rozita

    2014-01-01

    Existing nonviral gene delivery systems to lungs are inefficient and associated with dose limiting toxicity in mammalian cells. Therefore, carbonate apatite (CO3Ap) nanoparticles were examined as an alternative strategy for effective gene delivery to the lungs. This study aimed to (1) assess the gene delivery efficiency of CO3Ap in vitro and in mouse lungs, (2) evaluate the cytotoxicity effect of CO3Ap/pDNA in vitro, and (3) characterize the CO3Ap/pDNA complex formulations. A significantly high level of reporter gene expression was detected from the lung cell line transfected with CO3Ap/pDNA complex prepared in both serum and serum-free medium. Cytotoxicity analysis revealed that the percentage of the viable cells treated with CO3Ap to be almost similar to the untreated cells. Characterization analyses showed that the CO3Ap/pDNA complexes are in a nanometer range with aggregated spherical structures and tended to be more negatively charged. In the lung of mice, highest level of transgene expression was observed when CO3Ap (8 μL) was complexed with 40 μg of pDNA at day 1 after administration. Although massive reduction of gene expression was seen beyond day 1 post administration, the level of expression remained significant throughout the study period. PMID:25143941

  5. Gene gun delivery systems for cancer vaccine approaches.

    PubMed

    Aravindaram, Kandan; Yang, Ning Sun

    2009-01-01

    Gene-based immunization with transgenic DNA vectors expressing tumor-associated antigens (TAA), cytokines, or chemokines, alone or in combination, provides an attractive approach to increase the cytotoxic T cell immunity against various cancer diseases. With this consideration, particle-mediated or gene gun technology has been developed as a nonviral method for gene transfer into various mammalian tissues. It has been shown to induce both humoral and cell-mediated immune responses in both small and large experimental animals. A broad range of somatic cell types, including primary cultures and established cell lines, has been successfully transfected ex vivo or in vitro by gene gun technology, either as suspension or adherent cultures. Here, we show that protocols and techniques for use in gene gun-mediated transgene delivery system for skin vaccination against melanoma using tumor-associated antigen (TAA) human gpl00 and reporter gene assays as experimental systems.

  6. Baculovirus-mediated gene delivery and RNAi applications.

    PubMed

    Makkonen, Kaisa-Emilia; Airenne, Kari; Ylä-Herttulala, Seppo

    2015-04-22

    Baculoviruses are widely encountered in nature and a great deal of data is available about their safety and biology. Recently, these versatile, insect-specific viruses have demonstrated their usefulness in various biotechnological applications including protein production and gene transfer. Multiple in vitro and in vivo studies exist and support their use as gene delivery vehicles in vertebrate cells. Recently, baculoviruses have also demonstrated high potential in RNAi applications in which several advantages of the virus make it a promising tool for RNA gene transfer with high safety and wide tropism.

  7. Baculovirus-mediated Gene Delivery and RNAi Applications

    PubMed Central

    Makkonen, Kaisa-Emilia; Airenne, Kari; Ylä-Herttulala, Seppo

    2015-01-01

    Baculoviruses are widely encountered in nature and a great deal of data is available about their safety and biology. Recently, these versatile, insect-specific viruses have demonstrated their usefulness in various biotechnological applications including protein production and gene transfer. Multiple in vitro and in vivo studies exist and support their use as gene delivery vehicles in vertebrate cells. Recently, baculoviruses have also demonstrated high potential in RNAi applications in which several advantages of the virus make it a promising tool for RNA gene transfer with high safety and wide tropism. PMID:25912715

  8. Physical methods for gene transfer: improving the kinetics of gene delivery into cells.

    PubMed

    Mehier-Humbert, Sophie; Guy, Richard H

    2005-04-01

    One factor critical to successful gene therapy is the development of efficient delivery systems. Although advances in gene transfer technology, including viral and non-viral vectors, have been made, an ideal vector system has not yet been constructed. This review describes the basic principles behind various physical methods for gene transfer and assesses the advantages and performance of such approaches, compared to other transfection systems. In particular, the kinetics and efficiency of gene delivery, the toxicity, in vivo feasibility, and targeting ability of different physical methodologies are discussed and evaluated.

  9. The design of cationic lipids for gene delivery.

    PubMed

    Martin, B; Sainlos, M; Aissaoui, A; Oudrhiri, N; Hauchecorne, M; Vigneron, J-P; Lehn, J-M; Lehn, P

    2005-01-01

    Synthetic gene delivery vectors are gaining increasing importance in gene therapy as an alternative to recombinant viruses. Among the various types of non-viral vectors, cationic lipids are especially attractive as they can be prepared with relative ease and extensively characterised. Further, each of their constituent parts can be modified, thereby facilitating the elucidation of structure-activity relationships. In this forward-looking review, cationic lipid-mediated gene delivery will mainly be discussed in terms of the structure of the three basic constituent parts of any cationic lipid: the polar headgroup, hydrophobic moiety and linker. Particular emphasis will be placed on recent advances in the field as well as on our own original contributions. In addition to reviewing critical physicochemical features (such as headgroup hydration) of monovalent lipids, the use of headgroups with known nucleic-acid binding modes, such as linear and branched polyamines, aminoglycosides and guanidinium functions, will be comprehensively assessed. A particularly exciting innovation in linker design is the incorporation of environment-sensitive groups, the intracellular hydrolysis of which may lead to more controlled DNA delivery. Examples of pH-, redox- and enzyme-sensitive functional groups integrated into the linker are highlighted and the benefits of such degradable vectors can be evaluated in terms of transfection efficiency and cationic lipid-associated cytotoxicity. Finally, possible correlations between the length and type of hydrophobic moiety and transfection efficiency will be discussed. In conclusion it may be foreseen that in order to be successful, the future of cationic lipid-based gene delivery will probably require the development of sophisticated virus-like systems, which can be viewed as "programmed supramolecular systems" incorporating the various functions required to perform in a chronological order the different steps involved in gene transfection.

  10. Toxicity of cationic lipids and cationic polymers in gene delivery.

    PubMed

    Lv, Hongtao; Zhang, Shubiao; Wang, Bing; Cui, Shaohui; Yan, Jie

    2006-08-10

    Gene therapy, as a promising therapeutics to treat genetic or acquired diseases, has achieved exciting development in the past two decades. Appropriate gene vectors can be crucial for gene transfer. Cationic lipids and polymers, the most important non-viral vectors, have many advantages over viral ones as non-immunogenic, easy to produce and not oncogenic. They hold the promise to replace viral vectors to be used in clinic. However, the toxicity is still an obstacle to the application of non-viral vectors to gene therapy. For overcoming the problem, many new cationic compounds have been developed. This article provides a review with respect to toxicity of cationic lipids and polymers in gene delivery. We evaluate the structural features of cationic compounds and summarize the relationship of toxicity and structure and hope to provide available suggestions on the development of these cationic compounds.

  11. Adenovirus receptors and their implications in gene delivery

    PubMed Central

    Sharma, Anurag; Li, Xiaoxin; Bangari, Dinesh S.; Mittal, Suresh K.

    2010-01-01

    Adenoviruses (Ads) have gained popularity as gene delivery vectors for therapeutic and prophylactic applications. Ad entry into host cells involves specific interactions between cell surface receptors and viral capsid proteins. Several cell surface molecules have been identified as receptors for Ad attachment and entry. Tissue tropism of Ad vectors is greatly influenced by their receptor usage. A variety of strategies have been investigated to modify Ad vector tropism by manipulating the receptor-interacting moieties. Many such strategies are aimed at targeting and/or detargeting of Ad vectors. In this review, we discuss the various cell surface molecules that are implicated as receptors for virus attachment and internalization. Special emphasis is given to Ad types that are utilized as gene delivery vectors. Various strategies to modify Ad tropism using the knowledge of Ad receptors are also discussed. PMID:19647886

  12. Dendrimer type bio-reducible polymer for efficient gene delivery.

    PubMed

    Nam, Hye Yeong; Nam, Kihoon; Lee, Minhyung; Kim, Sung Wan; Bull, David A

    2012-06-28

    Arginine-grafted bio-reducible poly(disulfide amine) (ABP) was incorporated into the poly(amido amine) (PAMAM) dendrimer, creating a high molecular weight bio-reducible polymer, PAM-ABP, to overcome the limitations of the low molecular weight ABP. The newly synthesized PAM-ABP was studied to determine its efficacy as a gene delivery carrier. The PAM-ABP demonstrated superior condensing ability for plasmid DNA through the formation of compact nanosized polyplexes. These compact polyplexes enhanced cellular uptake and were less susceptible to reducing agents, resulting in greater transfection efficiency compared to ABP alone. Based on these results, this newly developed PAM-ABP polyplex is a promising delivery system for clinical gene therapy. PMID:22546681

  13. Characterization of novel cationic amphiphiles for gene delivery

    NASA Astrophysics Data System (ADS)

    Zhang, Xiaoxiang

    Gene therapy has drawn vast interest for treating, preventing, or controlling a myriad of diseases. The two most common methods for gene delivery use either synthetic or viral vectors. Viral vectors (infection) are by far the most effective and efficient means of DNA delivery, but their use is tempered by safety and immunogenicity concerns. Consequently, there has been a significant effort to develop and evaluate non- viral vectors, which include cationic amphiphiles and polymers, and more recently anionic amphiphiles. Non-viral vectors have the advantages of ease of production, better stability and low immunogenicity. At the same time, they also have a number of limitations, including low in vitro and in vivo transfection efficiencies, and cytotoxicity in many instances. My research project has been focused on design, development and characterizations of novel amphiphilic lipids for gene delivery. Through rational design and characterization of the amphiphile structures, it not only yielded vectors showing high transfection activities, but also provided information of the structure-activity relationship. These results provide us with a better understanding on the transfection process and future directions to further optimize the amphiphile structures. More specifically, my dissertation research included the following three parts: (i) characterization of novel lipopeptides possessing di- or tri- peptide head groups; (ii) determination of the effect of spacer (between the cationic domain and the hydrophobic domain of charge-reversal amphiphiles) length, rigidity and hydrophilicity on gene delivery; (iii) identification of the cellular uptake pathway and the transfection mechanism of a known enzyme-sensitive charge-reversal amphiphile.

  14. Chondroitin sulfate-based nanocarriers for drug/gene delivery.

    PubMed

    Zhao, Lili; Liu, Mengrui; Wang, Juan; Zhai, Guangxi

    2015-11-20

    In recent years, the naturally occurring polysaccharides captured an increasing amount of attention in the field of drug/gene delivery systems owing to their outstanding propensities, including biocompatibility, biodegradability, non-immunogenicity, extremely low toxicity, and so on. Chondroitin sulfate (ChS), a member of glycosaminoglycan family, consists of repeating disaccharide units of b-1,3-linked N-acetyl galactosamine (GalNAc) and b-1,4-linked d-glucuronic acid (GlcA) with certain position(s) sulfated, which has been widely applied in nano-sized carriers. This review will focus on shared and unique properties of ChS and its latest development in drug/gene delivery systems. In detail, the application of ChS as nanocarriers will be discussed in three dimensions: self-assembly of hydrophobically modified ChS, ChS decorated nanocarriers, and some other nanocarriers based on ChS. A discussion relating to the future perspectives of ChS-based nanocarriers for drug/gene delivery is also included.

  15. New serine-derived gemini surfactants as gene delivery systems.

    PubMed

    Cardoso, Ana M; Morais, Catarina M; Cruz, A Rita; Silva, Sandra G; do Vale, M Luísa; Marques, Eduardo F; de Lima, Maria C Pedroso; Jurado, Amália S

    2015-01-01

    Gemini surfactants have been extensively used for in vitro gene delivery. Amino acid-derived gemini surfactants combine the special aggregation properties characteristic of the gemini surfactants with high biocompatibility and biodegradability. In this work, novel serine-derived gemini surfactants, differing in alkyl chain lengths and in the linker group bridging the spacer to the headgroups (amine, amide and ester), were evaluated for their ability to mediate gene delivery either per se or in combination with helper lipids. Gemini surfactant-based DNA complexes were characterized in terms of hydrodynamic diameter, surface charge, stability in aqueous buffer and ability to protect DNA. Efficient formulations, able to transfect up to 50% of the cells without causing toxicity, were found at very low surfactant/DNA charge ratios (1/1-2/1). The most efficient complexes presented sizes suitable for intravenous administration and negative surface charge, a feature known to preclude potentially adverse interactions with serum components. This work brings forward a new family of gemini surfactants with great potential as gene delivery systems.

  16. Chondroitin sulfate-based nanocarriers for drug/gene delivery.

    PubMed

    Zhao, Lili; Liu, Mengrui; Wang, Juan; Zhai, Guangxi

    2015-11-20

    In recent years, the naturally occurring polysaccharides captured an increasing amount of attention in the field of drug/gene delivery systems owing to their outstanding propensities, including biocompatibility, biodegradability, non-immunogenicity, extremely low toxicity, and so on. Chondroitin sulfate (ChS), a member of glycosaminoglycan family, consists of repeating disaccharide units of b-1,3-linked N-acetyl galactosamine (GalNAc) and b-1,4-linked d-glucuronic acid (GlcA) with certain position(s) sulfated, which has been widely applied in nano-sized carriers. This review will focus on shared and unique properties of ChS and its latest development in drug/gene delivery systems. In detail, the application of ChS as nanocarriers will be discussed in three dimensions: self-assembly of hydrophobically modified ChS, ChS decorated nanocarriers, and some other nanocarriers based on ChS. A discussion relating to the future perspectives of ChS-based nanocarriers for drug/gene delivery is also included. PMID:26344295

  17. Viral Vectors for Gene Delivery to the Central Nervous System

    PubMed Central

    Lentz, Thomas B.; Gray, Steven J.; Samulski, R. Jude

    2011-01-01

    The potential benefits of gene therapy for neurological diseases such as Parkinson’s, Amyotrophic Lateral Sclerosis (ALS), Epilepsy, and Alzheimer’s are enormous. Even a delay in the onset of severe symptoms would be invaluable to patients suffering from these and other diseases. Significant effort has been placed in developing vectors capable of delivering therapeutic genes to the CNS in order to treat neurological disorders. At the forefront of potential vectors, viral systems have evolved to efficiently deliver their genetic material to a cell. The biology of different viruses offers unique solutions to the challenges of gene therapy, such as cell targeting, transgene expression and vector production. It is important to consider the natural biology of a vector when deciding whether it will be the most effective for a specific therapeutic function. In this review, we outline desired features of the ideal vector for gene delivery to the CNS and discuss how well available viral vectors compare to this model. Adeno-associated virus, retrovirus, adenovirus and herpesvirus vectors are covered. Focus is placed on features of the natural biology that have made these viruses effective tools for gene delivery with emphasis on their application in the CNS. Our goal is to provide insight into features of the optimal vector and which viral vectors can provide these features. PMID:22001604

  18. Dual delivery systems based on polyamine analog BENSpm as prodrug and gene delivery vectors

    NASA Astrophysics Data System (ADS)

    Zhu, Yu

    Combination drug and gene therapy shows promise in cancer treatment. However, the success of such strategy requires careful selection of the therapeutic agents, as well as development of efficient delivery vectors. BENSpm (N 1, N11-bisethylnorspermine), a polyamine analogue targeting the intracellular polyamine pathway, draws our special attention because of the following reasons: (1) polyamine pathway is frequently dysregulated in cancer; (2) BENSpm exhibits multiple functions to interfere with the polyamine pathway, such as to up-regulate polyamine metabolism enzymes and down-regulate polyamine biosynthesis enzymes. Therefore BENSpm depletes all natural polyamines and leads to apoptosis and cell growth inhibition in a wide range of cancers; (3) preclinical studies proved that BENSpm can act synergistically with various chemotherapy agents, making it a promising candidate in combination therapy; (4) multiple positive charges in BENSpm enable it as a suitable building block for cationic polymers, which can be further applied to gene delivery. In this dissertation, our goal was to design dual-function delivery vector based on BENSpm that can function as a gene delivery vector and, after intracellular degradation, as an active anticancer agent targeting dysregulated polyamine metabolism. We first demonstrated strong synergism between BENSpm and a potential therapeutic gene product TRAIL. Strong synergism was obtained in both estrogen-dependent MCF-7 breast cancer cells and triple-negative MDA-MB-231 breast cancer cells. Significant dose reduction of TRAIL in combination with BENSpm in MDA-MB-231 cells, together with the fact that BENSpm rendered MCF-7 cells more sensitive to TRAIL treatment verified our rationale of designing BENSpm-based delivery platform. This was expected to be beneficial for overcoming drug resistance in chemotherapy, as well as boosting the therapeutic effect of therapeutic genes. We first designed a lipid-based BENSpm dual vector (Lipo

  19. Modification of nanostructured calcium carbonate for efficient gene delivery.

    PubMed

    Zhao, Dong; Wang, Chao-Qun; Zhuo, Ren-Xi; Cheng, Si-Xue

    2014-06-01

    In this study, a facile method to modify nanostructured calcium carbonate (CaCO3) gene delivery systems by adding calcium phosphate (CaP) component was developed. CaCO3/CaP/DNA nanoparticles were prepared by the co-precipitation of Ca(2+) ions with plasmid DNA in the presence of carbonate and phosphate ions. For comparison, CaCO3/DNA nanoparticles and CaP/DNA co-precipitates were also prepared. The effects of carbonate ion/phosphate ion (CO3(2-)/PO4(3-)) ratio on the particle size and gene delivery efficiency were investigated. With an appropriate CO3(2-)/PO4(3-) ratio, the co-existence of carbonate and phosphate ions could control the size of co-precipitates effectively, and CaCO3/CaP/DNA nanoparticles with a decreased size and improved stability could be obtained. The in vitro gene transfections mediated by different nanoparticles in 293T cells and HeLa cells were carried out, using pGL3-Luc as a reporter plasmid. The gene transfection efficiency of CaCO3/CaP/DNA nanoparticles could be significantly improved as compared with CaCO3/DNA nanoparticles and CaP/DNA co-precipitates. The confocal microscopy study indicated that the cellular uptake and nuclear localization of CaCO3/CaP/DNA nanoparticles were significantly enhanced as compared with unmodified CaCO3/DNA nanoparticles.

  20. Gene therapy for cancer: dairy bacteria as delivery vectors.

    PubMed

    Tangney, Mark

    2010-09-01

    The prime obstacle to achieving an effective treatment for cancer is that of eradicating tumors without harming healthy organs and cells of the patient. The concept of utilizing biological agents for delivery of therapeutic genes to patients to kill cancer cells has been under investigation for two decades, which exploits the natural ability of disease causing microbes to invade human cells. Safety-modified versions of pathogenic viruses or bacteria can deposit genes and induce production of anti-cancer agents upon administration to tumors and promising clinical trial successes have been achieved with various types of gene delivery vehicles. Bacteria present an attractive class of gene vectors, possessing a natural ability to grow specifically within tumors following intravenous (IV) injection. Several species such as Clostridium and Salmonella have been examined in clinical trials. However, as foreign, disease-causing bugs, their inherent toxicity has outweighed therapeutic responses in patients, despite efforts to reduce toxicity through genetic modification. A promising alternative exploits non-pathogenic bacterial species that have an existing natural relationship with humans. Our recent study (Cronin et al., 2010) has demonstrated that IV injection or ingestion of a species of probiotic bacterium, Bifidobacterium breve, in high numbers, results in trafficking of the bacteria throughout the body and accumulation specifically within cancerous tissue.

  1. Retargeted adenoviruses for radiation-guided gene delivery

    PubMed Central

    Kaliberov, S A; Kaliberova, L N; Yan, H; Kapoor, V; Hallahan, D E

    2016-01-01

    The combination of radiation with radiosensitizing gene delivery or oncolytic viruses promises to provide an advantage that could improve the therapeutic results for glioblastoma. X-rays can induce significant molecular changes in cancer cells. We isolated the GIRLRG peptide that binds to radiation-inducible 78 kDa glucose-regulated protein (GRP78), which is overexpressed on the plasma membranes of irradiated cancer cells and tumor-associated microvascular endothelial cells. The goal of our study was to improve tumor-specific adenovirus-mediated gene delivery by selectively targeting the adenovirus binding to this radiation-inducible protein. We employed an adenoviral fiber replacement approach to conduct a study of the targeting utility of GRP78-binding peptide. We have developed fiber-modified adenoviruses encoding the GRP78-binding peptide inserted into the fiber-fibritin. We have evaluated the reporter gene expression of fiber-modified adenoviruses in vitro using a panel of glioma cells and a human D54MG tumor xenograft model. The obtained results demonstrated that employment of the GRP78-binding peptide resulted in increased gene expression in irradiated tumors following infection with fiber-modified adenoviruses, compared with untreated tumor cells. These studies demonstrate the feasibility of adenoviral retargeting using the GRP78-binding peptide that selectively recognizes tumor cells responding to radiation treatment. PMID:27492853

  2. Polyphosphazenes: Multifunctional, Biodegradable Vehicles for Drug and Gene Delivery

    PubMed Central

    Teasdale, Ian; Brüggemann, Oliver

    2014-01-01

    Poly[(organo)phosphazenes] are a unique class of extremely versatile polymers with a range of applications including tissue engineering and drug delivery, as hydrogels, shape memory polymers and as stimuli responsive materials. This review aims to divulge the basic principles of designing polyphosphazenes for drug and gene delivery and portray the huge potential of these extremely versatile materials for such applications. Polyphosphazenes offer a number of distinct advantages as carriers for bioconjugates; alongside their completely degradable backbone, to non-toxic degradation products, they possess an inherently and uniquely high functionality and, thanks to recent advances in their polymer chemistry, can be prepared with controlled molecular weights and narrow polydispersities, as well as self-assembled supra-molecular structures. Importantly, the rate of degradation/hydrolysis of the polymers can be carefully tuned to suit the desired application. In this review we detail the recent developments in the chemistry of polyphosphazenes, relevant to drug and gene delivery and describe recent investigations into their application in this field. PMID:24729871

  3. Nanomedicines for Back of the Eye Drug Delivery, Gene Delivery, and Imaging

    PubMed Central

    Kompella, Uday B.; Amrite, Aniruddha C.; Ravi, Rashmi Pacha; Durazo, Shelley A.

    2013-01-01

    Treatment and management of diseases of the posterior segment of the eye such as diabetic retinopathy, retinoblastoma, retinitis pigmentosa, and choroidal neovascularization is a challenging task due to the anatomy and physiology of ocular barriers. For instance, traditional routes of drug delivery for therapeutic treatment are hindered by poor intraocular penetration and/or rapid ocular elimination. One possible approach to improve ocular therapy is to employ nanotechnology. Nanomedicines, products of nanotechnology, having at least one dimension in the nanoscale include nanoparticles, micelles, nanotubes, and dendrimers, with and without targeting ligands, are making a significant impact in the fields of ocular drug delivery, gene delivery, and imaging, the focus of this review. Key applications of nanotechnology discussed in this review include a) bioadhesive nanomedicines; b) functionalized nanomedicines that enhance target recognition and/or cell entry; c) nanomedicines capable of controlled release of the payload; d) nanomedicines capable of enhancing gene transfection and duration of transfection; f) nanomedicines responsive to stimuli including light, heat, ultrasound, electrical signals, pH, and oxidative stress; g) diversely sized and colored nanoparticles for imaging, and h) nanowires for retinal prostheses. Additionally, nanofabricated delivery systems including implants, films, microparticles, and nanoparticles are described. Although the above nanomedicines may be administered by various routes including topical, intravitreal, intravenous, transscleral, suprachoroidal, and subretinal routes, each nanomedicine should be tailored for the disease, drug, and site of administration. In addition to the nature of materials used in nanomedicine design, depending on the site of nanomedicine administration, clearance and toxicity are expected to differ. PMID:23603534

  4. Nanoparticle-mediated delivery of suicide genes in cancer therapy.

    PubMed

    Vago, Riccardo; Collico, Veronica; Zuppone, Stefania; Prosperi, Davide; Colombo, Miriam

    2016-09-01

    Conventional chemotherapeutics have been employed in cancer treatment for decades due to their efficacy in killing the malignant cells, but the other side of the coin showed off-target effects, onset of drug resistance and recurrences. To overcome these limitations, different approaches have been investigated and suicide gene therapy has emerged as a promising alternative. This approach consists in the introduction of genetic materials into cancerous cells or the surrounding tissue to cause cell death or retard the growth of the tumor mass. Despite promising results obtained both in vitro and in vivo, this innovative approach has been limited, for long time, to the treatment of localized tumors, due to the suboptimal efficiency in introducing suicide genes into cancer cells. Nanoparticles represent a valuable non-viral delivery system to protect drugs in the bloodstream, to improve biodistribution, and to limit side effects by achieving target selectivity through surface ligands. In this scenario, the real potential of suicide genes can be translated into clinically viable treatments for patients. In the present review, we summarize the recent advances of inorganic nanoparticles as non-viral vectors in terms of therapeutic efficacy, targeting capacity and safety issues. We describe the main suicide genes currently used in therapy, with particular emphasis on toxin-encoding genes of bacterial and plant origin. In addition, we discuss the relevance of molecular targeting and tumor-restricted expression to improve treatment specificity to cancer tissue. Finally, we analyze the main clinical applications, limitations and future perspectives of suicide gene therapy.

  5. Poly(ethylenimine) conjugated bioreducible dendrimer for efficient gene delivery.

    PubMed

    Nam, Kihoon; Jung, Simhyun; Nam, Joung-Pyo; Kim, Sung Wan

    2015-12-28

    Branched poly(ethylenimine) (PEI) 25 kDa is an efficient gene delivery vector with outstanding gene condensation ability and great endosome escape activity. However, it also induces higher cytotoxicity. Transfection efficiency and toxicity of PEI are highly dependent upon their molecular weight and structure. We developed a bioreducible poly(ethylenimine) (PEI (-s-s-)) derived from low molecular weight PEI (1.8 kDa) for efficient gene delivery. Bioreducible core molecule is expected to increase molecular weight and reduce the cytotoxicity of the copolymer. PEI (-s-s-) polyplexes showed higher transfection efficiency and lower cytotoxicity compared to branched PEI 25 kDa, Lipofectamine® 2000 and, FuGENE® 6. In addition, PEI (-s-s-) derivative (16 kDa) formed stable polyplexes with a zeta-potential value of +34 mV and polyplex size of 61 nm. PEI (-s-s-) derivative (16 kDa) showed excellent transfection efficiency: 3.6 times higher than branched PEI 25 kDa in HeLa cells and 7.4 times higher than Lipofectamine® 2000 in H9C2 cell. The derivatives also showed lower cytotoxicity compared with Lipofectamine® 2000 and PEI 25 kDa in various cell types. In addition, newly synthesized PEI (-s-s-) derivatives have high reproducibility. PMID:26551343

  6. Dual delivery systems based on polyamine analog BENSpm as prodrug and gene delivery vectors

    NASA Astrophysics Data System (ADS)

    Zhu, Yu

    Combination drug and gene therapy shows promise in cancer treatment. However, the success of such strategy requires careful selection of the therapeutic agents, as well as development of efficient delivery vectors. BENSpm (N 1, N11-bisethylnorspermine), a polyamine analogue targeting the intracellular polyamine pathway, draws our special attention because of the following reasons: (1) polyamine pathway is frequently dysregulated in cancer; (2) BENSpm exhibits multiple functions to interfere with the polyamine pathway, such as to up-regulate polyamine metabolism enzymes and down-regulate polyamine biosynthesis enzymes. Therefore BENSpm depletes all natural polyamines and leads to apoptosis and cell growth inhibition in a wide range of cancers; (3) preclinical studies proved that BENSpm can act synergistically with various chemotherapy agents, making it a promising candidate in combination therapy; (4) multiple positive charges in BENSpm enable it as a suitable building block for cationic polymers, which can be further applied to gene delivery. In this dissertation, our goal was to design dual-function delivery vector based on BENSpm that can function as a gene delivery vector and, after intracellular degradation, as an active anticancer agent targeting dysregulated polyamine metabolism. We first demonstrated strong synergism between BENSpm and a potential therapeutic gene product TRAIL. Strong synergism was obtained in both estrogen-dependent MCF-7 breast cancer cells and triple-negative MDA-MB-231 breast cancer cells. Significant dose reduction of TRAIL in combination with BENSpm in MDA-MB-231 cells, together with the fact that BENSpm rendered MCF-7 cells more sensitive to TRAIL treatment verified our rationale of designing BENSpm-based delivery platform. This was expected to be beneficial for overcoming drug resistance in chemotherapy, as well as boosting the therapeutic effect of therapeutic genes. We first designed a lipid-based BENSpm dual vector (Lipo

  7. Peptide vectors for gene delivery: from single peptides to multifunctional peptide nanocarriers.

    PubMed

    Raad, Markus de; Teunissen, Erik A; Mastrobattista, Enrico

    2014-07-01

    The therapeutic use of nucleic acids relies on the availability of sophisticated delivery systems for targeted and intracellular delivery of these molecules. Such a gene delivery should possess essential characteristics to overcome several extracellular and intracellular barriers. Peptides offer an attractive platform for nonviral gene delivery, as several functional peptide classes exist capable of overcoming these barriers. However, none of these functional peptide classes contain all the essential characteristics required to overcome all of the barriers associated with successful gene delivery. Combining functional peptides into multifunctional peptide vectors will be pivotal for improving peptide-based gene delivery systems. By using combinatorial strategies and high-throughput screening, the identification of multifunctional peptide vectors will accelerate the optimization of peptide-based gene delivery systems.

  8. Gene Delivery into Plant Cells for Recombinant Protein Production

    PubMed Central

    Chen, Qiang

    2015-01-01

    Recombinant proteins are primarily produced from cultures of mammalian, insect, and bacteria cells. In recent years, the development of deconstructed virus-based vectors has allowed plants to become a viable platform for recombinant protein production, with advantages in versatility, speed, cost, scalability, and safety over the current production paradigms. In this paper, we review the recent progress in the methodology of agroinfiltration, a solution to overcome the challenge of transgene delivery into plant cells for large-scale manufacturing of recombinant proteins. General gene delivery methodologies in plants are first summarized, followed by extensive discussion on the application and scalability of each agroinfiltration method. New development of a spray-based agroinfiltration and its application on field-grown plants is highlighted. The discussion of agroinfiltration vectors focuses on their applications for producing complex and heteromultimeric proteins and is updated with the development of bridge vectors. Progress on agroinfiltration in Nicotiana and non-Nicotiana plant hosts is subsequently showcased in context of their applications for producing high-value human biologics and low-cost and high-volume industrial enzymes. These new advancements in agroinfiltration greatly enhance the robustness and scalability of transgene delivery in plants, facilitating the adoption of plant transient expression systems for manufacturing recombinant proteins with a broad range of applications. PMID:26075275

  9. Gene delivery into plant cells for recombinant protein production.

    PubMed

    Chen, Qiang; Lai, Huafang

    2015-01-01

    Recombinant proteins are primarily produced from cultures of mammalian, insect, and bacteria cells. In recent years, the development of deconstructed virus-based vectors has allowed plants to become a viable platform for recombinant protein production, with advantages in versatility, speed, cost, scalability, and safety over the current production paradigms. In this paper, we review the recent progress in the methodology of agroinfiltration, a solution to overcome the challenge of transgene delivery into plant cells for large-scale manufacturing of recombinant proteins. General gene delivery methodologies in plants are first summarized, followed by extensive discussion on the application and scalability of each agroinfiltration method. New development of a spray-based agroinfiltration and its application on field-grown plants is highlighted. The discussion of agroinfiltration vectors focuses on their applications for producing complex and heteromultimeric proteins and is updated with the development of bridge vectors. Progress on agroinfiltration in Nicotiana and non-Nicotiana plant hosts is subsequently showcased in context of their applications for producing high-value human biologics and low-cost and high-volume industrial enzymes. These new advancements in agroinfiltration greatly enhance the robustness and scalability of transgene delivery in plants, facilitating the adoption of plant transient expression systems for manufacturing recombinant proteins with a broad range of applications. PMID:26075275

  10. Arginine-Rich Polyplexes for Gene Delivery to Neuronal Cells

    PubMed Central

    Morris, Viola B.; Labhasetwar, Vinod

    2015-01-01

    Neuronal gene therapy potentially offers an effective therapeutic intervention to cure or slow the progression of neurological diseases. However, neuronal cells are difficult to transfect with nonviral vectors, and in vivo their transport across the blood-brain barrier (BBB) is inefficient. We synthesized a series of arginine-rich oligopeptides, grafted with polyethyleneimine (PEI) and modified with a short-chain polyethylene glycol (PEG). We hypothesized that the arginine would enhance cellular uptake and transport of these polyplexes across the BBB, with PEG imparting biocompatibility and “stealth” properties and PEI facilitating DNA condensation and gene transfection. The optimized composition of the polyplexes demonstrated hemocompatibility with red blood cells, causing no lysis or aggregation, and showed significantly better cytocompatibility than PEI in vitro. Polyplexes formulated with luciferase-expressing plasmid DNA could transfect rat primary astrocytes and neurons in vitro. Confocal imaging data showed efficient cellular uptake of DNA and its sustained intracellular retention and nuclear localization with polyplexes. Intravenous administration of the optimized polyplexes in mice led to gene expression in the brain, which upon further immunohistochemical analysis demonstrated gene expression in neurons. In conclusion, we have successfully designed a nonviral vector for in vitro and in vivo neuronal gene delivery. PMID:26000961

  11. [Development of gene delivery system using PLGA nanospheres].

    PubMed

    Tahara, Kohei; Yamamoto, Hiromitsu; Takeuchi, Hirofumi; Kawashima, Yoshiaki

    2007-10-01

    The development of nonviral vectors for the efficient and safe delivery to cells has long been awaited to facilitate gene therapy. Recently, many nonviral vectors modified with cationic lipids, cationic polymers, etc. have been reported. However, those nonviral vectors with cationic materials require improved stability, longer duration of gene expression, and reduced cytotoxicity. We successfully prepared mucoadhesive poly (lactide-co-glycolide) nanospheres (PLGA NS) by modifying the nanoparticulate surface with chitosan to improve mucosal peptide absorption after oral and pulmonary administration. Furthermore, we found that nucleic acid, which was not dispersed in the organic solvent, could be dispersed by forming a complex with cationic lipid. Using this phenomenon, polynucleic acids for gene therapy (plasmid DNA, antisense oligonucleotide, small interfering RNA, etc.) can be encapsulated into the matrix of the polymer particles with the emulsion solvent diffusion method. The advantages of this preparation method are its simple process and avoidance of an ultrasonication process for submicronization of particles. The resultant nanospheres show better cellular uptake and different gene therapeutic effects compared with conventional vectors due to their improved adherence to cells and sustained release of polynucleic acid in the cells. In conclusion, chitosan-coated PLGA NS can possibly be applied in nonviral vectors for gene therapy.

  12. Modified pectin-based carrier for gene delivery: Cellular barriers in gene delivery course

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The use of biodegradable and biocompatible polysaccharides as DNA carriers has high potential for gene therapy applications. Pectin is a structural plant polysaccharide heterogeneous with respect to its chemical structure. It contains branches rich in galactose residues which serve as potential liga...

  13. Ultrasound mediated delivery of drugs and genes to solid tumors

    PubMed Central

    Frenkel, Victor

    2008-01-01

    It has long been shown that therapeutic ultrasound can be used effectively to ablate solid tumors, and a variety of cancers are presently being treated in the clinic using these types of ultrasound exposures. There is, however, an ever-increasing body of preclinical literature that demonstrates how ultrasound energy can also be used non-destructively for increasing the efficacy of drugs and genes for improving cancer treatment. In this review, a summary of the most important ultrasound mechanisms will be given with a detailed description of how each one can be employed for a variety of applications. This includes the manner by which acoustic energy deposition can be used to create changes in tissue permeability for enhancing the delivery of conventional agents, as well as for deploying and activating drugs and genes via specially tailored vehicles and formulations. PMID:18474406

  14. Polyamine-DNA interactions and development of gene delivery vehicles.

    PubMed

    Thomas, T J; Tajmir-Riahi, H A; Thomas, Thresia

    2016-10-01

    Polyamines are positively charged organic cations under physiologic ionic and pH conditions and hence they interact with negatively charged macromolecules such as DNA and RNA. Although electrostatic interaction is the predominant mode of polyamine-nucleic acid interactions, site- and structure-specific binding has also been recognized. A major consequence of polyamine-DNA interaction is the collapse of DNA to nanoparticles of approximately 100 nm diameter. Electron and atomic force microscopic studies have shown that these nanoparticles are spheroids, toroids and rods. DNA transport to cells for gene therapy applications requires the condensation of DNA to nanoparticles and hence the study of polyamines and related compounds with nucleic acids has received technological importance. In addition to natural and synthetic polyamines, several amine-terminated or polyamine-substituted agents are under intense investigation for non-viral gene delivery vehicles.

  15. Fenton-treated functionalized diamond nanoparticles as gene delivery system.

    PubMed

    Martín, Roberto; Alvaro, Mercedes; Herance, José Raúl; García, Hermenegildo

    2010-01-26

    When raw diamond nanoparticles (Dnp, 7 nm average particle size) obtained from detonation are submitted to harsh Fenton-treatment, the resulting material becomes free of amorphous soot matter and the process maintains the crystallinity, reduces the particle size (4 nm average particle size), increases the surface OH population, and increases water solubility. All these changes are beneficial for subsequent Dnp covalent functionalization and for the ability of Dnp to cross cell membranes. Fenton-treated Dnps have been functionalized with thionine and the resulting sample has been observed in HeLa cell nuclei. A triethylammonium-functionalized Dnp pairs electrostatically with a plasmid having the green fluorescent protein gene and acts as gene delivery system permitting the plasmid to cross HeLa cell membrane, something that does not occur for the plasmid alone without assistance of polycationic Dnp. PMID:20047335

  16. Readministration of adenoviral gene delivery to dopamine neurons.

    PubMed

    Gonzalez, Sarah C; McMenamin, Margaret M; Charlton, Harry M; Goodman, James; Lantos, Tibor; Simpson, Christine; Wood, Matthew J A

    2007-10-01

    An approach currently being explored as treatment for Parkinson's disease is gene therapy. An important question concerns the duration of transgene expression in dopamine neurons and the issues of vector persistence, neuronal damage and the feasibility of readministering vector to the same neuronal population. We show, using an adenoviral vector expressing the LacZ reporter gene, that transgene expression declined over time but with minimal loss of dopamine neurons or vector DNA. Readministration of vector resulted in low levels of transgene delivery to the neurons. Moreover, the neurons to which vector had already been delivered were unable to transport the retrograde tracer fluorogold. Our findings indicate that transgene expression declined in dopamine neurons despite the persistence of virus, and the capacity to readminister vector to these neurons was limited. PMID:17885611

  17. Dual-Purpose Magnetic Micelles for MRI and Gene Delivery

    PubMed Central

    Wang, Chunyan; Ravi, Sowndharya; Martinez, Gary.V.; Chinnasamy, Vignesh; Raulji, Payal; Howell, Mark; Davis, Yvonne; Seehra, Mohindar S.; Mohapatra, Subhra

    2013-01-01

    Gene therapy is a promising therapeutic approach for treating disease, but the efficient delivery of genes to desired locations with minimal side effects remains a challenge. In addition to gene therapy, it is also highly desirable to provide sensitive imaging information in patients for disease diagnosis, screening and post-therapy monitoring. Here, we report on the development of dual-purpose chitosan and polyethyleneimine (PEI) coated magnetic micelles (CP-mag-micelles) that can deliver nucleic acid-based therapeutic agents and also provide magnetic resonance imaging (MRI). These ‘theranostic’ CP-mag-micelles are composed of monodisperse hydrophobic superparamagnetic iron oxide nanoparticles (SPIONs) loaded into the cores of micelles that are self-assembled from a block copolymer of poly (D, L-lactide) (PLA) and monomethoxy polyethylene glycol (mPEG). For efficient loading and protection of the nucleic acids the micelles were coated with cationic polymers, such as chitosan and PEI. The morphology and size distribution of the CP-mag-micelles were characterized and their potential for use as an MRI-probe was tested using an MRI scanner. The T2 relaxivity of micelles was similar to CP-mag-micelles confirming that coating with cationic polymers did not alter magnetism. Nanoparticles coated with chitosan:PEI at a weight ratio of 5:5 showed higher transfection efficiency in HEK293, 3T3 and PC3 cells than with weight ratios of 3:7 or 7:3. CP-mag-micelles are biocompatible, can be delivered to various organs and are safe. A single injection of CP-mag-micelles carrying reporter plasmids in vivo expressed genes for at least one week. Collectively, our results demonstrate that a structural reinforcement of SPIONs loaded in the core of an mPEG-PLA micelle coated with cationic polymers provides efficient DNA delivery and enhanced MRI potential, and affords a promising candidate for theranostics in the future. PMID:22561339

  18. Tumor targeting and microenvironment-responsive nanoparticles for gene delivery.

    PubMed

    Huang, Shixian; Shao, Kun; Kuang, Yuyang; Liu, Yang; Li, Jianfeng; An, Sai; Guo, Yubo; Ma, Haojun; He, Xi; Jiang, Chen

    2013-07-01

    A tumor targeting nanoparticle system has been successfully developed to response to the lowered tumor extracellular pH (pHe) and upregulated matrix metalloproteinase 2 (MMP2) in the tumor microenvironment. The nanoparticles are modified with activatable cell-penetrating peptide (designated as dtACPP) that's dual-triggered by the lowered pHe and MMP2. In dtACPP, the internalization function of cell-penetrating peptide (CPP) is quenched by a pH-sensitive masking peptide, linking by a MMP2 substrate. The masking peptide is negatively charged to quench the cationic CPP well after systemic administration. Hence, dtACPP-modified nanoparticles possesses passive tumor targetability via the enhanced permeability and retention (EPR) effect. Once reaching the tumor microenvironment, the pre-existing attraction would be eliminated due to the lowered pHe, accompanying the linker cleaved by MMP2, dtACPP would be activated to expose CPP to drive the nanoparticles' internalization into the intratumoral cells. The studies of plasmid DNA loading, toxicity assessment, cellular uptake, tumor targeting delivery, and gene transfection demonstrate that dtACPP-modified nanoparticle system is a potential candidate for tumor targeting gene delivery.

  19. Nanoparticle-mediated delivery of suicide genes in cancer therapy.

    PubMed

    Vago, Riccardo; Collico, Veronica; Zuppone, Stefania; Prosperi, Davide; Colombo, Miriam

    2016-09-01

    Conventional chemotherapeutics have been employed in cancer treatment for decades due to their efficacy in killing the malignant cells, but the other side of the coin showed off-target effects, onset of drug resistance and recurrences. To overcome these limitations, different approaches have been investigated and suicide gene therapy has emerged as a promising alternative. This approach consists in the introduction of genetic materials into cancerous cells or the surrounding tissue to cause cell death or retard the growth of the tumor mass. Despite promising results obtained both in vitro and in vivo, this innovative approach has been limited, for long time, to the treatment of localized tumors, due to the suboptimal efficiency in introducing suicide genes into cancer cells. Nanoparticles represent a valuable non-viral delivery system to protect drugs in the bloodstream, to improve biodistribution, and to limit side effects by achieving target selectivity through surface ligands. In this scenario, the real potential of suicide genes can be translated into clinically viable treatments for patients. In the present review, we summarize the recent advances of inorganic nanoparticles as non-viral vectors in terms of therapeutic efficacy, targeting capacity and safety issues. We describe the main suicide genes currently used in therapy, with particular emphasis on toxin-encoding genes of bacterial and plant origin. In addition, we discuss the relevance of molecular targeting and tumor-restricted expression to improve treatment specificity to cancer tissue. Finally, we analyze the main clinical applications, limitations and future perspectives of suicide gene therapy. PMID:27436147

  20. Intracellular delivery of potential therapeutic genes: prospects in cancer gene therapy.

    PubMed

    Bakhtiar, Athirah; Sayyad, Mustak; Rosli, Rozita; Maruyama, Atsushi; Chowdhury, Ezharul H

    2014-01-01

    Conventional therapies for malignant cancer such as chemotherapy and radiotherapy are associated with poor survival rates owing to the development of cellular resistance to cancer drugs and the lack of targetability, resulting in unwanted adverse effects on healthy cells and necessitating the lowering of therapeutic dose with consequential lower efficacy of the treatment. Gene therapy employing different types of viral and non-viral carriers to transport gene(s) of interest and facilitating production of the desirable therapeutic protein(s) has tremendous prospects in cancer treatments due to the high-level of specificity in therapeutic action of the expressed protein(s) with diminished off-target effects, although cancer cell-specific delivery of transgene(s) still poses some challenges to be addressed. Depending on the potential therapeutic target genes, cancer gene therapy could be categorized into tumor suppressor gene replacement therapy, immune gene therapy and enzyme- or prodrug-based therapy. This review would shed light on the current progress of delivery of potentially therapeutic genes into various cancer cells in vitro and animal models utilizing a variety of viral and non-viral vectors.

  1. Functionalized nanoparticles for AMF-induced gene and drug delivery

    NASA Astrophysics Data System (ADS)

    Biswas, Souvik

    The properties and broad applications of nano-magnetic colloids have generated much interest in recent years. Specially, Fe3O4 nanoparticles have attracted a great deal of attention since their magnetic properties can be used for hyperthermia treatment or drug targeting. For example, enhanced levels of intracellular gene delivery can be achieved using Fe3O4 nano-vectors in the presence of an external magnetic field, a process known as 'magnetofection'. The low cytotoxicity, tunable particle size, ease of surface functionalization, and ability to generate thermal energy using an external alternating magnetic field (AMF) are properties have propelled Fe3O4 research to the forefront of nanoparticle research. The strategy of nanoparticle-mediated, AMF-induced heat generation has been used to effect intracellular hyperthermia. One application of this 'magnetic hyperthermia' is heat activated local delivery of a therapeutic effector (e.g.; drug or polynucleotide). This thesis describes the development of a magnetic nano-vector for AMF-induced, heat-activated pDNA and small molecule delivery. The use of heat-inducible vectors, such as heat shock protein ( hsp) genes, is a promising mode of gene therapy that would restrict gene expression to a local region by focusing a heat stimulus only at a target region. We thus aimed to design an Fe3O4 nanoparticle-mediated gene transfer vehicle for AMF-induced localized gene expression. We opted to use 'click' oximation techniques to assemble the magnetic gene transfer vector. Chapter 2 describes the synthesis, characterization, and transfection studies of the oxime ether lipid-based nano-magnetic vectors MLP and dMLP. The synthesis and characterization of a novel series of quaternary ammonium aminooxy reagents (2.1--2.4) is described. These cationic aminooxy compounds were loaded onto nanoparticles for ligation with carbonyl groups and also to impart a net positive charge on the nanoparticle surface. Our studies indicated that the

  2. Magnetofection: A Reproducible Method for Gene Delivery to Melanoma Cells

    PubMed Central

    Prosen, Lara; Prijic, Sara; Music, Branka; Lavrencak, Jaka; Cemazar, Maja; Sersa, Gregor

    2013-01-01

    Magnetofection is a nanoparticle-mediated approach for transfection of cells, tissues, and tumors. Specific interest is in using superparamagnetic iron oxide nanoparticles (SPIONs) as delivery system of therapeutic genes. Magnetofection has already been described in some proof-of-principle studies; however, fine tuning of the synthesis of SPIONs is necessary for its broader application. Physicochemical properties of SPIONs, synthesized by the co-precipitation in an alkaline aqueous medium, were tested after varying different parameters of the synthesis procedure. The storage time of iron(II) sulfate salt, the type of purified water, and the synthesis temperature did not affect physicochemical properties of SPIONs. Also, varying the parameters of the synthesis procedure did not influence magnetofection efficacy. However, for the pronounced gene expression encoded by plasmid DNA it was crucial to functionalize poly(acrylic) acid-stabilized SPIONs (SPIONs-PAA) with polyethyleneimine (PEI) without the adjustment of its elementary alkaline pH water solution to the physiological pH. In conclusion, the co-precipitation of iron(II) and iron(III) sulfate salts with subsequent PAA stabilization, PEI functionalization, and plasmid DNA binding is a robust method resulting in a reproducible and efficient magnetofection. To achieve high gene expression is important, however, the pH of PEI water solution for SPIONs-PAA functionalization, which should be in the alkaline range. PMID:23862136

  3. Design of novel polysaccharidic nanostructures for gene delivery

    NASA Astrophysics Data System (ADS)

    de la Fuente, M.; Seijo, B.; Alonso, M. J.

    2008-02-01

    The goal of the present work was to develop a new synthetic nanosystem for gene delivery. For this purpose, we chose two polysaccharides, hyaluronic acid (HA) and chitosan (CS), as the main components of the nanocarrier. Nanoparticles with different hyaluronate:chitosan (HA:CS) mass ratios (0.5:1 and 1:1) and different polymer molecular weights (hyaluronate 170 (HA) or <10 kDa (HAO) and chitosan 125 (CS) or 10-12 (CSO) kDa) could be obtained using an ionic crosslinking method. These nanoparticles were loaded with pDNA and characterized for their size, zeta potential and pDNA association efficiency. Moreover, their toxicity and ability to transfect the model plasmid pEGFP-C1 were evaluated in the cell line HEK 293, as well as their intracellular fate. The results showed that HA:CS nanoparticles have a small size in the range of 110-230 nm, a positive zeta potential of +10 to +32 mV and a very high pDNA association efficiency of 87-99% (w/w). On the other hand, nanoparticles exhibited low cell toxicity and transfection levels up to 25% GFP expressing HEK 293 cells, lasting for the whole observation period of 10 days. We also provide basic information about the role of both polymers, HA and CS, and the effect of their molecular weight on the effectiveness of the resulting DNA nanocarrier, being the highest transfection levels observed with HAO:CSO 1:1 nanoparticles. In conclusion, HA:CS nanoparticles are promising carriers for gene delivery.

  4. Biological delivery approaches for gene therapy: strategies to potentiate efficacy and enhance specificity.

    PubMed

    Mohit, Elham; Rafati, Sima

    2013-12-01

    Nowadays many therapeutic agents such as suicide genes, anti-angiogenesis agents, cytokines, chemokines and other therapeutic genes were delivered to cancer cells. Various biological delivery systems have been applied for directing therapeutic gene to target cells. Some of these successful preclinical studies, steps forward to clinical trials and a few are examined in phase III clinical trials. In this review, the biological gene delivery systems were categorized into microorganism and cell based delivery systems. Viral, bacterial, yeast and parasite are among microorganism based delivery systems which are expanded in this review. In cell based approach, different strategies such as tumor cells, stem cells, dendritic cells and sertoli cells will be discussed. Different drawbacks are associated with each delivery system; therefore, many strategies have been improved and potentiated their direction toward specific target cells. Herein, further to the principle of each delivery system, the progresses of these approaches for development of newer generation are discussed.

  5. Solid Lipid Nanoparticles as Efficient Drug and Gene Delivery Systems: Recent Breakthroughs

    PubMed Central

    Ezzati Nazhad Dolatabadi, Jafar; Valizadeh, Hadi; Hamishehkar, Hamed

    2015-01-01

    In recent years, nanomaterials have been widely applied as advanced drug and gene delivery nanosystems. Among them, solid lipid nanoparticles (SLNs) have attracted great attention as colloidal drug delivery systems for incorporating hydrophilic or lipophilic drugs and various macromolecules as well as proteins and nucleic acids. Therefore, SLNs offer great promise for controlled and site specific drug and gene delivery. This article includes general information about SLN structures and properties, production procedures, characterization. In addition, recent progress on development of drug and gene delivery systems using SLNs was reviewed. PMID:26236652

  6. Therapeutic immunisation with COPV early genes by epithelial DNA delivery.

    PubMed

    Moore, Richard A; Walcott, Sarah; White, Kate L; Anderson, Davina M; Jain, Suchitra; Lloyd, Andrew; Topley, Peter; Thomsen, Lindy; Gough, Gerald W; Stanley, Margaret A

    2003-09-30

    Following challenge with COPV (canine oral papillomavirus), DNA plasmids encoding COPV L1, E1 or E2 protein were delivered into oral mucosal and cutaneous sites in beagles using particle-mediated immunotherapeutic delivery (PMID). Two weeks post-challenge, a priming dose of 8 microg DNA was delivered followed by a booster dose after a further two weeks. A group of control dogs were vaccinated using plasmid DNA encoding Hepatitis B virus surface (HBVs) gene. All of the control animals developed warts at the vast majority of sites (94%). All of the animals given wild type L1, E1, or E2 developed warts at most sites (88%, 75%, and 88%, respectively). The animals given codon optimised E2 however, were protected from wart growth with only one tiny lesion seen on a single animal that persisted for only a few days. The E1 codon optimised group was also significantly protected with a far lower number of smaller warts (48%) that persisted for a shorter duration. These data suggest that therapeutic immunisation by PMID with papillomavirus early genes is effective and emphasizes the importance of antigen load in the generation of protective responses to papillomavirus proteins. PMID:14554090

  7. A sight on the current nanoparticle-based gene delivery vectors

    PubMed Central

    2014-01-01

    Nowadays, gene delivery for therapeutic objects is considered one of the most promising strategies to cure both the genetic and acquired diseases of human. The design of efficient gene delivery vectors possessing the high transfection efficiencies and low cytotoxicity is considered the major challenge for delivering a target gene to specific tissues or cells. On this base, the investigations on non-viral gene vectors with the ability to overcome physiological barriers are increasing. Among the non-viral vectors, nanoparticles showed remarkable properties regarding gene delivery such as the ability to target the specific tissue or cells, protect target gene against nuclease degradation, improve DNA stability, and increase the transformation efficiency or safety. This review attempts to represent a current nanoparticle based on its lipid, polymer, hybrid, and inorganic properties. Among them, hybrids, as efficient vectors, are utilized in gene delivery in terms of materials (synthetic or natural), design, and in vitro/in vivo transformation efficiency. PMID:24936161

  8. A sight on the current nanoparticle-based gene delivery vectors

    NASA Astrophysics Data System (ADS)

    Dizaj, Solmaz Maleki; Jafari, Samira; Khosroushahi, Ahmad Yari

    2014-05-01

    Nowadays, gene delivery for therapeutic objects is considered one of the most promising strategies to cure both the genetic and acquired diseases of human. The design of efficient gene delivery vectors possessing the high transfection efficiencies and low cytotoxicity is considered the major challenge for delivering a target gene to specific tissues or cells. On this base, the investigations on non-viral gene vectors with the ability to overcome physiological barriers are increasing. Among the non-viral vectors, nanoparticles showed remarkable properties regarding gene delivery such as the ability to target the specific tissue or cells, protect target gene against nuclease degradation, improve DNA stability, and increase the transformation efficiency or safety. This review attempts to represent a current nanoparticle based on its lipid, polymer, hybrid, and inorganic properties. Among them, hybrids, as efficient vectors, are utilized in gene delivery in terms of materials (synthetic or natural), design, and in vitro/ in vivo transformation efficiency.

  9. PLGA Nanoparticles for Ultrasound-Mediated Gene Delivery to Solid Tumors

    PubMed Central

    Figueiredo, Marxa; Esenaliev, Rinat

    2012-01-01

    This paper focuses on novel approaches in the field of nanotechnology-based carriers utilizing ultrasound stimuli as a means to spatially target gene delivery in vivo, using nanoparticles made with either poly(lactic-co-glycolic acid) (PLGA) or other polymers. We specifically discuss the potential for gene delivery by particles that are echogenic (amenable to destruction by ultrasound) composed either of polymers (PLGA, polystyrene) or other contrast agent materials (Optison, SonoVue microbubbles). The use of ultrasound is an efficient tool to further enhance gene delivery by PLGA or other echogenic particles in vivo. Echogenic PLGA nanoparticles are an attractive strategy for ultrasound-mediated gene delivery since this polymer is currently approved by the US Food and Drug Administration for drug delivery and diagnostics in cancer, cardiovascular disease, and also other applications such as vaccines and tissue engineering. This paper will review recent successes and the potential of applying PLGA nanoparticles for gene delivery, which include (a) echogenic PLGA used with ultrasound to enhance local gene delivery in tumors or muscle and (b) PLGA nanoparticles currently under development, which could benefit in the future from ultrasound-enhanced tumor targeted gene delivery. PMID:22506124

  10. An efficient method for in vitro gene delivery via regulation of cellular endocytosis pathway

    PubMed Central

    Luo, Jing; Li, Caixia; Chen, Jianlin; Wang, Gang; Gao, Rong; Gu, Zhongwei

    2015-01-01

    Transfection efficiency was the primary goal for in vitro gene delivery mediated by nonviral gene carriers. Here, we report a modified gene transfection method that could greatly increase the efficiency of, and accelerate the process mediated by, 25 kDa branched polyethyleneimine and Lipofectamine™ 2000 in a broad range of cell strains, including tumor, normal, primary, and embryonic stem cells. In this method, the combination of transfection procedure with optimized complexation volume had a determinant effect on gene delivery result. The superiorities of the method were found to be related to the change of cellular endocytosis pathway and decrease of particle size. The efficient and simple method established in this study can be widely used for in vitro gene delivery into cultured cells. We think it may also be applicable for many more nonviral gene delivery materials than polyethyleneimine and liposome. PMID:25767387

  11. Nonviral gene delivery systems by the combination of bubble liposomes and ultrasound.

    PubMed

    Omata, Daiki; Negishi, Yoichi; Suzuki, Ryo; Oda, Yusuke; Endo-Takahashi, Yoko; Maruyama, Kazuo

    2015-01-01

    The combination of therapeutic ultrasound (US) and nano/microbubbles is an important system for establishing a novel and noninvasive gene delivery system. Genes are delivered more efficiently using this system compared with a conventional nonviral vector system such as the lipofection method, resulting in higher gene expression. This higher efficiency is due to the gene being delivered into the cytosol and bypassing the endocytosis pathway. Many in vivo studies have demonstrated US-mediated gene delivery with nano/microbubbles, and several gene therapy feasibility studies for various diseases have been reported. In addition, nano/microbubbles can deliver genes site specifically by the control of US exposure site. In the present review, we summarize the gene delivery systems by the combination of nano/microbubbles and US, describe their properties, and assess applications and challenges of US theranostics.

  12. Nonviral gene delivery to the rat kidney with polyethylenimine.

    PubMed

    Boletta, A; Benigni, A; Lutz, J; Remuzzi, G; Soria, M R; Monaco, L

    1997-07-01

    The aim of this study was to establish a nonviral method for gene delivery to the rat kidney. To this purpose, a panel of reagents was tested, including a monocationic lipid, DOTAP, a polycationic lipid, DOGS (or Transfectam), and three different forms of the cationic polymer polyethylenimine (PEI). A comparison among these compounds was performed in vivo, using luciferase as reporter gene. Complexes containing 10 microg of DNA were injected into the left renal artery of rats and allowed to remain in contact with the kidney for 10 min. Forty-eight hours later, luciferase expression levels in kidney extracts were measured. Kidneys injected with DNA complexed to the branched, 25-kD PEI polymer (PEI 25k) yielded activity levels significantly higher than control, sham-operated kidneys (2.7 x 10(4) vs. 5.2 x 10(3) RLU/kidney, respectively), whereas the other transfecting agents did not yield significant activity over controls. PEI 25k was therefore chosen for further optimization of transfection conditions. Dose-dependent luciferase expression was shown for 10, 50, and 100 microg of PEI-complexed DNA, reaching maximal levels of 2.4 x 10(5) RLU/kidney at 100 microg DNA. The optimal PEI nitrogen/DNA phosphate molar ratio was 10 equivalents. Luciferase activity peaked at 2 days, was still significantly higher than controls at 7 days, and was undetectable at 14 days post-injection. Using beta-galactosidase (beta-Gal) as a reporter, transgene expression was localized almost exclusively in proximal tubular cells.

  13. Discovery of Cationic Polymers for Non-viral Gene Delivery using Combinatorial Approaches

    PubMed Central

    Barua, Sutapa; Ramos, James; Potta, Thrimoorthy; Taylor, David; Huang, Huang-Chiao; Montanez, Gabriela; Rege, Kaushal

    2015-01-01

    Gene therapy is an attractive treatment option for diseases of genetic origin, including several cancers and cardiovascular diseases. While viruses are effective vectors for delivering exogenous genes to cells, concerns related to insertional mutagenesis, immunogenicity, lack of tropism, decay and high production costs necessitate the discovery of non-viral methods. Significant efforts have been focused on cationic polymers as non-viral alternatives for gene delivery. Recent studies have employed combinatorial syntheses and parallel screening methods for enhancing the efficacy of gene delivery, biocompatibility of the delivery vehicle, and overcoming cellular level barriers as they relate to polymer-mediated transgene uptake, transport, transcription, and expression. This review summarizes and discusses recent advances in combinatorial syntheses and parallel screening of cationic polymer libraries for the discovery of efficient and safe gene delivery systems. PMID:21843141

  14. Cationic liposome–nucleic acid complexes for gene delivery and gene silencing

    PubMed Central

    Ewert, Kai K.; Majzoub, Ramsey N.; Leal, Cecília

    2014-01-01

    Cationic liposomes (CLs) are studied worldwide as carriers of DNA and short interfering RNA (siRNA) for gene delivery and gene silencing, and related clinical trials are ongoing. Optimization of transfection efficiency and silencing efficiency by cationic liposome carriers requires a comprehensive understanding of the structures of CL–nucleic acid complexes and the nature of their interactions with cell membranes as well as events leading to release of active nucleic acids within the cytoplasm. Synchrotron x-ray scattering has revealed that CL–nucleic acid complexes spontaneously assemble into distinct liquid crystalline phases including the lamellar, inverse hexagonal, hexagonal, and gyroid cubic phases, and fluorescence microscopy has revealed CL–DNA pathways and interactions with cells. The combining of custom synthesis with characterization techniques and gene expression and silencing assays has begun to unveil structure–function relations in vitro. As a recent example, this review will briefly describe experiments with surface-functionalized PEGylated CL–DNA nanoparticles. The functionalization, which is achieved through custom synthesis, is intended to address and overcome cell targeting and endosomal escape barriers to nucleic acid delivery faced by PEGylated nanoparticles designed for in vivo applications. PMID:25587216

  15. Acidity-Activated Shielding Strategies of Cationic Gene Delivery for Cancer Therapy.

    PubMed

    Xia, Jialiang; Feng, Zongcai; Yang, Hongyan; Lin, Sanqing; Han, Bing

    2016-01-01

    Cationic gene vectors increased attractive for gene therapy. However, unstable systemic circulation due to the interaction of gene delivery system with blood cells limited the further application. Therefore, pH sensitive shielding systems were exploited, by which, the positive surface charge density of polyplexes was reduced, circulation time was improved and pH-triggered targeting delivery was promised. This mini review mainly focuses on the development of solid tumors pH environment activated shielding systems for cationic gene vectors. This shielding strategy shows great potential for enhancing efficient gene transporting and achieving better therapeutic effects in acidic tumor treatment.

  16. Delivery of nucleic acids for cancer gene therapy: overcoming extra- and intra-cellular barriers.

    PubMed

    McErlean, Emma M; McCrudden, Cian M; McCarthy, Helen O

    2016-09-01

    The therapeutic potential of cancer gene therapy has been limited by the difficulty of delivering genetic material to target sites. Various biological and molecular barriers exist which need to be overcome before effective nonviral delivery systems can be applied successfully in oncology. Herein, various barriers are described and strategies to circumvent such obstacles are discussed, considering both the extracellular and intracellular setting. Development of multifunctional delivery systems holds much promise for the progression of gene delivery, and a growing body of evidence supports this approach involving rational design of vectors, with a unique molecular architecture. In addition, the potential application of composite gene delivery platforms is highlighted which may provide an alternative delivery strategy to traditional systemic administration. PMID:27582234

  17. Current Progress in Gene Delivery Technology Based on Chemical Methods and Nano-carriers

    PubMed Central

    Jin, Lian; Zeng, Xin; Liu, Ming; Deng, Yan; He, Nongyue

    2014-01-01

    Gene transfer methods are promising in the field of gene therapy. Current methods for gene transfer include three major groups: viral, physical and chemical methods. This review mainly summarizes development of several types of chemical methods for gene transfer in vitro and in vivo by means of nano-carriers like; calcium phosphates, lipids, and cationic polymers including chitosan, polyethylenimine, polyamidoamine dendrimers, and poly(lactide-co-glycolide). This review also briefly introduces applications of these chemical methods for gene delivery. PMID:24505233

  18. Novel endosomolytic peptides for enhancing gene delivery in nanoparticles.

    PubMed

    Ahmad, Aqeel; Ranjan, Sanjeev; Zhang, Weikai; Zou, Jing; Pyykkö, Ilmari; Kinnunen, Paavo K J

    2015-02-01

    Trapping in the endosomes is currently believed to represent the main barrier for transfection. Peptides, which allow endosomal escape have been demonstrated to overcome this barrier, similarly to the entry of viruses. However, the design principles of such endosomolytic peptides remain unclear. We characterized three analogs derived from membrane disrupting antimicrobial peptides (AMP), viz. LL-37, melittin, and bombolitin V, with glutamic acid substituting for all basic residues. These analogs are pH-sensitive and cause negligible membrane permeabilization and insignificant cytotoxicity at pH7.4. However, at pH5.0, prevailing in endosomes, membrane binding and hemolysis of human erythrocytes become evident. We first condensed the emerald green fluorescent protein (emGFP) containing plasmid by protamine, yielding 115 nm diameter soluble nanoplexes. For coating of the nanoplex surface with a lipid bilayer we introduced a hydrophobic tether, stearyl-octa-arginine (SR8). The indicated peptides were dissolved in methanol and combined with lipid mixtures in chloroform, followed by drying at RT under a nitrogen flow. The dry residues were hydrated with nanoplexes in Hepes, pH7.4 yielding after a 30 min incubation at RT,rather monodisperse nanoparticles having an average diameter of 150-300 nm, measured by DLS and cryo-TEM. Studies with cell cultures showed the above peptides to yield expression levels comparable to those obtained using Lipofectamine 2000. However, unlike the polydisperse aggregates formed upon mixing Lipofectamine 2000 and plasmid, the procedure described yields soluble, and reasonably monodisperse nanoparticles, which can be expected to be suitable for gene delivery in vivo, using intravenous injection. PMID:25445677

  19. Complexation of oppositely charged polyelectrolytes in gene delivery and biology

    NASA Astrophysics Data System (ADS)

    Shklovskii, Boris

    2009-03-01

    Charge inversion of a DNA double helix by a positively charged flexible polymer (polyelectrolyte) is widely used to facilitate DNA contact with negative cell membranes for gene delivery. Motivated by this application in the first part of the talk I study the phase diagram a solution of long polyanions (PA) with a shorter polycations (PC) as a function the ratio of total charges of PC and PA in the solution, x, and the concentration of monovalent salt. Each PA attracts many PCs to form a complex. When x= 1, the complexes are neutral and condense in a macroscopic drop. When x is far away from 1, complexes are strongly charged and stable. PA are overcharged by PC at x > 1 and undercharged by PC at x < 1. As x approaches 1, PCs attached to PA disproportionate between complexes. Some complexes become neutral and condensed in a macroscopic drop while others become even stronger charged and stay free. The second part of the talk deals with biological example of PA -PC complexes namely self-assembly of vegetable viruses from long ss-RNA molecule paying role of scaffold and identical capsid proteins with long positive tails. I show that optimization Coulomb energy of the virus leads to the charge of RNA twice larger than the total charge of the capsid, in agreement with the experimental data. Then I discuss kinetics of the Coulomb complexation driven virus self-assembly. Capsid proteins stick to unassembled chain of ss RNA (which we call ``antenna'') and slide on it towards the assembly site. I show that at excess of capsid proteins such one-dimensional diffusion accelerates self-assembly more than ten times. On the other hand at excess of ss-RNA, antenna slows self-assembly down. Several experiments are proposed to verify the role of ss-RNA antenna in self-assembly.

  20. Effects of Fibrotic Tissue on Liver-targeted Hydrodynamic Gene Delivery.

    PubMed

    Kobayashi, Yuji; Kamimura, Kenya; Abe, Hiroyuki; Yokoo, Takeshi; Ogawa, Kohei; Shinagawa-Kobayashi, Yoko; Goto, Ryo; Inoue, Ryosuke; Ohtsuka, Masato; Miura, Hiromi; Kanefuji, Tsutomu; Suda, Takeshi; Tsuchida, Masanori; Aoyagi, Yutaka; Zhang, Guisheng; Liu, Dexi; Terai, Shuji

    2016-01-01

    Hydrodynamic gene delivery is a common method for gene transfer to the liver of small animals, and its clinical applicability in large animals has been demonstrated. Previous studies focused on functional analyses of therapeutic genes in animals with normal livers and little, however, is known regarding its effectiveness and safety in animals with liver fibrosis. Therefore, this study aimed to examine the effects of liver fibrosis on hydrodynamic gene delivery efficiency using a rat liver fibrosis model. We demonstrated for the first time, using pCMV-Luc plasmid, that this procedure is safe and that the amount of fibrotic tissue in the liver decreases gene delivery efficiency, resulting in decrease in luciferase activity depending on the volume of fibrotic tissue in the liver and the number of hepatocytes that are immunohistochemically stained positive for transgene product. We further demonstrate that antifibrotic gene therapy with matrix metalloproteinase-13 gene reduces liver fibrosis and improves efficiency of hydrodynamic gene delivery. These results demonstrate the negative effects of fibrotic tissue on hydrodynamic gene delivery and its recovery by appropriate antifibrotic therapy. PMID:27574785

  1. Effects of Fibrotic Tissue on Liver-targeted Hydrodynamic Gene Delivery

    PubMed Central

    Kobayashi, Yuji; Kamimura, Kenya; Abe, Hiroyuki; Yokoo, Takeshi; Ogawa, Kohei; Shinagawa-Kobayashi, Yoko; Goto, Ryo; Inoue, Ryosuke; Ohtsuka, Masato; Miura, Hiromi; Kanefuji, Tsutomu; Suda, Takeshi; Tsuchida, Masanori; Aoyagi, Yutaka; Zhang, Guisheng; Liu, Dexi; Terai, Shuji

    2016-01-01

    Hydrodynamic gene delivery is a common method for gene transfer to the liver of small animals, and its clinical applicability in large animals has been demonstrated. Previous studies focused on functional analyses of therapeutic genes in animals with normal livers and little, however, is known regarding its effectiveness and safety in animals with liver fibrosis. Therefore, this study aimed to examine the effects of liver fibrosis on hydrodynamic gene delivery efficiency using a rat liver fibrosis model. We demonstrated for the first time, using pCMV-Luc plasmid, that this procedure is safe and that the amount of fibrotic tissue in the liver decreases gene delivery efficiency, resulting in decrease in luciferase activity depending on the volume of fibrotic tissue in the liver and the number of hepatocytes that are immunohistochemically stained positive for transgene product. We further demonstrate that antifibrotic gene therapy with matrix metalloproteinase-13 gene reduces liver fibrosis and improves efficiency of hydrodynamic gene delivery. These results demonstrate the negative effects of fibrotic tissue on hydrodynamic gene delivery and its recovery by appropriate antifibrotic therapy. PMID:27574785

  2. Biodegradable poly(amine-co-ester) terpolymers for targeted gene delivery

    NASA Astrophysics Data System (ADS)

    Zhou, Jiangbing; Liu, Jie; Cheng, Christopher J.; Patel, Toral R.; Weller, Caroline E.; Piepmeier, Joseph M.; Jiang, Zhaozhong; Saltzman, W. Mark

    2012-01-01

    Many synthetic polycationic vectors for non-viral gene delivery show high efficiency in vitro, but their usually excessive charge density makes them toxic for in vivo applications. Here we describe the synthesis of a series of high molecular weight terpolymers with low charge density, and show that they exhibit efficient gene delivery, some surpassing the efficiency of the commercial transfection reagents Polyethylenimine and Lipofectamine 2000. The terpolymers were synthesized via enzyme-catalyzed copolymerization of lactone with dialkyl diester and amino diol, and their hydrophobicity adjusted by varying the lactone content and by selecting a lactone comonomer of specific ring size. Targeted delivery of the pro-apoptotic TRAIL gene to tumour xenografts by one of the terpolymers results in significant inhibition of tumour growth, with minimal toxicity both in vitro and in vivo. Our findings suggest that the gene delivery ability of the terpolymers stems from their high molecular weight and increased hydrophobicity, which compensates for their low charge density.

  3. MicroRNA-10 modulates Hox genes expression during Nile tilapia embryonic development.

    PubMed

    Giusti, Juliana; Pinhal, Danillo; Moxon, Simon; Campos, Camila Lovaglio; Münsterberg, Andrea; Martins, Cesar

    2016-05-01

    Hox gene clusters encode a family of transcription factors that govern anterior-posterior axis patterning during embryogenesis in all bilaterian animals. The time and place of Hox gene expression are largely determined by the relative position of each gene within its cluster. Furthermore, Hox genes were shown to have their expression fine-tuned by regulatory microRNAs (miRNAs). However, the mechanisms of miRNA-mediated regulation of these transcription factors during fish early development remain largely unknown. Here we have profiled three highly expressed miR-10 family members of Nile tilapia at early embryonic development, determined their genomic organization as well as performed functional experiments for validation of target genes. Quantitative analysis during developmental stages showed miR-10 family expression negatively correlates with the expression of HoxA3a, HoxB3a and HoxD10a genes, as expected for bona fide miRNA-mRNA interactions. Moreover, luciferase assays demonstrated that HoxB3a and HoxD10a are targeted by miR-10b-5p. Overall, our data indicate that the miR-10 family directly regulates members of the Hox gene family during Nile tilapia embryogenesis. PMID:26980108

  4. A visible, targeted high-efficiency gene delivery and transfection strategy

    PubMed Central

    2011-01-01

    Background To enhance myocardial angiogenic gene expression, a novel gene delivery strategy was tested. Direct intramyocardial injection of an angiogenic gene with microbubbles and insonation were applied in a dog animal model. Dogs received one of the four different treatments in conjunction with either the enhanced green fluorescence protein (EGFP) gene or the hepatocyte growth factor (HGF) gene: gene with microbubbles (MB) and ultrasound (US); gene with US; gene with MB; or the gene alone. Results Distribution of MB and the gene in the myocardium was visualized during the experiment. Compared with the EGFP gene group, an average 14.7-fold enhancement in gene expression was achieved in the EGFP+MB/US group (P < 0.01). Compared with the HGF gene group, an average 10.7-fold enhancement in gene expression was achieved in the HGF+MB/US group (P < 0.01). In addition, capillary density increased from 20.8 ± 3.4/mm2 in the HGF gene group to 146.7 ± 31.4/mm2 in HGF+MB/US group (P < 0.01). Conclusions Thus, direct intramyocardial injection of an angiogenic gene in conjunction with microbubbles plus insonation synergistically enhances angiogenesis. This method offers an observable gene delivery procedure with enhanced expression efficiency of the delivered gene. PMID:21600027

  5. Nanocarrier-mediated co-delivery of chemotherapeutic drugs and gene agents for cancer treatment.

    PubMed

    Kang, Lin; Gao, Zhonggao; Huang, Wei; Jin, Mingji; Wang, Qiming

    2015-05-01

    The efficacy of chemotherapeutic drug in cancer treatment is often hampered by drug resistance of tumor cells, which is usually caused by abnormal gene expression. RNA interference mediated by siRNA and miRNA can selectively knock down the carcinogenic genes by targeting specific mRNAs. Therefore, combining chemotherapeutic drugs with gene agents could be a promising strategy for cancer therapy. Due to poor stability and solubility associated with gene agents and drugs, suitable protective carriers are needed and have been widely researched for the co-delivery. In this review, we summarize the most commonly used nanocarriers for co-delivery of chemotherapeutic drugs and gene agents, as well as the advances in co-delivery systems. PMID:26579443

  6. Nanocarrier-mediated co-delivery of chemotherapeutic drugs and gene agents for cancer treatment

    PubMed Central

    Kang, Lin; Gao, Zhonggao; Huang, Wei; Jin, Mingji; Wang, Qiming

    2015-01-01

    The efficacy of chemotherapeutic drug in cancer treatment is often hampered by drug resistance of tumor cells, which is usually caused by abnormal gene expression. RNA interference mediated by siRNA and miRNA can selectively knock down the carcinogenic genes by targeting specific mRNAs. Therefore, combining chemotherapeutic drugs with gene agents could be a promising strategy for cancer therapy. Due to poor stability and solubility associated with gene agents and drugs, suitable protective carriers are needed and have been widely researched for the co-delivery. In this review, we summarize the most commonly used nanocarriers for co-delivery of chemotherapeutic drugs and gene agents, as well as the advances in co-delivery systems. PMID:26579443

  7. Asialoglycoprotein Receptor-Mediated Gene Delivery to Hepatocytes Using Galactosylated Polymers.

    PubMed

    Thapa, Bindu; Kumar, Piyush; Zeng, Hongbo; Narain, Ravin

    2015-09-14

    Highly efficient, specific, and nontoxic gene delivery vector is required for gene therapy to the liver. Hepatocytes exclusively express asialoglycoprotein receptor (ASGPR), which can recognize and bind to galactose or N-acetylgalactosamine. Galactosylated polymers are therefore explored for targeted gene delivery to the liver. A library of safe and stable galactose-based glycopolymers that can specifically deliver genes to hepatocytes were synthesized having different architectures, compositions, and molecular weights via the reversible addition-fragmentation chain transfer process. The physical and chemical properties of these polymers have a great impact on gene delivery efficacy into hepatocytes, as such block copolymers are found to form more stable complexes with plasmid and have high gene delivery efficiency into ASGPR expressing hepatocytes. Transfection efficiency and uptake of polyplexes with these polymers decreased significantly by preincubation of hepatocytes with free asialofetuin or by adding free asialofetuin together with polyplexes into hepatocytes. The results confirmed that polyplexes with these polymers were taken up specifically by hepatocytes via ASGPR-mediated endocytosis. The results from transfection efficiency and uptake of these polymers in cells without ASGPR, such as SK Hep1 and HeLa cells, further support this mechanism. Since in vitro cytotoxicity assays prove these glycopolymers to be nontoxic, they may be useful for delivery of clinically important genes specifically to the liver.

  8. Parameters Affecting Image-guided, Hydrodynamic Gene Delivery to Swine Liver

    PubMed Central

    Kamimura, Kenya; Suda, Takeshi; Zhang, Guisheng; Aoyagi, Yutaka; Liu, Dexi

    2013-01-01

    Development of a safe and effective method for gene delivery to hepatocytes is a critical step toward gene therapy for liver diseases. Here, we assessed the parameters for gene delivery to the livers of large animals (pigs, 40–65 kg) using an image-guided hydrodynamics-based procedure that involves image-guided catheter insertion into the lobular hepatic vein and hydrodynamic injection of reporter plasmids using a computer-controlled injector. We demonstrated that injection parameters (relative position of the catheter in the hepatic vasculature, intravascular pressure upon injection, and injection volume) are directly related to the safety and efficiency of the procedure. By optimizing these parameters, we explored for the first time, the advantage of the procedure for sequential injections to multiple lobes in human-sized pigs. The optimized procedure resulted in sustained expression of the human α-1 antitrypsin gene in livers for more than 2 months after gene delivery. In addition, repeated hydrodynamic gene delivery was safely conducted and no adverse events were seen in the entire period of the study. Our results support the clinical applicability of the image-guided hydrodynamic gene delivery method for the treatment of liver diseases. PMID:24129227

  9. Portal Vein Delivery of Viral Vectors for Gene Therapy for Hemophilia

    PubMed Central

    Sherman, Alexandra; Schlachterman, Alexander; Cooper, Mario; Merricks, Elizabeth P.; Raymer, Robin A.; Bellinger, Dwight A.; Herzog, Roland W.; Nichols, Timothy C.

    2014-01-01

    The liver is a very complex organ with a large variety of functions, making it an attractive organ for gene replacement therapy. Many genetic disorders can be corrected by delivering gene products directly into the liver using viral vectors. In this chapter, we will describe gene delivery via portal vein administration in mice and dogs to correct the blood coagulation disorder hemophilia B. Although there are multiple delivery routes for both viral and non-viral vectors in animals, portal vein administration delivers vectors directly and efficiently into the liver. Complete correction of murine hemophilia B and multi-year near-correction of canine hemophilia B have been achieved following portal vein delivery of adeno-associated viral (AAV) vectors expressing factor IX from hepatocyte-specific promoters. Peripheral vein injection can lead to increased vector dissemination to off-target organ such as the lung and spleen. Below, we will describe portal vein injection delivery route via laparotomy. PMID:24557919

  10. Hydroxyl PAMAM dendrimer-based gene vectors for transgene delivery to human retinal pigment epithelial cells

    NASA Astrophysics Data System (ADS)

    Mastorakos, Panagiotis; Kambhampati, Siva P.; Mishra, Manoj K.; Wu, Tony; Song, Eric; Hanes, Justin; Kannan, Rangaramanujam M.

    2015-02-01

    Ocular gene therapy holds promise for the treatment of numerous blinding disorders. Despite the significant progress in the field of viral and non-viral gene delivery to the eye, significant obstacles remain in the way of achieving high-level transgene expression without adverse effects. The retinal pigment epithelium (RPE) is involved in the pathogenesis of retinal diseases and is a key target for a number of gene-based therapeutics. In this study, we addressed the inherent drawbacks of non-viral gene vectors and combined different approaches to design an efficient and safe dendrimer-based gene-delivery platform for delivery to human RPE cells. We used hydroxyl-terminated polyamidoamine (PAMAM) dendrimers functionalized with various amounts of amine groups to achieve effective plasmid compaction. We further used triamcinolone acetonide (TA) as a nuclear localization enhancer for the dendrimer-gene complex and achieved significant improvement in cell uptake and transfection of hard-to-transfect human RPE cells. To improve colloidal stability, we further shielded the gene vector surface through incorporation of PEGylated dendrimer along with dendrimer-TA for DNA complexation. The resultant complexes showed improved stability while minimally affecting transgene delivery, thus improving the translational relevance of this platform.Ocular gene therapy holds promise for the treatment of numerous blinding disorders. Despite the significant progress in the field of viral and non-viral gene delivery to the eye, significant obstacles remain in the way of achieving high-level transgene expression without adverse effects. The retinal pigment epithelium (RPE) is involved in the pathogenesis of retinal diseases and is a key target for a number of gene-based therapeutics. In this study, we addressed the inherent drawbacks of non-viral gene vectors and combined different approaches to design an efficient and safe dendrimer-based gene-delivery platform for delivery to human RPE

  11. Bioplex technology: novel synthetic gene delivery pharmaceutical based on peptides anchored to nucleic acids.

    PubMed

    Simonson, Oscar E; Svahn, Mathias G; Törnquist, Elisabeth; Lundin, Karin E; Smith, C I E

    2005-01-01

    Non-viral gene delivery is an important approach in order to establish safe in vivo gene therapy in the clinic. Although viral vectors currently exhibit superior gene transfer efficacy, the safety aspect of viral gene delivery is a concern. In order to improve non-viral in vivo gene delivery we have designed a pharmaceutical platform called Bioplex (biological complex). The concept of Bioplex is to link functional entities via hybridising anchors, such as Peptide Nucleic Acids (PNA), directly to naked DNA. In order to promote delivery functional entities consisting of biologically active peptides or carbohydrates, are linked to the PNA anchor. The PNA acts as genetic glue and hybridises with DNA in a sequence specific manner. By using functional entities, which elicit receptor-mediated endocytosis, improved endosomal escape and enhance nuclear entry we wish to improve the transfer of genetic material into the cell. An important aspect is that the functional entities should also have tissue-targeting properties in vivo. Examples of functional entities investigated to date are the Simian virus 40 nuclear localisation signal to improve nuclear uptake and different carbohydrate ligands in order to achieve receptor specific uptake. The delivery system is also endowed with regulatory capability, since the release of functional entities can be controlled. The aim is to create a safe, pharmaceutically defined and stable delivery system for nucleic acids with enhanced transfection properties that can be used in the clinic.

  12. Microbubbles in Ultrasound-Triggered Drug and Gene Delivery

    PubMed Central

    Hernot, Sophie; Klibanov, Alexander L.

    2008-01-01

    Ultrasound contrast agents, in the form of gas-filled microbubbles, are becoming popular in perfusion monitoring; they are employed as molecular imaging agents. Microbubbles are manufactured from biocompatible materials, they can be injected intravenously, and some are approved for clinical use. Microbubbles can be destroyed by ultrasound irradiation. This destruction phenomenon can be applied to targeted drug delivery and enhancement of drug action. The ultrasonic field can be focused at the target tissues and organs; thus, selectivity of the treatment can be improved, reducing undesirable side effects. Microbubbles enhance ultrasound energy deposition in the tissues and serve as cavitation nuclei, increasing intracellular drug delivery. DNA delivery and successful tissue transfection is observed in the areas of the body where ultrasound is applied after intravascular administration of microbubbles and plasmid DNA. Accelerated blood clot dissolution in the areas of insonation by cooperative action of thrombolytic agents and microbubbles is demonstrated in several clinical trials. PMID:18486268

  13. Magnetic nanoparticles for targeted therapeutic gene delivery and magnetic-inducing heating on hepatoma

    NASA Astrophysics Data System (ADS)

    Yuan, Chenyan; An, Yanli; Zhang, Jia; Li, Hongbo; Zhang, Hao; Wang, Ling; Zhang, Dongsheng

    2014-08-01

    Gene therapy holds great promise for treating cancers, but their clinical applications are being hampered due to uncontrolled gene delivery and expression. To develop a targeted, safe and efficient tumor therapy system, we constructed a tissue-specific suicide gene delivery system by using magnetic nanoparticles (MNPs) as carriers for the combination of gene therapy and hyperthermia on hepatoma. The suicide gene was hepatoma-targeted and hypoxia-enhanced, and the MNPs possessed the ability to elevate temperature to the effective range for tumor hyperthermia as imposed on an alternating magnetic field (AMF). The tumoricidal effects of targeted gene therapy associated with hyperthermia were evaluated in vitro and in vivo. The experiment demonstrated that hyperthermia combined with a targeted gene therapy system proffer an effective tool for tumor therapy with high selectivity and the synergistic effect of hepatoma suppression.

  14. Up-Scaled Synthesis and Characterization of Nonviral Gene Delivery Particles for Transient In Vitro and In Vivo Transgene Expression.

    PubMed

    Taschauer, Alexander; Geyer, Antonia; Gehrig, Sebastian; Maier, Julia; Sami, Haider; Ogris, Manfred

    2016-06-01

    Polyethylenimine-based polyplexes are promising nonviral gene delivery systems for preclinical and clinical applications. Pipette-based polyplexing is associated with several disadvantages, such as batch-to-batch variability, restriction to smaller volumes, and variable gene delivery results. The present protocol describes syringe-pump-mediated upscaled synthesis of well-defined gene delivery nanoparticles capable of efficient in vitro and in vivo gene delivery. Syringe-pump-based synthesis ensures controlled mixing, upscaling, and reproducible gene delivery. Nanoparticle tracking analysis of the upscaled formulations involved single nanoparticle tracking, thereby generating highly resolved biophysical characterization. Gene delivery performance was investigated by luciferase gene expression in cells and three-dimensional bioluminescence imaging in mice. PMID:27169568

  15. Cationic liposome-nucleic acid nanoparticle assemblies with applications in gene delivery and gene silencing.

    PubMed

    Majzoub, Ramsey N; Ewert, Kai K; Safinya, Cyrus R

    2016-07-28

    Cationic liposomes (CLs) are synthetic carriers of nucleic acids in gene delivery and gene silencing therapeutics. The introduction will describe the structures of distinct liquid crystalline phases of CL-nucleic acid complexes, which were revealed in earlier synchrotron small-angle X-ray scattering experiments. When mixed with plasmid DNA, CLs containing lipids with distinct shapes spontaneously undergo topological transitions into self-assembled lamellar, inverse hexagonal, and hexagonal CL-DNA phases. CLs containing cubic phase lipids are observed to readily mix with short interfering RNA (siRNA) molecules creating double gyroid CL-siRNA phases for gene silencing. Custom synthesis of multivalent lipids and a range of novel polyethylene glycol (PEG)-lipids with attached targeting ligands and hydrolysable moieties have led to functionalized equilibrium nanoparticles (NPs) optimized for cell targeting, uptake or endosomal escape. Very recent experiments are described with surface-functionalized PEGylated CL-DNA NPs, including fluorescence microscopy colocalization with members of the Rab family of GTPases, which directly reveal interactions with cell membranes and NP pathways. In vitro optimization of CL-DNA and CL-siRNA NPs with relevant primary cancer cells is expected to impact nucleic acid therapeutics in vivo. This article is part of the themed issue 'Soft interfacial materials: from fundamentals to formulation'. PMID:27298431

  16. Polymeric Carriers for Gene Delivery: Chitosan and Poly(amidoamine) Dendrimers

    PubMed Central

    Xu, Qingxing; Wang, Chi-Hwa; Pack, Daniel Wayne

    2012-01-01

    Gene therapy is a potential medical solution that promises new treatments and may hold the cure for many different types of diseases and disorders of the human race. However, gene therapy is still a growing medical field and the technology is still in its infancy. The main challenge for gene therapy is to find safe and effective vectors that are able to deliver genes to the specific cells and get them to express inside the cells. Due to safety concerns, synthetic delivery systems, rather than viral vectors, are preferred for gene delivery and significant efforts have been focused on the development of this field. However, we are faced with problems like low gene transfer efficiency, cytotoxicity and lack of cell-targeting capability for these synthetic delivery systems. Over the years, we have seen a variety of new and effective polymers which have been designed and synthesized specifically for gene delivery. Moreover, various strategies that aimed at enhancing their physicochemical properties, improving transfection efficiency, reducing cytotoxicity as well as incorporating functional groups that offer better targetability and higher cellular uptake are established. Here, we look at two potential polymeric carriers, chitosan and poly(amidoamine) dendrimers, which have been widely reported for gene delivery. For chitosan, the interest arises from their availability, excellent non-cytotoxicity profile, biodegradability and ease of modification. For poly(amidoamine) dendrimers, the interest arises from their ease of synthesis with controlled structure and size, minimal cytotoxicity, biodegradability and high transfection efficiencies. The latest developments on these polymers for gene delivery will be the main focus of this article. PMID:20618156

  17. Modulating polymer chemistry to enhance non-viral gene delivery inside hydrogels with tunable matrix stiffness.

    PubMed

    Keeney, Michael; Onyiah, Sheila; Zhang, Zhe; Tong, Xinming; Han, Li-Hsin; Yang, Fan

    2013-12-01

    Non-viral gene delivery holds great promise for promoting tissue regeneration, and offers a potentially safer alternative than viral vectors. Great progress has been made to develop biodegradable polymeric vectors for non-viral gene delivery in 2D culture, which generally involves isolating and modifying cells in vitro, followed by subsequent transplantation in vivo. Scaffold-mediated gene delivery may eliminate the need for the multiple-step process in vitro, and allows sustained release of nucleic acids in situ. Hydrogels are widely used tissue engineering scaffolds given their tissue-like water content, injectability and tunable biochemical and biophysical properties. However, previous attempts on developing hydrogel-mediated non-viral gene delivery have generally resulted in low levels of transgene expression inside 3D hydrogels, and increasing hydrogel stiffness further decreased such transfection efficiency. Here we report the development of biodegradable polymeric vectors that led to efficient gene delivery inside poly(ethylene glycol) (PEG)-based hydrogels with tunable matrix stiffness. Photocrosslinkable gelatin was maintained constant in the hydrogel network to allow cell adhesion. We identified a lead biodegradable polymeric vector, E6, which resulted in increased polyplex stability, DNA protection and achieved sustained high levels of transgene expression inside 3D PEG-DMA hydrogels for at least 12 days. Furthermore, we demonstrated that E6-based polyplexes allowed efficient gene delivery inside hydrogels with tunable stiffness ranging from 2 to 175 kPa, with the peak transfection efficiency observed in hydrogels with intermediate stiffness (28 kPa). The reported hydrogel-mediated gene delivery platform using biodegradable polyplexes may serve as a local depot for sustained transgene expression in situ to enhance tissue engineering across broad tissue types.

  18. Efficient intranuclear gene delivery by CdSe aqueous quantum dots electrostatically-coated with polyethyleneimine

    NASA Astrophysics Data System (ADS)

    Au, Giang H. T.; Y Shih, Wan; Shih, Wei-Heng

    2015-01-01

    Quantum dots (QDs) are semiconducting nanoparticles with photoluminescence properties that do not photobleach. Due to these advantages, using QDs for non-viral gene delivery has the additional benefit of being able to track the delivery of the genes in real time as it happens. We investigate the efficacy of mercaptopropionic acid (MPA)-capped CdSe aqueous quantum dots (AQDs) electrostatically complexed with branched polyethyleneimine (PEI) both as a non-viral gene delivery vector and as a fluorescent probe for tracking the delivery of genes into nuclei. The MPA-capped CdSe AQDs that were completely synthesized in water were the model AQDs. A nominal MPA:Cd:Se = 4:3:1 was chosen for optimal photoluminescence and zeta potential. The gene delivery study was carried out in vitro using a human colon cancer cell line, HT29 (ATCC). The model gene was a plasmid DNA (pDNA) that can express red fluorescent protein (RFP). Positively charged branched PEI was employed to provide a proton buffer to the AQDs to allow for endosomal escape. It is shown that by using a PEI-AQD complex with a PEI/AQD molar ratio of 300 and a nominal pDNA/PEI-AQD ratio of 6, we can achieve 75 ± 2.6% RFP expression efficiency with cell vitality remaining at 78 ± 4% of the control.

  19. Gene delivery to the neurulating embryo during culture

    EPA Science Inventory

    Modulating expression of specific genes during embryogenesis will help elucidate their role in development. Transient overexpression of specific genes can be accomplished by adding additional copies, or else antisense transcripts can be used to block expression. Manipulation of g...

  20. Design and fabrication of N-alkyl-polyethylenimine-stabilized iron oxide nanoclusters for gene delivery.

    PubMed

    Liu, Gang; Wang, Zhiyong; Lee, Seulki; Ai, Hua; Chen, Xiaoyuan

    2012-01-01

    With the rapid development of nanotechnology, inorganic magnetic nanoparticles, especially iron oxide nanoparticles (IOs), have emerged as great vehicles for biomedical diagnostic and therapeutic applications. In order to rationally design IO-based gene delivery nanovectors, surface modification is essential and determines the loading and release of the gene of interest. Here we highlight the basic concepts and applications of nonviral gene delivery vehicles based on low molecular weight N-alkyl polyethylenimine-stabilized IOs. The experimental protocols related to these topics are described in this chapter. PMID:22568910

  1. Microvesicle-associated AAV Vector as a Novel Gene Delivery System

    PubMed Central

    Maguire, Casey A; Balaj, Leonora; Sivaraman, Sarada; Crommentuijn, Matheus HW; Ericsson, Maria; Mincheva-Nilsson, Lucia; Baranov, Vladimir; Gianni, Davide; Tannous, Bakhos A; Sena-Esteves, Miguel; Breakefield, Xandra O; Skog, Johan

    2012-01-01

    Adeno-associated virus (AAV) vectors have shown remarkable efficiency for gene delivery to cultured cells and in animal models of human disease. However, limitations to AAV vectored gene transfer exist after intravenous transfer, including off-target gene delivery (e.g., liver) and low transduction of target tissue. Here, we show that during production, a fraction of AAV vectors are associated with microvesicles/exosomes, termed vexosomes (vector-exosomes). AAV capsids associated with the surface and in the interior of microvesicles were visualized using electron microscopy. In cultured cells, vexosomes outperformed conventionally purified AAV vectors in transduction efficiency. We found that purified vexosomes were more resistant to a neutralizing anti-AAV antibody compared to conventionally purified AAV. Finally, we show that vexosomes bound to magnetic beads can be attracted to a magnetized area in cultured cells. Vexosomes represent a unique entity which offers a promising strategy to improve gene delivery. PMID:22314290

  2. Hyaluronic acid and fibrin hydrogels with concentrated DNA/PEI polyplexes for local gene delivery.

    PubMed

    Lei, Yuguo; Rahim, Maha; Ng, Quinn; Segura, Tatiana

    2011-08-10

    Local delivery of DNA through a hydrogel scaffold would increase the applicability of gene therapy in tissue regeneration and cancer therapy. However, the delivery of DNA/cationic polymer nanoparticles (polyplexes) using hydrogels is challenging due to the aggregation and inactivation of polyplexes during their incorporation into hydrogel scaffolds. We developed a novel process (termed caged nanoparticle encapsulation or CnE) to load concentrated and unaggregated non-viral gene delivery nanoparticles into various hydrogels. Previously, we showed that PEG hydrogels loaded with DNA/PEI polyplexes through this process were able to deliver genes both in vitro and in vivo. In this study, we found that hyaluronic acid and fibrin hydrogels with concentrated and unaggregated polyplexes loaded through CnE were able to deliver genes in vivo as well, demonstrating the universality of the process.

  3. Development of a Targeted Gene-Delivery System Using Escherichia coli.

    PubMed

    Chiang, Chung-Jen; Chang, Chih-Hsiang; Chao, Yun-Peng; Kao, Ming-Ching

    2016-01-01

    A gene-delivery system based on microbes is useful for development of targeted gene therapy of non-phagocytic cancer cells. Here, the feasibility of the delivery system is illustrated by targeted delivery of a transgene (i.e., eukaryotic GFP) by Escherichia coli to HER2/neu-positive cancer cells. An E. coli strain was engineered with surface display of the anti-HER2/neu affibody. To release the gene cargo, a programmed lysis system based on phage ϕX174 gene E was introduced into the E. coli strain. As a result, 3 % of HER2/neu-positive cells that were infected with engineered E. coli were able to express the GFP. PMID:26846805

  4. Role of nanotechnology and gene delivery systems in TRAIL-based therapies

    PubMed Central

    Naoum, George E; Tawadros, Fady; Farooqi, Ammad Ahmad; Qureshi, Muhammad Zahid; Tabassum, Sobia; Buchsbaum, Donald J; Arafat, Waleed

    2016-01-01

    Since its identification as a member of the tumour necrosis factor (TNF) family, TRAIL (TNF-related apoptosis-inducing ligand) has emerged as a new avenue in apoptosis-inducing cancer therapies. Its ability to circumvent the chemoresistance of conventional therapeutics and to interact with cancer stem cells (CSCs) self-renewal pathways, amplified its potential as a cancer apoptotic agent. Many recombinant preparations of this death ligand and monoclonal antibodies targeting its death receptors have been tested in monotherapy and combinational clinical trials. Gene therapy is a new approach for cancer treatment which implies viral or non-viral functional transgene induction of apoptosis in cancer cells or repair of the underlying genetic abnormality on a molecular level. The role of this approach in overcoming the traditional barriers of radiation and chemotherapeutics systemic toxicity, risk of recurrence, and metastasis made it a promising platform for cancer treatment. The recent first Food Drug Administration (FDA) approved oncolytic herpes virus for melanoma treatment brings forth the potency of the cancer gene therapy approach in the future. Many gene delivery systems have been studied for intratumoural TRAIL gene delivery alone or in combination with chemotherapeutic agents to produce synergistic cancer cytotoxicity. However, there still remain many obstacles to be conquered for this different gene delivery systems. Nanomedicine on the other hand offers a new frontier for clinical trials and biomedical research. The FDA approved nanodrugs motivates horizon exploration for other nanoscale designed particles’ implications in gene delivery. In this review we aim to highlight the molecular role of TRAIL in apoptosis and interaction with cancer stem cells (CSCs) self-renewal pathways. Finally, we also aim to discuss the different roles of gene delivery systems, mesenchymal cells, and nanotechnology designs in TRAIL gene delivery. PMID:27594905

  5. Role of nanotechnology and gene delivery systems in TRAIL-based therapies.

    PubMed

    Naoum, George E; Tawadros, Fady; Farooqi, Ammad Ahmad; Qureshi, Muhammad Zahid; Tabassum, Sobia; Buchsbaum, Donald J; Arafat, Waleed

    2016-01-01

    Since its identification as a member of the tumour necrosis factor (TNF) family, TRAIL (TNF-related apoptosis-inducing ligand) has emerged as a new avenue in apoptosis-inducing cancer therapies. Its ability to circumvent the chemoresistance of conventional therapeutics and to interact with cancer stem cells (CSCs) self-renewal pathways, amplified its potential as a cancer apoptotic agent. Many recombinant preparations of this death ligand and monoclonal antibodies targeting its death receptors have been tested in monotherapy and combinational clinical trials. Gene therapy is a new approach for cancer treatment which implies viral or non-viral functional transgene induction of apoptosis in cancer cells or repair of the underlying genetic abnormality on a molecular level. The role of this approach in overcoming the traditional barriers of radiation and chemotherapeutics systemic toxicity, risk of recurrence, and metastasis made it a promising platform for cancer treatment. The recent first Food Drug Administration (FDA) approved oncolytic herpes virus for melanoma treatment brings forth the potency of the cancer gene therapy approach in the future. Many gene delivery systems have been studied for intratumoural TRAIL gene delivery alone or in combination with chemotherapeutic agents to produce synergistic cancer cytotoxicity. However, there still remain many obstacles to be conquered for this different gene delivery systems. Nanomedicine on the other hand offers a new frontier for clinical trials and biomedical research. The FDA approved nanodrugs motivates horizon exploration for other nanoscale designed particles' implications in gene delivery. In this review we aim to highlight the molecular role of TRAIL in apoptosis and interaction with cancer stem cells (CSCs) self-renewal pathways. Finally, we also aim to discuss the different roles of gene delivery systems, mesenchymal cells, and nanotechnology designs in TRAIL gene delivery. PMID:27594905

  6. Role of nanotechnology and gene delivery systems in TRAIL-based therapies

    PubMed Central

    Naoum, George E; Tawadros, Fady; Farooqi, Ammad Ahmad; Qureshi, Muhammad Zahid; Tabassum, Sobia; Buchsbaum, Donald J; Arafat, Waleed

    2016-01-01

    Since its identification as a member of the tumour necrosis factor (TNF) family, TRAIL (TNF-related apoptosis-inducing ligand) has emerged as a new avenue in apoptosis-inducing cancer therapies. Its ability to circumvent the chemoresistance of conventional therapeutics and to interact with cancer stem cells (CSCs) self-renewal pathways, amplified its potential as a cancer apoptotic agent. Many recombinant preparations of this death ligand and monoclonal antibodies targeting its death receptors have been tested in monotherapy and combinational clinical trials. Gene therapy is a new approach for cancer treatment which implies viral or non-viral functional transgene induction of apoptosis in cancer cells or repair of the underlying genetic abnormality on a molecular level. The role of this approach in overcoming the traditional barriers of radiation and chemotherapeutics systemic toxicity, risk of recurrence, and metastasis made it a promising platform for cancer treatment. The recent first Food Drug Administration (FDA) approved oncolytic herpes virus for melanoma treatment brings forth the potency of the cancer gene therapy approach in the future. Many gene delivery systems have been studied for intratumoural TRAIL gene delivery alone or in combination with chemotherapeutic agents to produce synergistic cancer cytotoxicity. However, there still remain many obstacles to be conquered for this different gene delivery systems. Nanomedicine on the other hand offers a new frontier for clinical trials and biomedical research. The FDA approved nanodrugs motivates horizon exploration for other nanoscale designed particles’ implications in gene delivery. In this review we aim to highlight the molecular role of TRAIL in apoptosis and interaction with cancer stem cells (CSCs) self-renewal pathways. Finally, we also aim to discuss the different roles of gene delivery systems, mesenchymal cells, and nanotechnology designs in TRAIL gene delivery.

  7. Self-assembled pentablock copolymers for selective and sustained gene delivery

    SciTech Connect

    Zhang, Bingqi

    2011-05-15

    The poly(diethylaminoethyl methacrylate) (PDEAEM) - Pluronic F127 - PDEAEM pentablock copolymer (PB) gene delivery vector system has been found to possess an inherent selectivity in transfecting cancer cells over non-cancer cells in vitro, without attaching any targeting ligands. In order to understand the mechanism of this selective transfection, three possible intracellular barriers to transfection were investigated in both cancer and non-cancer cells. We concluded that escape from the endocytic pathway served as the primary intracellular barrier for PB-mediated transfection. Most likely, PB vectors were entrapped and rendered non-functional in acidic lysosomes of non-cancer cells, but survived in less acidic lysosomes of cancer cells. The work highlights the importance of identifying intracellular barriers for different gene delivery systems and provides a new paradigm for designing targeting vectors based on intracellular differences between cell types, rather than through the use of targeting ligands. The PB vector was further developed to simultaneously deliver anticancer drugs and genes, which showed a synergistic effect demonstrated by significantly enhanced gene expression in vitro. Due to the thermosensitive gelation behavior, the PB vector packaging both drug and gene was also investigated for its in vitro sustained release properties by using polyethylene glycol diacrylate as a barrier gel to mimic the tumor matrix in vivo. Overall, this work resulted in the development of a gene delivery vector for sustained and selective gene delivery to tumor cells for cancer therapy.

  8. A novel cationic liposome formulation for efficient gene delivery via a pulmonary route

    NASA Astrophysics Data System (ADS)

    Li, Peng; Liu, Donghua; Sun, Xiaoli; Liu, Chunxi; Liu, Yongjun; Zhang, Na

    2011-06-01

    The clinical success of gene therapy for lung cancer is not only dependent on efficient gene carriers but also on a suitable delivery route. A pulmonary delivery route can directly deliver gene vectors to the lung which is more efficient than a systemic delivery route. For gene carriers, cationic liposomes have recently emerged as leading non-viral vectors in worldwide gene therapy clinical trials. However, cytotoxic effects or apoptosis are often observed which is mostly dependent on the cationic lipid used. Therefore, an efficient and safe cationic lipid, 6-lauroxyhexyl lysinate (LHLN), previously synthesized by our group was first used to prepare cationic liposomes. Physicochemical and biological properties of LHLN-liposomes were investigated. LHLN-liposome/DNA complexes showed positive surface charge, spherical morphology, a relatively narrow particle size distribution and strong DNA binding capability. Compared with Lipofectamine2000, the new cationic liposome formulation using LHLN exhibited not only lower cytotoxicity (P < 0.05) but also similar transfection efficiency in A549 and HepG2 lung cancer cells for in vitro tests. When administered by intratracheal instillation into rat lungs for in vivo evaluation, LHLN-liposome/DNA complexes exhibited higher pulmonary gene transfection efficiency than Lipofectamine2000/DNA complexes (P < 0.05). These results suggested that LHLN-liposomes may have great potential for efficient pulmonary gene delivery.

  9. Self-assembled pentablock copolymers for selective and sustained gene delivery

    NASA Astrophysics Data System (ADS)

    Zhang, Bingqi

    The poly(diethylaminoethyl methacrylate) (PDEAEM) - Pluronic F127 - PDEAEM pentablock copolymer (PB) gene delivery vector system has been found to possess an inherent selectivity in transfecting cancer cells over non-cancer cells in vitro, without attaching any targeting ligands. In order to understand the mechanism of this selective transfection, three possible intracellular barriers to transfection were investigated in both cancer and non-cancer cells. We concluded that escape from the endocytic pathway served as the primary intracellular barrier for PB-mediated transfection. Most likely, PB vectors were entrapped and rendered non-functional in acidic lysosomes of non-cancer cells, but survived in less acidic lysosomes of cancer cells. The work highlights the importance of identifying intracellular barriers for different gene delivery systems and provides a new paradigm for designing targeting vectors based on intracellular differences between cell types, rather than through the use of targeting ligands. The PB vector was further developed to simultaneously deliver anticancer drugs and genes, which showed a synergistic effect demonstrated by significantly enhanced gene expression in vitro. Due to the thermosensitive gelation behavior, the PB vector packaging both drug and gene was also investigated for its in vitro sustained release properties by using polyethylene glycol diacrylate as a barrier gel to mimic the tumor matrix in vivo . Overall, this work resulted in the development of a gene delivery vector for sustained and selective gene delivery to tumor cells for cancer therapy.

  10. Nacystelyn enhances adenoviral vector-mediated gene delivery to mouse airways.

    PubMed

    Kushwah, R; Oliver, J R; Cao, H; Hu, J

    2007-08-01

    Adenoviral vector-mediated gene delivery has been vastly investigated for cystic fibrosis (CF) gene therapy; however, one of its drawbacks is the low efficiency of gene transfer, which is due to basolateral colocalization of viral receptors, immune responses to viral vectors and the presence of a thick mucus layer in the airways of CF patients. Therefore, enhancement of gene transfer can lead to reduction in the viral dosage, which could further reduce the acute toxicity associated with the use of adenoviral vectors. Nacystelyn (NAL) is a mucolytic agent with anti-inflammatory and antioxidant properties, and has been used clinically in CF patients to reduce mucus viscosity in the airways. In this study, we show that pretreatment of the airways with NAL followed by administration of adenoviral vectors in complex with DEAE-Dextran can significantly enhance gene delivery to the airways of mice without any harmful effects. Moreover, NAL pretreatment can reduce the airway inflammation, which is normally observed after delivery of adenoviral particles. Taken together, these results indicate that NAL pretreatment followed by adenoviral vector-mediated gene delivery can be beneficial to CF patients by increasing the efficiency of gene transfer to the airways, and reducing the acute toxicity associated with the administration of adenoviral vectors. PMID:17525704

  11. Multifunctional nanocarrier based on clay nanotubes for efficient intracellular siRNA delivery and gene silencing.

    PubMed

    Wu, Hui; Shi, Yinfeng; Huang, Chusen; Zhang, Yang; Wu, Jiahui; Shen, Hebai; Jia, Nengqin

    2014-04-01

    RNA interference-mediated gene silencing relating to disease has recently emerged as a powerful method in gene therapy. Despite the promises, effective transport of siRNA with minimal side effects remains a challenge. Halloysites are cheap and naturally available aluminosilicate clay nanotubes with high mechanical strength and biocompatibility. In this study, a novel multifunctional nanocarrier based on functionalized halloysite nanotubes (f-HNTs) has been developed via electrostatic layer-by-layer assembling approach for loading and intracellular delivery of therapeutic antisurvivin siRNA and simultaneously tracking their intracellular transport, in which PEI-modified HNTs are used as gene vector, antisurvivin siRNA as gene therapeutic agent, and mercaptoacetic acid-capped CdSe quantum dots as fluorescent labeling probes. The successful assembly of the f-HNTs-siRNA complexes was systematically characterized by transmission electron microscopy (TEM), UV-visible spectrophotometry, Zeta potential measurement, fluorescence spectrophotometry, and electrochemical impedance spectroscopy. Confocal microscopy, biological TEM, and flow cytometry studies revealed that the complexes enabled the efficient intracellular delivery of siRNA for cell-specific gene silencing. MTT assays exhibited that the complexes can enhance antitumor activity. Furthermore, Western blot analysis showed that f-HNTs-mediated siRNA delivery effectively knocked down gene expression of survivin and thereby decreased the levels of target proteins of PANC-1 cells. Therefore, this study suggested that the synthesized f-HNTs were a new effective drug delivery system for potential application in cancer gene therapy.

  12. Nacystelyn enhances adenoviral vector-mediated gene delivery to mouse airways.

    PubMed

    Kushwah, R; Oliver, J R; Cao, H; Hu, J

    2007-08-01

    Adenoviral vector-mediated gene delivery has been vastly investigated for cystic fibrosis (CF) gene therapy; however, one of its drawbacks is the low efficiency of gene transfer, which is due to basolateral colocalization of viral receptors, immune responses to viral vectors and the presence of a thick mucus layer in the airways of CF patients. Therefore, enhancement of gene transfer can lead to reduction in the viral dosage, which could further reduce the acute toxicity associated with the use of adenoviral vectors. Nacystelyn (NAL) is a mucolytic agent with anti-inflammatory and antioxidant properties, and has been used clinically in CF patients to reduce mucus viscosity in the airways. In this study, we show that pretreatment of the airways with NAL followed by administration of adenoviral vectors in complex with DEAE-Dextran can significantly enhance gene delivery to the airways of mice without any harmful effects. Moreover, NAL pretreatment can reduce the airway inflammation, which is normally observed after delivery of adenoviral particles. Taken together, these results indicate that NAL pretreatment followed by adenoviral vector-mediated gene delivery can be beneficial to CF patients by increasing the efficiency of gene transfer to the airways, and reducing the acute toxicity associated with the administration of adenoviral vectors.

  13. DOPC-Detergent Conjugates: Fusogenic Carriers for Improved In Vitro and In Vivo Gene Delivery.

    PubMed

    Pierrat, Philippe; Casset, Anne; Kereselidze, Dimitri; Lux, Marie; Pons, Françoise; Lebeau, Luc

    2016-07-01

    Phospholipid-detergent conjugates are proposed as fusogenic carriers for gene delivery. Eleven compounds are prepared and their properties are investigated. The ability of the conjugates to promote fusion with a negatively charged model membrane is determined. Their DNA delivery efficiency and cytotoxicity are assessed in vitro. Lipoplexes are administered in the mouse lung, and transgene expression Indeterminate inflammatory activity are measured. The results show that conjugation of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) with C12 E4 produces a carrier that can efficiently deliver DNA to cells, with negligible -associated toxicity. Fusogenicity of the conjugates shows good correlation with in vitro transfection efficiency and crucially depends on the length of the polyether moiety of the detergent. Finally, DOPC-C12 E4 reveals highly potent for in vivo DNA delivery and favorably compares to GL67A, the current golden standard for gene delivery to the airway, opening the way for further promising developments. PMID:26990218

  14. DOPC-Detergent Conjugates: Fusogenic Carriers for Improved In Vitro and In Vivo Gene Delivery.

    PubMed

    Pierrat, Philippe; Casset, Anne; Kereselidze, Dimitri; Lux, Marie; Pons, Françoise; Lebeau, Luc

    2016-07-01

    Phospholipid-detergent conjugates are proposed as fusogenic carriers for gene delivery. Eleven compounds are prepared and their properties are investigated. The ability of the conjugates to promote fusion with a negatively charged model membrane is determined. Their DNA delivery efficiency and cytotoxicity are assessed in vitro. Lipoplexes are administered in the mouse lung, and transgene expression Indeterminate inflammatory activity are measured. The results show that conjugation of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) with C12 E4 produces a carrier that can efficiently deliver DNA to cells, with negligible -associated toxicity. Fusogenicity of the conjugates shows good correlation with in vitro transfection efficiency and crucially depends on the length of the polyether moiety of the detergent. Finally, DOPC-C12 E4 reveals highly potent for in vivo DNA delivery and favorably compares to GL67A, the current golden standard for gene delivery to the airway, opening the way for further promising developments.

  15. A chemistry/physics pathway with nanofibrous scaffolds for gene delivery.

    PubMed

    Wan, Fen; Tang, Zhaohui; He, Weidong; Chu, Benjamin

    2010-10-21

    This perspective is to introduce a new pathway for non-viral gene delivery by taking advantage of nanofibrous scaffolds as gene storage devices, gene carriers and homing devices. During gene delivery to the target, the DNA has to be protected in order to pass through a set of barriers before reaching the nucleus. The DNA can form a complex with polycations, and numerous publications exist on how to stabilize the DNA fragments by natural and synthetic materials. Electrospun nanofibrous scaffolds can be used to store the DNA, especially in the form of a more stabilized polyplex, and then to deliver the DNA (polyplex) to cells that are attached to the scaffold. While each essential step has been tested experimentally, the overall yet untested process, especially for in vivo experiments, may lead to a promising specific approach for gene/drug storage and delivery. The pathway described herein is based mainly on our understanding of the physics and chemistry of gene storage and delivery processes, in contrast to using pure biological concepts. Novel biodegradable, biocompatible nanofibrous materials with imbedded DNA (e.g., in the polyplex form) can then be designed to fabricate an intelligent scaffold for gene delivery. To achieve the above goal, the first step is to stabilize the DNA so that it can be incorporated into nanofibrous scaffolds. In this respect, we shall discuss the different methods of DNA/gene condensation and complex formation, and then explain the strategy used to incorporate DNA into electrospun nanofibers. Solvent-induced DNA condensation and then encapsulation were achieved. However, the released naked DNA was not sufficiently protected for gene transfection in cells. The objective of the current perspective is to suggest that, instead of the solvent-induced DNA condensation, one can combine the recently developed polyplex formation by using branched polyethyleneimine (bPEI). More importantly, free bPEI can be incorporated into the nanofibers

  16. Micro- and nanobubbles: a versatile non-viral platform for gene delivery.

    PubMed

    Cavalli, Roberta; Bisazza, Agnese; Lembo, David

    2013-11-18

    Micro- and nanobubbles provide a promising non-viral strategy for ultrasound mediated gene delivery. Microbubbles are spherical gas-filled structures with a mean diameter of 1-8 μm, characterised by their core-shell composition and their ability to circulate in the bloodstream following intravenous injection. They undergo volumetric oscillations or acoustic cavitation when insonified by ultrasound and, most importantly, they are able to resonate at diagnostic frequencies. It is due to this behaviour that microbubbles are currently being used as ultrasound contrast agents, but their use in therapeutics is still under investigation. For example, microbubbles could play a role in enhancing gene delivery to cells: when combined with clinical ultrasound exposure, microbubbles are able to favour gene entry into cells by cavitation. Two different delivery strategies have been used to date: DNA can be co-administered with the microbubbles (i.e. the contrast agent) or 'loaded' in purposed-built bubble systems - indeed a number of different technological approaches have been proposed to associate genes within microbubble structures. Nanobubbles, bubbles with sizes in the nanometre order of magnitude, have also been developed with the aim of obtaining more efficient gene delivery systems. Their small sizes allow the possibility of extravasation from blood vessels into the surrounding tissues and ultrasound-targeted site-specific release with minimal invasiveness. In contrast, microbubbles, due to their larger sizes, are unable to extravasate, thus and their targeting capacity is limited to specific antigens present within the vascular lumen. This review provides an overview of the use of microbubbles as gene delivery systems, with a specific focus on recent research into the development of nanosystems. In particular, ultrasound delivery mechanisms, formulation parameters, gene-loading approaches and the advantages of nanometric systems will be described. PMID:24008081

  17. Potent spinal parenchymal AAV9-mediated gene delivery by subpial injection in adult rats and pigs

    PubMed Central

    Miyanohara, Atsushi; Kamizato, Kota; Juhas, Stefan; Juhasova, Jana; Navarro, Michael; Marsala, Silvia; Lukacova, Nada; Hruska-Plochan, Marian; Curtis, Erik; Gabel, Brandon; Ciacci, Joseph; Ahrens, Eric T; Kaspar, Brian K; Cleveland, Don; Marsala, Martin

    2016-01-01

    Effective in vivo use of adeno-associated virus (AAV)-based vectors to achieve gene-specific silencing or upregulation in the central nervous system has been limited by the inability to provide more than limited deep parenchymal expression in adult animals using delivery routes with the most clinical relevance (intravenous or intrathecal). Here, we demonstrate that the spinal pia membrane represents the primary barrier limiting effective AAV9 penetration into the spinal parenchyma after intrathecal AAV9 delivery. We develop a novel subpial AAV9 delivery technique and AAV9-dextran formulation. We use these in adult rats and pigs to show (i) potent spinal parenchymal transgene expression in white and gray matter including neurons, glial and endothelial cells after single bolus subpial AAV9 delivery; (ii) delivery to almost all apparent descending motor axons throughout the length of the spinal cord after cervical or thoracic subpial AAV9 injection; (iii) potent retrograde transgene expression in brain motor centers (motor cortex and brain stem); and (iv) the relative safety of this approach by defining normal neurological function for up to 6 months after AAV9 delivery. Thus, subpial delivery of AAV9 enables gene-based therapies with a wide range of potential experimental and clinical utilizations in adult animals and human patients. PMID:27462649

  18. Antheraea pernyi silk fibroin for targeted gene delivery of VEGF165-Ang-1 with PEI.

    PubMed

    Ma, Caili; Lv, Linlin; Liu, Yu; Yu, Yanni; You, Renchuan; Yang, Jicheng; Li, Mingzhong

    2014-06-01

    Vascularization is a crucial challenge in tissue engineering. One solution for this problem is to implant scaffolds that contain functional genes that promote vascularization by providing angiogenic growth factors via a gene delivery carrier. Poly(ethylenimine) (PEI) is a gene delivery carrier with high transfection efficiency but with cytotoxicity. To solve this problem, we utilized Antheraea pernyi silk fibroin (ASF), which has favorable cytocompatibility and biodegradability, RGD sequences and a negative charge, in conjunction with PEI, as the delivery vector for vascular endothelial growth factor (VEGF) 165-angiopoietin-1 (Ang-1) dual gene simultaneous expression plasmid, creating an ASF/PEI/pDNA complex. The results suggested that the zeta potential of the ASF/PEI/pDNA complex was significantly lower than that of the PEI/pDNA complex. Decreased nitrogen and increased oxygen on the surface of the complex demonstrated that the ASF had successfully combined with the surface of the PEI/pDNA. Furthermore, the complexes resisted digestion by nucleic acid enzymes and degradation by serum. L929 cells were cultured and transfected in vitro and improved cytotoxicity was found when the cells were transfected with ASF/PEI/pDNA compared with PEI/pDNA. In addition, the transfection efficiency and VEGF secretion increased. In general, this study provides a novel method for decreasing the cytotoxicity of PEI gene delivery vectors and increasing transfection efficiency of angiogenesis-related genes.

  19. Understanding Intracellular Transport Processes Pertinent to Synthetic Gene Delivery via Stochastic Simulations and Sensitivity Analyses

    PubMed Central

    Dinh, Anh-Tuan; Pangarkar, Chinmay; Theofanous, Theo; Mitragotri, Samir

    2007-01-01

    A major challenge in synthetic gene delivery is to quantitatively predict the optimal design of polymer-based gene carriers (polyplexes). Here, we report a consistent, integrated, and fundamentally grounded computational methodology to address this challenge. This is achieved by accurately representing the spatio-temporal dynamics of intracellular structures and by describing the interactions between gene carriers and cellular components at a discrete, nanoscale level. This enables the applications of systems tools such as optimization and sensitivity analysis to search for the best combination of systems parameters. We validate the approach using DNA delivery by polyethylenimine as an example. We show that the cell topology (e.g., size, circularity, and dimensionality) strongly influences the spatiotemporal distribution of gene carriers, and consequently, their optimal intracellular pathways. The model shows that there exists an upper limit on polyplexes' intracellular delivery efficiency due to their inability to protect DNA until nuclear entry. The model predicts that even for optimally designed polyethylenimine vectors, only ∼1% of total DNA is delivered to the nucleus. Based on comparison with gene delivery by viruses, the model suggests possible strategies to significantly improve transfection efficiencies of synthetic gene vectors. PMID:17085500

  20. Gene Delivery from Supercharged Coiled-coil Protein and Cationic Lipid Hybrid Complex

    PubMed Central

    More, Haresh T.; Frezzo, Joseph A.; Dai, Jisen; Yamano, Seiichi; Montclare, Jin K.

    2014-01-01

    A lipoproteoplex comprised of an engineered supercharged coiled-coil protein (CSP) bearing multiple arginines and the cationic lipid formulation FuGENE HD (FG) was developed for effective condensation and delivery of nucleic acids. The CSP was able to maintain helical structure and self-assembly properties while exhibiting binding to plasmid DNA. The ternary CSP•DNA(8:1)•FG lipoproteoplex complex demonstrated enhanced transfection of β-galactosidase DNA into MC3T3-E1 mouse preosteoblasts. The lipoproteoplexes showed significant increases in transfection efficiency when compared to conventional FG and an mTat•FG lipopolyplex with a 6- and 2.5-fold increase in transfection, respectively. The CSP•DNA(8:1)•FG lipoproteoplex assembled into spherical particles with a net positive surface charge, enabling efficient gene delivery. These results support the application of lipoproteoplexes with protein engineered CSP for non-viral gene delivery. PMID:24875765

  1. Pullulan-protamine as efficient haemocompatible gene delivery vector: synthesis and in vitro characterization.

    PubMed

    Priya, S S; Rekha, M R; Sharma, Chandra P

    2014-02-15

    Biodegradable non-viral vectors with good transfection efficiency is essential for successful gene delivery. The purpose of this study was to design a non-viral vector by conjugating protamine to pullulan and elucidate the potential use of pullulan protamine conjugate (PPA) as an effective, non toxic and haemocompatible gene delivery system. The particle size and surface charge were measured using Nanosizer. Derivatization was confirmed by NMR, FTIR and DSC analyses. Acid base titration revealed the buffering behaviour of the conjugate. The protection of DNA from nuclease enzyme and interaction of plasma components on the stability of nanoplexes were also analysed. The uptake studies confirmed the plasmid delivery into the nucleus and the inhibitor studies determined the uptake mechanism. Transfection experiments revealed the capability of PPA to cellular uptake in C6 cells and facilitate high gene expression. Thus, PPA proves to be a promising non-viral vector.

  2. Rapid and efficient gene delivery into the adult mouse brain via focal electroporation

    PubMed Central

    Nomura, Tadashi; Nishimura, Yusuke; Gotoh, Hitoshi; Ono, Katsuhiko

    2016-01-01

    In vivo gene delivery is required for studying the cellular and molecular mechanisms of various biological events. Virus-mediated gene transfer or generation of transgenic animals is widely used; however, these methods are time-consuming and expensive. Here we show an improved electroporation technique for acute gene delivery into the adult mouse brain. Using a syringe-based microelectrode, local DNA injection and the application of electric current can be performed simultaneously; this allows rapid and efficient gene transduction of adult non-neuronal cells. Combining this technique with various expression vectors that carry specific promoters resulted in targeted gene expression in astrocytic cells. Our results constitute a powerful strategy for the genetic manipulation of adult brains in a spatio-temporally controlled manner. PMID:27430903

  3. Rapid and efficient gene delivery into the adult mouse brain via focal electroporation.

    PubMed

    Nomura, Tadashi; Nishimura, Yusuke; Gotoh, Hitoshi; Ono, Katsuhiko

    2016-01-01

    In vivo gene delivery is required for studying the cellular and molecular mechanisms of various biological events. Virus-mediated gene transfer or generation of transgenic animals is widely used; however, these methods are time-consuming and expensive. Here we show an improved electroporation technique for acute gene delivery into the adult mouse brain. Using a syringe-based microelectrode, local DNA injection and the application of electric current can be performed simultaneously; this allows rapid and efficient gene transduction of adult non-neuronal cells. Combining this technique with various expression vectors that carry specific promoters resulted in targeted gene expression in astrocytic cells. Our results constitute a powerful strategy for the genetic manipulation of adult brains in a spatio-temporally controlled manner. PMID:27430903

  4. Transient Gene Expression in Epidermal Cells of Plant Leaves by Biolistic DNA Delivery

    PubMed Central

    Ueki, Shoko; Magori, Shimpei; Lacroix, Benoît; Citovsky, Vitaly

    2013-01-01

    Transient gene expression is a useful approach for studying the functions of gene products. In the case of plants, Agrobacterium infiltration is a method of choice for transient introduction of genes for many species. However, this technique does not work efficiently in some species, such as Arabidopsis thaliana. Moreover, the infection of Agrobacterium is known to induce dynamic changes in gene expression patterns in the host plants, possibly affecting the function and localization of the proteins to be tested. These problems can be circumvented by biolistic delivery of the genes of interest. Here, we present an optimized protocol for biolistic delivery of plasmid DNA into epidermal cells of plant leaves, which can be easily performed using the Bio-Rad Helios gene gun system. This protocol allows efficient and reproducible transient expression of diverse genes in Arabidopsis, Nicotiana benthamiana and N. tabacum, and is suitable for studies of the biological function and subcellular localization of the gene products directly in planta. The protocol also can be easily adapted to other species by optimizing the delivery gas pressure. PMID:23104330

  5. Non-viral gene delivery strategies for cancer therapy, tissue engineering and regenerative medicine

    NASA Astrophysics Data System (ADS)

    Bhise, Nupura S.

    Gene therapy involves the delivery of deoxyribonucleic acid (DNA) into cells to override or replace a malfunctioning gene for treating debilitating genetic diseases, including cancer and neurodegenerative diseases. In addition to its use as a therapeutic, it can also serve as a technology to enable regenerative medicine strategies. The central challenge of the gene therapy research arena is developing a safe and effective delivery agent. Since viral vectors have critical immunogenic and tumorogenic safety issues that limit their clinical use, recent efforts have focused on developing non-viral biomaterial based delivery vectors. Cationic polymers are an attractive class of gene delivery vectors due to their structural versatility, ease of synthesis, biodegradability, ability to self-complex into nanoparticles with negatively charged DNA, capacity to carry large cargo, cellular uptake and endosomal escape capacity. In this thesis, we hypothesized that developing a biomaterial library of poly(betaamino esters) (PBAE), a newer class of cationic polymers consisting of biodegradable ester groups, would allow investigating vector design parameters and formulating effective non-viral gene delivery strategies for cancer drug delivery, tissue engineering and stem cell engineering. Consequently, a high-throughput transfection assay was developed to screen the PBAE-based nanoparticles in hard to transfect fibroblast cell lines. To gain mechanistic insights into the nanoparticle formulation process, biophysical properties of the vectors were characterized in terms of molecular weight (MW), nanoparticle size, zeta potential and plasmid per particle count. We report a novel assay developed for quantifying the plasmid per nanoparticle count and studying its implications for co-delivery of multiple genes. The MW of the polymers ranged from 10 kDa to 100 kDa, nanoparticle size was about 150 run, zeta potential was about 30 mV in sodium acetate buffer (25 mM, pH 5) and 30 to 100

  6. Ionic α-Helical Polypeptides towards Non-Viral Gene Delivery

    PubMed Central

    Zhang, Rujing; Song, Ziyuan; Yin, Lichen; Zheng, Nan; Tang, Haoyu; Lu, Hua; Gabrielson, Nathan P.; Lin, Yao; Kim, Kyung; Cheng, Jianjun

    2014-01-01

    The advent of polymeric materials has significantly promoted the development and rapid growth of various technologies in biomedical applications, such as tissue engineering and controlled drug and gene delivery. Water-soluble polypeptides bearing functional side chains and adopting stable secondary structures are a new class of functional polymeric materials of potentially broad applications in medicine and biotechnology. In this article, we summarize our recent effort on the design and synthesis of the water-soluble α-helical ionic polypeptides originally developed in our laboratory and highlight their applications in cell membrane penetration and non-viral gene/siRNA delivery. PMID:25377262

  7. Sui generis: gene therapy and delivery systems for the treatment of glioblastoma

    PubMed Central

    Kane, J. Robert; Miska, Jason; Young, Jacob S.; Kanojia, Deepak; Kim, Julius W.; Lesniak, Maciej S.

    2015-01-01

    Gene therapy offers a multidimensional set of approaches intended to treat and cure glioblastoma (GBM), in combination with the existing standard-of-care treatment (surgery and chemoradiotherapy), by capitalizing on the ability to deliver genes directly to the site of neoplasia to yield antitumoral effects. Four types of gene therapy are currently being investigated for their potential use in treating GBM: (i) suicide gene therapy, which induces the localized generation of cytotoxic compounds; (ii) immunomodulatory gene therapy, which induces or augments an enhanced antitumoral immune response; (iii) tumor-suppressor gene therapy, which induces apoptosis in cancer cells; and (iv) oncolytic virotherapy, which causes the lysis of tumor cells. The delivery of genes to the tumor site is made possible by means of viral and nonviral vectors for direct delivery of therapeutic gene(s), tumor-tropic cell carriers expressing therapeutic gene(s), and “intelligent” carriers designed to increase delivery, specificity, and tumoral toxicity against GBM. These vehicles are used to carry genetic material to the site of pathology, with the expectation that they can provide specific tropism to the desired site while limiting interaction with noncancerous tissue. Encouraging preclinical results using gene therapies for GBM have led to a series of human clinical trials. Although there is limited evidence of a therapeutic benefit to date, a number of clinical trials have convincingly established that different types of gene therapies delivered by various methods appear to be safe. Due to the flexibility of specialized carriers and genetic material, the technology for generating new and more effective therapies already exists. PMID:25746089

  8. Prodrug converting enzyme gene delivery by L. monocytogenes

    PubMed Central

    Stritzker, Jochen; Pilgrim, Sabine; Szalay, Aladar A; Goebel, Werner

    2008-01-01

    Background Listeria monocytogenes is a highly versatile bacterial carrier system for introducing protein, DNA and RNA into mammalian cells. The delivery of tumor antigens with the help of this carrier into tumor-bearing animals has been successfully carried out previously and it was recently reported that L. monocytogenes is able to colonize and replicate within solid tumors after local or even systemic injection. Methods Here we report on the delivery of two prodrug converting enzymes, purine-deoxynucleoside phosphorylase (PNP) and a fusion protein consisting of yeast cytosine deaminase and uracil phosphoribosyl transferase (FCU1) into cancer cells in culture by L. monocytogenes. Transfer of the prodrug converting enzymes was achieved by bacterium mediated transfer of eukaryotic expression plasmids or by secretion of the proteins directly into the host cell cytosol by the infecting bacteria. Results The results indicate that conversion of appropriate prodrugs to toxic drugs in the cancer cells occured after both procedures although L. monocytogenes-mediated bactofection proved to be more efficient than enzyme secretion 4T1, B16 and COS-1 tumor cells. Exchanging the constitutively PCMV-promoter with the melanoma specific P4xTETP-promoter resulted in melanoma cell-specific expression of the prodrug converting enzymes but reduced the efficiencies. Conclusion These experiments open the way for bacterium mediated tumor specific activation of prodrugs in live animals with tumors. PMID:18402662

  9. Gastrointestinal gene delivery by cyclodextrins--in vitro quantification of extracellular barriers.

    PubMed

    O'Neill, Martin J; O'Mahony, Aoife M; Byrne, Colin; Darcy, Raphael; O'Driscoll, Caitriona M

    2013-11-18

    Local gene delivery represents a promising therapeutic approach for diseases of the intestine. However, the gastrointestinal tract poses significant challenges to successful gene delivery. Cyclodextrins (CDs) have been extensively investigated as non-viral vectors. Here, we assessed the suitability of an amphiphilic cationic CD for intestinal gene transfer, with particular focus on extracellular barriers. Stability and transfection efficiency of CD·DNA complexes were assessed post incubation in simulated gastric and intestinal fluids, bile salts and mucin, or with intestinal enzymes to represent extracellular barriers to intestinal gene delivery. Stability was determined by gel electrophoresis and transfection was measured by luciferase expression in intestinal epithelial cells (Caco-2). Transfection efficiency of CD·DNA complexes was enhanced after incubation in bile salts but was reduced after incubation in gastric and intestinal fluids and mucin. CD·DNA complexes were stable after incubation with pancreatic enzymes and with a model lower intestinal enzyme. Furthermore, the CD protected pDNA from degradation by DNase. In summary, physiologically relevant in vitro models were established and used to quantify the barriers posed by the intestinal extracellular environment to gene delivery. This systematic assessment identified the advantages and limitations of the CD vector and facilitated the proposal of formulation strategies to overcome these barriers.

  10. Gastrointestinal gene delivery by cyclodextrins--in vitro quantification of extracellular barriers.

    PubMed

    O'Neill, Martin J; O'Mahony, Aoife M; Byrne, Colin; Darcy, Raphael; O'Driscoll, Caitriona M

    2013-11-18

    Local gene delivery represents a promising therapeutic approach for diseases of the intestine. However, the gastrointestinal tract poses significant challenges to successful gene delivery. Cyclodextrins (CDs) have been extensively investigated as non-viral vectors. Here, we assessed the suitability of an amphiphilic cationic CD for intestinal gene transfer, with particular focus on extracellular barriers. Stability and transfection efficiency of CD·DNA complexes were assessed post incubation in simulated gastric and intestinal fluids, bile salts and mucin, or with intestinal enzymes to represent extracellular barriers to intestinal gene delivery. Stability was determined by gel electrophoresis and transfection was measured by luciferase expression in intestinal epithelial cells (Caco-2). Transfection efficiency of CD·DNA complexes was enhanced after incubation in bile salts but was reduced after incubation in gastric and intestinal fluids and mucin. CD·DNA complexes were stable after incubation with pancreatic enzymes and with a model lower intestinal enzyme. Furthermore, the CD protected pDNA from degradation by DNase. In summary, physiologically relevant in vitro models were established and used to quantify the barriers posed by the intestinal extracellular environment to gene delivery. This systematic assessment identified the advantages and limitations of the CD vector and facilitated the proposal of formulation strategies to overcome these barriers. PMID:24016741

  11. Local delivery of gene-modifying triplex-forming molecules to epidermis

    PubMed Central

    Rogers, Faye A.; Hu, Rong-Hua; Milstone, Leonard M.

    2012-01-01

    Epidermal keratinocytes are particularly suitable candidates for in situ gene correction. Intraperitoneal administration of a triplex-forming oligonucleotide (TFO) was shown previously to introduce DNA base changes in a reporter gene in skin, without identifying which cells had been targeted. We extend those previous experiments using two triplex-forming molecules (TFMs), a peptide nucleic acid (PNA-Antp) and a TFO (AG30), and two lines of transgenic mice that have the chromosomally integrated λsupFG1 shuttle-reporter transgene. Successful in vivo genomic modification occurs in epidermis and dermis in CD1 transgenic mice following either intraperitoneal or intradermal delivery of the PNA-Antennapedia conjugate. FITC-PNA-Antp accumulates in nuclei of keratinocytes and, after intradermal delivery of the PNA-Antp, chromosomally modified, keratin 5 positive basal keratinocytes persist for at least 10 days. In hairless (SKH1) mice with the λsupFG1 transgene, intradermal delivery of the TFO, AG30, introduces gene modifications in both tail and back skin and those chromosomal modifications persist in basal keratinocytes for 10 days. Hairless mice should facilitate comparison of various targeting agents and methods of delivery. Gene targeting by repeated local administration of oligonucleotides may prove clinically useful for judiciously selected disease-causing genes in the epidermis. PMID:23014335

  12. Early osteoblastic differentiation induced by dexamethasone enhances adenoviral gene delivery to marrow stromal cells.

    PubMed

    Blum, Jeremy S; Parrott, M Brandon; Mikos, Antonios G; Barry, Michael A

    2004-03-01

    We investigated the implications of induced osteogenic differentiation on gene delivery in multipotent rat marrow stromal cells (MSCs). Prior to genetic manipulation cells were cultured with or without osteogenic supplements (5x10(-8) M dexamethasone, 160 microM l-ascorbic acid 2-phosphate, and 10 mM beta-glycerophosphate). Comparison of liposome, retroviral, and adenoviral vectors demonstrated that all three vectors could mediate gene delivery to primary rat MSCs. When these vectors were applied in the absence or presence of osteogenic supplements, we found that MSCs differentiated prior to transduction with adenovirus type 5 vectors produced a 300% increase in transgene expression compared to MSCs that were not exposed to osteogenic supplements. This differentiation effect appeared specific to adenoviral mediated gene delivery, since there was minimal increase in retroviral gene delivery and no increase in liposome gene delivery when MSCs were treated with osteogenic supplements. In addition, we also determined this increase in transgene production to occur at a higher concentration of dexamethasone (5x10(-8) M) in the culture medium of MSCs prior to adenoviral transduction. We found that this increased transgene production could be extended to the osteogenic protein, human bone morphogenetic protein 2 (hBMP-2). When delivered by an adenoviral vector, hBMP-2 transgene production could be increased from 1.4 ng/10(5) cells/3 days to 4.3 ng/10(5) cells/3 days by culture of MSCs with osteogenic supplements prior to transduction. These results indicate that the utility of MSCs as a therapeutic protein delivery mechanism through genetic manipulation can be enhanced by pre-culture of these cells with dexamethasone. PMID:15013104

  13. Biodegradable Poly(aminoester)-Mediated p53 Gene Delivery for Cancer Therapy.

    PubMed

    Shen, He; Liu, Min; Zhang, Zhijun

    2016-03-01

    Gene therapy is a promising strategy in cancer treatment. However, efficient gene translation still remains challenging. In the previous work, a hydrolytically degradable poly(aminoester) with good biocompatibility was synthesized. Herein, the poly(aminoester) was explored as a vector for gene delivery and cancer therapy. The experiments revealed that the poly(aminoester) condensed plasmid DNA into nanosized particles via electrostatic interaction. The pEGFP-N1 and pGL-3 were first used as two reporter genes to study intracellular transfection. The poly(aminoester) showed higher GFP expression (33%) than PEI 25 kDa (21%). Intracellular trafficking of Cy3-labelled pGL-3 also indicated that the poly(aminoester) showed superior DNA delivery ability to nucleus compared to PEI 25 kDa. Furthermore, the therapeutic gene (p53) was translated into the breast cancer cell line (MCF-7), and then induced cell apoptosis. These results suggested that the degradable poly(aminoester) is a promising and efficient gene delivery vector for gene therapeutic applications.

  14. Biodegradable Poly(aminoester)-Mediated p53 Gene Delivery for Cancer Therapy.

    PubMed

    Shen, He; Liu, Min; Zhang, Zhijun

    2016-03-01

    Gene therapy is a promising strategy in cancer treatment. However, efficient gene translation still remains challenging. In the previous work, a hydrolytically degradable poly(aminoester) with good biocompatibility was synthesized. Herein, the poly(aminoester) was explored as a vector for gene delivery and cancer therapy. The experiments revealed that the poly(aminoester) condensed plasmid DNA into nanosized particles via electrostatic interaction. The pEGFP-N1 and pGL-3 were first used as two reporter genes to study intracellular transfection. The poly(aminoester) showed higher GFP expression (33%) than PEI 25 kDa (21%). Intracellular trafficking of Cy3-labelled pGL-3 also indicated that the poly(aminoester) showed superior DNA delivery ability to nucleus compared to PEI 25 kDa. Furthermore, the therapeutic gene (p53) was translated into the breast cancer cell line (MCF-7), and then induced cell apoptosis. These results suggested that the degradable poly(aminoester) is a promising and efficient gene delivery vector for gene therapeutic applications. PMID:27455620

  15. Mechanical oscillations enhance gene delivery into suspended cells

    PubMed Central

    Zhou, Z. L.; Sun, X. X.; Ma, J.; Man, C. H.; Wong, A. S. T.; Leung, A. Y.; Ngan, A. H. W.

    2016-01-01

    Suspended cells are difficult to be transfected by common biochemical methods which require cell attachment to a substrate. Mechanical oscillations of suspended cells at certain frequencies are found to result in significant increase in membrane permeability and potency for delivery of nano-particles and genetic materials into the cells. Nanomaterials including siRNAs are found to penetrate into suspended cells after subjecting to short-time mechanical oscillations, which would otherwise not affect the viability of the cells. Theoretical analysis indicates significant deformation of the actin-filament network in the cytoskeleton cortex during mechanical oscillations at the experimental frequency, which is likely to rupture the soft phospholipid bilayer leading to increased membrane permeability. The results here indicate a new method for enhancing cell transfection. PMID:26956215

  16. Lignin nanotubes as vehicles for gene delivery into human cells.

    PubMed

    Ten, Elena; Ling, Chen; Wang, Yuan; Srivastava, Arun; Dempere, Luisa Amelia; Vermerris, Wilfred

    2014-01-13

    Lignin nanotubes (LNTs) synthesized from the aromatic plant cell wall polymer lignin in a sacrificial alumina membrane template have as useful features their flexibility, ease of functionalization due to the availability of many functional groups, label-free detection by autofluorescence, and customizable optical properties. In this report we show that the physicochemical properties of LNTs can be varied over a wide range to match requirements for specific applications by using lignin with different subunit composition, a function of plant species and genotype, and by choosing the lignin isolation method (thioglycolic acid, phosphoric acid, sulfuric acid (Klason), sodium hydroxide lignin), which influences the size and reactivity of the lignin fragments. Cytotoxicity studies with human HeLa cells showed that concentrations of up to 90 mg/mL are tolerated, which is a 10-fold higher concentration than observed for single- or multiwalled carbon nanotubes (CNTs). Confocal microscopy imaging revealed that all LNT formulations enter HeLa cells without auxiliary agents and that LNTs made from NaOH-lignin penetrate the cell nucleus. We further show that DNA can adsorb to LNTs. Consequently, exposure of HeLa cells to LNTs coated with DNA encoding the green fluorescent protein (GFP) leads to transfection and expression of GFP. The highest transfection efficiency was obtained with LNTs made from NaOH-lignin due to a combination of high DNA binding capacity and DNA delivery directly into the nucleus. These combined features of LNTs make LNTs attractive as smart delivery vehicles of DNA without the cytotoxicity associated with CNTs or the immunogenicity of viral vectors.

  17. Mesoporous Silica Nanoparticles as Controlled Release Drug Delivery and Gene Transfection Carriers

    SciTech Connect

    Igor I. Slowing; Juan L. Viveo-Escoto; Chia-Wen Wu; Victor S. Y. Lin

    2008-04-10

    In this review, we highlight the recent research developments of a series of surface-functionalized mesoporous silica nanoparticle (MSN) materials as efficient drug delivery carriers. The synthesis of this type of MSN materials is described along with the current methods for controlling the structural properties and chemical functionalization for biotechnological and biomedical applications. We summarized the advantages of using MSN for several drug delivery applications. The recent investigations of the biocompatibility of MSN in vitro are discussed. We also describe the exciting progress on using MSN to penetrate various cell membranes in animal and plant cells. The novel concept of gatekeeping is introduced and applied to the design of a variety of stimuli-responsive nanodevices. We envision that these MSN-based systems have a great potential for a variety of drug delivery applications, such as the site-specific delivery and intracellular controlled release of drugs, genes, and other therapeutic agents.

  18. Gene delivery in bone tissue engineering: progress and prospects using viral and nonviral strategies.

    PubMed

    Partridge, Kris A; Oreffo, Richard O C

    2004-01-01

    Bone tissue loss as a consequence of the natural aging process or as a result of trauma and degenerative disease has led to the need for procedures to generate cartilage and bone for a variety of orthopedic applications. The ability to transfer genes into multipotential mesenchymal stem cells, while still in its infancy, offers considerable therapeutic hope in a variety of musculoskeletal disorders. However, the choice of gene delivery method is key. This review examines the various techniques and methods currently available to enable gene transfer into a target population from viral methods (transduction) to nonviral (transfection) methods and the limitations associated with each method. The potential applications and current understanding of each method are presented. Given the demographic challenge of an aging population, the ultimate goal remains the development of simple, safe, and reproducible strategies for gene delivery that will address the pressing orthopedic clinical imperatives of many.

  19. Lipopolyplex for Therapeutic Gene Delivery and Its Application for the Treatment of Parkinson’s Disease

    PubMed Central

    Chen, Wei; Li, Hui; Liu, Zhenguo; Yuan, Weien

    2016-01-01

    Lipopolyplex is a core-shell structure composed of nucleic acid, polycation and lipid. As a non-viral gene delivery vector, lipopolyplex combining the advantages of polyplex and lipoplex has shown superior colloidal stability, reduced cytotoxicity, extremely high gene transfection efficiency. Following intravenous administration, there are many strategies based on lipopolyplex to overcome the complex biological barriers in systemic gene delivery including condensation of nucleic acids into nanoparticles, long circulation, cell targeting, endosomal escape, release to cytoplasm and entry into cell nucleus. Parkinson’s disease (PD) is the second most common neurodegenerative disorder and severely influences the patients’ life quality. Current gene therapy clinical trials for PD employing viral vectors didn’t achieve satisfactory efficacy. However, lipopolyplex may become a promising alternative approach owing to its stability in blood, ability to cross the blood-brain barrier (BBB) and specific targeting to diseased brain cells. PMID:27092073

  20. Gene therapy for cardiovascular disease: advances in vector development, targeting, and delivery for clinical translation.

    PubMed

    Rincon, Melvin Y; VandenDriessche, Thierry; Chuah, Marinee K

    2015-10-01

    Gene therapy is a promising modality for the treatment of inherited and acquired cardiovascular diseases. The identification of the molecular pathways involved in the pathophysiology of heart failure and other associated cardiac diseases led to encouraging preclinical gene therapy studies in small and large animal models. However, the initial clinical results yielded only modest or no improvement in clinical endpoints. The presence of neutralizing antibodies and cellular immune responses directed against the viral vector and/or the gene-modified cells, the insufficient gene expression levels, and the limited gene transduction efficiencies accounted for the overall limited clinical improvements. Nevertheless, further improvements of the gene delivery technology and a better understanding of the underlying biology fostered renewed interest in gene therapy for heart failure. In particular, improved vectors based on emerging cardiotropic serotypes of the adeno-associated viral vector (AAV) are particularly well suited to coax expression of therapeutic genes in the heart. This led to new clinical trials based on the delivery of the sarcoplasmic reticulum Ca(2+)-ATPase protein (SERCA2a). Though the first clinical results were encouraging, a recent Phase IIb trial did not confirm the beneficial clinical outcomes that were initially reported. New approaches based on S100A1 and adenylate cyclase 6 are also being considered for clinical applications. Emerging paradigms based on the use of miRNA regulation or CRISPR/Cas9-based genome engineering open new therapeutic perspectives for treating cardiovascular diseases by gene therapy. Nevertheless, the continuous improvement of cardiac gene delivery is needed to allow the use of safer and more effective vector doses, ultimately bringing gene therapy for heart failure one step closer to reality.

  1. Systemic gene delivery to the central nervous system using Adeno-associated virus

    PubMed Central

    Bourdenx, Mathieu; Dutheil, Nathalie; Bezard, Erwan; Dehay, Benjamin

    2014-01-01

    Adeno-associated virus (AAV)-mediated gene delivery has emerged as an effective and safe tool for both preclinical and clinical studies of neurological disorders. The recent discovery that several serotypes are able to cross the blood–brain barrier when administered systemically has been a real breakthrough in the field of neurodegenerative diseases. Widespread transgene expression after systemic injection could spark interest as a therapeutic approach. Such strategy will avoid invasive brain surgery and allow non-focal gene therapy promising for CNS diseases affecting large portion of the brain. Here, we will review the recent results achieved through different systemic routes of injection generated in the last decade using systemic AAV-mediated delivery and propose a brief assessment of their values. In particular, we emphasize how the methods used for virus engineering could improve brain transduction after peripheral delivery. PMID:24917785

  2. Calcium Gluconate in Phosphate Buffered Saline Increases Gene Delivery with Adenovirus Type 5

    PubMed Central

    Ahonen, Marko T.; Diaconu, Iulia; Pesonen, Sari; Kanerva, Anna; Baumann, Marc; Parviainen, Suvi T.; Spiller, Brad

    2010-01-01

    Background Adenoviruses are attractive vectors for gene therapy because of their stability in vivo and the possibility of production at high titers. Despite exciting preclinical data with various approaches, there are only a few examples of clear efficacy in clinical trials. Effective gene delivery to target cells remains the key variable determining efficacy and thus enhanced transduction methods are important. Methods/Results We found that heated serum could enhance adenovirus 5 mediated gene delivery up to twentyfold. A new protein-level interaction was found between fiber knob and serum transthyretin, but this was not responsible for the observed effect. Instead, we found that heating caused the calcium and phosphate present in the serum mix to precipitate, and this was responsible for enhanced gene delivery. This finding could have relevance for designing preclinical experiments with adenoviruses, since calcium and phosphate are present in many solutions. To translate this into an approach potentially testable in patients, we used calcium gluconate in phosphate buffered saline, both of which are clinically approved, to increase adenoviral gene transfer up to 300-fold in vitro. Gene transfer was increased with or without heating and in a manner independent from the coxsackie-adenovirus receptor. In vivo, in mouse studies, gene delivery was increased 2-, 110-, 12- and 13-fold to tumors, lungs, heart and liver and did not result in increased pro-inflammatory cytokine induction. Antitumor efficacy of a replication competent virus was also increased significantly. Conclusion In summary, adenoviral gene transfer and antitumor efficacy can be enhanced by calcium gluconate in phosphate buffered saline. PMID:20927353

  3. Autophagy and formation of tubulovesicular autophagosomes provide a barrier against nonviral gene delivery.

    PubMed

    Roberts, Rebecca; Al-Jamal, Wafa' T; Whelband, Matthew; Thomas, Paul; Jefferson, Matthew; van den Bossche, Jeroen; Powell, Penny P; Kostarelos, Kostas; Wileman, Thomas

    2013-05-01

    Cationic liposome (lipoplex) and polymer (polyplex)-based vectors have been developed for nonviral gene delivery. These vectors bind DNA and enter cells via endosomes, but intracellular transfer of DNA to the nucleus is inefficient. Here we show that lipoplex and polyplex vectors enter cells in endosomes, activate autophagy and generate tubulovesicular autophagosomes. Activation of autophagy was dependent on ATG5, resulting in lipidation of LC3, but did not require the PtdIns 3-kinase activity of PIK3C3/VPS34. The autophagosomes generated by lipoplex fused with each other, and with endosomes, resulting in the delivery of vectors to large tubulovesicular autophagosomes, which accumulated next to the nucleus. The tubulovesicular autophagosomes contained autophagy receptor protein SQSTM1/p62 and ubiquitin, suggesting capture of autophagy cargoes, but fusion with lysosomes was slow. Gene delivery and expression from both lipoplex and polyplex increased 8-fold in atg5 (-/-) cells unable to generate tubulovesicular autophagosomes. Activation of autophagy and capture within tubulovesicular autophagosomes therefore provides a new cellular barrier against efficient gene transfer and should be considered when designing efficient nonviral gene delivery vectors.

  4. Non-Condensing Polymeric Nanoparticles for Targeted Gene and siRNA Delivery

    PubMed Central

    Xu, Jing; Ganesh, Shanthi; Amiji, Mansoor

    2011-01-01

    Gene therapy has shown a tremendous potential to benefit patients in a variety of disease conditions. However, finding a safe and effective systemic delivery system is the major obstacle in this area. Although viral vectors showed promise for high transfection rate, the immunogenicity associated with these systems has hindered further development. As an alternative to viral gene delivery, this review focuses on application of novel safe and effective non-condensing polymeric systems that have shown high transgene expression when administered systemically or by the oral route. Type B gelatin-based engineered nanocarriers were evaluated for passive and active tumor-targeted delivery and transfection using both reporter and therapeutic plasmid DNA. Additionally, we have shown that nanoparticles-in-microsphere oral system (NiMOS) can efficiently deliver reporter and therapeutic gene constructs in the gastrointestinal tract. Additionally, there has been a significant recent interest in the use small interfering RNA (siRNA) as a therapeutic system for gene silencing. Both gelatin nanoparticles and NiMOS have shown activity in systemic and oral delivery of siRNA, respectively. PMID:21621597

  5. Development of Therapeutic Microbubbles for Enhancing Ultrasound-Mediated Gene Delivery

    PubMed Central

    Sun, Ryan R.; Noble, Misty L.; Sun, Samuel S.; Song, Shuxian; Miao, Carol H.

    2014-01-01

    Ultrasound (US)-mediated gene delivery has emerged as a promising non-viral method for safe and selective gene delivery. When enhanced by the cavitation of microbubbles (MBs), US exposure can induce sonoporation that transiently increases cell membrane permeability for localized delivery of DNA. The present study explores the effect of generalizable MB customizations on MB facilitation of gene transfer compared to Definity®, a clinically available contrast agent. These modifications are 1) increased MB shell acyl chain length (RN18) for elevated stability and 2) addition of positive charge on MB (RC5K) for greater DNA associability. The MB types were compared in their ability to facilitate transfection of luciferase and GFP reporter plasmid DNA in vitro and in vivo under various conditions of US intensity, MB dosage, and pretreatment MB-DNA incubation. The results indicated that both RN18 and RC5K were more efficient than Definity®, and that the cationic RC5K can induce even greater transgene expression by increasing payload capacity with prior DNA incubation without compromising cell viability. These findings could be applied to enhance MB functions in a wide range of therapeutic US/MB gene and drug delivery approach. With further designs, MB customizations have the potential to advance this technology closer to clinical application. PMID:24650644

  6. Dependence of PEI and PAMAM Gene Delivery on Clathrin- and Caveolin-Dependent Trafficking Pathways

    PubMed Central

    Hwang, Mark E.; Keswani, Rahul K.; Pack, Daniel W.

    2014-01-01

    Purpose Non-viral gene delivery vehicles such as polyethylenimine and polyamidoamine dendrimer effectively condense plasmid DNA, facilitate endocytosis, and deliver nucleic acid cargo to the nucleus in vitro. Better understanding of intracellular trafficking mechanisms involved in polymeric gene delivery is a prerequisite to clinical application. This study investigates the role of clathrin and caveolin endocytic pathways in cellular uptake and subsequent vector processing. Methods We formed 25-kD polyethylenimine (PEI) and generation 4 (G4) polyamidoamine (PAMAM) polyplexes at N/P 10 and evaluated internalization pathways and gene delivery in HeLa cells. Clathrin- and caveolin-dependent endocytosis inhibitors were used at varying concentrations to elucidate the roles of these important pathways. Results PEI and PAMAM polyplexes were internalized by both pathways. However, the amount of polyplex internalized poorly correlated with transgene expression. While the caveolin-dependent pathway generally led to effective gene delivery with both polymers, complete inhibition of the clathrin-dependent pathway was also deleterious to transfection with PEI polyplexes. Inhibition of one endocytic pathway may lead to an overall increase in uptake via unaffected pathways, suggesting the existence of compensatory endocytic mechanisms. Conclusions The well-studied clathrin- and caveolin-dependent endocytosis pathways are not necessarily independent, and perturbing one mechanism of trafficking influences the larger trafficking network. PMID:25511918

  7. Controlling mesenchymal stem cell gene expression using polymer-mediated delivery of siRNA

    PubMed Central

    Benoit, Danielle S.W.; Boutin, Molly E.

    2012-01-01

    siRNA treatment has great promise to specifically control gene expression and select cell behaviors but have delivery challenges limiting their use. Particularly for applications in regenerative medicine, uniform and consistent delivery of siRNA to control gene expression and subsequent stem cell functions, such as differentiation, is paramount. Therefore, a diblock copolymer was examined for its ability to effective delivery siRNA to mesenchymal stem cells (MSCs). The diblock copolymers, which are composed of cationic blocks for siRNA complexation, protection, and uptake and pH-responsive blocks for endosomal escape, were shown to facilitate nearly 100% MSC uptake of siRNA, which is vastly superior to a commercially-available control, DharmaFECT, which resulted in only ~60% siRNA positive MSCs. Moreover, the diblock copolymer, at conditions that result in excellent knockdown (down to ~10% of control gene expression), is cytocompatible, causing no negative effects on MSC survivability. In contrast, DharmaFECT:siRNA treatment results in only ~60% survivability of MSCs. Longitudinal knockdown after siRNA treatment was examined and protein knockdown persists for ~6 days regardless of delivery system (diblock copolymer or DharmaFECT). Finally, MSC phenotype and differentiation capacity was examined after treatment with control siRNA. There is no statistically significant differences on cell surface markers of diblock copolymer:siRNA or DharmaFECT:siRNA treated or cells measured 2 weeks after siRNA delivery compared to untreated cells. Upon differentiation with typical media/culture conditions to adipogenic, chondrogenic, and osteogenic lineages and examination of histological staining markers, there is no discernable differences between treated and untreated cells, regardless of delivery mechanism. Thus, diblock copolymers examined herein facilitate uniform siRNA treatment of MSCs, inducing siRNA-specific gene and protein knockdown without adversely affecting MSC

  8. Cationic surface modification of PLG nanoparticles offers sustained gene delivery to pulmonary epithelial cells.

    PubMed

    Baoum, Abdulgader; Dhillon, Navneet; Buch, Shilpa; Berkland, Cory

    2010-05-01

    Biodegradable polymeric nanoparticles are currently being explored as a nonviral gene delivery system; however, many obstacles impede the translation of these nanomaterials. For example, nanoparticles delivered systemically are inherently prone to adsorbing serum proteins and agglomerating as a result of their large surface/volume ratio. What is desired is a simple procedure to prepare nanoparticles that may be delivered locally and exhibit minimal toxicity while improving entry into cells for effectively delivering DNA. The objective of this study was to optimize the formulation of poly(D,L-lactide-co-glycolide) (PLG) nanoparticles for gene delivery performance to a model of the pulmonary epithelium. Using a simple solvent diffusion technique, the chemistry of the particle surface was varied by using different coating materials that adsorb to the particle surface during formation. A variety of cationic coating materials were studied and compared to more conventional surfactants used for PLG nanoparticle fabrication. Nanoparticles (approximately 200 nm) efficiently encapsulated plasmids encoding for luciferase (80-90%) and slowly released the same for 2 weeks. In A549 alveolar lung epithelial cells, high levels of gene expression appeared at day 5 for certain positively charged PLG particles and gene expression was maintained for at least 2 weeks. In contrast, PEI gene expression ended at day 5. PLG particles were also significantly less cytotoxic than PEI suggesting the use of these vehicles for localized, sustained gene delivery to the pulmonary epithelium.

  9. Synthesis of Electroneutralized Amphiphilic Copolymers with Peptide Dendrons for Intramuscular Gene Delivery.

    PubMed

    Pu, Linyu; Wang, Jiali; Li, Na; Chai, Qiuxia; Irache, Juan M; Wang, Gang; Tang, James Zhenggui; Gu, Zhongwei

    2016-06-01

    Intramuscular gene delivery materials are of great importance in plasmid-based gene therapy system, but there is limited information so far on how to design and synthesize them. A previous study showed that the peptide dendron-based triblock copolymer with its components arranged in a reversed biomembrane architecture could significantly increase intramuscular gene delivery and expression. Herein, we wonder whether copolymers with biomembrane-mimicking arrangement may have similar function on intramuscular gene delivery. Meanwhile, it is of great significance to uncover the influence of electric charge and molecular structure on the function of the copolymers. To address the issues, amphiphilic triblock copolymers arranged in hydrophilic-hydrophobic-hydrophilic structure were constructed despite the paradoxical characteristics and difficulties in synthesizing such hydrophilic but electroneutral molecules. The as-prepared two copolymers, dendronG2(l-lysine-OH)-poly propylene glycol2k(PPG2k)-dendronG2(l-lysine-OH) (rL2PL2) and dendronG3(l-lysine-OH)-PPG2k-dendronG3(l-lysine-OH) (rL3PL3), were in similar structure but had different hydrophilic components and surface charges, thus leading to different capabilities in gene delivery and expression in skeletal muscle. rL2PL2 was more efficient than Pluronic L64 and rL3PL3 when mediating luciferase, β-galactosidase, and fluorescent protein expressions. Furthermore, rL2PL2-mediated growth-hormone-releasing hormone expression could significantly induce mouse body weight increase in the first 21 days after injection. In addition, both rL2PL2 and rL3PL3 showed good in vivo biosafety in local and systemic administration. Altogether, rL2PL2-mediated gene expression in skeletal muscle exhibited applicable potential for gene therapy. The study revealed that the molecular structure and electric charge were critical factors governing the function of the copolymers for intramuscular gene delivery. It can be concluded that, combined

  10. Viral vectors and delivery strategies for CNS gene therapy

    PubMed Central

    Gray, Steven J; Woodard, Kenton T; Samulski, R Jude

    2015-01-01

    This review aims to provide a broad overview of the targets, challenges and potential for gene therapy in the CNS, citing specific examples. There are a broad range of therapeutic targets, with very different requirements for a suitable viral vector. By utilizing different vector tropisms, novel routes of administration and engineered promoter control, transgenes can be targeted to specific therapeutic applications. Viral vectors have proven efficacious in preclinical models for several disease applications, spurring several clinical trials. While the field has pushed the limits of existing adeno-associated virus-based vectors, a next generation of vectors based on rational engineering of viral capsids should expand the application of gene therapy to be more effective in specific therapeutic applications. PMID:22833965

  11. Split vector systems for ultra-targeted gene delivery: a contrivance to achieve ethical assurance of somatic gene therapy in vivo.

    PubMed

    Tolmachov, Oleg E

    2014-08-01

    Tightly controlled spatial localisation of therapeutic gene delivery is essential to maximize the benefits of somatic gene therapy in vivo and to reduce its undesired effects on the 'bystander' cell populations, most importantly germline cells. Indeed, complete ethical assurance of somatic gene therapy can only be achieved with ultra-targeted gene delivery, which excludes the risk of inadvertent germline gene transfer. Thus, it is desired to supplement existing strategies of physical focusing and biological (cell-specific) targeting of gene delivery with an additional principle for the rigid control over spread of gene transfer within the body. In this paper I advance the concept of 'combinatorial' targeting of therapeutic gene transfer in vivo. I hypothesize that it is possible to engineer complex gene delivery vector systems consisting of several components, each one of them capable of independent spread within the human body but incapable of independent facilitation of gene transfer. As the gene delivery augmented by such split vector systems would be reliant on the simultaneous availability of all the vector system components at a predetermined body site, it is envisaged that higher order reaction kinetics required for the assembly of the functional gene transfer configuration would sharpen spatial localisation of gene transfer via curtailing the blurring effect of the vector spread within the body. A particular implementation of such split vector system could be obtained through supplementing a viral therapeutic gene vector with a separate auxiliary vector carrying a non-integrative and non-replicative form of a gene (e.g., mRNA) coding for a cellular receptor of the therapeutic vector component. Gene-transfer-enabling components of the vector system, which would be delivered separately from the vector component loaded with the therapeutic gene cargo, could also be cell-membrane-insertion-proficient receptors, elements of artificial transmembrane channels

  12. The expanding role of aerosols in systemic drug delivery, gene therapy, and vaccination.

    PubMed

    Laube, Beth L

    2005-09-01

    Aerosolized medications have been used for centuries to treat respiratory diseases. Until recently, inhalation therapy focused primarily on the treatment of asthma and chronic obstructive pulmonary disease, and the pressurized metered-dose inhaler was the delivery device of choice. However, the role of aerosol therapy is clearly expanding beyond that initial focus. This expansion has been driven by the Montreal protocol and the need to eliminate chlorofluorocarbons (CFCs) from traditional metered-dose inhalers, by the need for delivery devices and formulations that can efficiently and reproducibly target the systemic circulation for the delivery of proteins and peptides, and by developments in medicine that have made it possible to consider curing lung diseases with aerosolized gene therapy and preventing epidemics of influenza and measles with aerosolized vaccines. Each of these drivers has contributed to a decade or more of unprecedented research and innovation that has altered how we think about aerosol delivery and has expanded the role of aerosol therapy into the fields of systemic drug delivery, gene therapy, and vaccination. During this decade of innovation, we have witnessed the coming of age of dry powder inhalers, the development of new soft mist inhalers, and improved pressurized metered-dose inhaler delivery as a result of the replacement of CFC propellants with hydrofluoroalkane. The continued expansion of the role of aerosol therapy will probably depend on demonstration of the safety of this route of administration for drugs that have their targets outside the lung and are administered long term (eg, insulin aerosol), on the development of new drugs and drug carriers that can efficiently target hard-to-reach cell populations within the lungs of patients with disease (eg, patients with cystic fibrosis or lung cancer), and on the development of devices that improve aerosol delivery to infants, so that early intervention in disease processes with aerosol

  13. Lipid nanoparticles as drug/gene delivery systems to the retina.

    PubMed

    del Pozo-Rodríguez, Ana; Delgado, Diego; Gascón, Alicia R; Solinís, Maria Ángeles

    2013-03-01

    This review highlights the application of lipid nanoparticles (Solid Lipid Nanoparticles, Nanostructured Lipid Carriers, or Lipid Drug Conjugates) as effective drug/gene delivery systems for retinal diseases. Most drug products for ocular disease treatment are marketed as eye drop formulations but, due to ocular barriers, the drug concentration in the retina hardly ever turns out to be effective. Up to this date, several delivery systems have been designed to deliver drugs to the retina. Drug delivery strategies may be classified into 3 groups: noninvasive techniques, implants, and colloidal carriers. The best known systems for drug delivery to the posterior eye are intravitreal implants; in fact, some of them are being clinically used. However, their long-term accumulation might impact the patient's vision. On the contrary, colloidal drug delivery systems (microparticles, liposomes, or nanoparticles) can be easily administered in a liquid form. Nanoparticular systems diffuse rapidly and are better internalized in ocular tissues than microparticles. In comparison with liposomes, nanoparticles have a higher loading capacity and are more stable in biological fluids and during storage. In addition, their capacity to adhere to the ocular surface and interact with the endothelium makes these drug delivery systems interesting as new therapeutic tools in ophthalmology. Within the group of nanoparticles, those composed of lipids (Solid Lipid Nanoparticles, Nanostructred Lipid Carriers, and Lipid Drug Conjugates) are more biocompatible, easy to produce at large scale, and they may be autoclaved or sterilized. The present review summarizes scientific results that evidence the potential application of lipid nanoparticles as drug delivery systems for the retina and also as nonviral vectors in gene therapy of retina disorders, although much more effort is still needed before these lipidic systems could be available in the market. PMID:23286300

  14. Enhancement of the efficiency of non-viral gene delivery by application of pulsed magnetic field

    PubMed Central

    Kamau, Sarah W.; Hassa, Paul O.; Steitz, Benedikt; Petri-Fink, Alke; Hofmann, Heinrich; Hofmann-Amtenbrink, Margarethe; von Rechenberg, Brigitte; Hottiger, Michael O.

    2006-01-01

    New approaches to increase the efficiency of non-viral gene delivery are still required. Here we report a simple approach that enhances gene delivery using permanent and pulsating magnetic fields. DNA plasmids and novel DNA fragments (PCR products) containing sequence encoding for green fluorescent protein were coupled to polyethylenimine coated superparamagnetic nanoparticles (SPIONs). The complexes were added to cells that were subsequently exposed to permanent and pulsating magnetic fields. Presence of these magnetic fields significantly increased the transfection efficiency 40 times more than in cells not exposed to the magnetic field. The transfection efficiency was highest when the nanoparticles were sedimented on the permanent magnet before the application of the pulsating field, both for small (50 nm) and large (200–250 nm) nanoparticles. The highly efficient gene transfer already within 5 min shows that this technique is a powerful tool for future in vivo studies, where rapid gene delivery is required before systemic clearance or filtration of the gene vectors occurs. PMID:16540591

  15. Multifunctional Spider Silk Polymers for Gene Delivery to Human Mesenchymal Stem Cells

    PubMed Central

    Tokareva, Olena; Glettig, Dean; Abbott, Rosalyn D.; Kaplan, David L.

    2014-01-01

    Non-viral gene delivery systems are important transport vehicles that can be safe and effective alternatives to currently available viral systems. A new family of multifunctional spider silk-based gene carriers was bioengineered and found capable of targeting human mesenchymal stem cells (hMSCs). These carriers successfully delivered DNA to the nucleus of these mammalian cells. The presence of specific functional sequences in the recombinant proteins, such as a nuclear localization sequence (NLS) of the large tumor (T) antigen of the Simian virus 40 (SV40), an hMSC high affinity binding peptide (HAB), and a translocation motif (TLM) of the hepatitis-B virus surface protein (PreS2), and their roles in mitigation and enhancement of gene transfection efficiency towards hMSCs were characterized. The results demonstrate that these bioengineered spider silk proteins serve as effective carriers, without the well-known complications associated with viral delivery systems. PMID:25399785

  16. Tailoring the Surface of a Gene Delivery Vector with Carboxymethylated Dextran: A Systematic Analysis.

    PubMed

    Fortier, Charles; Louvier, Elodie; Durocher, Yves; De Crescenzo, Gregory

    2015-06-01

    Polymeric nanocarriers are attractive nonviral vectors for gene delivery purposes in vivo. For such applications, numerous physiological and subcellular bottlenecks have to be overcome. In that endeavor, each structural feature of nanocarriers can be optimized with respect to its corresponding challenges. Here, we focused on the interface between a model gene delivery nanocarrier and relevant constituents of the physiological environment. We screened a library of carboxymethylated dextrans (CMD) for the electrostatic coating of positively charged nanocarriers. We evaluated the jointed influence of the CMD molecular weight and charge density upon nanocarrier coating with respect to DNase, small ions, plasma proteins, red blood cells, and target cells. A total of 4 out of 26 CMD coated nanocarriers successfully passed every screening assay, but did not yield increased reporter gene expression in target cells compared to uncoated nanocarriers. The fine-tuning of CMD for nanocarrier coating yielded a relevant shortlist of candidates that will be further tested in vivo. PMID:25876439

  17. Formation of liposome by microfluidic flow focusing and its application in gene delivery

    NASA Astrophysics Data System (ADS)

    Wi, Rinbok; Oh, Yeonsu; Chae, Chanhee; Kim, Do Hyun

    2012-06-01

    We report the formation of liposomes in a simple procedure using a microfluidic hydrodynamic flow focusing method for the application in gene delivery. We fabricated microfluidic device using soft lithography and polydimethylsiloxane (PDMS) molding technique. Lipid-containing stream was surrounded by aqueous stream and liposomes were formed at the lipid-water interface. Size distribution of liposomes and zeta potential of liposome dispersion were investigated under various flow rate ratio (FRR) and processing temperature. Size distributions of liposomes were measured by dynamic light scattering (DLS), and zeta potential was measured to quantify the colloidal stability. Prepared liposomes were used as a vehicle for gene delivery, and the successful expression of delivered gene was observed by fluorescent microscope.

  18. Bacterial spores as particulate carriers for gene gun delivery of plasmid DNA.

    PubMed

    Aps, Luana R M M; Tavares, Milene B; Rozenfeld, Julio H K; Lamy, M Teresa; Ferreira, Luís C S; Diniz, Mariana O

    2016-06-20

    Bacillus subtilis spores represent a suitable platform for the adsorption of proteins, enzymes and viral particles at physiological conditions. In the present work, we demonstrate that purified spores can also adsorb DNA on their surface after treatment with cationic molecules. In addition, we demonstrate that DNA-coated B. subtilis spores can be used as particulate carriers and act as an alternative to gold microparticles for the biolistic (gene gun) administration of plasmid DNA in mice. Gene gun delivery of spores pre-treated with DODAB (dioctadecyldimethylammonium bromide) allowed efficient plasmid DNA absorption and induced protein expression levels similar to those obtained with gold microparticles. More importantly, we demonstrated that a DNA vaccine adsorbed on spores can be loaded into biolistic cartridges and efficiently delivered into mice, which induced specific cellular and antibody responses. Altogether, these data indicate that B. subtilis spores represent a simple and low cost alternative for the in vivo delivery of DNA vaccines by the gene gun technology.

  19. Preparation and characterization of gelatin-based mucoadhesive nanocomposites as intravesical gene delivery scaffolds.

    PubMed

    Liu, Ching-Wen; Chang, Li-Ching; Lin, Kai-Jen; Yu, Tsan-Jung; Tsai, Ching-Chung; Wang, Hao-Kuang; Tsai, Tong-Rong

    2014-01-01

    This study aimed to develop optimal gelatin-based mucoadhesive nanocomposites as scaffolds for intravesical gene delivery to the urothelium. Hydrogels were prepared by chemically crosslinking gelatin A or B with glutaraldehyde. Physicochemical and delivery properties including hydration ratio, viscosity, size, yield, thermosensitivity, and enzymatic degradation were studied, and scanning electron microscopy (SEM) was carried out. The optimal hydrogels (H), composed of 15% gelatin A175, displayed an 81.5% yield rate, 87.1% hydration ratio, 42.9 Pa·s viscosity, and 125.8 nm particle size. The crosslinking density of the hydrogels was determined by performing pronase degradation and ninhydrin assays. In vitro lentivirus (LV) release studies involving p24 capsid protein analysis in 293T cells revealed that hydrogels containing lentivirus (H-LV) had a higher cumulative release than that observed for LV alone (3.7-, 2.3-, and 2.3-fold at days 1, 3, and 5, resp.). Lentivirus from lentivector constructed green fluorescent protein (GFP) was then entrapped in hydrogels (H-LV-GFP). H-LV-GFP showed enhanced gene delivery in AY-27 cells in vitro and to rat urothelium by intravesical instillation in vivo. Cystometrogram showed mucoadhesive H-LV reduced peak micturition and threshold pressure and increased bladder compliance. In this study, we successfully developed first optimal gelatin-based mucoadhesive nanocomposites as intravesical gene delivery scaffolds. PMID:25580433

  20. Versatile types of polysaccharide-based supramolecular polycation/pDNA nanoplexes for gene delivery

    NASA Astrophysics Data System (ADS)

    Hu, Yang; Zhao, Nana; Yu, Bingran; Liu, Fusheng; Xu, Fu-Jian

    2014-06-01

    Different polysaccharide-based supramolecular polycations were readily synthesized by assembling multiple β-cyclodextrin-cored star polycations with an adamantane-functionalized dextran via host-guest interaction in the absence or presence of bioreducible linkages. Compared with nanoplexes of the starting star polycation and pDNA, the supramolecular polycation/pDNA nanoplexes exhibited similarly low cytotoxicity, improved cellular internalization and significantly higher gene transfection efficiencies. The incorporation of disulfide linkages imparted the supramolecular polycation/pDNA nanoplexes with the advantage of intracellular bioreducibility, resulting in better gene delivery properties. In addition, the antitumor properties of supramolecular polycation/pDNA nanoplexes were also investigated using a suicide gene therapy system. The present study demonstrates that the proper assembly of cyclodextrin-cored polycations with adamantane-functionalized polysaccharides is an effective strategy for the production of new nanoplex delivery systems.Different polysaccharide-based supramolecular polycations were readily synthesized by assembling multiple β-cyclodextrin-cored star polycations with an adamantane-functionalized dextran via host-guest interaction in the absence or presence of bioreducible linkages. Compared with nanoplexes of the starting star polycation and pDNA, the supramolecular polycation/pDNA nanoplexes exhibited similarly low cytotoxicity, improved cellular internalization and significantly higher gene transfection efficiencies. The incorporation of disulfide linkages imparted the supramolecular polycation/pDNA nanoplexes with the advantage of intracellular bioreducibility, resulting in better gene delivery properties. In addition, the antitumor properties of supramolecular polycation/pDNA nanoplexes were also investigated using a suicide gene therapy system. The present study demonstrates that the proper assembly of cyclodextrin-cored polycations

  1. High capacity nanoporous silicon carrier for systemic delivery of gene silencing therapeutics.

    PubMed

    Shen, Jianliang; Xu, Rong; Mai, Junhua; Kim, Han-Cheon; Guo, Xiaojing; Qin, Guoting; Yang, Yong; Wolfram, Joy; Mu, Chaofeng; Xia, Xiaojun; Gu, Jianhua; Liu, Xuewu; Mao, Zong-Wan; Ferrari, Mauro; Shen, Haifa

    2013-11-26

    Gene silencing agents such as small interfering RNA (siRNA) and microRNA offer the promise to modulate expression of almost every gene for the treatment of human diseases including cancer. However, lack of vehicles for effective systemic delivery to the disease organs has greatly limited their in vivo applications. In this study, we developed a high capacity polycation-functionalized nanoporous silicon (PCPS) platform comprised of nanoporous silicon microparticles functionalized with arginine-polyethyleneimine inside the nanopores for effective delivery of gene silencing agents. Incubation of MDA-MB-231 human breast cancer cells with PCPS loaded with STAT3 siRNA (PCPS/STAT3) or GRP78 siRNA (PCPS/GRP78) resulted in 91 and 83% reduction of STAT3 and GRP78 gene expression in vitro. Treatment of cells with a microRNA-18a mimic in PCPS (PCPS/miR-18) knocked down 90% expression of the microRNA-18a target gene ATM. Systemic delivery of PCPS/STAT3 siRNA in murine model of MDA-MB-231 breast cancer enriched particles in tumor tissues and reduced STAT3 expression in cancer cells, causing significant reduction of cancer stem cells in the residual tumor tissue. At the therapeutic dosage, PCPS/STAT3 siRNA did not trigger acute immune response in FVB mice, including changes in serum cytokines, chemokines, and colony-stimulating factors. In addition, weekly dosing of PCPS/STAT3 siRNA for four weeks did not cause signs of subacute toxicity based on changes in body weight, hematology, blood chemistry, and major organ histology. Collectively, the results suggest that we have developed a safe vehicle for effective delivery of gene silencing agents.

  2. Design and characterization of novel recombinant listeriolysin O-protamine fusion proteins for enhanced gene delivery.

    PubMed

    Kim, Na Hyung; Provoda, Chester; Lee, Kyung-Dall

    2015-02-01

    To improve the efficiency of gene delivery for effective gene therapy, it is essential that the vector carries functional components that can promote overcoming barriers in various steps leading to the transport of DNA from extracellular to ultimately nuclear compartment. In this study, we designed genetically engineered fusion proteins as a platform to incorporate multiple functionalities in one chimeric protein. Prototypes of such a chimera tested here contain two domains: one that binds to DNA; the other that can facilitate endosomal escape of DNA. The fusion proteins are composed of listeriolysin O (LLO), the endosomolytic pore-forming protein from Listeria monocytogenes, and a 22 amino acid sequence of the DNA-condensing polypeptide protamine (PN), singly or as a pair: LLO-PN and LLO-PNPN. We demonstrate dramatic enhancement of the gene delivery efficiency of protamine-condensed DNA upon incorporation of a small amount of LLO-PN fusion protein and further improvement with LLO-PNPN in vitro using cultured cells. Additionally, the association of anionic liposomes with cationic LLO-PNPN/protamine/DNA complexes, yielding a net negative surface charge, resulted in better in vitro transfection efficiency in the presence of serum. An initial, small set of data in mice indicated that the observed enhancement in gene expression could also be applicable to in vivo gene delivery. This study suggests that incorporation of a recombinant fusion protein with multiple functional components, such as LLO-protamine fusion protein, in a nonviral vector is a promising strategy for various nonviral gene delivery systems.

  3. 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.

  4. Gene delivery in conjunction with gold nanoparticle and tumor treating electric field

    NASA Astrophysics Data System (ADS)

    Tiwari, Pawan K.; Soo Lee, Yeon

    2013-08-01

    The advances in electrotherapy to treat the diseased biological cell instigate its extension in gene therapy through the delivery of gene into the nucleus. The objective of this study is to investigate the application of moderate intensity alternating electric field, also known as tumor treating electric field on a carrier system consisting of a charged gene complex conjugated to the surface of a gold nanoparticle. The gene delivery mechanism relies on the magnitude and direction of the induced electric field inside the cytoplasm in presence of carrier system. The induced electric field strength is significant in breaking the gene complex-gold nanoparticle bonding, and exerting an electric force pushing the charged gene into the nucleus. The electric force orientation is dependent on the aspect ratio (AR) of the gold nanoparticle and a relationship between them is studied via Maxwell two-dimensional (2D) finite element simulation analyzer. The development of charge density on the surface of carrier system and the required electric field strength to break the bonding are investigated utilizing the Gouy-Chapman-Grahame-Stern (GCGS) theoretical model. A carrier system having the aspect ratio of the gold nanoparticle in the range 1 < AR ≤ 5 and AR = 1 are substantial delivering cationic and anionic genes into the nucleus, respectively.

  5. Delivery.

    PubMed

    Miller, Thomas A

    2013-11-01

    Enthusiasm greeted the development of synthetic organic insecticides in the mid-twentieth century, only to see this give way to dismay and eventually scepticism and outright opposition by some. Regardless of how anyone feels about this issue, insecticides and other pesticides have become indispensable, which creates something of a dilemma. Possibly as a result of the shift in public attitude towards insecticides, genetic engineering of microbes was first met with scepticism and caution among scientists. Later, the development of genetically modified crop plants was met with an attitude that hardened into both acceptance and hard-core resistance. Transgenic insects, which came along at the dawn of the twenty-first century, encountered an entrenched opposition. Those of us responsible for studying the protection of crops have been affected more or less by these protagonist and antagonistic positions, and the experiences have often left one thoughtfully mystified as decisions are made by non-participants. Most of the issues boil down to concerns over delivery mechanisms.

  6. Delivery

    PubMed Central

    Miller, Thomas A

    2013-01-01

    Enthusiasm greeted the development of synthetic organic insecticides in the mid-twentieth century, only to see this give way to dismay and eventually scepticism and outright opposition by some. Regardless of how anyone feels about this issue, insecticides and other pesticides have become indispensable, which creates something of a dilemma. Possibly as a result of the shift in public attitude towards insecticides, genetic engineering of microbes was first met with scepticism and caution among scientists. Later, the development of genetically modified crop plants was met with an attitude that hardened into both acceptance and hard-core resistance. Transgenic insects, which came along at the dawn of the twenty-first century, encountered an entrenched opposition. Those of us responsible for studying the protection of crops have been affected more or less by these protagonist and antagonistic positions, and the experiences have often left one thoughtfully mystified as decisions are made by non-participants. Most of the issues boil down to concerns over delivery mechanisms. © 2013 Society of Chemical Industry PMID:23852646

  7. Synthesis and characterization of polyaminoacidic polycations for gene delivery.

    PubMed

    Licciardi, Mariano; Campisi, Monica; Cavallaro, Gennara; Cervello, Melchiorre; Azzolina, Antonina; Giammona, Gaetano

    2006-03-01

    The properties as non viral gene vector of a protein-like polymer, the alpha,beta-poly(N-2-hydroxyethyl)-d,l-aspartamide (PHEA) were exploited after its derivatization with 3-(carboxypropyl)trimethyl-ammonium chloride (CPTA) as molecule bearing a cationic group, in order to obtain stable polycations able to condense DNA. PHEA was firstly functionalized with aminic pendant groups by reaction with ethylenediamine (EDA) obtaining the alpha,beta-poly(N-2-hydroxyethyl)(2-aminoethylcarbamate)-d,l-aspartamide (PHEA-EDA) copolymer. We demonstrated that polymer functionalization degree is easily modulable by varying reaction conditions, so allowing to produce two PHEA-EDA derivatives at different molar percentage of amine groups. Subsequently, the condensation reaction of PHEA-EDA copolymers with CPTA yielded alpha,beta-poly(N-2-hydroxyethyl)(2-[3-(trimethylammonium chloride)propylamide]-amidoethylcarbamate)-d,l-aspartamide (PHEA-EDA-CPTA) polycation derivatives. In vitro studies were carried out to evaluate polycations ability to complex DNA and to protect it from nuclease degradation. Obtained results demonstrated the good ability of our new PHEA polycationic derivatives, PHEA-EDA-CPTA, to complex and condense genomic material, neutralizing its anionic charge even at very low polycation/DNA weight ratio. Finally, PHEA-EDA-CPTA polycations were characterized by in vitro cytotoxicity studies to evaluate their effects on the viability of HuH-6 human hepatocellular carcinoma cells by MTS assay. No cytotoxicity was evidenced by both polycationic derivatives after 48h of incubation at all tested concentrations.

  8. New development and application of ultrasound targeted microbubble destruction in gene therapy and drug delivery.

    PubMed

    Chen, Zhi-Yi; Yang, Feng; Lin, Yan; Zhang, Jin-Shan; Qiu, Ri-Xiang; Jiang, Lan; Zhou, Xing-Xing; Yu, Jiang-Xiu

    2013-08-01

    Ultrasound is a common used technique for clinical imaging. In recent years, with the advances in preparation technology of microbubbles and the innovations in ultrasound imaging, ultrasound is no longer confined to detection of tissue perfusion, but extends to specific ultrasound molecular imaging and target therapy gradually. With the development of research, ultrasound molecular imaging and target therapy have made great progresses. Targeted microbubbles for molecular imaging are achieved by binding target molecules, specific antibody or ligand to the surface of microbubbles to obtain specific imaging by attaching to target tissues. Meanwhile, it can also achieve targeting gene therapy or drug delivery by ultrasound targeted microbubble destruction (UTMD) mediating genes or drugs to specific target sites. UTMD has a number of advantages, such as target-specific, highly effective, non-invasivity, relatively low-cost and no radiation, and has broad application prospects, which is regarded as one hot spot in medical studies. We reviewed the new development and application of UTMD in gene therapy and drug delivery in this paper. With further development of technology and research, the gene or drug delivery system and related methods will be widely used in application and researches.

  9. Non-Viral Transfection Methods Optimized for Gene Delivery to a Lung Cancer Cell Line

    PubMed Central

    Salimzadeh, Loghman; Jaberipour, Mansooreh; Hosseini, Ahmad; Ghaderi, Abbas

    2013-01-01

    Background Mehr-80 is a newly established adherent human large cell lung cancer cell line that has not been transfected until now. This study aims to define the optimal transfection conditions and effects of some critical elements for enhancing gene delivery to this cell line by utilizing different non-viral transfection Procedures. Methods In the current study, calcium phosphate (CaP), DEAE-dextran, superfect, electroporation and lipofection transfection methods were used to optimize delivery of a plasmid construct that expressed Green Fluorescent Protein (GFP). Transgene expression was detected by fluorescent microscopy and flowcytometry. Toxicities of the methods were estimated by trypan blue staining. In order to evaluate the density of the transfected gene, we used a plasmid construct that expressed the Stromal cell-Derived Factor-1 (SDF-1) gene and measured its expression by real-time PCR. Results Mean levels of GFP-expressing cells 48 hr after transfection were 8.4% (CaP), 8.2% (DEAE-dextran), 4.9% (superfect), 34.1% (electroporation), and 40.1% (lipofection). Lipofection had the highest intense SDF-1 expression of the analyzed methods. Conclusion This study has shown that the lipofection and electroporation methods were more efficient at gene delivery to Mehr-80 cells. The quantity of DNA per transfection, reagent concentration, and incubation time were identified as essential factors for successful transfection in all of the studied methods. PMID:23799175

  10. Trastuzumab-targeted gene delivery to Her2-overexpressing breast cancer cells

    PubMed Central

    Mann, K; Kullberg, M

    2016-01-01

    We describe a novel gene delivery system that specifically targets human epidermal growth factor receptor 2 (Her2)-overexpressing breast cancer cells. The targeting complexes consist of a PEGylated polylysine core that is bound to DNA molecules coding for either green fluorescent protein or shrimp luciferase. The complex is disulfide linked to the monoclonal antibody trastuzumab and to a pore-forming protein, Listeriolysin O (LLO). Trastuzumab is responsible for specific targeting of Her2 receptors and uptake of the gene delivery complex into endosomes of recipient cells, whereas LLO ensures that the DNA molecules are capable of transit from the endosomes into the cytoplasm. Omission of either trastuzumab or LLO from the nanocomplexes results in minimal gene product in targeted cells. Treatment of isogeneic MCF7 and MCF7/Her18 cell lines, differing only in number of Her2 receptors, with the complete gene delivery system results in a 30-fold greater expression of luciferase activity in the Her2-overexpressing MCF7/Her18 cells. Our nanocomplexes are small (150–250 nm), stable to storage, nontoxic and generic in make-up such that any plasmid DNA or antibody specific for cell-surface receptors can be coupled to the PEGylated polylysine core. PMID:27199219

  11. Mucus-penetrating nanoparticles for drug and gene delivery to mucosal tissues

    PubMed Central

    Lai, Samuel K.; Wang, Ying-Ying; Hanes, Justin

    2009-01-01

    Mucus is a viscoelastic and adhesive gel that protects the lung airways, gastrointestinal (GI) tract, vagina, eye and other mucosal surfaces. Most foreign particulates, including conventional particle-based drug delivery systems, are efficiently trapped in human mucus layers by steric obstruction and/or adhesion. Trapped particles are typically removed from the mucosal tissue within seconds to a few hours depending on anatomical location, thereby strongly limiting the duration of sustained drug delivery locally. A number of debilitating diseases could be treated more effectively and with fewer side effects if drugs and genes could be more efficiently delivered to the underlying mucosal tissues in a controlled manner. This review first describes the tenacious mucus barrier properties that have precluded the efficient penetration of therapeutic particles. It then reviews the design and development of new mucus-penetrating particles that may avoid rapid mucus clearance mechanisms, and thereby provide targeted or sustained drug delivery for localized therapies in mucosal tissues. PMID:19133304

  12. Polyethylenimine-cyclodextrin-tegafur conjugate shows anti-cancer activity and a potential for gene delivery*

    PubMed Central

    Hu, Qi-da; Fan, Hui; Lou, Wei-jian; Wang, Qing-qing; Tang, Gu-ping

    2011-01-01

    Polyethylenimine-cyclodextrin-tegafur (PEI-CyD-tegafur) conjugate was synthesized as a novel multifunctional prodrug of tegafur for co-delivery of chemotherapeutic agent tegafur and enhanced green fluorescent protein (EGFP) reporter plasmid DNA. Conjugation of tegafur to PEI-CyD via chemical linkage was characterized by 1H NMR spectrometry and ultraviolet (UV) spectrometry. PEI-CyD-tegafur was able to condense plasmid DNA into complexes of around 150 nm with positive charge at the N/P ratio of 25, in accordance with electron microscopy observation of compact and monodisperse nanoparticles. The results of in vitro experiments showed enhanced cytotoxicity and considerable transfection efficiency in B16F10 cell line. Therefore, PEI-CyD-tegafur may have great potential as a co-delivery system with anti-cancer activity and potential for gene delivery. PMID:21887847

  13. Hepatic gene therapy: efficient gene delivery and expression in primary hepatocytes utilizing a conjugated adenovirus-DNA complex.

    PubMed Central

    Cristiano, R J; Smith, L C; Kay, M A; Brinkley, B R; Woo, S L

    1993-01-01

    Receptor-mediated endocytosis is an effective method for gene delivery into target cells. We have previously shown that DNA molecules complexed with asialoglycoprotein can be efficiently endocytosed by primary hepatocytes and the internalized DNA can be released from endosomes by the use of a replication-defective adenovirus. Because the DNA and virus enter target cells independently, activity enhancement requires high concentrations of adenoviral particles. In this study, adenoviral particles were chemically conjugated to poly(L-lysine) and bound ionically to DNA molecules. Quantitative delivery to primary hepatocytes was achieved with significantly reduced viral titer when the asialoorosomucoid-poly(L-lysine) conjugate was included in the complex. The conjugated adenovirus was used to deliver a DNA vector containing canine factor IX to mouse hepatocytes, resulting in the expression of significant concentrations of canine factor IX in the culture medium. The results suggest that receptor-mediated endocytosis coupled with an efficient endosomal lysis vector should permit the application of targeted and efficient gene delivery into the liver for gene therapy of hepatic deficiencies. Images Fig. 2 Fig. 4 PMID:8265587

  14. Surface-Engineered Viral Vectors for Selective and Cell Type-Specific Gene Delivery.

    PubMed

    Buchholz, Christian J; Friedel, Thorsten; Büning, Hildegard

    2015-12-01

    Recent progress in gene transfer technology enables the delivery of genes precisely to the application-relevant cell type ex vivo on cultivated primary cells or in vivo on local or systemic administration. Gene vectors based on lentiviruses or adeno-associated viruses can be engineered such that they use a cell surface marker of choice for cell entry instead of their natural receptors. Binding to the surface marker is mediated by a targeting ligand displayed on the vector particle surface, which can be a peptide, single-chain antibody, or designed ankyrin repeat protein. Examples include vectors that deliver genes to specialized endothelial cells or lymphocytes, tumor cells, or particular cells of the nervous system with potential applications in gene function studies and molecular medicine.

  15. Cell-Targeting Cationic Gene Delivery System Based on a Modular Design Rationale.

    PubMed

    Liu, Jia; Xu, Luming; Jin, Yang; Qi, Chao; Li, Qilin; Zhang, Yunti; Jiang, Xulin; Wang, Guobin; Wang, Zheng; Wang, Lin

    2016-06-01

    En route to target cells, a gene carrier faces multiple extra- and intracellular hurdles that would affect delivery efficacy. Although diverse strategies have been proposed to functionalize gene carriers for individually overcoming these barriers, it is challenging to generate a single multifunctional gene carrier capable of surmounting all these barriers. Aiming at this challenge, we have developed a supramolecular modular approach to fabricate a multifunctional cationic gene delivery system. It consists of two prefunctionalized modules: (1) a host module: a polymer (PCD-SS-PDMAEMA) composed of poly(β-cyclodextrin) backbone and disulfide-linked PDMAEMA arms, expectedly acting to compact DNA and release DNA upon cleavage of disulfide linkers in reductive microenvironment; and (2) a guest module: adamantyl and folate terminated PEG (Ad-PEG-FA), expectedly functioning to reduce nonspecific interactions, improve biocompatibility, and provide folate-mediated cellular targeting specificity. Through the host-guest interaction between β-cyclodextrin units of the "host" module and adamantyl groups of the "guest" module, the PCD-SS-PDMAEMA-1 (host) and Ad-PEG-FA (guest) self-assemble forming a supramolecular pseudocopolymer (PCD-SS-PDMAEMA-1/PEG-FA). Our comprehensive analyses demonstrate that the functions preassigned to the two building modules are well realized. The gene carrier effectively compacts DNA into stable nanosized polyplexes resistant to enzymatic digestion, triggers DNA release in reducing environment, possesses significantly improved hemocompatibility, and specifically targets folate-receptor positive cells. Most importantly, endowed with these predesigned functions, the PCD-SS-PDMAEMA-1/PEG-FA supramolecular gene carrier exhibits excellent transfection efficacy for both pDNA and siRNA. Thus, this work represents a proof-of-concept example showing the efficiency and convenience of an adaptable, modular approach for conferring multiple functions to a single

  16. Real-time fluorescence tracking of gene delivery via multifunctional nanocomposites.

    PubMed

    Bai, Min; Bai, Xilin; Wang, Leyu

    2014-11-18

    Fluorescence imaging of transduced cells and tissues is valuable in the development of gene vectors and the evaluation of gene therapy efficacy. We report here the simple and rational design of multifunctional nanocomposites (NCs) for simultaneous gene delivery and fluorescence tracking based on ZnS:Mn(2+) quantum dots (QDs) and positively charged polymer coating. The positively charged imidazole in the as-synthesized amphiphilic copolymer can be used for gene loading via electrostatic interaction. While the introduced poly(ethylene glycol) (PEG) can be used to reduce the binding of plasma proteins to nanovectors and minimize clearance by the reticuloendothelial system after intravenous administration. Most importantly, these multifunctional nanovectors showed much lower cellular toxicity than the commercial polyethylenimine (PEI) transfection vectors. On the basis of the red fluorescence of QDs, we can real-time track the gene delivery in cells, and the transfection efficacy of pDNA encoding enhanced green fluorescence protein (pEGFP) was monitored via the green fluorescence of the GFP expressed by the pDNA delivered into the nuclei. Fluorescence imaging analysis confirmed that the QDs-based nanovectors delivered pDNA into HepG2 cells efficiently. These new insights and capabilities pave a new way toward nanocomposite engineering for fluorescence imaging tracking of gene therapy.

  17. Avidin-biotin interaction mediated peptide assemblies as efficient gene delivery vectors for cancer therapy.

    PubMed

    Qu, Wei; Chen, Wei-Hai; Kuang, Ying; Zeng, Xuan; Cheng, Si-Xue; Zhou, Xiang; Zhuo, Ren-Xi; Zhang, Xian-Zheng

    2013-01-01

    Gene therapy offers a bright future for the treatment of cancers. One of the research highlights focuses on smart gene delivery vectors with good biocompatibility and tumor-targeting ability. Here, a novel gene vector self-assembled through avidin-biotin interaction with optimized targeting functionality, biotinylated tumor-targeting peptide/avidin/biotinylated cell-penetrating peptide (TAC), was designed and prepared to mediate the in vitro and in vivo delivery of p53 gene. TAC exhibited efficient DNA-binding ability and low cytotoxicity. In in vitro transfection assay, TAC/p53 complexes showed higher transfection efficiency and expression amount of p53 protein in MCF-7 cells as compared with 293T and HeLa cells, primarily due to the specific recognition between tumor-targeting peptides and receptors on MCF-7 cells. Additionally, by in situ administration of TAC/p53 complexes into tumor-bearing mice, the expression of p53 gene was obviously upregulated in tumor cells, and the tumor growth was significantly suppressed. This study provides an alternative and unique strategy to assemble functionalized peptides, and the novel self-assembled vector TAC developed is a promising gene vector for cancer therapy.

  18. Evaluation and mechanism studies of PEGylated dendrigraft poly-L-lysines as novel gene delivery vectors

    NASA Astrophysics Data System (ADS)

    Huang, Rongqin; Liu, Shuhuan; Shao, Kun; Han, Liang; Ke, Weilun; Liu, Yang; Li, Jianfeng; Huang, Shixian; Jiang, Chen

    2010-07-01

    Dendrimers have attracted great interest in the field of gene delivery due to their synthetic controllability and excellent gene transfection efficiency. In this work, dendrigraft poly-L-lysines (DGLs) were evaluated as a novel gene vector for the first time. Derivatives of DGLs (generation 2 and 3) with different extents of PEGylation were successfully synthesized and used to compact pDNA as complexes. The result of gel retardation assay showed that pDNA could be effectively packed by all the vectors at a DGLs to pDNA weight ratio greater than 2. An increase in the PEGylation extent of vectors resulted in a decrease in the incorporation efficiency and cytotoxicity of complexes in 293 cells, which also decreased the zeta potential a little but did not affect the mean diameter of complexes. Higher generation of DGLs could mediate higher gene transfection in vitro. Confocal microscopy and cellular uptake inhibition studies demonstrated that caveolae-mediated process and macropinocytosis were involved in the cellular uptake of DGLs-based complexes. Also the results indicate that proper PEGylated DGLs could mediate efficient gene transfection, showing their potential as an alternate biodegradable vector in the field of nonviral gene delivery.

  19. Adenovirus-mediated delivery of antiangiogenic genes as an antitumor approach.

    PubMed

    Régulier, E; Paul, S; Marigliano, M; Kintz, J; Poitevin, Y; Ledoux, C; Roecklin, D; Cauet, G; Calenda, V; Homann, H E

    2001-01-01

    Based on the observation that the growth of solid tumors is dependent on the formation of new blood vessels, therapeutic strategies aimed at inhibiting angiogenesis have been proposed. A number of proteins with angiostatic activity have been described, but their development as therapeutic agents has been hampered by difficulties in their production and their poor pharmacokinetics. These limitations may be resolved using a gene therapy approach whereby the genes are delivered and expressed in vivo. Here we compared adenoviral delivery of endostatin, proliferin-related protein (PRP), and interferon-inducible protein 10 (IP10) genes. Recombinant adenoviruses carrying the three angiostatic genes express biologically active gene products as determined in vitro in endothelial cell proliferation and migration assays, and in vivo by inhibition of neoangiogenesis in rat chambers. Eradication of established tumors in vivo, in the murine B16F10 melanoma model in immunocompetent mice, was not achieved by intratumoral injection of the different vectors. However, the combination of intravenous plus intratumoral injections allowed rejection of tumors. Ad-PRP or Ad-IP10 were significantly more efficient than Ad-endostatin, leading to complete tumor rejection and prolonged survival in a high proportion of treated animals. These data support the use of in vivo gene delivery approaches to produce high-circulating and local levels of antiangiogenic agents for the therapy of local and metastatic human tumors. PMID:11219493

  20. The potential of adeno-associated viral vectors for gene delivery to muscle tissue

    PubMed Central

    Nahid, M Abu; Gao, Guangping

    2014-01-01

    Introduction Muscle-directed gene therapy is rapidly gaining attention primarily because muscle is an easily accessible target tissue and is also associated with various severe genetic disorders. Localized and systemic delivery of recombinant adeno-associated virus (rAAV) vectors of several serotypes results in very efficient transduction of skeletal and cardiac muscles, which has been achieved in both small and large animals, as well as in humans. Muscle is the target tissue in gene therapy for many muscular dystrophy diseases, and may also be exploited as a biofactory to produce secretory factors for systemic disorders. Current limitations of using rAAVs for muscle gene transfer include vector size restriction, potential safety concerns such as off-target toxicity and the immunological barrier composing of pre-existing neutralizing antibodies and CD8+ T-cell response against AAV capsid in humans. Areas covered In this article, we will discuss basic AAV vector biology and its application in muscle-directed gene delivery, as well as potential strategies to overcome the aforementioned limitations of rAAV for further clinical application. Expert opinion Delivering therapeutic genes to large muscle mass in humans is arguably the most urgent unmet demand in treating diseases affecting muscle tissues throughout the whole body. Muscle-directed, rAAV-mediated gene transfer for expressing antibodies is a promising strategy to combat deadly infectious diseases. Developing strategies to circumvent the immune response following rAAV administration in humans will facilitate clinical application. PMID:24386892

  1. Carbon nanotubes part II: a remarkable carrier for drug and gene delivery

    PubMed Central

    Karimi, Mahdi; Solati, Navid; Ghasemi, Amir; Estiar, Mehrdad Asghari; Hashemkhani, Mahshid; Kiani, Parnian; Mohamed, Elmira; Saeidi, Ahad; Taheri, Mahdiar; Avci, Pinar; Aref, Amir R; Amiri, Mohammad; Baniasadi, Fazel; Hamblin, Michael R

    2015-01-01

    Introduction Carbon nanotubes (CNT) have recently been studied as novel and versatile drug and gene delivery vehicles. When CNT are suitably functionalized, they can interact with various cell types and are taken up by endocytosis. Areas covered Anti-cancer drugs cisplatin and doxorubicin have been delivered by CNT, as well as methotrexate, taxol and gemcitabine. The delivery of the antifungal compound amphotericin B and the oral administration of erythropoietin have both been assisted using CNT. Frequently, targeting moieties such as folic acid, epidermal growth factor or various antibodies are attached to the CNT-drug nanovehicle. Different kinds of functionalization (e.g., polycations) have been used to allow CNT to act as gene delivery vectors. Plasmid DNA, small interfering RNA and micro-RNA have all been delivered by CNT vehicles. Significant concerns are raised about the nanotoxicology of the CNT and their potentially damaging effects on the environment. Expert opinion CNT-mediated drug delivery has been studied for over a decade, and both in vitro and in vivo studies have been reported. The future success of CNTs as vectors in vivo and in clinical application will depend on achievement of efficacious therapy with minimal adverse effects and avoidance of possible toxic and environmentally damaging effects. PMID:25613837

  2. New cyclodextrin derivative containing poly(L-lysine) dendrons for gene and drug co-delivery.

    PubMed

    Ma, Dong; Zhang, Hong-Bin; Chen, Yu-Yun; Lin, Jian-Tao; Zhang, Li-Ming

    2013-09-01

    To develop a multifunctional polymeric carrier for gene and drug co-delivery, a new cyclodextrin derivative containing poly(L-lysine) dendrons was prepared by the click conjugation of per-6-azido-β-cyclodextrin with propargyl focal point poly(L-lysine) dendron of third generation and then characterized by FTIR, (1)H NMR, and GPC analyses. It was found that such a conjugate could form colloidally stable nanocomplexes with plasmid DNA in aqueous system and exhibited high gene transfection efficiency. Moreover, it could load efficiently methotrexate drug with anticancer activity and showed a sustained release behavior. Different from commonly used amphiphilic copolymers with cationic character, the as obtained cyclodextrin derivative may be used directly for the combinatorial delivery of nucleic acid and lipophilic anticancer drugs without a complicated micellization process. PMID:23769303

  3. Asymmetric 1-alkyl-2-acyl phosphatidylcholine: a helper lipid for enhanced non-viral gene delivery.

    PubMed

    Huang, Zhaohua; Li, Weijun; Szoka, Francis C

    2012-05-01

    Rationally designed asymmetrical alkylacyl phosphatidylcholines (APC) have been synthesized and evaluated as helper lipids for non-viral gene delivery. A long aliphatic chain (C22-C24) was introduced at the 1-position of glycerol backbone, a branched lipid chain (C18) at the 2-position, and a phosphocholine head group at the 3-position. The fusogenicity of APC depends on the length and degree of saturation of the alkyl chain. Cationic lipids were formulated with APC as either lipoplexes or nanolipoparticles, and evaluated for their stability, transfection efficiency, and cytotoxicity. APC mediated high in vitro transfection efficiency, and had low cytotoxicity. Small nanolipoparticles (less than 100 nm) can be obtained with APC by applying as low as 0.1% PEG-lipid. Our study extends the type of helper lipids that are suitable for gene transfer and points the way to improve non-viral nucleic acid delivery system other than the traditional cationic lipids optimization.

  4. Early pregnancy peripheral blood gene expression and risk of preterm delivery: a nested case control study

    PubMed Central

    2009-01-01

    Background Preterm delivery (PTD) is a significant public health problem associated with greater risk of mortality and morbidity in infants and mothers. Pathophysiologic processes that may lead to PTD start early in pregnancy. We investigated early pregnancy peripheral blood global gene expression and PTD risk. Methods As part of a prospective study, ribonucleic acid was extracted from blood samples (collected at 16 weeks gestational age) from 14 women who had PTD (cases) and 16 women who delivered at term (controls). Gene expressions were measured using the GeneChip® Human Genome U133 Plus 2.0 Array. Student's T-test and fold change analysis were used to identify differentially expressed genes. We used hierarchical clustering and principle components analysis to characterize signature gene expression patterns among cases and controls. Pathway and promoter sequence analyses were used to investigate functions and functional relationships as well as regulatory regions of differentially expressed genes. Results A total of 209 genes, including potential candidate genes (e.g. PTGDS, prostaglandin D2 synthase 21 kDa), were differentially expressed. A set of these genes achieved accurate pre-diagnostic separation of cases and controls. These genes participate in functions related to immune system and inflammation, organ development, metabolism (lipid, carbohydrate and amino acid) and cell signaling. Binding sites of putative transcription factors such as EGR1 (early growth response 1), TFAP2A (transcription factor AP2A), Sp1 (specificity protein 1) and Sp3 (specificity protein 3) were over represented in promoter regions of differentially expressed genes. Real-time PCR confirmed microarray expression measurements of selected genes. Conclusions PTD is associated with maternal early pregnancy peripheral blood gene expression changes. Maternal early pregnancy peripheral blood gene expression patterns may be useful for better understanding of PTD pathophysiology and PTD risk

  5. Integrating mitosis, toxicity, and transgene expression in a telecommunications packet-switched network model of lipoplex-mediated gene delivery.

    PubMed

    Martin, Timothy M; Wysocki, Beata J; Beyersdorf, Jared P; Wysocki, Tadeusz A; Pannier, Angela K

    2014-08-01

    Gene delivery systems transport exogenous genetic information to cells or biological systems with the potential to directly alter endogenous gene expression and behavior with applications in functional genomics, tissue engineering, medical devices, and gene therapy. Nonviral systems offer advantages over viral systems because of their low immunogenicity, inexpensive synthesis, and easy modification but suffer from lower transfection levels. The representation of gene transfer using models offers perspective and interpretation of complex cellular mechanisms,including nonviral gene delivery where exact mechanisms are unknown. Here, we introduce a novel telecommunications model of the nonviral gene delivery process in which the delivery of the gene to a cell is synonymous with delivery of a packet of information to a destination computer within a packet-switched computer network. Such a model uses nodes and layers to simplify the complexity of modeling the transfection process and to overcome several challenges of existing models. These challenges include a limited scope and limited time frame, which often does not incorporate biological effects known to affect transfection. The telecommunication model was constructed in MATLAB to model lipoplex delivery of the gene encoding the green fluorescent protein to HeLa cells. Mitosis and toxicity events were included in the model resulting in simulation outputs of nuclear internalization and transfection efficiency that correlated with experimental data. A priori predictions based on model sensitivity analysis suggest that increasing endosomal escape and decreasing lysosomal degradation, protein degradation, and GFP-induced toxicity can improve transfection efficiency by three-fold. Application of the telecommunications model to nonviral gene delivery offers insight into the development of new gene delivery systems with therapeutically relevant transfection levels.

  6. Novel Therapeutic Approaches for Various Cancer Types Using a Modified Sleeping Beauty-Based Gene Delivery System

    PubMed Central

    Kim, Hyun-Pyo

    2014-01-01

    Successful gene therapy largely depends on the selective introduction of therapeutic genes into the appropriate target cancer cells. One of the most effective and promising approaches for targeting tumor tissue during gene delivery is the use of viral vectors, which allow for high efficiency gene delivery. However, the use of viral vectors is not without risks and safety concerns, such as toxicities, a host immune response towards the viral antigens or potential viral recombination into the host's chromosome; these risks limit the clinical application of viral vectors. The Sleeping Beauty (SB) transposon-based system is an attractive, non-viral alternative to viral delivery systems. SB may be less immunogenic than the viral vector system due to its lack of viral sequences. The SB-based gene delivery system can stably integrate into the host cell genome to produce the therapeutic gene product over the lifetime of a cell. However, when compared to viral vectors, the non-viral SB-based gene delivery system still has limited therapeutic efficacy due to the lack of long-lasting gene expression potential and tumor cell specific gene transfer ability. These limitations could be overcome by modifying the SB system through the introduction of the hTERT promoter and the SV40 enhancer. In this study, a modified SB delivery system, under control of the hTERT promoter in conjunction with the SV40 enhancer, was able to successfully transfer the suicide gene (HSV-TK) into multiple types of cancer cells. The modified SB transfected cancer cells exhibited a significantly increased cancer cell specific death rate. These data suggest that our modified SB-based gene delivery system can be used as a safe and efficient tool for cancer cell specific therapeutic gene transfer and stable long-term expression. PMID:24466025

  7. [Gene therapy: nucleic acids as drugs. Action mechanisms and delivery into the cell].

    PubMed

    Cavagnari, Brian M

    2011-06-01

    Gene therapy involves the transference of new genetic material to the cell in order to obtain a therapeutic benefit, offering a new option for the treatment of various diseases. In this article, some of these nucleic acid-based drugs, such as plasmids, aptamers, oligonucleotides, ribozymes and small interfering ribonucleic acid, are presented. Their mechanism and level of action is commented and several delivery systems, such as liposomes, cationic polymers, direct nucleic acid transfer and viral vectors, are also discussed.

  8. Evaluation of polymeric gene delivery nanoparticles by nanoparticle tracking analysis and high-throughput flow cytometry.

    PubMed

    Shmueli, Ron B; Bhise, Nupura S; Green, Jordan J

    2013-03-01

    Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases(1) and as a technology for regenerative medicine(2). Unlike viruses, which have significant safety issues, polymeric nanoparticles can be designed to be non-toxic, non-immunogenic, non-mutagenic, easier to synthesize, chemically versatile, capable of carrying larger nucleic acid cargo and biodegradable and/or environmentally responsive. Cationic polymers self-assemble with negatively charged DNA via electrostatic interaction to form complexes on the order of 100 nm that are commonly termed polymeric nanoparticles. Examples of biomaterials used to form nanoscale polycationic gene delivery nanoparticles include polylysine, polyphosphoesters, poly(amidoamines)s and polyethylenimine (PEI), which is a non-degradable off-the-shelf cationic polymer commonly used for nucleic acid delivery(1,3) . Poly(beta-amino ester)s (PBAEs) are a newer class of cationic polymers(4) that are hydrolytically degradable(5,6) and have been shown to be effective at gene delivery to hard-to-transfect cell types such as human retinal endothelial cells (HRECs)(7), mouse mammary epithelial cells(8), human brain cancer cells(9) and macrovascular (human umbilical vein, HUVECs) endothelial cells(10). A new protocol to characterize polymeric nanoparticles utilizing nanoparticle tracking analysis (NTA) is described. In this approach, both the particle size distribution and the distribution of the number of plasmids per particle are obtained(11). In addition, a high-throughput 96-well plate transfection assay for rapid screening of the transfection efficacy of polymeric nanoparticles is presented. In this protocol, poly(beta-amino ester)s (PBAEs) are used as model polymers and human retinal endothelial cells (HRECs) are used as model human cells. This protocol can be easily adapted to evaluate any polymeric nanoparticle and any cell type of interest in a multi

  9. Lipid peptide nanocomplexes for gene delivery and magnetic resonance imaging in the brain

    PubMed Central

    Writer, Michele J.; Kyrtatos, Panagiotis G.; Bienemann, Alison S.; Pugh, John A.; Lowe, Andrew S.; Villegas-Llerena, Claudio; Kenny, Gavin D.; White, Edward A.; Gill, Steven S.; McLeod, Cameron W.; Lythgoe, Mark F.; Hart, Stephen L.

    2012-01-01

    Gadolinium-labelled nanocomplexes offer prospects for the development of real-time, non-invasive imaging strategies to visualise the location of gene delivery by MRI. In this study, targeted nanoparticle formulations were prepared comprising a cationic liposome (L) containing a Gd-chelated lipid at 10, 15 and 20% by weight of total lipid, a receptor-targeted, DNA-binding peptide (P) and plasmid DNA (D), which electrostatically self-assembled into LPD nanocomplexes. The LPD formulation containing the liposome with 15% Gd-chelated lipid displayed optimal peptide-targeted, transfection efficiency. MRI conspicuity peaked at 4 h after incubation of the nanocomplexes with cells, suggesting enhancement by cellular uptake and trafficking. This was supported by time course confocal microscopy analysis of transfections with fluorescently-labelled LPD nanocomplexes. Gd-LPD nanocomplexes delivered to rat brains by convection-enhanced delivery were visible by MRI at 6 h, 24 h and 48 h after administration. Histological brain sections analysed by laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) confirmed that the MRI signal was associated with the distribution of Gd3 + moieties and differentiated MRI signals due to haemorrhage. The transfected brain cells near the injection site appeared to be mostly microglial. This study shows the potential of Gd-LPD nanocomplexes for simultaneous delivery of contrast agents and genes for real-time monitoring of gene therapy in the brain. PMID:22800579

  10. A self-assembled system for tumor-targeted co-delivery of drug and gene.

    PubMed

    Wang, Cheng; Li, Min; Yang, Tie; Ding, Xuefang; Bao, Xiuli; Ding, Yang; Xiong, Hui; Wu, Ying; Wang, Wei; Zhou, Jianping

    2015-11-01

    A new cationic polymer eprosartan-graft-PEI (ESP) containing eprosartan (ES) and polyethylenimine 1.8K was successfully developed and employed as a safe gene vector to assemble a drug (ES) and gene co-delivery complex (ESP/pDNA). Chondroitin sulfate (CS) was then used as a coating polymer to shield the surface charge of ESP/pDNA complexes, as well as a tumor targeting entity to ensure specific delivery of CS/ESP/pDNA complexes. The CS/ESP/pDNA complexes with desirable particle size and zeta potential, exhibited amidase-responsive drug release and CS-mediated endocytosis in vitro. As compared with ESP/pDNA complexes, in vivo imaging results demonstrated decreased reticuloendothelial system uptake and remarkably increased tumor accumulation of CS/ESP/pDNA complexes. All these findings indicated the potential of CS/ESP/pDNA as a promising tumor-targeted drug and gene co-delivery system. PMID:26249591

  11. Gene Delivery Mediated by Recombinant Silk Proteins Containing Cationic and Cell Binding Motifs

    PubMed Central

    Numata, Keiji; Hamasaki, Juliana; Subramanian, Balajikarthick; Kaplan, David L

    2010-01-01

    Silk proteins are biodegradable and biocompatible, and can also be tailored to contain additional features via genetic engineering, suggesting utility for gene delivery. In the present study, novel silk-based block copolymers were bioengineered both with poly(L-lysine) domains to interact with plasmid DNA (pDNA) and RGD, to enhance cell-binding and transfection efficiency. Ionic complexes of these silk-polylysine-RGD block copolymers with pDNA were prepared, characterized and utilized for gene delivery to HeLa cells and human embryonic kidney (HEK) cells. The material systems were characterized by agarose gel electrophoresis, zeta-potentialmeter, atomic force microscopy, and dynamic light scattering. Sizes and charges of the pDNA complexes were regulated by the polymer/nucleotide molar ratio. Samples with 30-lysine residues and 11 RGD sequences, prepared at the ratio of number of amines/phosphates from pDNA (N/P) of 2, had an average solution diameter of 186 nm and showed the highest transfection efficiency. The intracellular distribution of complexes of Cy5-labeled pDNA was investigated by confocal laser scanning microscopy. The Cy5-labeled pDNA was distributed near the cell membrane and around the nuclei, indicating that the pDNA was transferred near the nucleus. The results demonstrated the potential of bioengineered silk proteins with additional functional features as a new family of highly tailored gene delivery systems. PMID:20457191

  12. Preparation and characterization of liposome-encapsulated plasmid DNA for gene delivery.

    PubMed

    Levine, Rachel M; Pearce, Timothy R; Adil, Maroof; Kokkoli, Efrosini

    2013-07-23

    The success of common nonviral gene delivery vehicles, lipoplexes and polyplexes, is limited by the toxicity and instability of these charged molecules. Stealth liposomes could provide a stable, safe alternative to cationic DNA complexes for effective gene delivery. DNA encapsulations in three stealth liposomal formulations prepared by thin film, reverse phase evaporation, and asymmetric liposome formation were compared, and the thin film method was found to produce the highest yields of encapsulated DNA. A DNA quantification method appropriate for DNA encapsulated within liposomes was also developed and verified for accuracy. The effect of initial lipid and DNA concentrations on the encapsulation yield and fraction of DNA-filled liposomes was evaluated. Higher encapsulation yields were achieved by higher lipid contents, while a higher fraction of DNA-filled liposomes was produced by either lower lipid content or higher DNA concentration. Control of these parameters allows for the design of gene delivery nanoparticles with high DNA encapsulation yields or higher fraction of DNA-filled liposomes. PMID:23837701

  13. Particle tracking in drug and gene delivery research: State-of-the-art applications and methods.

    PubMed

    Schuster, Benjamin S; Ensign, Laura M; Allan, Daniel B; Suk, Jung Soo; Hanes, Justin

    2015-08-30

    Particle tracking is a powerful microscopy technique to quantify the motion of individual particles at high spatial and temporal resolution in complex fluids and biological specimens. Particle tracking's applications and impact in drug and gene delivery research have greatly increased during the last decade. Thanks to advances in hardware and software, this technique is now more accessible than ever, and can be reliably automated to enable rapid processing of large data sets, thereby further enhancing the role that particle tracking will play in drug and gene delivery studies in the future. We begin this review by discussing particle tracking-based advances in characterizing extracellular and cellular barriers to therapeutic nanoparticles and in characterizing nanoparticle size and stability. To facilitate wider adoption of the technique, we then present a user-friendly review of state-of-the-art automated particle tracking algorithms and methods of analysis. We conclude by reviewing technological developments for next-generation particle tracking methods, and we survey future research directions in drug and gene delivery where particle tracking may be useful.

  14. Biopolymer-Based Nanoparticles for Drug/Gene Delivery and Tissue Engineering

    PubMed Central

    Nitta, Sachiko Kaihara; Numata, Keiji

    2013-01-01

    There has been a great interest in application of nanoparticles as biomaterials for delivery of therapeutic molecules such as drugs and genes, and for tissue engineering. In particular, biopolymers are suitable materials as nanoparticles for clinical application due to their versatile traits, including biocompatibility, biodegradability and low immunogenicity. Biopolymers are polymers that are produced from living organisms, which are classified in three groups: polysaccharides, proteins and nucleic acids. It is important to control particle size, charge, morphology of surface and release rate of loaded molecules to use biopolymer-based nanoparticles as drug/gene delivery carriers. To obtain a nano-carrier for therapeutic purposes, a variety of materials and preparation process has been attempted. This review focuses on fabrication of biocompatible nanoparticles consisting of biopolymers such as protein (silk, collagen, gelatin, β-casein, zein and albumin), protein-mimicked polypeptides and polysaccharides (chitosan, alginate, pullulan, starch and heparin). The effects of the nature of the materials and the fabrication process on the characteristics of the nanoparticles are described. In addition, their application as delivery carriers of therapeutic drugs and genes and biomaterials for tissue engineering are also reviewed. PMID:23344060

  15. A pH-sensitive multifunctional gene carrier assembled via layer-by-layer technique for efficient gene delivery

    PubMed Central

    Li, Peng; Liu, Donghua; Miao, Lei; Liu, Chunxi; Sun, Xiaoli; Liu, Yongjun; Zhang, Na

    2012-01-01

    Background The success of gene therapy asks for the development of multifunctional vectors that could overcome various gene delivery barriers, such as the cell membrane, endosomal membrane, and nuclear membrane. Layer-by-layer technique is an efficient method with easy operation which can be used for the assembly of multifunctional gene carriers. This work describes a pH-sensitive multifunctional gene vector that offered long circulation property but avoided the inhibition of tumor cellular uptake of gene carriers associated with the use of polyethylene glycol. Methods Deoxyribonucleic acid (DNA) was firstly condensed with protamine into a cationic core which was used as assembly template. Then, additional layers of anionic DNA, cationic liposomes, and o-carboxymethyl-chitosan (CMCS) were alternately adsorbed onto the template via layer-by-layer technique and finally the multifunctional vector called CMCS-cationic liposome-coated DNA/protamine/DNA complexes (CLDPD) was constructed. For in vitro test, the cytotoxicity and transfection investigation was carried out on HepG2 cell line. For in vivo evaluation, CMCS-CLDPD was intratumorally injected into tumor-bearing mice and the tumor cells were isolated for fluorescence determination of transfection efficiency. Results CMCS-CLDPD had ellipsoidal shapes and showed “core-shell” structure which showed stabilization property in serum and effective protection of DNA from nuclease degradation. In vitro and in vivo transfection results demonstrated that CMCS-CLDPD had pH-sensitivity and the outermost layer of CMCS fell off in the tumor tissue, which could not only protect CMCS- CLDPD from serum interaction but also enhance gene transfection efficiency. Conclusion These results demonstrated that multifunctional CMCS-CLDPD had pH- sensitivity, which may provide a new approach for the antitumor gene delivery. PMID:22393290

  16. Protein antigen delivery by gene gun-mediated epidermal antigen incorporation (EAI).

    PubMed

    Scheiblhofer, Sandra; Ritter, Uwe; Thalhamer, Josef; Weiss, Richard

    2013-01-01

    The gene gun technology can not only be employed for efficient transfer of gene vaccines into upper layers of the skin, but also for application of protein antigens. As a tissue rich in professional antigen presenting cells, the skin represents an attractive target for immunizations. In this chapter we present a method for delivery of the model antigen ovalbumin into the skin of mice termed epidermal antigen incorporation and describe in detail how antigen-specific proliferation in draining lymph nodes can be followed by flow cytometry.

  17. Polydnaviruses of Parasitic Wasps: Domestication of Viruses To Act as Gene Delivery Vectors

    PubMed Central

    Burke, Gaelen R.; Strand, Michael R.

    2012-01-01

    Symbiosis is a common phenomenon in which associated organisms can cooperate in ways that increase their ability to survive, reproduce, or utilize hostile environments. Here, we discuss polydnavirus symbionts of parasitic wasps. These viruses are novel in two ways: (1) they have become non-autonomous domesticated entities that cannot replicate outside of wasps; and (2) they function as a delivery vector of genes that ensure successful parasitism of host insects that wasps parasitize. In this review we discuss how these novelties may have arisen, which genes are potentially involved, and what the consequences have been for genome evolution. PMID:26467950

  18. Mesoporous silica nanoparticles with controlled loading of cationic dendrimer for gene delivery

    NASA Astrophysics Data System (ADS)

    Lin, Jian-Tao; Wang, Chao; Zhao, Yi; Wang, Guan-Hai

    2014-09-01

    In this work, a series of polyamidoamine (PAMAM) dendrimer-functionalized mesoporous silica nanoparticles (MSNs) with predictable and adjustable cationic charge densities for gene delivery were designed, synthesized and characterized. The ‘clickable’ MSNs with controlled and randomly distributed azide groups were synthesized by co-condensation method, and PAMAM dendrimer was conjugated to MSNs via quantitative click modification. The structures of PAMAM-functionalized MSNs were characterized by FTIR, XRD and TEM analyses. Dendrimer-functionalized MSNs formed complexes with plasmid DNA (pDNA), and the complexes were successfully transfected into human kidney cell 293 T. The in vitro cytotoxicity and gene transfection efficacy were also investigated.

  19. A novel gene delivery composite system based on biodegradable folate-poly (ester amine) polymer and thermosensitive hydrogel for sustained gene release

    PubMed Central

    Yang, Yi; Zhao, Hang; Jia, YanPeng; Guo, QingFa; Qu, Ying; Su, Jing; Lu, XiaoLing; Zhao, YongXiang; Qian, ZhiYong

    2016-01-01

    Local anti-oncogene delivery providing high local concentration of gene, increasing antitumor effect and decreasing systemic side effects is currently attracting interest in cancer therapy. In this paper, a novel local sustained anti-oncogene delivery system, PECE thermoresponsive hydrogel containing folate-poly (ester amine) (FA-PEA) polymer/DNA (tumor suppressor) complexes, is demonstrated. First, a tumor-targeted biodegradable folate-poly (ester amine) (FA-PEA) polymer based on low-molecular-weight polyethyleneimine (PEI) was synthesized and characterized, and the application for targeted gene delivery was investigated. The polymer had slight cytotoxicity and high transfection efficiency in vitro compared with PEI 25k, which indicated that FA-PEA was a potential vector for targeted gene delivery. Meanwhile, we successfully prepared a thermoresponsive PECE hydrogel composite containing FA-PEA/DNA complexes which could contain the genes and slowly release the genes into cells. We concluded the folate-poly (ester amine) (FA-PEA) polymer would be useful for targeted gene delivery, and the novel gene delivery composite based on biodegradable folate-poly (ester amine) polymer and thermosensitive PECE hydrogel showed potential for sustained gene release. PMID:26883682

  20. A novel gene delivery composite system based on biodegradable folate-poly (ester amine) polymer and thermosensitive hydrogel for sustained gene release

    NASA Astrophysics Data System (ADS)

    Yang, Yi; Zhao, Hang; Jia, Yanpeng; Guo, Qingfa; Qu, Ying; Su, Jing; Lu, Xiaoling; Zhao, Yongxiang; Qian, Zhiyong

    2016-02-01

    Local anti-oncogene delivery providing high local concentration of gene, increasing antitumor effect and decreasing systemic side effects is currently attracting interest in cancer therapy. In this paper, a novel local sustained anti-oncogene delivery system, PECE thermoresponsive hydrogel containing folate-poly (ester amine) (FA-PEA) polymer/DNA (tumor suppressor) complexes, is demonstrated. First, a tumor-targeted biodegradable folate-poly (ester amine) (FA-PEA) polymer based on low-molecular-weight polyethyleneimine (PEI) was synthesized and characterized, and the application for targeted gene delivery was investigated. The polymer had slight cytotoxicity and high transfection efficiency in vitro compared with PEI 25k, which indicated that FA-PEA was a potential vector for targeted gene delivery. Meanwhile, we successfully prepared a thermoresponsive PECE hydrogel composite containing FA-PEA/DNA complexes which could contain the genes and slowly release the genes into cells. We concluded the folate-poly (ester amine) (FA-PEA) polymer would be useful for targeted gene delivery, and the novel gene delivery composite based on biodegradable folate-poly (ester amine) polymer and thermosensitive PECE hydrogel showed potential for sustained gene release.

  1. Prevention of adverse events of interferon γ gene therapy by gene delivery of interferon γ-heparin-binding domain fusion protein in mice.

    PubMed

    Ando, Mitsuru; Takahashi, Yuki; Yamashita, Takuma; Fujimoto, Mai; Nishikawa, Makiya; Watanabe, Yoshihiko; Takakura, Yoshinobu

    2014-01-01

    Sustained gene delivery of interferon (IFN) γ can be an effective treatment, but our previous study showed high levels of IFNγ-induced adverse events, including the loss of body weight. These unwanted events could be reduced by target-specific delivery of IFNγ after in vivo gene transfer. To achieve this, we selected the heparin-binding domain (HBD) of extracellular superoxide dismutase as a molecule to anchor IFNγ to the cell surface. We designed three IFNγ derivatives, IFNγ-HBD1, IFNγ-HBD2, and IFNγ-HBD3, each of which had 1, 2, or 3 HBDs, respectively. Each plasmid-encoding fusion proteins was delivered to the liver, a model target in this study, by hydrodynamic tail vein injection. The serum concentration of IFNγ-HBD2 and IFNγ-HBD3 after gene delivery was lower than that of IFNγ or IFNγ-HBD1. Gene delivery of IFNγ-HBD2, but not of IFNγ-HBD3, effectively increased the mRNA expression of IFNγ-inducible genes in the liver, suggesting liver-specific distribution of IFNγ-HBD2. Gene delivery of IFNγ-HBD2-suppressed tumor growth in the liver as efficiently as that of IFNγ with much less symptoms of adverse effects. These results indicate that the adverse events of IFNγ gene transfer can be prevented by gene delivery of IFNγ-HBD2, a fusion protein with high cell surface affinity. PMID:26015966

  2. Enhanced gene delivery of low molecular weight PEI by flower-like ZnO microparticles.

    PubMed

    Chen, Ming; Tang, Yaqin; Wang, Tingting; Long, Qipeng; Zeng, Ziying; Chen, Houwen; Feng, Xuli

    2016-12-01

    Low molecular weight (1.8 kDa) branched polyethylenimine (PEI) has been used as non-viral vector for gene delivery because of its low toxicity, however, its further application in biomedical field has been restricted due to its low gene transfection efficiency. Herein, ZnO microflowers were prepared to increase the gene expression level mediated by PEI. Four methods have been applied to tune the shape of ZnO microstructures. Scanning electron microscopy (SEM) demonstrated the successful preparation of four kinds of flower like ZnO microparticles. By loading PEI/pDNA into ZnO microparticles, the formed new complexes showed enhanced gene transfection compared to PEI/pDNA alone. Cell uptaking experiments explained a possible mechanism that the tips of ZnO microflowers penetrated into the surface of cells, thus facilitating the entry of gene cargo into cells. These findings highlight the potential of needle like microstructure as adjuvant for efficient biomacromolecular delivery. PMID:27612838

  3. Human Artificial Chromosomes for Gene Delivery and the Development of Animal Models

    PubMed Central

    Kazuki, Yasuhiro; Oshimura, Mitsuo

    2011-01-01

    Random integration of conventional gene delivery vectors such as viruses, plasmids, P1 phage-derived artificial chromosomes, bacterial artificial chromosomes and yeast artificial chromosomes can be associated with transgene silencing. Furthermore, integrated viral sequences can activate oncogenes adjacent to the insertion site resulting in cancer. Various human artificial chromosomes (HACs) exhibit several potential characteristics desired for an ideal gene delivery vector, including stable episomal maintenance and the capacity to carry large genomic loci with their regulatory elements, thus allowing the physiological regulation of the introduced gene in a manner similar to that of native chromosomes. HACs have been generated mainly using either a “top-down approach” (engineered chromosomes), or a “bottom-up approach” (de novo artificial chromosomes). The recent emergence of stem cell–based tissue engineering has opened up new avenues for gene and cell therapies. This review describes the lessons learned and prospects identified mainly from studies in the construction of HACs and HAC-mediated gene expression systems in cultured cells, as well as in animals. PMID:21750534

  4. Dextran-protamine polycation: an efficient nonviral and haemocompatible gene delivery system.

    PubMed

    Thomas, Jane Joy; Rekha, M R; Sharma, Chandra P

    2010-11-01

    Despite the remarkable progress in the field of gene therapy with viral vectors, nonviral vectors have attracted great interests due to their unique properties. Imparting desired characteristics to nonviral gene delivery systems requires the development of cationic polymers. The purpose of this work was to design a cationic derivative (Dex-P) of dextran using protamine in order to assert target specific cellular binding. Our objective was to elucidate the potential use of Dex-P as a haemocompatible, nontoxic and efficient nonviral candidate for gene therapy. Nanoplexes were prepared with calf thymus DNA and Dex-P. Derivatization was confirmed by FTIR, gel permeation chromatography and TNBS assay. Dynamic light scattering and TEM studies determined the size and morphology of the nanoplex. The buffering behaviour was assessed by acid base titration. Complexation stability was evaluated using agarose gel electrophoresis and EtBr displacement assay. The protection of ctDNA from nuclear digestion and the effect of plasma components towards stability of the nanoplexes were also analyzed. Various haemocompatible studies were performed to check haemolysis, aggregation, clotting time, and complement activation. Transfection and cytotoxicity experiments were performed in vitro. The nanosize, spherical shape and stability of nanoplexes were affirmed. Various experiments conducted confirmed Dex-P to be nontoxic and haemocompatible. Transfection experiments revealed the capability of Dex-P to facilitate high gene expression and cellular uptake in HepG2 cells. With the improved physicochemical, biological and transfection properties, Dex-P seems to be a promising gene delivery system.

  5. Development of hybrid baculovirus vectors for artificial MicroRNA delivery and prolonged gene suppression.

    PubMed

    Chen, Chiu-Ling; Luo, Wen-Yi; Lo, Wen-Hsin; Lin, Kun-Ju; Sung, Li-Yu; Shih, Yung-Shen; Chang, Yu-Han; Hu, Yu-Chen

    2011-12-01

    MicroRNA (miRNA) plays essential roles in regulating gene expression, but miRNA delivery remains a hurdle, thus entailing a vector system for efficient transfer. Baculovirus emerges as a promising gene delivery vector but its inherent transient expression restricts its applications in some scenarios. Therefore, this study primarily aimed to develop baculovirus as a miRNA expression vector for prolonged gene suppression. We constructed recombinant baculoviruses carrying artificial egfp-targeting miRNA sequences within the miR155 backbone, which after expression by the cytomegalovirus promoter could knockdown the enhanced green fluorescent protein (EGFP) expression in a sequence- and dose-dependent manner. By swapping the mature miRNA sequences, the baculovirus miRNA shuttle effectively repressed the overexpression of endogenous TNF-α in arthritic synoviocytes without inducing apoptosis. To prolong the baculovirus-mediated expression, we further developed a hybrid baculovirus vector that exploited the Sleeping Beauty (SB) transposon for gene integration and sustained miRNA expression. The hybrid baculovirus vector that combined the miR155 scaffold and SB transposon effectively repressed the transgene expression for a prolonged period of time, hence diversifying the applications of baculovirus to indications necessitating prolonged gene regulation such as arthritis. PMID:21732325

  6. Galactose-functionalized cationic polycarbonate diblock copolymer for targeted gene delivery to hepatocytes.

    PubMed

    Ong, Zhan Yuin; Yang, Chuan; Gao, Shu Jun; Ke, Xi-Yu; Hedrick, James L; Yan Yang, Yi

    2013-11-01

    To mediate selective gene delivery to hepatocytes via the asialoglycoprotein receptors (ASGP-Rs), we designed and synthesized well-defined and narrowly dispersed galactose- and glucose-functionalized cationic polycarbonate diblock copolymers (designated as Gal-APC and Glu-APC, respectively) using organocatalytic ring-opening polymerization of functionalized carbonate monomers, with a subsequent quaternization step using bis-tertiary amines to confer quaternary and tertiary amines for DNA binding and endosomal buffering, respectively. The sugar-functionalized diblock copolymers effectively bound and condensed DNA to form positively charged nanoparticles (<100 nm in diameter and ≈30 mV zeta-potential) that were stable under high physiological salt conditions. In comparison to the control Glu-APC/DNA complexes, Gal-APC/DNA complexes mediated significantly higher gene expression in ASGP-R positive HepG2 cells with no significant difference observed in ASGP-R negative HeLa cells. The co-incubation of Gal-APC/DNA complexes with a natural ASGP-R ligand effectively led to a decrease in gene expression, hence providing evidence for the ASGP-R mediated endocytosis of the polyplexes. Importantly, the Gal-APC/DNA complexes induced minimal cytotoxicities in HepG2 cells at the N/P ratios tested. Taken together, the galactose-functionalized cationic polycarbonate diblock copolymer has potential for use as a non-viral gene vector for the targeted delivery of therapeutic genes to hepatocytes in the treatment of liver diseases.

  7. A novel magnetic crystal-lipid nanostructure for magnetically guided in vivo gene delivery

    NASA Astrophysics Data System (ADS)

    Namiki, Yoshihisa; Namiki, Tamami; Yoshida, Hiroshi; Ishii, Yukiko; Tsubota, Akihito; Koido, Shigeo; Nariai, Kouichi; Mitsunaga, Makoto; Yanagisawa, Satoru; Kashiwagi, Hideyuki; Mabashi, Yasuo; Yumoto, Yoko; Hoshina, Sadayori; Fujise, Kiyotaka; Tada, Norio

    2009-09-01

    Cancer gene therapy requires a safe and effective gene delivery system. Polymer- and lipid-coated magnetic nanocrystals have been used to deliver silencing RNA, but synthesizing these magnetic vectors is difficult. Here, we show that a new nanoparticle formulation can be magnetically guided to deliver and silence genes in cells and tumours in mice. This formulation, termed LipoMag, consists of an oleic acid-coated magnetic nanocrystal core and a cationic lipid shell. When compared with the commercially available PolyMag formulation, LipoMag displayed more efficient gene silencing in 9 of 13 cell lines, and better anti-tumour effects when systemically administered to mice bearing gastric tumours. By delivering an optimized sequence of a silencing RNA that targets the epidermal growth factor receptor of tumour vessels, the intended therapeutic benefit was achieved with no evident adverse immune reaction or untoward side effects.

  8. Magnetic Microbubbles: Magnetically Targeted and Ultrasound-Triggered Vectors for Gene Delivery in Vitro

    NASA Astrophysics Data System (ADS)

    Vlaskou, Dialechti; Pradhan, Pallab; Bergemann, Christian; Klibanov, Alexander L.; Hensel, Karin; Schmitz, Georg; Plank, Christian; Mykhaylyk, Olga

    2010-12-01

    Based on the concept of magnetofection, we prepared lipid shell microbubbles loaded with highly positively charged iron oxide magnetic nanoparticles through electrostatic and matrix affinity interactions. These magnetic microbubbles showed strong ultrasound contrast. When the magnetic microbubbles were mixed with plasmid DNA encoding a reporter gene, gene delivery to HeLa cells was achieved only when ultrasound was applied. Gene transfer efficiency strongly depended on the application of a gradient magnetic field. Treatment of HeLa cells with the microbubbles and ultrasound resulted in strong concentration-dependent cytotoxic effects, whereas ultrasound alone, lipid microbubbles alone, magnetic nanoparticles or magnetic microbubbles alone did not significantly affect cell viability. These magnetic microbubbles could be used as magnetically targeted diagnostic agents for real-time ultrasound imaging or for cancer therapy, therapy of vascular thrombosis and gene therapy.

  9. Targeted delivery of genes to endothelial cells and cell- and gene-based therapy in pulmonary vascular diseases.

    PubMed

    Suen, Colin M; Mei, Shirley H J; Kugathasan, Lakshmi; Stewart, Duncan J

    2013-10-01

    Pulmonary arterial hypertension (PAH) is a devastating disease that, despite significant advances in medical therapies over the last several decades, continues to have an extremely poor prognosis. Gene therapy is a method to deliver therapeutic genes to replace defective or mutant genes or supplement existing cellular processes to modify disease. Over the last few decades, several viral and nonviral methods of gene therapy have been developed for preclinical PAH studies with varying degrees of efficacy. However, these gene delivery methods face challenges of immunogenicity, low transduction rates, and nonspecific targeting which have limited their translation to clinical studies. More recently, the emergence of regenerative approaches using stem and progenitor cells such as endothelial progenitor cells (EPCs) and mesenchymal stem cells (MSCs) have offered a new approach to gene therapy. Cell-based gene therapy is an approach that augments the therapeutic potential of EPCs and MSCs and may deliver on the promise of reversal of established PAH. These new regenerative approaches have shown tremendous potential in preclinical studies; however, large, rigorously designed clinical studies will be necessary to evaluate clinical efficacy and safety.

  10. Stable Somatic Gene Expression in Mouse Lungs Following Electroporation-mediated Tol2 Transposon Delivery.

    PubMed

    Muliawan, Hary Sakti; Nakayama, Kazuhiko; Yagi, Keiko; Ikeda, Koji; Yagita, Kazuhiro; Hirata, Ken-ichi; Emoto, Noriaki

    2015-10-07

    Gene delivery to the lung has rapidly progressed as an important method for studying various chronic lung diseases. Viral vectors, albeit highly efficient, are limited by the host immune response. Electroporation, a well-known non-viral method, can efficiently deliver genes to the lung, but is unable to induce stable gene expression. The Tol2 transposon is another non-viral method that can induce stable gene expression by reinserting its genes into the host genome. In this study, we combined electroporation and Tol2 transposons to obtain stable, high-level gene expression in the mouse lung. Tol2 transposon plasmids (pT2A-EGFP; Tol2, pCAGGS-TP; transposase) were optimized in vitro, and the electroporation procedure (pCAG-EGFP) was optimized in mouse lungs. After optimization, a combination of electroporation plus the Tol2 transposon was used in a comparative analysis with electroporation plus pCAG-EGFP. GFP expression levels were quantified and visualized on days 4 and 7 post-electroporation. We successfully reproduced the Tol2 transposon system in vitro and the electroporation procedure in vivo. We observed sustainable GFP expression using electroporation plus the Tol2 transposon on days 4 and 7, while electroporation plus pCAG-EGFP resulted in decreased GFP expression on day 7. We were able to induce high-level, stable gene expression in mouse lungs using a combination of electroporation and the Tol2 transposon. This represents a safer method for lung gene delivery that can be used as an alternative to viral vectors.

  11. Depletion of autophagy receptor p62/SQSTM1 enhances the efficiency of gene delivery in mammalian cells.

    PubMed

    Tsuchiya, Megumi; Ogawa, Hidesato; Koujin, Takako; Kobayashi, Shouhei; Mori, Chie; Hiraoka, Yasushi; Haraguchi, Tokuko

    2016-08-01

    Novel methods that increase the efficiency of gene delivery to cells will have many useful applications. Here, we report a simple approach involving depletion of p62/SQSTM1 to enhance the efficiency of gene delivery. The efficiency of reporter gene delivery was remarkably higher in p62-knockout murine embryonic fibroblast (MEF) cells compared with normal MEF cells. This higher efficiency was partially attenuated by ectopic expression of p62. Furthermore, siRNA-mediated knockdown of p62 clearly increased the efficiency of transfection of murine embryonic stem (mES) cells and human HeLa cells. These data indicate that p62 acts as a key regulator of gene delivery. PMID:27317902

  12. Development of a gene delivery system in Streptococcus gordonii using thymidylate synthase as a selection marker.

    PubMed

    Lee, Song F; Hulbah, Maram; Halperin, Scott A

    2016-06-01

    Streptococcus gordonii, a commensal bacterium of the human oral cavity, is a potential live vaccine vector. In this study, we have developed a system that delivers a vaccine antigen gene onto the chromosome of S. gordonii. The system consisted of a recipient strain, that is a thymidine auxotroph constructed by deletion of a portion of thyA gene, and a linear gene delivery construct, composed of the functional thyA gene, the vaccine antigen gene, and a DNA fragment immediately downstream of thyA. The construct is assembled by a ligation and polymerase chain reaction strategy. Upon introduction into the thyA mutant, the vaccine antigen gene integrated into the chromosome via a double crossing-over event. Using the above strategy, a test vaccine antigen gene coding for a fusion protein composed of the Bordetella pertussis filamentous hemagglutinin type I domain and the single chain antibody against complement receptor 1 was successfully delivered to S. gordonii. The resulting S. gordonii expressed the fusion protein and the delivered gene was stable in the bacterium in vitro and in a mouse colonization experiment. Mice colonized by the fusion protein-expressing S. gordonii developed antibodies that recognized the native filamentous hemagglutinin protein suggesting that an immune response was elicited. PMID:27062990

  13. BMP2 gene delivery to bone mesenchymal stem cell by chitosan-g-PEI nonviral vector

    NASA Astrophysics Data System (ADS)

    Yue, Jianhui; Wu, Jun; Liu, Di; Zhao, Xiaoli; Lu, William W.

    2015-04-01

    Nanotechnology has made a significant impact on the development of nanomedicine. Nonviral vectors have been attracting more attention for the advantage of biosafety in gene delivery. Polyethylenimine (PEI)-conjugated chitosan (chitosan-g-PEI) emerged as a promising nonviral vector and has been demonstrated in many tumor cells. However, there is a lack of study focused on the behavior of this vector in stem cells which hold great potential in regenerative medicine. Therefore, in this study, in vitro gene delivering effect of chitosan-g-PEI was investigated in bone marrow stem cells. pIRES2-ZsGreen1-hBMP2 dual expression plasmid containing both the ZsGreen1 GFP reporter gene and the BMP2 functional gene was constructed for monitoring the transgene expression level. Chitosan-g-PEI-mediated gene transfer showed 17.2% of transfection efficiency and more than 80% of cell viability in stem cells. These values were higher than that of PEI. The expression of the delivered BMP2 gene in stem cells enhanced the osteogenic differentiation. These results demonstrated that chitosan-g-PEI is capable of applying in delivering gene to stem cells and providing potential applications in stem cell-based gene therapy.

  14. Spider Silk-Based Gene Carriers for Tumor Cell-Specific Delivery

    PubMed Central

    Numata, Keiji; Reagan, Michaela R; Goldstein, Robert H; Rosenblatt, Michael; Kaplan, David L

    2011-01-01

    The present study demonstrates pDNA complexes of recombinant silk proteins containing poly(L-lysine) and tumor-homing peptides (THPs), which are globular and approximately 150–250 nm in diameter, show significant enhancement of target specificity to tumor cells by additions of F3 and CGKRK THPs. We report herein the preparation and study of novel nano-scale silk-based ionic complexes containing pDNA able to home specifically to tumor cells. Particular focus was on how the THP, F3 (KDEPQRRSARLSAKPAPPKPEPKPKKAPAKK) and CGKRK, enhanced transfection specificity to tumor cells. Genetically engineered silk proteins containing both poly(L-lysine) domains to interact with pDNA and the THP to bind to specific tumor cells for target-specific pDNA delivery were prepared using Escherichia coli, followed by in-vitro and in-vivo transfection experiments into MDA-MB-435 melanoma cells and highly metastatic human breast tumor MDA-MB-231 cells. Non-tumorigenic MCF-10A breast epithelial cells were used as a control cell line for in-vitro tumor-specific delivery studies. These results demonstrate that combination of the bioengineered silk delivery systems and THP can serve as a versatile and useful new platform for non-viral gene delivery. PMID:21739966

  15. Modulation of Gene Expression by Polymer Nanocapsule Delivery of DNA Cassettes Encoding Small RNAs.

    PubMed

    Yan, Ming; Wen, Jing; Liang, Min; Lu, Yunfeng; Kamata, Masakazu; Chen, Irvin S Y

    2015-01-01

    Small RNAs, including siRNAs, gRNAs and miRNAs, modulate gene expression and serve as potential therapies for human diseases. Delivery to target cells remains the fundamental limitation for use of these RNAs in humans. To address this challenge, we have developed a nanocapsule delivery technology that encapsulates small DNA molecules encoding RNAs into a small (30 nm) polymer nanocapsule. For proof of concept, we transduced DNA expression cassettes for three small RNAs. In one application, the DNA cassette encodes an shRNA transcriptional unit that downregulates CCR5 and protects from HIV-1 infection. The DNA cassette nanocapsules were further engineered for timed release of the DNA cargo for prolonged knockdown of CCR5. Secondly, the nanocapsules provide an efficient means for delivery of gRNAs in the CRISPR/Cas9 system to mutate integrated HIV-1. Finally, delivery of microRNA-125b to mobilized human CD34+ cells enhances survival and expansion of the CD34+ cells in culture. PMID:26035832

  16. Intrathecal polymer-based interleukin-10* gene delivery for neuropathic pain

    PubMed Central

    MILLIGAN, ERIN D.; SODERQUIST, RYAN G.; MALONE, STEPHANIE M.; MAHONEY, JOHN H.; HUGHES, TRAVIS S.; LANGER, STEPHEN J.; SLOANE, EVAN M.; MAIER, STEVEN F.; LEINWAND, LESLIE A.; WATKINS, LINDA R.; MAHONEY, MELISSA J.

    2007-01-01

    Research on communication between glia and neurons has increased in the past decade. The onset of neuropathic pain, a major clinical problem that is not resolved by available therapeutics, involves activation of spinal cord glia through the release of proinflammatory cytokines in acute animal models of neuropathic pain. Here, we demonstrate for the first time that the spinal action of the proinflammatory cytokine, interleukin 1 (IL-1) is involved in maintaining persistent (2 months) allodynia induced by chronic-constriction injury (CCI). The anti-inflammatory cytokine IL-10 can suppress proinflammatory cytokines and spinal cord glial amplification of pain. Given that IL-1 is a key mediator of neuropathic pain, developing a clinically viable means of long-term delivery of IL-10 to the spinal cord is desirable. High doses of intrathecal IL-10-gene therapy using naked plasmid DNA (free pDNA-IL-10) is effective, but the dose required limits its potential clinical utility. Here we show that intrathecal gene therapy for neuropathic pain is improved sufficiently using two, distinct synthetic polymers, poly(lactic-co-glycolic) and polyethylenimine, that substantially lower doses of pDNA-IL-10 are effective. In conclusion, synthetic polymers used as i.t. gene-delivery systems are well-tolerated and improve the long-duration efficacy of pDNA-IL-10 gene therapy. PMID:18079973

  17. Self-Immolative Polycations as Gene Delivery Vectors and Prodrugs Targeting Polyamine Metabolism in Cancer

    PubMed Central

    2015-01-01

    Polycations are explored as carriers to deliver therapeutic nucleic acids. Polycations are conventionally pharmacological inert with the sole function of delivering therapeutic cargo. This study reports synthesis of a self-immolative polycation (DSS-BEN) based on a polyamine analogue drug N1,N11-bisethylnorspermine (BENSpm). The polycation was designed to function dually as a gene delivery carrier and a prodrug targeting dysregulated polyamine metabolism in cancer. Using a combination of NMR and HPLC, we confirm that the self-immolative polycation undergoes intracellular degradation into the parent drug BENSpm. The released BENSpm depletes cellular levels of spermidine and spermine and upregulates polyamine catabolic enzymes spermine/spermidine N1-acetyltransferase (SSAT) and spermine oxidase (SMO). The synthesized polycations form polyplexes with DNA and facilitate efficient transfection. Taking advantage of the ability of BENSpm to sensitize cancer cells to TNFα-induced apoptosis, we show that DSS-BEN enhances the cell killing activity of TNFα gene therapy. The reported findings validate DSS-BEN as a dual-function delivery system that can deliver a therapeutic gene and improve the outcome of gene therapy as a result of the intracellular degradation of DSS-BEN to BENSpm and the subsequent beneficial effect of BENSpm on dysregulated polyamine metabolism in cancer. PMID:25153488

  18. Enhancing gene delivery of adeno-associated viruses by cell-permeable peptides

    PubMed Central

    Liu, Yarong; Kim, Young Joo; Ji, Man; Fang, Jinxu; Siriwon, Natnaree; Zhang, Li I; Wang, Pin

    2014-01-01

    Adeno-associated virus type 2 (AAV2) is considered a promising gene delivery vector and has been extensively applied in several disease models; however, inefficient transduction in various cells and tissues has limited its widespread application in many areas of gene therapy. In this study, we have developed a general, but efficient, strategy to enhance viral transduction, both in vitro and in vivo, by incubating viral particles with cell-permeable peptides (CPPs). We show that CPPs increase internalization of viral particles into cells by facilitating both energy-independent and energy-dependent endocytosis. Moreover, CPPs can significantly enhance the endosomal escape process of viral particles, thus enhancing viral transduction to those cells that have exhibited very low permissiveness to AAV2 infection as a result of impaired intracellular viral processing. We also demonstrated that this approach could be applicable to other AAV serotypes. Thus, the membrane-penetrating ability of CPPs enables us to generate an efficient method for enhanced gene delivery of AAV vectors, potentially facilitating its applicability to human gene therapy. PMID:26015948

  19. Progranulin Gene Delivery Protects Dopaminergic Neurons in a Mouse Model of Parkinson’s Disease

    PubMed Central

    Van Kampen, Jackalina M.; Baranowski, David; Kay, Denis G.

    2014-01-01

    Parkinson’s disease (PD) is a progressive neurodegenerative disorder characterized by tremor, rigidity and akinesia/bradykinesia resulting from the progressive loss of nigrostriatal dopaminergic neurons. To date, only symptomatic treatment is available for PD patients, with no effective means of slowing or stopping the progression of the disease. Progranulin (PGRN) is a 593 amino acid multifunction protein that is widely distributed throughout the CNS, localized primarily in neurons and microglia. PGRN has been demonstrated to be a potent regulator of neuroinflammation and also acts as an autocrine neurotrophic factor, important for long-term neuronal survival. Thus, enhancing PGRN expression may strengthen the cells resistance to disease. In the present study, we have used the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of PD to investigate the possible use of PGRN gene delivery as a therapy for the prevention or treatment of PD. Viral vector delivery of the PGRN gene was an effective means of elevating PGRN expression in nigrostriatal neurons. When PGRN expression was elevated in the SNC, nigrostriatal neurons were protected from MPTP toxicity in mice, along with a preservation of striatal dopamine content and turnover. Further, protection of nigrostriatal neurons by PGRN gene therapy was accompanied by reductions in markers of MPTP-induced inflammation and apoptosis as well as a complete preservation of locomotor function. We conclude that PGRN gene therapy may have beneficial effects in the treatment of PD. PMID:24804730

  20. Structure and function studies of cationic lipid non-viral gene delivery systems

    NASA Astrophysics Data System (ADS)

    Slack, Nelle Lynn

    Gene and drug delivery systems incorporating lipids mimic the biological environment and thus offer many advantages. In order to design successful gene or drug delivery vehicles based on lipid molecules an understanding of the affects of biopolymers on lipid membranes is necessary. We have examined the structures and interactions involved in two systems based on model biomembrane/biopolymer mixtures that are relevant for gene and drug delivery research. Liposomes incorporating PEG-lipids have shown great promise as drug carriers since they have proven to increase blood circulation times by evading the immune system. Previously, it was found that the addition of single-end-anchored (SEA)-PEG-surfactants to fluid lamellar membrane systems induces a novel hydrogel. As an extension of this work, we examine the affects of adding double-end-anchored (DEA)-PEG-surfactants to fluid membrane systems. The DEA-PEG-surfactants can adopt either a looping or bridging configuration which could deeply alter the microstructure of lamellar phases. We show that the DEA-PEG-surfactants induce gelation of fluid Lalpha phases in a way similar to that previously reported for SEA-PEG-surfactants. We also show, via x-ray diffraction, two striking differences between the SEA-and DEA-PEG-surfactant systems which demonstrate the existence of a large concentration of bridging polymers in the DEA system. The use of cationic liposomes (formed by mixtures of cationic and neutral lipids) as carriers of DNA for delivery in cells is a promising alternative to viral carriers for gene therapy. Using x-ray diffraction and biological assays, we show key parameters for optimizing gene transfer with these systems that are mediated by properties of the lipid. We have found cationic liposomes complexed with supercoiled plasmid DNA in solution self-assemble into a lamellar or an inverted hexagonal structure depending on lipid composition. Transfection efficiencies, determined by X-Gal and Luciferase assays

  1. SOCS-1 gene delivery cooperates with cisplatin plus pemetrexed to exhibit preclinical antitumor activity against malignant pleural mesothelioma.

    PubMed

    Iwahori, Kota; Serada, Satoshi; Fujimoto, Minoru; Ripley, Barry; Nomura, Shintaro; Mizuguchi, Hiroyuki; Shimada, Kazuki; Takahashi, Tsuyoshi; Kawase, Ichiro; Kishimoto, Tadamitsu; Naka, Tetsuji

    2013-01-15

    Malignant pleural mesothelioma (MPM) is an aggressive tumor with poor prognosis for which an effective therapy remains to be established. This study investigated the therapeutic potential of gene delivery using suppressor of cytokine signaling 1 (SOCS-1), an endogenous inhibitor of intracellular signaling pathways, for the treatment of MPM. We infected MPM cells (MESO-4, H28 and H226) with adenovirus-expressing SOCS-1 vector to examine the effect of SOCS-1 overexpression on MPM cells. We evaluated the antitumor effect of SOCS-1 gene delivery combined with cisplatin plus pemetrexed by cell proliferation, apoptosis and invasion assay. We also investigated the regulation of NF-κB and STAT3 signaling related to apoptotic pathways. Furthermore, we evaluated the inhibition of tumor growth by SOCS-1 gene delivery combined with cisplatin plus pemetrexed in vivo. SOCS-1 gene delivery cooperated with cisplatin plus pemetrexed to inhibit cell proliferation, invasiveness and induction of apoptosis in MPM cells. SOCS-1 regulated NF-κB and STAT3 signaling to induce apoptosis in MESO-4 and H226 cells. Furthermore, SOCS-1 gene delivery cooperated with cisplatin plus pemetrexed to regulate NF-κB signaling and significantly inhibit tumor growth of MPM in vivo. These results suggest that SOCS-1 gene delivery has a potent antitumor effect against MPM and a potential for clinical use in combination with cisplatin plus pemetrexed.

  2. Multi-layered nanoparticles for combination gene and drug delivery to tumors

    PubMed Central

    Ediriwickrema, Asiri; Zhou, Jiangbing; Deng, Yang; Saltzman, Mark

    2014-01-01

    Drug resistance and toxicity are major obstacles in cancer chemotherapy. Combination therapies can overcome resistance, and synergies can minimize dosing. Polymer nanocarriers are interesting vehicles for cancer therapeutics for their delivery and tumor targeting abilities. We synthesized a multilayered polymer nanoparticle (MLNP), comprising of poly(lactic-co-glycolic acid) with surface polyethyleneimine and functional peptides, for targeted drug and gene delivery. We confirmed the particle’s ability to inhibit tumor growth through synergistic action of the drug and gene product. MLNPs achieved transfection levels similar to lipofectamine, while maintaining minimal cytotoxicity. The particles delivered camptothecin (CPT), and plasmid encoding TNF related apoptosis inducing ligand (pTRAIL) (CT MLNPs), and synergistically inhibited growth of multiple cancer cells in vitro. The synergy of co-delivering CPT and pTRAIL via CT MLNPs was confirmed using the Chou-Talalay method: the combination index (CI) values at 50% inhibition ranged between 0.31–0.53 for all cell lines. Further, co-delivery with MLNPs resulted in a 3.1–15 fold reduction in CPT and 4.7–8.0 fold reduction in pTRAIL dosing. CT MLNPs obtained significant HCT116 growth inhibition in vivo compared to monotherapy. These results support our hypothesis that MLNPs can deliver both small molecules and genetic agents towards synergistically inhibiting tumor growth. PMID:25112935

  3. Intracellular Protein Delivery and Gene Transfection by Electroporation Using a Microneedle Electrode Array

    PubMed Central

    Choi, Seong-O; Kim, Yeu-Chun; Lee, Jeong Woo; Park, Jung-Hwan

    2012-01-01

    The impact of many biopharmaceuticals, including protein- and gene-based therapies, has been limited by the need for better methods of delivery into cells within tissues. Here, we present intracellular delivery of molecules and transfection with plasmid DNA by electroporation using a novel microneedle electrode array designed for targeted treatment of skin and other tissue surfaces. The microneedle array is molded out of polylactic acid. Electrodes and circuitry required for electroporation are applied to the microneedle array surface by a new metal-transfer micromolding method. The microneedle array maintains mechanical integrity after insertion into pig cadaver skin and is able to electroporate human prostate cancer cells in vitro. Quantitative measurements show that increasing electroporation pulse voltage increases uptake efficiency of calcein and bovine serum albumin, whereas increasing pulse length has lesser effects over the range studied. Uptake of molecules by up to 50 % of cells and transfection of 12 % of cells with a gene for green fluorescent protein is demonstrated at high cell viability. We conclude that the microneedle electrode array is able to electroporate cells, resulting in intracellular uptake of molecules, and has potential applications to improve intracellular delivery of proteins, DNA and other biopharmaceuticals. PMID:22328093

  4. The effect of cholesterol domains on PEGylated liposomal gene delivery in vitro

    PubMed Central

    Xu, Long; Wempe, Michael F; Anchordoquy, Thomas J

    2011-01-01

    Aim PEGylated components have been widely used to reduce particle aggregation in serum and extend circulation lifetime for lipid- and polymer-based gene-delivery systems. However, PEGylation is known to interfere with cell interaction and intracellular trafficking, resulting in decreased biological activity. In the present study, the effect of cholesterol domains on PEGylated liposome-mediated gene delivery was evaluated by PEGylating formulations with and without a cholesterol domain, and also by altering the location of PEG on the particle surface (i.e., within or excluded from the domain). Materials and methods Lipoplexes formulated with PEG–cholesterol or PEG–diacyl lipid were used to transfect various cell lines, including human and mouse cancer cells. Cellular uptake of lipoplexes was also quantified and compared with the transfection results. Results Our findings are consistent with previous work demonstrating that PEGylation reduces transfection rates; however, formulations in which PEG was incorporated into the cholesterol domain did not exhibit this detrimental effect. In some cell lines, the incorporation of PEG into the domain actually increased transfection rates, despite no enhancement of cellular uptake. Discussion These results suggest that the adverse alterations in intracellular trafficking that are a consequence of PEGylation may be avoided by utilizing delivery vehicles that allow PEG to partition into a cholesterol domain. PMID:22428082

  5. Efficient gene silencing by delivery of locked nucleic acid antisense oligonucleotides, unassisted by transfection reagents.

    PubMed

    Stein, C A; Hansen, J Bo; Lai, Johnathan; Wu, SiJian; Voskresenskiy, Anatoliy; Høg, Anja; Worm, Jesper; Hedtjärn, Maj; Souleimanian, Naira; Miller, Paul; Soifer, Harris S; Castanotto, Daniella; Benimetskaya, Luba; Ørum, Henrik; Koch, Troels

    2010-01-01

    For the past 15-20 years, the intracellular delivery and silencing activity of oligodeoxynucleotides have been essentially completely dependent on the use of a delivery technology (e.g. lipofection). We have developed a method (called 'gymnosis') that does not require the use of any transfection reagent or any additives to serum whatsoever, but rather takes advantage of the normal growth properties of cells in tissue culture in order to promote productive oligonucleotide uptake. This robust method permits the sequence-specific silencing of multiple targets in a large number of cell types in tissue culture, both at the protein and mRNA level, at concentrations in the low micromolar range. Optimum results were obtained with locked nucleic acid (LNA) phosphorothioate gap-mers. By appropriate manipulation of oligonucleotide dosing, this silencing can be continuously maintained with little or no toxicity for >240 days. High levels of oligonucleotide in the cell nucleus are not a requirement for gene silencing, contrary to long accepted dogma. In addition, gymnotic delivery can efficiently deliver oligonucleotides to suspension cells that are known to be very difficult to transfect. Finally, the pattern of gene silencing of in vitro gymnotically delivered oligonucleotides correlates particularly well with in vivo silencing. The establishment of this link is of particular significance to those in the academic research and drug discovery and development communities.

  6. Multi-layered nanoparticles for combination gene and drug delivery to tumors.

    PubMed

    Ediriwickrema, Asiri; Zhou, Jiangbing; Deng, Yang; Saltzman, W Mark

    2014-11-01

    Drug resistance and toxicity are major obstacles in cancer chemotherapy. Combination therapies can overcome resistance, and synergies can minimize dosing. Polymer nanocarriers are interesting vehicles for cancer therapeutics for their delivery and tumor targeting abilities. We synthesized a multi-layered polymer nanoparticle (MLNP), comprising of poly(lactic-co-glycolic acid) with surface polyethyleneimine and functional peptides, for targeted drug and gene delivery. We confirmed the particle's ability to inhibit tumor growth through synergistic action of the drug and gene product. MLNPs achieved transfection levels similar to lipofectamine, while maintaining minimal cytotoxicity. The particles delivered camptothecin (CPT), and plasmid encoding TNF related apoptosis inducing ligand (pTRAIL) (CT MLNPs), and synergistically inhibited growth of multiple cancer cells in vitro. The synergy of co-delivering CPT and pTRAIL via CT MLNPs was confirmed using the Chou-Talalay method: the combination index (CI) values at 50% inhibition ranged between 0.31 and 0.53 for all cell lines. Further, co-delivery with MLNPs resulted in a 3.1-15 fold reduction in CPT and 4.7-8.0 fold reduction in pTRAIL dosing. CT MLNPs obtained significant HCT116 growth inhibition in vivo compared to monotherapy. These results support our hypothesis that MLNPs can deliver both small molecules and genetic agents towards synergistically inhibiting tumor growth.

  7. A new liposome-based gene delivery system targeting lung epithelial cells using endothelin antagonist.

    PubMed

    Allon, Nahum; Saxena, Ashima; Chambers, Carolyn; Doctor, Bhupendra P

    2012-06-10

    We formulated a new gene delivery system based on targeted liposomes. The efficacy of the delivery system was demonstrated in in vitro and in vivo models. The targeting moiety consists of a high-affinity 7-amino-acid peptide, covalently and evenly conjugated to the liposome surface. The targeting peptide acts as an endothelin antagonist, and accelerates liposome binding and internalization. It is devoid of other biological activity. Liposomes with high phosphatidyl serine (PS) were specially formulated to help their fusion with the endosomal membrane at low pH and enable release of the liposome payload into the cytoplasm. A DNA payload, pre-compressed by protamine, was encapsulated into the liposomes, which directed the plasmid into the cell's nucleus. Upon exposure to epithelial cells, binding of the liposomes occurred within 5-10 min, followed by facilitated internalization of the complex. Endosomal escape was complete within 30 min, followed by DNA accumulation in the nucleus 2h post-transfection. A549 lung epithelial cells transfected with plasmid encoding for GFP encapsulated in targeted liposomes expressed significantly more protein than those transfected with plasmid complexed with Lipofectamine. The intra-tracheal instillation of plasmid encoding for GFP encapsulated in targeted liposomes into rat lungs resulted in the expression of GFP in bronchioles and alveoli within 5 days. These results suggest that this delivery system has great potential in targeting genes to lungs.

  8. Polydioxanone-based bio-materials for tissue engineering and drug/gene delivery applications.

    PubMed

    Goonoo, Nowsheen; Jeetah, Roubeena; Bhaw-Luximon, Archana; Jhurry, Dhanjay

    2015-11-01

    Since the commercialization of polydioxanone (PDX) as a biodegradable monofilament suture by Ethicon in 1981, the polymer has received only limited interest until recently. The limitations of polylactide-co-glycolide (PLGA) coupled with the growing need for materials with enhanced features and the advent of new fabrication techniques such as electrospinning have revived interest for PDX in medical devices, tissue engineering and drug delivery applications. Electrospun PDX mats show comparable mechanical properties as the major structural components of native vascular extracellular matrix (ECM) i.e. collagen and elastin. In addition, PDX's unique shape memory property provides rebound and kink resistance when fabricated into vascular conduits. The synthesis of methyl dioxanone (MeDX) monomer and copolymers of dioxanone (DX) and MeDX have opened up new perspectives for poly(ester-ether)s, enabling the design of the next generation of tissue engineering scaffolds for application in regenerating such tissues as arteries, peripheral nerve and bone. Tailoring of polymer properties and their formulation as nanoparticles, nanomicelles or nanofibers have brought along important developments in the area of controlled drug or gene delivery. This paper reviews the synthesis of PDX and its copolymers and provides for the first time an exhaustive account of its applications in the (bio)medical field with focus on tissue engineering and drug/gene delivery. PMID:26614558

  9. Intracellular protein delivery and gene transfection by electroporation using a microneedle electrode array.

    PubMed

    Choi, Seong-O; Kim, Yeu-Chun; Lee, Jeong Woo; Park, Jung-Hwan; Prausnitz, Mark R; Allen, Mark G

    2012-04-10

    The impact of many biopharmaceuticals, including protein- and gene-based therapies, has been limited by the need for better methods of delivery into cells within tissues. Here, intracellular delivery of molecules and transfection with plasmid DNA by electroporation is presented using a novel microneedle electrode array designed for the targeted treatment of skin and other tissue surfaces. The microneedle array is molded out of polylactic acid. Electrodes and circuitry required for electroporation are applied to the microneedle array surface by a new metal-transfer micromolding method. The microneedle array maintains mechanical integrity after insertion into pig cadaver skin and is able to electroporate human prostate cancer cells in vitro. Quantitative measurements show that increasing electroporation pulse voltage increases uptake efficiency of calcein and bovine serum albumin, whereas increasing pulse length has lesser effects over the range studied. Uptake of molecules by up to 50% of cells and transfection of 12% of cells with a gene for green fluorescent protein is demonstrated at high cell viability. It is concluded that the microneedle electrode array is able to electroporate cells, resulting in intracellular uptake of molecules, and has potential applications to improve intracellular delivery of proteins, DNA, and other biopharmaceuticals. PMID:22328093

  10. Synthetic vectors for gene delivery: An overview of their evolution depending on routes of administration.

    PubMed

    Belmadi, Nawal; Midoux, Patrick; Loyer, Pascal; Passirani, Catherine; Pichon, Chantal; Le Gall, Tony; Jaffres, Paul-Alain; Lehn, Pierre; Montier, Tristan

    2015-09-01

    Nucleic acid delivery constitutes an emerging therapeutic strategy to cure various human pathologies. This therapy consists of introducing genetic material into the whole body or isolated cells to correct a cellular abnormality or disfunction. As with any drug, the main objective of nucleic acid delivery is to establish optimal balance between efficacy and tolerance. The methods of administration and the vectors used are selected depending on whether the goal of treatment is the production of an active protein; the replacement of a missing or inactive gene; or the combat of acquired diseases, such as cancer or AIDS. In that sense, synthetic vectors represent a valuable solution because they are well characterized, their structure can be fine tuned, and their potential toxicity can be reduced, since toxicity depends on the composition of the formulations. Here we review various synthetic vectors for gene delivery and address the question of their biodistribution as a function of the route of administration. We highlight the modifications to vectors structure and formulations necessary to overcome the major hurdles limiting the effectiveness of nucleic acid therapies.

  11. Polydioxanone-based bio-materials for tissue engineering and drug/gene delivery applications.

    PubMed

    Goonoo, Nowsheen; Jeetah, Roubeena; Bhaw-Luximon, Archana; Jhurry, Dhanjay

    2015-11-01

    Since the commercialization of polydioxanone (PDX) as a biodegradable monofilament suture by Ethicon in 1981, the polymer has received only limited interest until recently. The limitations of polylactide-co-glycolide (PLGA) coupled with the growing need for materials with enhanced features and the advent of new fabrication techniques such as electrospinning have revived interest for PDX in medical devices, tissue engineering and drug delivery applications. Electrospun PDX mats show comparable mechanical properties as the major structural components of native vascular extracellular matrix (ECM) i.e. collagen and elastin. In addition, PDX's unique shape memory property provides rebound and kink resistance when fabricated into vascular conduits. The synthesis of methyl dioxanone (MeDX) monomer and copolymers of dioxanone (DX) and MeDX have opened up new perspectives for poly(ester-ether)s, enabling the design of the next generation of tissue engineering scaffolds for application in regenerating such tissues as arteries, peripheral nerve and bone. Tailoring of polymer properties and their formulation as nanoparticles, nanomicelles or nanofibers have brought along important developments in the area of controlled drug or gene delivery. This paper reviews the synthesis of PDX and its copolymers and provides for the first time an exhaustive account of its applications in the (bio)medical field with focus on tissue engineering and drug/gene delivery.

  12. Spermine-modified Antheraea pernyi silk fibroin as a gene delivery carrier

    PubMed Central

    Yu, Yanni; Hu, Yongpei; Li, Xiufang; Liu, Yu; Li, Mingzhong; Yang, Jicheng; Sheng, Weihua

    2016-01-01

    The development of a novel cationized polymer used as a gene delivery carrier that can conveniently and effectively transfect cells resulting in a stably expressed target gene remains a challenge. Antheraea pernyi silk fibroin (ASF) is a cytocompatible and biodegradable natural polymer, and it possesses Arg–Gly–Asp sequences but a negative charge. In order to render ASF amenable to packaging plasmid DNA (pDNA), spermine was used to modify ASF to synthesize cationized ASF (CASF), which was used as a gene delivery carrier. CASF was characterized using trinitrobenzene sulfonic acid assay, the zeta potential determination, and a Fourier transform infrared analysis, and the results of these characterizations indicated that the –NH2 in spermine effectively reacts with the –COOH in the side chains of ASF. Spermine grafted to the side chains of ASF resulted in the conversion of the negative charge of ASF to a positive charge. CASF packaged pDNA and formed CASF/pDNA complexes, which exhibited spherical morphology with average particle sizes of 215–281 nm and zeta potential of approximately +3.0 mV to +3.2 mV. The results of the MTT assay, confocal laser scanning microscopy, and flow cytometry analysis in a human endothelial cell line revealed that CASF/pDNA complexes exhibited lower cytotoxicity and higher transfection efficiency compared to the pDNA complexes of polyethyleneimine. These results indicate that our synthesized CASF, a cationized polymer, is a potential gene delivery carrier with the advantages of biodegradability and low cytotoxicity. PMID:27042056

  13. Aerosol delivery of DNA/liposomes to the lung for cystic fibrosis gene therapy.

    PubMed

    Davies, Lee A; Nunez-Alonso, Graciela A; McLachlan, Gerry; Hyde, Stephen C; Gill, Deborah R

    2014-06-01

    Abstract Lung gene therapy is being evaluated for a range of acute and chronic diseases, including cystic fibrosis (CF). As these therapies approach clinical realization, it is becoming increasingly clear that the ability to efficiently deliver gene transfer agents (GTAs) to target cell populations within the lung may prove just as critical as the gene therapy formulation itself in terms of generating positive clinical outcomes. Key to the success of any aerosol gene therapy is the interaction between the GTA and nebulization device. We evaluated the effects of aerosolization on our preferred formulation, plasmid DNA (pDNA) complexed with the cationic liposome GL67A (pDNA/GL67A) using commercially available nebulizer devices. The relatively high viscosity (6.3±0.1 cP) and particulate nature of pDNA/GL67A formulations hindered stable aerosol generation in ultrasonic and vibrating mesh nebulizers but was not problematic in the jet nebulizers tested. Aerosol size characteristics varied significantly between devices, but the AeroEclipse II nebulizer operating at 50 psi generated stable pDNA/GL67A aerosols suitable for delivery to the CF lung (mass median aerodynamic diameter 3.4±0.1 μm). Importantly, biological function of pDNA/GL67A formulations was retained after nebulization, and although aerosol delivery rate was lower than that of other devices (0.17±0.01 ml/min), the breath-actuated AeroEclipse II nebulizer generated aerosol only during the inspiratory phase and as such was more efficient than other devices with 83±3% of generated aerosol available for patient inhalation. On the basis of these results, we have selected the AeroEclipse II nebulizer for the delivery of pDNA/GL67A formulations to the lungs of CF patients as part of phase IIa/b clinical studies. PMID:24865497

  14. Optimization of Brush-like Cationic Copolymers for Non-viral Gene Delivery

    PubMed Central

    Wei, Hua; Pahang, JoshuelA; Pun, Suzie H.

    2012-01-01

    Polyethylenimine (PEI) is one of the most broadly used polycations for gene delivery due to its high transfection efficiency and commercial availability but materials are cytotoxic and often polydisperse. The goal of current work is to develop an alternative family of polycations based on controlled living radical polymerization (CLRP) and to optimize the polymer structure for efficient gene delivery. In this study, well-defined poly(glycidyl methacrylate)(P(GMA)) homopolymers were synthesized using reversible addition fragmentation chain transfer (RAFT) polymerization followed by decoration using three different types of oligoamines, i.e., tetraethylenepentamine (TEPA), pentaethylenehexamine (PEHA), and tris(2-aminoethyl)amine (TREN), respectively, to generate various P(GMA-oligoamine) homopolycations. The effect of P(GMA) backbone length and structure of oligoamine on gene transfer efficiency was then determined. The optimal polymer, P(GMA-TEPA)50, provided comparable transfection efficiency but lower cytotoxicity than PEI. P(GMA-TEPA)50 was then used as the cationic block in di-block copolymers containing hydrophilic N-(2-hydroxypropyl) methacrylamide (HPMA) and oligo(ethylene glycol) monomethyl ether methacrylate (OEGMA). Polyplexes of block copolymers were stable against aggregation in physiological salt condition and in Opti-MEM due to the shielding effect of P(HPMA) and P(OEGMA). However, the presence of the HPMA/OEGMA block significantly decreased the transfection efficacy of P(GMA-TEPA)50homopolycation. To compensate for reduced cell uptake caused by the hydrophilic shell of polyplex, the integrin-binding peptide, RGD, was conjugated to the hydrophilic chain end of P(OEGMA)15-b-P(GMA-TEPA)50 copolymer by Michael-type addition reaction. At low polymer to DNA ratios, the RGD-functionalized polymer showed increased gene delivery efficiency to HeLa cells compared to analogous polymers lacking RGD. PMID:23240866

  15. Mesenchymal Stem Cells as Therapeutics and Vehicles for Gene and Drug Delivery

    PubMed Central

    Porada, Christopher D.; Almeida-Porada, Graça

    2010-01-01

    Mesenchymal stem cells (MSCs) possess a set of several fairly unique properties which make them ideally suited both for cellular therapies/regenerative medicine, and as vehicles for gene and drug delivery. These include: 1) relative ease of isolation; 2) the ability to differentiate into a wide variety of seemingly functional cell types of both mesenchymal and non-mesenchymal origin; 3) the ability to be extensively expanded in culture without a loss of differentiative capacity; 4) they are not only hypoimmunogenic, but they produce immunosuppression upon transplantation; 5) their pronounced anti-inflammatory properties; and 6) their ability to home to damaged tissues, tumors, and metastases following in vivo administration. In this review, we summarize the latest research in the use of mesenchymal stem cells in regenerative medicine, as immunomodulatory/anti-inflammatory agents, and as vehicles for transferring both therapeutic genes in genetic disease and genes designed to destroy malignant cells. PMID:20828588

  16. Silk-based nanocomplexes with tumor-homing peptides for tumor-specific gene delivery.

    PubMed

    Numata, Keiji; Mieszawska-Czajkowska, Aneta J; Kvenvold, Laura A; Kaplan, David L

    2012-01-01

    Nanoscale complexes of recombinant silk molecules containing THPs with DNA are designed as less cytotoxic and highly target-specific gene carriers. Genetically engineered silk proteins containing poly(L-lysine) domains to interact with pDNA and the THP to bind to specific tumorigenic cells for target-specific pDNA delivery are prepared, followed by in vitro transfection into MDA-MB-435 melanoma cells, highly metastatic human breast tumor MDA-MB-231 cells, and non-tumorigenic MCF-10A breast epithelial cells. The silk/poly(L-lysine) block copolymer containing Lyp1 (ML-Lyp1) shows significant differences from silk/poly(L-lysine) block copolymer containing F3 (ML-F3) in cytotoxicity to MCF10A cells. ML-F3 is the most promising candidate for target delivery into tumorigenic cells.

  17. Generation of stable cell line by using chitosan as gene delivery system.

    PubMed

    Şalva, Emine; Turan, Suna Özbaş; Ekentok, Ceyda; Akbuğa, Jülide

    2016-08-01

    Establishing stable cell lines are useful tools to study the function of various genes and silence or induce the expression of a gene of interest. Nonviral gene transfer is generally preferred to generate stable cell lines in the manufacturing of recombinant proteins. In this study, we aimed to establish stable recombinant HEK-293 cell lines by transfection of chitosan complexes preparing with pDNA which contain LacZ and GFP genes. Chitosan which is a cationic polymer was used as gene delivery system. Stable HEK-293 cell lines were established by transfection of cells with complexes which were prepared with chitosan and pVitro-2 plasmid vector that contains neomycin drug resistance gene, beta gal and GFP genes. The transfection efficiency was shown with GFP expression in the cells using fluorescence microscopy. Beta gal protein expression in stable cells was examined by beta-galactosidase assay as enzymatically and X-gal staining method as histochemically. Full complexation was shown in the above of 1/1 ratio in the chitosan/pDNA complexes. The highest beta-galactosidase activity was obtained with transfection of chitosan complexes. Beta gal gene expression was 15.17 ng/ml in the stable cells generated by chitosan complexes. In addition, intensive blue color was observed depending on beta gal protein expression in the stable cell line with X-gal staining. We established a stable HEK-293 cell line that can be used for recombinant protein production or gene expression studies by transfecting the gene of interest.

  18. Topical Non-Invasive Gene Delivery using Gemini Nanoparticles in Interferon-gamma-deficient Mice

    SciTech Connect

    Badea,I.; Wettig, S.; Verrall, R.; Foldvari, M.

    2007-01-01

    Cutaneous gene therapy, although a promising approach for many dermatologic diseases, has not progressed to the stage of clinical trials, mainly due to the lack of an effective gene delivery system. The main objective of this study was to construct and evaluate gemini nanoparticles as a topical formulation for the interferon gamma (IFN-{gamma}) gene in an IFN-{gamma}-deficient mouse model. Nanoparticles based on the gemini surfactant 16-3-16 (NP16-DNA) and another cationic lipid cholesteryl 3{beta}-(-N-[dimethylamino-ethyl] carbamate) [Dc-chol] (NPDc-DNA) were prepared and characterized. Zetasizer measurement indicated a bimodal distribution of 146 and 468 nm average particle sizes for the NP16-DNA ({zeta}-potential +51 mV) nanoparticles and monomodal distribution of 625 nm ({zeta}-potential +44 mV) for the NPDc-DNA. Circular dichroism studies showed that the gemini surfactant compacted the plasmid more efficiently compared to the Dc-chol. Small-angle X-ray scattering measurements revealed structural polymorphism in the NP16-DNA nanoparticles, with lamellar and Fd3m cubic phases present, while for the NPDc-DNA two lamellar phases could be distinguished. In vivo, both topically applied nanoparticles induced higher gene expression compared to untreated control and naked DNA (means of 0.480 and 0.398 ng/cm{sup 2} vs 0.067 and 0.167 ng/cm{sup 2}). However, treatment with NPDc-DNA caused skin irritation, and skin damage, whereas NP16-DNA showed no skin toxicity. In this study, we demonstrated that topical cutaneous gene delivery using gemini surfactant-based nanoparticles in IFN-{gamma}-deficient mice was safe and may provide increased gene expression in the skin due to structural complexity of NP16 nanoparticles (lamellar-cubic phases).

  19. Efficacious redox-responsive gene delivery in serum by ferrocenylated monomeric and dimeric cationic cholesterols.

    PubMed

    Vulugundam, Gururaja; Kumar, Krishan; Kondaiah, Paturu; Bhattacharya, Santanu

    2015-04-14

    Herein, we present the design and synthesis of new redox-active monomeric and dimeric (gemini) cationic lipids based on ferrocenylated cholesterol derivatives for gene delivery. The cationic cholesterols are shown to be transfection efficient after being formulated with the neutral helper lipid DOPE in the presence of serum (FBS). The redox activity of the resulting co-liposomes and their lipoplexes could be regulated using the alkanyl ferrocene moiety attached to the ammonium head groups of the cationic cholesterols. Atomic force microscopy (AFM), dynamic light scattering (DLS) and zeta potential measurements were performed to characterize the co-liposomal aggregates and their complexes with pDNA. The transfection efficiency of lipoplexes could be tuned by changing the oxidation state of the ferrocene moiety. The gene transfection capability was assayed in terms of green fluorescence protein (GFP) expression using pEGFP-C3 plasmid DNA in three cell lines of different origins, namely Caco-2, HEK293T and HeLa, in the presence of serum. The vesicles possessing ferrocene in the reduced state induced an efficient transfection, even better than a commercial reagent Lipofectamine 2000 (Lipo 2000) as evidenced by flow cytometry and fluorescence microscopy. All the co-liposomes containing the oxidized ferrocene displayed diminished levels of gene expression. Gene transfection events from the oxidized co-liposomes were further potentiated by introducing ascorbic acid (AA) as a reducing agent during lipoplex incubation with cells, leading to the resumption of transfection activity. Assessment of transfection capability of both reduced and oxidized co-liposomes was also undertaken following cellular internalization of labelled pDNA using confocal microscopy and flow cytometry. Overall, we demonstrate here controlled gene transfection activities using redox-driven, transfection efficient cationic monomeric and dimeric cholesterol lipids. Such systems could be used in gene

  20. Synthesis and characterization of an octaarginine functionalized graphene oxide nano-carrier for gene delivery applications.

    PubMed

    Imani, Rana; Emami, Shahriar Hojjati; Faghihi, Shahab

    2015-03-01

    The success of gene therapy is largely dependent on the development of a gene carrier. Recently cell-penetrating peptides (CPPs) have been employed for enhancing the gene and drug delivery efficacy of nano-particles. The feasibility of octaarginine (R8) functionalized graphene oxide (GO) as a novel nano-carrier for gene delivery is investigated. A DNA plasmid expressing enhanced green fluorescent protein (pEGFP) is used as a model gene to study the R8-GO transfection ability into mammalian cells. R8 peptide is conjugated in different ratios (0.1-1.5 μmol per mg of GO) to carboxylated graphene oxide by a two step amidation process. The process of peptide conjugation is analyzed by Fourier transform infrared (FTIR), atomic force microscopy (AFM), UV-vis spectroscopy and X-ray diffraction (XRD). In order to obtain the highest transfection of pEGFP into the cells, the amount of peptide bound to GO is optimized which is evidenced by dynamic light scattering (DLS), zeta potential, TNBS and gel retardation assays. The cytotoxicity of R8-functionalized GO is also tested by MTT assay. The results confirm the successful attachment of R8 peptide to GO. The AFM and XRD results show a significant increase in the thickness of nano graphene oxide sheets (NGOS) from 0.8 to 2-7 nm as well as an increase in the GO interlying space after the R8-functionalization process. A reduction in nano-carrier stability in both aqueous solution and cell culture media is observed when the amount of peptide is increased to more than 1 μmol mg(-1). Gel electrophoresis analysis shows the highest DNA loading on the peptide functionalized GO at the ratios of 0.5 and 1 μmol mg(-1). As a result, the conjugated peptide sample with a peptide molar ratio of 1 μmol per mg of GO shows the highest conjugational efficiency and EGFP gene expression along with improved dispersibility and biocompatibility. Overall, the findings reveal the importance of peptide density on the surface of NGOS in order to obtain

  1. Enteral siRNA delivery technique for therapeutic gene silencing in the liver via the lymphatic route.

    PubMed

    Murakami, Masahiro; Nishina, Kazutaka; Watanabe, Chie; Yoshida-Tanaka, Kie; Piao, Wenying; Kuwahara, Hiroya; Horikiri, Yuji; Miyata, Kanjiro; Nishiyama, Nobuhiro; Kataoka, Kazunori; Yoshida, Masayuki; Mizusawa, Hidehiro; Yokota, Takanori

    2015-11-23

    An efficient targeting delivery technology is needed for functional oligonucleotides to exert their potential effect on the target gene without an adverse effect in vivo. Development of enteral delivery systems for nucleic acids is a major challenge because of their large molecular size and instability. Here, we describe a new enteral delivery technique that enables small interfering RNA (siRNA) selectively delivered to the liver to silence its target Apolipoprotein B gene expression. A nuclease-resistant synthetic siRNA was conjugated with α-tochopherol and administered as lipid nanoparticle to the large intestine of the mice in a postprandial state. The selective transport into the liver, effective gene silence, and consequently significant reduction in serum low density lipoprotein-cholesterol level, were demonstrated. The chylomicron-mediated pathway via the lymphatic route was suggested as major mechanism. This unique approach may provide a basis for developing oral and rectal delivery systems for nucleic acids targeting liver.

  2. Enteral siRNA delivery technique for therapeutic gene silencing in the liver via the lymphatic route

    PubMed Central

    Murakami, Masahiro; Nishina, Kazutaka; Watanabe, Chie; Yoshida-Tanaka, Kie; Piao, Wenying; Kuwahara, Hiroya; Horikiri, Yuji; Miyata, Kanjiro; Nishiyama, Nobuhiro; Kataoka, Kazunori; Yoshida, Masayuki; Mizusawa, Hidehiro; Yokota, Takanori

    2015-01-01

    An efficient targeting delivery technology is needed for functional oligonucleotides to exert their potential effect on the target gene without an adverse effect in vivo. Development of enteral delivery systems for nucleic acids is a major challenge because of their large molecular size and instability. Here, we describe a new enteral delivery technique that enables small interfering RNA (siRNA) selectively delivered to the liver to silence its target Apolipoprotein B gene expression. A nuclease-resistant synthetic siRNA was conjugated with α-tochopherol and administered as lipid nanoparticle to the large intestine of the mice in a postprandial state. The selective transport into the liver, effective gene silence, and consequently significant reduction in serum low density lipoprotein-cholesterol level, were demonstrated. The chylomicron-mediated pathway via the lymphatic route was suggested as major mechanism. This unique approach may provide a basis for developing oral and rectal delivery systems for nucleic acids targeting liver. PMID:26593819

  3. Delivery of human NKG2D-IL-15 fusion gene by chitosan nanoparticles to enhance antitumor immunity

    SciTech Connect

    Yan, Chen; Jie, Leng; Yongqi, Wang; Weiming, Xiao; Juqun, Xi; Yanbing, Ding; Li, Qian; Xingyuan, Pan; Mingchun, Ji; Weijuan, Gong

    2015-07-31

    Nanoparticles are becoming promising carriers for gene delivery because of their high capacity in gene loading and low cell cytotoxicity. In this study, a chitosan-based nanoparticle encapsulated within a recombinant pcDNA3.1-dsNKG2D-IL-15 plasmid was generated. The fused dsNKG2D-IL-15 gene fragment consisted of double extracellular domains of NKG2D with IL-15 gene at downstream. The average diameter of the gene nanoparticles ranged from 200 nm to 400 nm, with mean zeta potential value of 53.8 ± 6.56 mV. The nanoparticles which were loaded with the dsNKG2D-IL-15 gene were uptaken by tumor cells with low cytotoxicity. Tumor cells pre-transfected by gene nanopartilces stimulated NK and T cells in vitro. Intramuscular injection of gene nanoparticles suppressed tumor growth and prolonged survival of tumor-bearing mice through activation of NK and CD8{sup +} T cells. Thus, chitosan-based nanoparticle delivery of dsNKG2D-IL-15 gene vaccine can be potentially used for tumor therapy. - Highlights: • Generation of a nanoparticle for delivery of dsNKG2D-IL-15 gene. • Characterization of the gene nanoparticle. • Antitumor activity mediated by the gene nanoparticle.

  4. Macrophages in gene therapy: cellular delivery vehicles and in vivo targets.

    PubMed

    Burke, B; Sumner, S; Maitland, N; Lewis, C E

    2002-09-01

    The appearance and activation of macrophages are thought to be rapid events in the development of many pathological lesions, including malignant tumors, atherosclerotic plaques, and arthritic joints. This has prompted recent attempts to use macrophages as novel cellular vehicles for gene therapy, in which macrophages are genetically modified ex vivo and then reintroduced into the body with the hope that a proportion will then home to the diseased site. Here, we critically review the efficacy of various gene transfer methods (viral, bacterial, protozoan, and various chemical and physical methods) in transfecting macrophages in vitro, and the results obtained when transfected macrophages are used as gene delivery vehicles. Finally, we discuss the use of various viral and nonviral methods to transfer genes to macrophages in vivo. As will be seen, definitive evidence for the use of macrophages as gene transfer vehicles has yet to be provided and awaits detailed trafficking studies in vivo. Moreover, although methods for transfecting macrophages have improved considerably in efficiency in recent years, targeting of gene transfer specifically to macrophages in vivo remains a problem. However, possible solutions to this include placing transgenes under the control of macrophage-specific promoters to limit expression to macrophages or stably transfecting CD34(+) precursors of monocytes/macrophages and then differentiating these cells into monocytes/macrophages ex vivo. The latter approach could conceivably lead to the bone marrow precursor cells of patients with inherited genetic disorders being permanently fortified or even replaced with genetically modified cells. PMID:12223508

  5. Effective delivery of large genes to the retina by dual AAV vectors

    PubMed Central

    Trapani, Ivana; Colella, Pasqualina; Sommella, Andrea; Iodice, Carolina; Cesi, Giulia; de Simone, Sonia; Marrocco, Elena; Rossi, Settimio; Giunti, Massimo; Palfi, Arpad; Farrar, Gwyneth J; Polishchuk, Roman; Auricchio, Alberto

    2014-01-01

    Retinal gene therapy with adeno-associated viral (AAV) vectors is safe and effective in humans. However, AAV's limited cargo capacity prevents its application to therapies of inherited retinal diseases due to mutations of genes over 5 kb, like Stargardt's disease (STGD) and Usher syndrome type IB (USH1B). Previous methods based on ‘forced’ packaging of large genes into AAV capsids may not be easily translated to the clinic due to the generation of genomes of heterogeneous size which raise safety concerns. Taking advantage of AAV's ability to concatemerize, we generated dual AAV vectors which reconstitute a large gene by either splicing (trans-splicing), homologous recombination (overlapping), or a combination of the two (hybrid). We found that dual trans-splicing and hybrid vectors transduce efficiently mouse and pig photoreceptors to levels that, albeit lower than those achieved with a single AAV, resulted in significant improvement of the retinal phenotype of mouse models of STGD and USH1B. Thus, dual AAV trans-splicing or hybrid vectors are an attractive strategy for gene therapy of retinal diseases that require delivery of large genes. PMID:24150896

  6. Gene delivery efficiency and cytotoxicity of heterocyclic amine-modified PAMAM and PPI dendrimers.

    PubMed

    Hashemi, Maryam; Tabatabai, Seyed Meghdad; Parhiz, Hamideh; Milanizadeh, Soroush; Amel Farzad, Sara; Abnous, Khalil; Ramezani, Mohammad

    2016-04-01

    Poly-(amidoamine) (PAMAM) and poly-(propylenimine) (PPI) are the two most widely investigated dendrimers for drug and gene delivery. In order to enhance DNA transfection activity of these dendrimers, generation 3 and 4 PAMAM and generation 4 and 5 PPI were modified by partial substitution of surface primary amines with histidine, pyridine, and piperazine, which have buffering capacity properties. It was shown that higher dendrimer generations and higher grafting percentages (30% and 50% of primary amines) were associated with higher transfection activity. Pyridine was the most effective substituent for PPI, while piperazine-modified PAMAM dendrimers showed the best transfection efficiency among PAMAM-based vectors in murine neuroblastoma (Neuro-2a) cells. None of the modified carriers showed remarkable cytotoxicity in vitro. Pretreatment of cells with bafilomycin A indicated that endosomal buffering capacity is the main mechanism of endosomal escape. In conclusion, PAMAM and PPI may exhibit different gene delivery efficiency and cytotoxicity profiles with the same chemical modifications. These modified dendrimers could be considered as efficient and safe gene carriers in neuroblastoma cells in vitro. PMID:26838910

  7. Close-field electroporation gene delivery using the cochlear implant electrode array enhances the bionic ear.

    PubMed

    Pinyon, Jeremy L; Tadros, Sherif F; Froud, Kristina E; Y Wong, Ann C; Tompson, Isabella T; Crawford, Edward N; Ko, Myungseo; Morris, Renée; Klugmann, Matthias; Housley, Gary D

    2014-04-23

    The cochlear implant is the most successful bionic prosthesis and has transformed the lives of people with profound hearing loss. However, the performance of the "bionic ear" is still largely constrained by the neural interface itself. Current spread inherent to broad monopolar stimulation of the spiral ganglion neuron somata obviates the intrinsic tonotopic mapping of the cochlear nerve. We show in the guinea pig that neurotrophin gene therapy integrated into the cochlear implant improves its performance by stimulating spiral ganglion neurite regeneration. We used the cochlear implant electrode array for novel "close-field" electroporation to transduce mesenchymal cells lining the cochlear perilymphatic canals with a naked complementary DNA gene construct driving expression of brain-derived neurotrophic factor (BDNF) and a green fluorescent protein (GFP) reporter. The focusing of electric fields by particular cochlear implant electrode configurations led to surprisingly efficient gene delivery to adjacent mesenchymal cells. The resulting BDNF expression stimulated regeneration of spiral ganglion neurites, which had atrophied 2 weeks after ototoxic treatment, in a bilateral sensorineural deafness model. In this model, delivery of a control GFP-only vector failed to restore neuron structure, with atrophied neurons indistinguishable from unimplanted cochleae. With BDNF therapy, the regenerated spiral ganglion neurites extended close to the cochlear implant electrodes, with localized ectopic branching. This neural remodeling enabled bipolar stimulation via the cochlear implant array, with low stimulus thresholds and expanded dynamic range of the cochlear nerve, determined via electrically evoked auditory brainstem responses. This development may broadly improve neural interfaces and extend molecular medicine applications.

  8. Water soluble cationic dextran derivatives containing poly(amidoamine) dendrons for efficient gene delivery.

    PubMed

    Mai, Kaijin; Zhang, Shanshan; Liang, Bing; Gao, Cong; Du, Wenjun; Zhang, Li-Ming

    2015-06-01

    To develop new dextran derivatives for efficient gene delivery, the conjugation of poly(amidoamine) dendrons onto biocompatible dextran was carried out by a Cu(I)-catalyzed azide-alkyne cycloaddition, as confirmed by FTIR and (1)H NMR analyses. For resultant dextran conjugates with various generations of poly(amidoamine) dendrons, their buffering capacity and in vitro cytotoxicity were evaluated by acid-base titration and MTT tests, respectively. In particular, their physicochemical characteristics for the complexation with plasmid DNA were investigated by the combined analyses from agarose gel electrophoresis, zeta potential, particle size, transmission electron microscopy and fluorescence emission spectra. Moreover, their complexes with plasmid DNA were studied with respect to their transfection efficiency in human embryonic kidney (HEK293) cell lines. In the case of a higher generation of poly(amidoamine) dendrons, such a dextran conjugate was found to have much lower cytotoxicity and better gene delivery capability when compared to branched polyethylenimine (bPEI, 25kDa), a commonly used gene vector. PMID:25843855

  9. Cholesterol derived cationic lipids as potential non-viral gene delivery vectors and their serum compatibility.

    PubMed

    Ju, Jia; Huan, Meng-Lei; Wan, Ning; Hou, Yi-Lin; Ma, Xi-Xi; Jia, Yi-Yang; Li, Chen; Zhou, Si-Yuan; Zhang, Bang-Le

    2016-05-15

    Cholesterol derivatives M1-M6 as synthetic cationic lipids were designed and the biological evaluation of the cationic liposomes based on them as non-viral gene delivery vectors were described. Plasmid pEGFP-N1, used as model gene, was transferred into 293T cells by cationic liposomes formed with M1-M6 and transfection efficiency and GFP expression were tested. Cationic liposomes prepared with cationic lipids M1-M6 exhibited good transfection activity, and the transfection activity was parallel (M2 and M4) or superior (M1 and M6) to that of DC-Chol derived from the same backbone. Among them, the transfection efficiency of cationic lipid M6 was parallel to that of the commercially available Lipofectamine2000. The optimal formulation of M1 and M6 were found to be at a mol ratio of 1:0.5 for cationic lipid/DOPE, and at a N/P charge mol ratio of 3:1 for liposome/DNA. Under optimized conditions, the efficiency of M1 and M6 is greater than that of all the tested commercial liposomes DC-Chol and Lipofectamine2000, even in the presence of serum. The results indicated that M1 and M6 exhibited low cytotoxicity, good serum compatibility and efficient transfection performance, having the potential of being excellent non-viral vectors for gene delivery. PMID:27072908

  10. Development of Non-Viral, Trophoblast-Specific Gene Delivery for Placental Therapy.

    PubMed

    Abd Ellah, Noura; Taylor, Leeanne; Troja, Weston; Owens, Kathryn; Ayres, Neil; Pauletti, Giovanni; Jones, Helen

    2015-01-01

    Low birth weight is associated with both short term problems and the fetal programming of adult onset diseases, including an increased risk of obesity, diabetes and cardiovascular disease. Placental insufficiency leading to intrauterine growth restriction (IUGR) contributes to the prevalence of diseases with developmental origins. Currently there are no therapies for IUGR or placental insufficiency. To address this and move towards development of an in utero therapy, we employ a nanostructure delivery system complexed with the IGF-1 gene to treat the placenta. IGF-1 is a growth factor critical to achieving appropriate placental and fetal growth. Delivery of genes to a model of human trophoblast and mouse placenta was achieved using a diblock copolymer (pHPMA-b-pDMAEMA) complexed to hIGF-1 plasmid DNA under the control of trophoblast-specific promoters (Cyp19a or PLAC1). Transfection efficiency of pEGFP-C1-containing nanocarriers in BeWo cells and non-trophoblast cells was visually assessed via fluorescence microscopy. In vivo transfection and functionality was assessed by direct placental-injection into a mouse model of IUGR. Complexes formed using pHPMA-b-pDMAEMA and CYP19a-923 or PLAC1-modified plasmids induce trophoblast-selective transgene expression in vitro, and placental injection of PLAC1-hIGF-1 produces measurable RNA expression and alleviates IUGR in our mouse model, consequently representing innovative building blocks towards human placental gene therapies.

  11. Interleukin-27 Gene Delivery for Modifying Malignant Interactions Between Prostate Tumor and Bone

    PubMed Central

    Zolochevska, Olga; Ellis, Jayne; Parelkar, Sangram; Chan-Seng, Delphine; Emrick, Todd; Wei, Jingna; Patrikeev, Igor; Motamedi, Massoud

    2013-01-01

    Abstract We have examined the role of a novel cytokine, interleukin-27 (IL-27), in mediating interactions between prostate cancer and bone. IL-27 is the most recently characterized member of the family of heterodimeric IL-12-related cytokines and has shown promise in halting tumor growth and mediating tumor regression in several cancer models, including prostate cancer. Prostate cancer is frequently associated with metastases to the bone, where the tumor induces a vicious cycle of communication with osteoblasts and osteoclasts to induce bone lesions, which are a significant cause of pain and skeletal-related events for patients, including a high fracture risk. We describe our findings in the effects of IL-27 gene delivery on prostate cancer cells, osteoblasts, and osteoclasts at different stages of differentiation. We applied the IL-27 gene delivery protocol in vivo utilizing sonoporation (sonodelivery) with the goal of treating and reducing the growth of prostate cancer at a bone metastatic site in vivo. We used a new model of immune-competent prostate adenocarcinoma and characterized the tumor growth reduction, gene expression, and effector cellular profiles. Our results suggest that IL-27 can be effective in reducing tumor growth, can help normalize bone structure, and can promote enhanced accumulation of effector cells in prostate tumors. These results are promising, because they are relevant to developing a novel IL-27-based strategy that can treat both the tumor and the bone, by using this simple and effective sonodelivery method for treating prostate tumor bone metastases. PMID:24028178

  12. Characterization and Investigation of Redox-Sensitive Liposomes for Gene Delivery.

    PubMed

    Pezzoli, Daniele; Tallarita, Elena; Rosini, Elena; Candiani, Gabriele

    2016-01-01

    A number of smart nonviral gene delivery vectors relying on bioresponsiveness have been introduced in the past few years to overcome the limits of the first generation of gene carriers. Among them, redox-sensitive lipidic and polymeric vectors exploit the presence of disulfide bonds in their structure to take advantage of the highly reductive intracellular milieu and to promote complex unpacking and nucleic acids release after cellular uptake (disulfide linker strategy). Glutathione (GSH) has been often identified as the leading actor in the intracellular reduction of bioreducible vectors but their actual mechanisms of action have been rarely investigated in depth and doubts about the real effectiveness of the disulfide linker strategy have been raised. Herein, we outline a simple protocol for the preparation and investigation of nano-sized reducible cationic liposomes, focusing on their thorough characterization and optimization as gene delivery vectors. In addition, we carefully describe the techniques and procedures necessary for the assessment of the bioreducibility of the vectors and to demonstrate that the GSH-mediated intracellular cleavage of disulfide bonds is a pivotal step in their transfection process. Liposomes composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE), and of the reducible cationic lipid SS14 are reported as a practical example but the proposed protocol can be easily shifted to other formulations of reducible lipids/liposomes and to reducible polymers. PMID:27436322

  13. Gene delivery by surface immobilization of plasmid to tissue-engineering scaffolds.

    PubMed

    Salvay, D M; Zelivyanskaya, M; Shea, L D

    2010-09-01

    Biomaterial scaffolds that serve as vehicles for gene delivery to promote expression of inductive factors have numerous regenerative medicine applications. In this report, we investigate plasmid delivery from biomaterial scaffolds using a surface immobilization strategy. Porous scaffolds were fabricated from poly(D,L-lactide-co-glycolide) (PLG), and plasmids were immobilized by drying. In vitro plasmid release indicated that the majority (>70%) of adsorbed plasmids were released within 24 h and >98% within 3 days; however, in vivo implantation of the scaffolds at the subcutaneous site yielded transgene expression that persisted for at least 28 weeks and was localized to the site of implantation. Histological analysis of DNA-adsorbed scaffolds indicated that macrophages at the scaffold were transfected in the first 2 weeks after implantation, whereas muscle cells adjacent to the implant primarily expressed the transgene at 4 weeks. In addition to localized gene expression, a secreted protein (human factor IX) was retained at the implant site and not available systemically after 3 days, indicating minimal off-target effects. These findings show that surface immobilization of plasmid onto microporous PLG scaffolds can produce localized and long-term gene expression in vivo, which may be used to enhance the bioactivity of scaffolds used for regenerative medicine.

  14. Improved Gene Delivery to Intestinal Mucosa by Adenoviral Vectors Bearing Subgroup B and D Fibers

    PubMed Central

    Lecollinet, S.; Gavard, F.; Havenga, M. J. E.; Spiller, O. B.; Lemckert, A.; Goudsmit, J.; Eloit, M.; Richardson, J.

    2006-01-01

    A major obstacle to successful oral vaccination is the lack of antigen delivery systems that are both safe and highly efficient. Conventional replication-incompetent adenoviral vectors, derived from human adenoviruses of subgroup C, are poorly efficient in delivering genetic material to differentiated intestinal epithelia. To date, 51 human adenovirus serotypes have been identified and shown to recognize different cellular receptors with different tissue distributions. This natural diversity was exploited in the present study to identify suitable adenoviral vectors for efficient gene delivery to the human intestinal epithelium. In particular, we compared the capacities of a library of adenovirus type 5-based vectors pseudotyped with fibers of several human serotypes for transduction, binding, and translocation toward the basolateral pole in human and murine tissue culture models of differentiated intestinal epithelia. In addition, antibody-based inhibition was used to gain insight into the molecular interactions needed for efficient attachment. We found that vectors differing merely in their fiber proteins displayed vastly different capacities for gene transfer to differentiated human intestinal epithelium. Notably, vectors bearing fibers derived from subgroup B and subgroup D serotypes transduced the apical pole of human epithelium with considerably greater efficiency than a subgroup C vector. Such efficiency was correlated with the capacity to use CD46 or sialic acid-containing glycoconjugates as opposed to CAR as attachment receptors. These results suggest that substantial gains could be made in gene transfer to digestive epithelium by exploiting the tropism of existing serotypes of human adenoviruses. PMID:16501084

  15. Development of TMTP-1 targeted designer biopolymers for gene delivery to prostate cancer.

    PubMed

    McBride, John W; Massey, Ashley S; McCaffrey, J; McCrudden, Cian M; Coulter, Jonathan A; Dunne, Nicholas J; Robson, Tracy; McCarthy, Helen O

    2016-03-16

    Designer biopolymers (DBPs) represent state of the art genetically engineered biomacromolecules designed to condense plasmid DNA, and overcome intra- and extra- cellular barriers to gene delivery. Three DBPs were synthesized, each with the tumor molecular targeting peptide-1 (TMTP-1) motif to specifically target metastases. Each DBP was complexed with a pEGFP-N1 reporter plasmid to permit physiochemical and biological assay analysis. Results indicated that two of the biopolymers (RMHT and RM3GT) effectively condensed pEGFP-N1 into cationic nanoparticles <100 nm and were capable of transfecting PC-3 metastatic prostate cancer cells. Conversely the anionic RMGT DBP nanoparticles could not transfect PC-3 cells. RMHT and RM3GT nanoparticles were stable in the presence of serum and protected the cargo from degradation. Additionally it was concluded that cell viability could recover post-transfection with these DBPs, which were less toxic than the commercially available transfection reagent Lipofectamine(®) 2000. With both DBPs, a higher transfection efficacy was observed in PC-3 cells than in the moderately metastatic, DU145, and normal, PNT2-C2, cell lines. Blocking of the TMTP-1 receptors inhibited gene transfer indicating internalization via this receptor. In conclusion RMHT and RM3GT are fully functional DBPs that address major obstacles to gene delivery and target metastatic cells expressing the TMTP-1 receptor.

  16. Nanoparticle-Mediated Target Delivery of TRAIL as Gene Therapy for Glioblastoma.

    PubMed

    Wang, Kui; Kievit, Forrest M; Jeon, Mike; Silber, John R; Ellenbogen, Richard G; Zhang, Miqin

    2015-12-01

    Human tumor necrosis factor α-related apoptosis-inducing ligand (TRAIL) is an attractive cancer therapeutic because of its ability to induce apoptosis in tumor cells while having a negligible effect on normal cells. However, the short serum half-life of TRAIL and lack of efficient in vivo administration approaches have largely hindered its clinical use. Using nanoparticles (NPs) as carriers in gene therapy is considered as an alternative approach to increase TRAIL delivery to tumors as transfected cells would be induced to secrete TRAIL into the tumor microenvironment. To enable effective delivery of plasmid DNA encoding TRAIL into glioblastoma (GBM), we developed a targeted iron oxide NP coated with chitosan-polyethylene glycol-polyethyleneimine copolymer and chlorotoxin (CTX) and evaluated its effect in delivering TRAIL in vitro and in vivo. NP-TRAIL successfully delivers TRAIL into human T98G GBM cells and induces secretion of 40 pg mL(-1) of TRAIL in vitro. Transfected cells show threefold increased apoptosis as compared to the control DNA bound NPs. Systemic administration of NP-TRAIL-CTX to mice bearing T98G-derived flank xenografts results in near-zero tumor growth and induces apoptosis in tumor tissue. Our results suggest that NP-TRAIL-CTX can potentially serve as a targeted anticancer therapeutic for more efficient TRAIL delivery to GBM. PMID:26498165

  17. Nanoparticle-Mediated Target Delivery of TRAIL as Gene Therapy for Glioblastoma.

    PubMed

    Wang, Kui; Kievit, Forrest M; Jeon, Mike; Silber, John R; Ellenbogen, Richard G; Zhang, Miqin

    2015-12-01

    Human tumor necrosis factor α-related apoptosis-inducing ligand (TRAIL) is an attractive cancer therapeutic because of its ability to induce apoptosis in tumor cells while having a negligible effect on normal cells. However, the short serum half-life of TRAIL and lack of efficient in vivo administration approaches have largely hindered its clinical use. Using nanoparticles (NPs) as carriers in gene therapy is considered as an alternative approach to increase TRAIL delivery to tumors as transfected cells would be induced to secrete TRAIL into the tumor microenvironment. To enable effective delivery of plasmid DNA encoding TRAIL into glioblastoma (GBM), we developed a targeted iron oxide NP coated with chitosan-polyethylene glycol-polyethyleneimine copolymer and chlorotoxin (CTX) and evaluated its effect in delivering TRAIL in vitro and in vivo. NP-TRAIL successfully delivers TRAIL into human T98G GBM cells and induces secretion of 40 pg mL(-1) of TRAIL in vitro. Transfected cells show threefold increased apoptosis as compared to the control DNA bound NPs. Systemic administration of NP-TRAIL-CTX to mice bearing T98G-derived flank xenografts results in near-zero tumor growth and induces apoptosis in tumor tissue. Our results suggest that NP-TRAIL-CTX can potentially serve as a targeted anticancer therapeutic for more efficient TRAIL delivery to GBM.

  18. Integration of Drug, Protein, and Gene Delivery Systems with Regenerative Medicine

    PubMed Central

    Lorden, Elizabeth R.; Levinson, Howard M.; Leong, Kam W.

    2013-01-01

    Regenerative medicine has the potential to drastically change the field of health care from reactive to preventative and restorative. Exciting advances in stem cell biology and cellular reprogramming have fueled the progress of this field. Biochemical cues in the form of small molecule drugs, growth factors, zinc finger protein transcription factors and nucleases, transcription activator-like effector nucleases, monoclonal antibodies, plasmid DNA, aptamers, or RNA interference agents can play an important role to influence stem cell differentiation and the outcome of tissue regeneration. Many of these biochemical factors are fragile and must act intracellularly at the molecular level. They require an effective delivery system, which can take the form of a scaffold (e.g. hydrogels and electrospun fibers), carrier (viral and nonviral), nano- and micro-particle, or genetically modified cell. In this review, we will discuss the history and current technologies of drug, protein and gene delivery in the context of regenerative medicine. Next we will present case examples of how delivery technologies are being applied to promote angiogenesis in non-healing wounds or prevent angiogenesis in age related macular degeneration. Finally, we will conclude with a brief discussion of the regulatory pathway from bench-to-bedside for the clinical translation of these novel therapeutics. PMID:25787742

  19. Subretinal delivery of recombinant AAV serotype 8 vector in dogs results in gene transfer to neurons in the brain.

    PubMed

    Stieger, Knut; Colle, Marie-Anne; Dubreil, Laurence; Mendes-Madeira, Alexandra; Weber, Michel; Le Meur, Guylène; Deschamps, Jack Yves; Provost, Nathalie; Nivard, Delphine; Cherel, Yan; Moullier, Philippe; Rolling, Fabienne

    2008-05-01

    Recombinant adeno-associated virus (rAAV) vectors are among the most efficient gene delivery vehicles for gene transfer to the retina. This study evaluates the behavior of the rAAV8 serotype vector with regard to intraocular delivery in rats and dogs. Subretinal delivery of an AAV2/8.gfp vector results in efficient gene transfer in the retinal pigment epithelium (RPE), the photoreceptors and, surprisingly, in the cells of the inner nuclear layer as well as in ganglion cells. Most importantly, in dogs, gene transfer also occurred distal to the injection site in neurons of the lateral geniculate nucleus of the brain. Because green fluorescent protein (GFP) was detected along the visual pathway within the brain, we analyzed total DNA extracted from various brain slices using PCR. Vector sequences were detected in many parts of the brain, but chiefly in the contralateral hemisphere.

  20. Novel hyperbranched polyamidoamine nanoparticle based gene delivery: transfection, cytotoxicity and in vitro evaluation.

    PubMed

    Zhu, Kai; Guo, Changfa; Lai, Hao; Yang, Wuli; Wang, Chunsheng

    2012-02-28

    In this study, hyperbranched polyamidoamine (hPAMAM) was developed as a novel non-viral gene vector for the first time. The hPAMAM was synthesized using a modified "one-pot" method. DNA was then bound to hPAMAM at different weight ratios (w(hPAMAM)/w(DNA)). The higher weight ratio could bring larger particle size and higher zeta potential of hPAMAM-DNA complexes. The encapsulated DNA was protected by hPAMAM from degradation for over 3h. Under the optimal condition, high gene transfection efficiency could be achieved in COS7 (47.47 ± 1.42%) and HEK293 (40.8 ± 0.98%) cell lines. And hPAMAM showed rather minor cytotoxicity in vitro (cell viability=91.38 ± 0.46% in COS7 and 92.38 ± 0.61% in HEK293). The hPAMAM mediated human vascular endothelial growth factor 165 (hVEGF(165)) gene transfected cells could express hVEGF(165) stably for 14 days, with the peak expression at day 2. In conclusion, hPAMAM based gene delivery was economical, effective and biocompatible, and may serve as a promising non-viral vehicle for gene therapy.

  1. Synergistically combined gene delivery for enhanced VEGF secretion and anti-apoptosis

    PubMed Central

    Won, Young-Wook; Lee, Minhyung; Kim, Hyun Ah; Nam, Kihoon; Bull, David A.; Kim, Sung Wan

    2013-01-01

    With current pharmacological treatments, preventing the remodeling of the left ventricle and the progression to heart failure is a difficult task. Gene therapy is considered to provide a direct treatment to the long-term complications of ischemic heart diseases. Although current gene therapies that use single molecular targets seem potentially possible, they have not achieved a success in the treatment of ischemic diseases. With an efficient polymeric gene carrier, PAM-ABP, we designed a synergistically combined gene delivery strategy to enhance vascular endothelial growth factor (VEGF) secretion and prolong anti-apoptotic effects. A hypoxia-inducible plasmid expressing both hypoxia-inducible heme oxygenase-1 (HO-1) and the Src homology domain-2 containing tyrosine phosphatase-1 microRNA (miSHP 1) and a hypoxia-responsive VEGF plasmid were combined in this study. The positive feedback circuit between HO-1 and VEGF, and the negative regulatory role of SHP-1 in angiogenesis enhance VEGF secretion synergistically. The synergy in VEGF secretion as a consequence of the gene combination and the prolonged HO-1 activity was confirmed in hypoxic cardiomyocytes and cardiomyocyte apoptosis under hypoxia, and was decreased synergistically. These results suggest that the synergistic combination of VEGF, HO-1, and miSHP-1 may be promising for the clinical treatment of ischemic diseases. PMID:24007285

  2. Delivery of cationic polymer-siRNA nanoparticles for gene therapies in neural regeneration

    SciTech Connect

    Liang, Yanran; Liu, Zhonglin; Shuai, Xintao; Wang, Weiwei; Liu, Jun; Bi, Wei; Wang, Chuanming; Jing, Xiuna; Liu, Yunyun; Tao, Enxiang

    2012-05-18

    Highlights: Black-Right-Pointing-Pointer Nogo receptor can inhibit growth of injured axons, thus affecting neural regeneration. Black-Right-Pointing-Pointer The delivery of siRNA is crucial to inhibit NgR expression in NSCs. Black-Right-Pointing-Pointer Non-viral vector PEG-PEI condensed siRNA targeting NgR into nanoscale particles. Black-Right-Pointing-Pointer PEG-PEI/siRNA at N/P = 15 displayed high transfection efficiency and low cytotoxicity. Black-Right-Pointing-Pointer PEG-PEI has great potential in carrying siRNA to diminish the gene expression in NSCs. -- Abstract: The therapeutic applications of neural stem cells (NSCs) have potential to promote recovery in many obstinate diseases in central nervous system. Regulation of certain gene expressions using siRNA may have significant influence on the fate of NSC. To achieve the optimum gene silencing effect of siRNA, non-viral vector polyethylene glycol-polyethyleneimine (PEG-PEI) was investigated in the delivery of siRNA to NSCs. The characteristics of PEG-PEI/siRNA polyplexes were detected by scanning electron microscopy (SEM). The effects of nanoparticles on cell viability were measured via CCK-8 assay. In addition, the transfection efficiency was evaluated by fluorescence microscope and flow cytometry, and real-time PCR and Western Blot were employed to detect the gene inhibition effect of siRNA delivered by PEG-PEI. The SEM micrographs showed that PEG-PEI could condense siRNA to form diffuse and spherical nanoparticles. The cytotoxicity of PEG-PEI/siRNA nanocomplexes (N/P = 15) was significantly lower when compared with that of Lipofectamine 2000/siRNA (P < 0.05). Moreover, the highest transfection efficiency of PEG-PEI/siRNA nanoparticles was obtained at an N/P ratio of 15, which was better than that achieved in the transfection using Lipofectamine 2000 (P < 0.05). Finally, the gene knockdown effect of PEG-PEI/siRNA nanoparticles was verified at the levels of mRNA and protein. These results suggest that

  3. Ultrasound-Triggered Phase-Transition Cationic Nanodroplets for Enhanced Gene Delivery.

    PubMed

    Gao, Di; Xu, Ming; Cao, Zhong; Gao, Jinbiao; Chen, Ya; Li, Yingqin; Yang, Zhe; Xie, Xiaoyan; Jiang, Qing; Wang, Wei; Liu, Jie

    2015-06-24

    Ultrasound as an external stimulus for enhanced gene transfection represents a safe, noninvasive, cost-effective delivery strategy for gene therapy. Herein, we have developed an ultrasound-triggered phase-transition cationic nanodroplet based on a novel perfluorinated amphiphilic poly(amino acid), which could simultaneously load perfluoropentane (PFP) and nucleic acids. The heptadecafluoroundecylamine (C11F17-NH2) was chosen to initiate β-benzyl-L-aspartate N-carboxyanhydride (BLA-NCA) ring-opening polymerization to prepare C11F17-poly(β-benzyl-L-aspartate) (C11F17-PBLA). Subsequently, C11F17-poly{N-[N'-(2-aminoethyl)]aspartamide} [C11F17-PAsp(DET)] was synthesized by aminolysis reaction of C11F17-PBLA with diethylenetriamine (DET). PFP/pDNA-loaded nanodroplets PFP-TNDs [PFP/C11F17-PAsp(DET)/LucDNA/γ-PGA or poly(glutamic acid)-g-MeO-poly(ethylene glycol) (PGA-g-mPEG) ternary nanodroplets] were primarily formulated by an oil/water emulsification method, followed by surface modification with PGA-g-mPEG. The average diameter of PFP-TNDs ranged from 300 to 400 nm, and transmission electron microscopy images showed that the nanodroplets were nearly spherical in shape. The ζ potential of the nanodroplets dramatically decreased from +54.3 to +15.3 mV after modification with PGA-g-mPEG, resulting in a significant increase of the stability of the nanodroplets in the serum-containing condition. With ultrasound irradiation, the gene transfection efficiency was enhanced 14-fold on HepG2 cells, and ultrasound-triggered phase-transition cationic nanodroplets also displayed a good ultrasound contrast effect. These results suggest that the PFP/DNA-loaded phase-transition cationic nanodroplets can be utilized as efficient theranostic agents for targeting gene delivery. PMID:26016606

  4. AAV9-mediated central nervous system–targeted gene delivery via cisterna magna route in mice

    PubMed Central

    Lukashchuk, Vera; Lewis, Katherine E; Coldicott, Ian; Grierson, Andrew J; Azzouz, Mimoun

    2016-01-01

    Current barriers to the use of adeno-associated virus serotype 9 (AAV9) in clinical trials for treating neurological disorders are its high expression in many off-target tissues such as liver and heart, and lack of cell specificity within the central nervous system (CNS) when using ubiquitous promoters such as human cytomegalovirus (CMV) or chicken-β-actin hybrid (CAG). To enhance targeting the transgene expression in CNS cells, self-complementary (sc) AAV9 vectors, scAAV9-GFP vectors carrying neuronal Hb9 and synapsin 1, and nonspecific CMV and CAG promoters were constructed. We demonstrate that synapsin 1 and Hb9 promoters exclusively targeted neurons in vitro, although their strengths were up to 10-fold lower than that of CMV. In vivo analyses of mouse tissue after scAAV9-GFP vector delivery via the cisterna magna revealed a significant advantage of synapsin 1 promoter over both Hb9 variants in targeting neurons throughout the brain, since Hb9 promoters were driving gene expression mainly within the motor-related areas of the brain stem. In summary, this study demonstrates that cisterna magna administration is a safe alternative to intracranial or intracerebroventricular vector delivery route using scAAV9, and introduces a novel utility of the Hb9 promoter for the targeted gene expression for both in vivo and in vitro applications. PMID:26942208

  5. A Simple Zn2+ Complex-Based Composite System for Efficient Gene Delivery.

    PubMed

    Zhang, Zhe; Zhao, Yanjie; Meng, Xianggao; Zhao, Dan; Zhang, Dan; Wang, Li; Liu, Changlin

    2016-01-01

    Metal complexes might become a new type of promising gene delivery systems because of their low cytotoxicity, structural diversity, controllable aqua- and lipo-solubility, and appropriate density and distribution of positive charges. In this study, Zn2+ complexes (1-10) formed with a series of ligands contained benzimidazole(bzim)were prepared and characterized. They were observed to have different affinities for DNA, dependent on their numbers of positive charges, bzim groups, and coordination structures around Zn2+. The binding induced DNA to condensate into spherical nanoparticles with ~ 50 nm in diameter. The cell transfection efficiency of the DNA nanoparticles was poor, although they were low toxic. The sequential addition of the cell-penetrating peptide (CPP) TAT(48-60) and polyethylene glycol (PEG) resulted in the large DNA condensates (~ 100 nm in diameter) and the increased cellular uptake. The clathrin-mediated endocytosis was found to be a key cellular uptake pathway of the nanoparticles formed with or without TAT(48-60) or/and PEG. The DNA nanoparticles with TAT(48-60) and PEG was found to have the cell transfection efficiency up to 20% of the commercial carrier Lipofect. These results indicated that a simple Zn2+-bzim complex-based composite system can be developed for efficient and low toxic gene delivery through the combination with PEG and CPPs such as TAT. PMID:27433798

  6. A Simple Zn2+ Complex-Based Composite System for Efficient Gene Delivery

    PubMed Central

    Zhang, Zhe; Zhao, Yanjie; Meng, Xianggao; Zhao, Dan; Zhang, Dan; Wang, Li; Liu, Changlin

    2016-01-01

    Metal complexes might become a new type of promising gene delivery systems because of their low cytotoxicity, structural diversity, controllable aqua- and lipo-solubility, and appropriate density and distribution of positive charges. In this study, Zn2+ complexes (1–10) formed with a series of ligands contained benzimidazole(bzim)were prepared and characterized. They were observed to have different affinities for DNA, dependent on their numbers of positive charges, bzim groups, and coordination structures around Zn2+. The binding induced DNA to condensate into spherical nanoparticles with ~ 50 nm in diameter. The cell transfection efficiency of the DNA nanoparticles was poor, although they were low toxic. The sequential addition of the cell-penetrating peptide (CPP) TAT(48–60) and polyethylene glycol (PEG) resulted in the large DNA condensates (~ 100 nm in diameter) and the increased cellular uptake. The clathrin-mediated endocytosis was found to be a key cellular uptake pathway of the nanoparticles formed with or without TAT(48–60) or/and PEG. The DNA nanoparticles with TAT(48–60) and PEG was found to have the cell transfection efficiency up to 20% of the commercial carrier Lipofect. These results indicated that a simple Zn2+-bzim complex-based composite system can be developed for efficient and low toxic gene delivery through the combination with PEG and CPPs such as TAT. PMID:27433798

  7. Mapping the AAV Capsid Host Antibody Response toward the Development of Second Generation Gene Delivery Vectors

    PubMed Central

    Tseng, Yu-Shan; Agbandje-McKenna, Mavis

    2013-01-01

    The recombinant adeno-associated virus (rAAV) gene delivery system is entering a crucial and exciting phase with the promise of more than 20 years of intense research now realized in a number of successful human clinical trials. However, as a natural host to AAV infection, anti-AAV antibodies are prevalent in the human population. For example, ~70% of human sera samples are positive for AAV serotype 2 (AAV2). Furthermore, low levels of pre-existing neutralizing antibodies in the circulation are detrimental to the efficacy of corrective therapeutic AAV gene delivery. A key component to overcoming this obstacle is the identification of regions of the AAV capsid that participate in interactions with host immunity, especially neutralizing antibodies, to be modified for neutralization escape. Three main approaches have been utilized to map antigenic epitopes on AAV capsids. The first is directed evolution in which AAV variants are selected in the presence of monoclonal antibodies (MAbs) or pooled human sera. This results in AAV variants with mutations on important neutralizing epitopes. The second is epitope searching, achieved by peptide scanning, peptide insertion, or site-directed mutagenesis. The third, a structure biology-based approach, utilizes cryo-electron microscopy and image reconstruction of AAV capsids complexed to fragment antibodies, which are generated from MAbs, to directly visualize the epitopes. In this review, the contribution of these three approaches to the current knowledge of AAV epitopes and success in their use to create second generation vectors will be discussed. PMID:24523720

  8. Synthesis and Characterization of New Multivalent Cationic Lipids for Applications in Gene Delivery

    NASA Astrophysics Data System (ADS)

    Ahmad, A.; Ewert, K.; Evans, Heather M.; Schmidt, H. W.; Safinya, C. R.

    2002-03-01

    Cationic lipid mediated delivery of foreign DNA into cells is a promising alternative to viral based gene therapy, although its comparatively low efficiency may result from a lack of understanding of these DNA-lipid complexes. Understanding the effect of different components of the lipid, such as size and charge of the head group, tail length, etc., is essential to the design of an efficient carrier for use in gene delivery. Here we investigate a new class of multivalent lipids (MVLs) to examine the role of the charge of the head group, providing a short description of the synthesis of these lipids and characterization of the lamellar structure of complexes of these lipids with DNA through x-ray diffraction and optical microscopy. Cationic lipid:DNA(CL:DNA) complexes containing mixtures of these MVLs and the neutral lipid DOPC are compared with CL:DNA complexes containing the monovalent lipid DOTAP and DOPC. We demonstrate that due to the higher membrane charge density, certain MVLs have a structural advantage resulting in a higher transfection efficiency at smaller ratios of cationic to neutral lipid. Funding: NIH GM59288, NSF DMR-9972246

  9. Supramolecular anchoring of DNA polyplexes in cyclodextrin-based polypseudorotaxane hydrogels for sustained gene delivery.

    PubMed

    Li, Zibiao; Yin, Hui; Zhang, Zhongxing; Liu, Kerh Li; Li, Jun

    2012-10-01

    A cyclodextrin-based supramolecular hydrogel system with supramolecularly anchored active cationic copolymer/plasmid DNA (pDNA) polyplexes was studied as a sustained gene delivery carrier. A few biodegradable triblock copolymers of methoxy-poly(ethylene glycol)-b-poly(ε-caprolactone)-b-poly[2-(dimethylamino)ethyl methacrylate] (MPEG-PCL-PDMAEMA) with well-defined cationic block lengths were prepared to condense pDNA. The MPEG-PCL-PDMAEMA copolymers exhibit good ability to condense pDNA into 275-405 nm polyplexes with hydrophilic MPEG in the outer corona. The MPEG corona imparted greater stability to the pDNA polyplexes and also served as an anchoring segment when the pDNA polyplexes were encapsulated in α-CD-based supramolecular polypseudorotaxane hydrogels. More interestingly, the resultant hydrogels were able to sustain release of pDNA up to 6 days. The pDNA was released in the form of polyplex nanoparticles as it was bound electrostatically to the cationic segment of the MPEG-PCL-PDMAEMA copolymers. The bioactivity of the released pDNA polyplexes at various durations was further investigated. Protein expression level of pDNA polyplexes released over the durations was comparable to that of freshly prepared PEI polyplexes. Being thixotropic and easily prepared without using organic solvent, this supramolecular in situ gelling system has immense potential as an injectable carrier for sustained gene delivery.

  10. 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.

  11. Safety and angiogenic effects of systemic gene delivery of a modified erythropoietin.

    PubMed

    de Lucas Cerrillo, A M; Bond, W S; Rex, T S

    2015-05-01

    Erythropoietin (EPO) is critical for red blood cell production and is also an effective neuroprotective agent. However, it may contribute to pathological angiogenesis. Here we investigate the angiogenic potential of EPO and a mutant form with attenuated erythropoietic activity, EPO-R76E, on primary human retinal microvascular endothelial cells (HRMECs) and in the adult retina. Assays of death, proliferation and tube formation were performed on HRMECs exposed to EPO, EPO-R76E or media alone. Postnatal day-9 wild-type mice were injected intramuscularly with adeno-associated virus vectors expressing either enhanced green fluorescent protein or EpoR76E. At 3 months, levels of EPO-R76E in the eye were quantified, and the health of the retinal vasculature was assessed by fluorescein angiography and isolectin immunolabeling. Immunohistochemistry, histology and electroretinogram (ERG) assessments were performed as measures of retinal health. Neither EPO nor EPO-R76E induced proliferation or tube formation in HRMECs under the conditions used. EPO-R76E decreased HRMEC death in a dose-dependent manner. Long-term systemic gene delivery of EPO-R76E was safe in terms of retinal vasculature, histology and the ERG in vivo. Our results show that EPO-R76E can block HRMEC death, consistent with its role in erythropoiesis and neuroprotection. In addition, long-term gene delivery of EPO-R76E is safe in the adult retina. PMID:25716531

  12. Chitosan enhanced gene delivery of cationic liposome via non-covalent conjugation.

    PubMed

    Wang, Bing; Zhang, Shubiao; Cui, Shaohui; Yang, Baoling; Zhao, Yinan; Chen, Huiying; Hao, Xiaomin; Shen, Qiong; Zhou, Jiti

    2012-01-01

    Two new types of stable ternary complexes were formed by mixing chitosan with DOTAP/pDNA lipoplex and DOTAP with chitosan/pDNA polyplex via non-covalent conjugation for the efficient delivery of plasmid DNA. They were characterized by atomic force microscopy, gel retarding, and dynamic light scattering. The DOTAP/CTS/pDNA complexes were in compacted spheroids and irregular lump of larger aggregates in structure, while the short rod- and toroid-like and donut shapes were found in CTS/DOTAP/pDNA complexes. The transfection efficiency of the lipopolyplexes showed higher GFP gene expression than DOTAP/pDNA and CTS/pDNA controls in Hep-2 and Hela cells, and luciferase gene expression 2-3-fold than DOTAP/pDNA control and 70-120-fold than CTS/pDNA control in Hep-2 cells. The intracellular trafficking was examined by confocal laser scanning microscopy. Rapid pDNA delivery to the nucleus enchanced by chitosan was achieved after 4 h transfection. PMID:22009568

  13. Physical Factors Affecting Plasmid DNA Compaction in Stearylamine-Containing Nanoemulsions Intended for Gene Delivery

    PubMed Central

    Silva, André Leandro; Júnior, Francisco Alexandrino; Verissimo, Lourena Mafra; Agnez-Lima, Lucymara Fassarella; Egito, Lucila Carmem Monte; de Oliveira, Anselmo Gomes; do Egito, Eryvaldo Socrates Tabosa

    2012-01-01

    Cationic lipids have been used in the development of non-viral gene delivery systems as lipoplexes. Stearylamine, a cationic lipid that presents a primary amine group when in solution, is able to compact genetic material by electrostatic interactions. In dispersed systems such as nanoemulsions this lipid anchors on the oil/water interface confering a positive charge to them. The aim of this work was to evaluate factors that influence DNA compaction in cationic nanoemulsions containing stearylamine. The influence of the stearylamine incorporation phase (water or oil), time of complexation, and different incubation temperatures were studied. The complexation rate was assessed by electrophoresis migration on agarose gel 0.7%, and nanoemulsion and lipoplex characterization was done by Dynamic Light Scattering (DLS). The results demonstrate that the best DNA compaction process occurs after 120 min of complexation, at low temperature (4 ± 1 °C), and after incorporation of the cationic lipid into the aqueous phase. Although the zeta potential of lipoplexes was lower than the results found for basic nanoemulsions, the granulometry did not change. Moreover, it was demonstrated that lipoplexes are suitable vehicles for gene delivery. PMID:24281666

  14. Influence of molecular weight upon mannosylated bio-synthetic hybrids for targeted antigen presenting cell gene delivery.

    PubMed

    Jones, Charles H; Gollakota, Akhila; Chen, Mingfu; Chung, Tai-Chun; Ravikrishnan, Anitha; Zhang, Guojian; Pfeifer, Blaine A

    2015-07-01

    Given the rise of antibiotic resistant microbes, genetic vaccination is a promising prophylactic strategy that enables rapid design and manufacture. Facilitating this process is the choice of vector, which is often situationally-specific and limited in engineering capacity. Furthermore, these shortcomings are usually tied to an incomplete understanding of the structure-function relationships driving vector-mediated gene delivery. Building upon our initial report of a hybrid bacterial-biomaterial gene delivery vector, a comprehensive structure-function assessment was completed using a class of mannosylated poly(beta-amino esters). Through a top-down screening methodology, an ideal polymer was selected on the basis of gene delivery efficacy and then used for the synthesis of a stratified molecular weight polymer library. By eliminating contributions of polymer chemical background, we were able to complete an in-depth assessment of gene delivery as a function of (1) polymer molecular weight, (2) relative mannose content, (3) polymer-membrane biophysical properties, (4) APC uptake specificity, and (5) serum inhibition. In summary, the flexibility and potential of the hybrid design featured in this work highlights the ability to systematically probe vector-associated properties for the development of translational gene delivery candidates.

  15. Cancer Screening by Systemic Administration of a Gene Delivery Vector Encoding Tumor-Selective Secretable Biomarker Expression

    PubMed Central

    Browne, Andrew W.; Leddon, Jennifer L.; Currier, Mark A.; Williams, Jon P.; Frischer, Jason S.; Collins, Margaret H.; Ahn, Chong H.; Cripe, Timothy P.

    2011-01-01

    Cancer biomarkers facilitate screening and early detection but are known for only a few cancer types. We demonstrated the principle of inducing tumors to secrete a serum biomarker using a systemically administered gene delivery vector that targets tumors for selective expression of an engineered cassette. We exploited tumor-selective replication of a conditionally replicative Herpes simplex virus (HSV) combined with a replication-dependent late viral promoter to achieve tumor-selective biomarker expression as an example gene delivery vector. Virus replication, cytotoxicity and biomarker production were low in quiescent normal human foreskin keratinocytes and high in cancer cells in vitro. Following intravenous injection of virus >90% of tumor-bearing mice exhibited higher levels of biomarker than non-tumor-bearing mice and upon necropsy, we detected virus exclusively in tumors. Our strategy of forcing tumors to secrete a serum biomarker could be useful for cancer screening in high-risk patients, and possibly for monitoring response to therapy. In addition, because oncolytic vectors for tumor specific gene delivery are cytotoxic, they may supplement our screening strategy as a “theragnostic” agent. The cancer screening approach presented in this work introduces a paradigm shift in the utility of gene delivery which we foresee being improved by alternative vectors targeting gene delivery and expression to tumors. Refining this approach will usher a new era for clinical cancer screening that may be implemented in the developed and undeveloped world. PMID:21589655

  16. Combinational Spinal GAD65 Gene Delivery and Systemic GABA-Mimetic Treatment for Modulation of Spasticity

    PubMed Central

    Kakinohana, Osamu; Hefferan, Michael P.; Miyanohara, Atsushi; Nejime, Tetsuya; Marsala, Silvia; Juhas, Stefan; Juhasova, Jana; Motlik, Jan; Kucharova, Karolina; Strnadel, Jan; Platoshyn, Oleksandr; Lazar, Peter; Galik, Jan; Vinay, Laurent; Marsala, Martin

    2012-01-01

    Background Loss of GABA-mediated pre-synaptic inhibition after spinal injury plays a key role in the progressive increase in spinal reflexes and the appearance of spasticity. Clinical studies show that the use of baclofen (GABAB receptor agonist), while effective in modulating spasticity is associated with major side effects such as general sedation and progressive tolerance development. The goal of the present study was to assess if a combined therapy composed of spinal segment-specific upregulation of GAD65 (glutamate decarboxylase) gene once combined with systemic treatment with tiagabine (GABA uptake inhibitor) will lead to an antispasticity effect and whether such an effect will only be present in GAD65 gene over-expressing spinal segments. Methods/Principal Findings Adult Sprague-Dawley (SD) rats were exposed to transient spinal ischemia (10 min) to induce muscle spasticity. Animals then received lumbar injection of HIV1-CMV-GAD65 lentivirus (LVs) targeting ventral α-motoneuronal pools. At 2–3 weeks after lentivirus delivery animals were treated systemically with tiagabine (4, 10, 20 or 40 mg/kg or vehicle) and the degree of spasticity response measured. In a separate experiment the expression of GAD65 gene after spinal parenchymal delivery of GAD65-lentivirus in naive minipigs was studied. Spastic SD rats receiving spinal injections of the GAD65 gene and treated with systemic tiagabine showed potent and tiagabine-dose-dependent alleviation of spasticity. Neither treatment alone (i.e., GAD65-LVs injection only or tiagabine treatment only) had any significant antispasticity effect nor had any detectable side effect. Measured antispasticity effect correlated with increase in spinal parenchymal GABA synthesis and was restricted to spinal segments overexpressing GAD65 gene. Conclusions/Significance These data show that treatment with orally bioavailable GABA-mimetic drugs if combined with spinal-segment-specific GAD65 gene overexpression can represent a novel

  17. Establishment of medakafish as a model for stem cell-based gene therapy: efficient gene delivery and potential chromosomal integration by baculoviral vectors.

    PubMed

    Yan, Yan; Du, Juan; Chen, Tiansheng; Yi, Meisheng; Li, Mingyou; Wang, Shu; Li, Chang Ming; Hong, Yunhan

    2009-08-01

    Viral vectors hold promise and challenges in gene therapy. Specifically, we have previously shown that baculoviral (BV) vectors have a high efficiency of gene delivery in human embryonic stem (ES) cells. Here we report the development of a complementary system to further our evaluation by utilizing the laboratory fish medaka that has ES cell lines and tools for experimental analyses in vitro and in vivo. We show that BV vectors can give rise to almost 100% of transient gene delivery in the medaka ES cell line MES1. BV-transduced MES1 cells reproducibly (at approximately 10(-5)) produce GFP-expressing colonies that, upon manual isolation, develop into stable clones during 300 days of culture. Surprisingly, BV transduction can also mediate efficient gene integration in the medaka genome, as fluorescent in situ hybridization revealed the presence of the BV-delivered gfp transgene in multiple locations in nuclei and on various chromosomes of metaphase spreads. We show that BV transduction does not compromise the genome stability and pluripotency of MES1 cells. We conclude that BV can efficiently mediate gene delivery and chromosomal integration in medaka ES cells. Therefore, medaka provides a powerful system for analyzing the potential of BV-mediated gene delivery in stem cells and gene therapy.

  18. A Review of Gene Delivery and Stem Cell Based Therapies for Regenerating Inner Ear Hair Cells

    PubMed Central

    Devarajan, Keerthana; Staecker, Hinrich; Detamore, Michael S.

    2011-01-01

    Sensory neural hearing loss and vestibular dysfunction have become the most common forms of sensory defects, affecting millions of people worldwide. Developing effective therapies to restore hearing loss is challenging, owing to the limited regenerative capacity of the inner ear hair cells. With recent advances in understanding the developmental biology of mammalian and non-mammalian hair cells a variety of strategies have emerged to restore lost hair cells are being developed. Two predominant strategies have developed to restore hair cells: transfer of genes responsible for hair cell genesis and replacement of missing cells via transfer of stem cells. In this review article, we evaluate the use of several genes involved in hair cell regeneration, the advantages and disadvantages of the different viral vectors employed in inner ear gene delivery and the insights gained from the use of embryonic, adult and induced pluripotent stem cells in generating inner ear hair cells. Understanding the role of genes, vectors and stem cells in therapeutic strategies led us to explore potential solutions to overcome the limitations associated with their use in hair cell regeneration. PMID:24956306

  19. Gene delivery to neuroblastoma cells by poly (l-lysine)-grafted low molecular weight polyethylenimine copolymers.

    PubMed

    Askarian, Saeedeh; Abnous, Khalil; Darroudi, Majid; Oskuee, Reza Kazemi; Ramezani, Mohammad

    2016-07-01

    Polyethylenimine (PEI) and poly (l-lysine) (PLL) are among the most investigated non-viral gene carriers. However, both polymers contain deficiencies that restrict their applications. In the present study, we synthesized PLL-alkyl-PEI conjugates via 6-carbon alkyl linker and investigated their possible advantages in gene delivery. Four PLL copolymers were synthesized with different molecular weights and ratios of PEI. The physiochemical properties of synthesized conjugates such as size, zeta potential, DNA condensation ability, buffering capacity and cytotoxicity were investigated. Renilla luciferase assay was employed to evaluate the gene transfection efficiency of pDNA-polymer to Neuro2A cell line. DNA condensation and particle size measurements showed that new PLL-PEI conjugates could form polyplexes in nano-scale size in the range of 99-122 nm and were able to condense DNA at low concentration. While cytotoxicity reduced in some groups, the transfection efficiency increased about 2.8 and 4 fold as compared to the unmodified PEI 1.8 kDa and 10 kDa, respectively. The results of the present study showed that the chemical modifications of PEI with PLL could significantly improve transfection efficiency and PLP10-10% shows the most promise as a new gene carrier. PMID:27118207

  20. TITER AND PRODUCT AFFECTS THE DISTRIBUTION OF GENE EXPRESSION AFTER INTRAPUTAMINAL CONVECTION-ENHANCED DELIVERY

    PubMed Central

    Emborg, Marina E.; Hurley, Samuel A.; Joers, Valerie; Tromp, Do P.M.; Swanson, Christine R.; Ohshima-Hosoyama, Sachiko; Bondarenko, Viktorya; Cummisford, Kyle; Sonnemans, Marc; Hermening, Stephan; Blits, Bas; Alexander, Andrew L.

    2014-01-01

    Background Efficacy and safety of intracerebral gene therapy for brain disorders, like Parkinson’s disease, depends on appropriate distribution of gene expression. Objectives To assess if the distribution of gene expression is affected by vector titer and protein type. Methods Four adult macaque monkeys seronegative for adeno-associated virus 5 (AAV5) received in the right and left ventral postcommisural putamen 30μl inoculation of a high or low titer suspension of AAV5 encoding glial derived neurotrophic factor (GDNF) or green fluorescent protein (GFP). Inoculations were performed using convection enhanced delivery and intraoperative MRI (IMRI). Results IMRI confirmed targeting and infusion cloud irradiating from the catheter tip into surrounding area. Postmortem analysis six weeks after surgery revealed GFP and GDNF expression ipsilateral to the injection side that had a titer-dependent distribution. GFP and GDNF expression was also observed in fibers in the Substantia Nigra (SN) pars reticulata (pr), demonstrating anterograde transport. Few GFP-positive neurons were present in the SN pars compacta (pc), possibly by direct retrograde transport of the vector. GDNF was present in many SNpc and SNpr neurons. Conclusions After controlling for target and infusate volume, intracerebral distribution of gene product is affected by vector titer and product biology. PMID:24943657

  1. Magnetic nanoparticles as gene delivery agents: enhanced transfection in the presence of oscillating magnet arrays

    NASA Astrophysics Data System (ADS)

    McBain, S. C.; Griesenbach, U.; Xenariou, S.; Keramane, A.; Batich, C. D.; Alton, E. W. F. W.; Dobson, J.

    2008-10-01

    Magnetic nanoparticle-based gene transfection has been shown to be effective in combination with both viral vectors and with non-viral agents. In these systems, therapeutic or reporter genes are attached to magnetic nanoparticles which are then focused to the target site/cells via high-field/high-gradient magnets. The technique has been shown to be efficient and rapid for in vitro transfection and compares well with cationic lipid-based reagents, producing good overall transfection levels with lower doses and shorter transfection times. In spite of its potential advantages (particularly for in vivo targeting), the overall transfection levels do not generally exceed those of other non-viral agents. In order to improve the overall transfection levels while maintaining the advantages inherent in this technique, we have developed a novel, oscillating magnet array system which adds lateral motion to the particle/gene complex in order to promote transfection. Experimental results indicate that the system significantly enhances overall in vitro transfection levels in human airway epithelial cells compared to both static field techniques (p<0.005) and the cationic lipids (p<0.001) tested. In addition, it has the previously demonstrated advantages of magnetofection—rapid transfection times and requiring lower levels of DNA than cationic lipid-based transfection agents. This method shows potential for non-viral gene delivery both in vitro and in vivo.

  2. Controlled Striatal DOPA Production From a Gene Delivery System in a Rodent Model of Parkinson's Disease

    PubMed Central

    Cederfjäll, Erik; Broom, Lauren; Kirik, Deniz

    2015-01-01

    Conventional symptomatic treatment for Parkinson's disease (PD) with long-term L-3,4-dihydroxyphenylalanine (DOPA) is complicated with development of drug-induced side effects. In vivo viral vector-mediated gene expression encoding tyrosine hydroxylase (TH) and GTP cyclohydrolase 1 (GCH1) provides a drug delivery strategy of DOPA with distinct advantages over pharmacotherapy. Since the brain alterations made with current gene transfer techniques are irreversible, the therapeutic approaches taken to the clinic should preferably be controllable to match the needs of each individual during the course of their disease. We used a recently described tunable gene expression system based on the use of destabilized dihydrofolate reductase (DD) and generated a N-terminally coupled GCH1 enzyme (DD-GCH1) while the TH enzyme was constitutively expressed, packaged in adeno-associated viral (AAV) vectors. Expression of DD-GCH1 was regulated by the activating ligand trimethoprim (TMP) that crosses the blood–brain barrier. We show that the resulting intervention provides a TMP-dose-dependent regulation of DOPA synthesis that is closely linked to the magnitude of functional effects. Our data constitutes the first proof of principle for controlled reconstitution of dopamine capacity in the brain and suggests that such next-generation gene therapy strategies are now mature for preclinical development toward use in patients with PD. PMID:25592335

  3. Human Erythropoietin Gene Delivery Using an Arginine-grafted Bioreducible Polymer System

    PubMed Central

    Lee, Youngsook; Nam, Hye Yeong; Kim, Jaesung; Lee, Minhyung; Yockman, James W; Shin, Sug Kyun; Kim, Sung Wan

    2012-01-01

    Erythropoiesis-stimulating agents are widely used to treat anemia for chronic kidney disease (CKD) and cancer, however, several clinical limitations impede their effectiveness. Nonviral gene therapy systems are a novel solution to these problems as they provide stable and low immunogenic protein expression levels. Here, we show the application of an arginine-grafted bioreducible poly(disulfide amine) (ABP) polymer gene delivery system as a platform for in vivo transfer of human erythropoietin plasmid DNA (phEPO) to produce long-term, therapeutic erythropoiesis. A single systemic injection of phEPO/ABP polyplex led to higher hematocrit levels over a 60-day period accompanied with reticulocytosis and high hEPO protein expression. In addition, we found that the distinct temporal and spatial distribution of phEPO/ABP polyplexes contributed to increased erythropoietic effects compared to those of traditional EPO therapies. Overall, our study suggests that ABP polymer-based gene therapy provides a promising clinical strategy to reach effective therapeutic levels of hEPO gene. PMID:22472948

  4. Anti-interleukin-6 therapy through application of a monogenic protein inhibitor via gene delivery

    PubMed Central

    Görtz, Dieter; Braun, Gerald S.; Maruta, Yuichi; Djudjaj, Sonja; van Roeyen, Claudia R.; Martin, Ina V.; Küster, Andrea; Schmitz-Van de Leur, Hildegard; Scheller, Jürgen; Ostendorf, Tammo; Floege, Jürgen; Müller-Newen, Gerhard

    2015-01-01

    Anti-cytokine therapies have substantially improved the treatment of inflammatory and autoimmune diseases. Cytokine-targeting drugs are usually biologics such as antibodies or other engineered proteins. Production of biologics, however, is complex and intricate and therefore expensive which might limit therapeutic application. To overcome this limitation we developed a strategy that involves the design of an optimized, monogenic cytokine inhibitor and the protein producing capacity of the host. Here, we engineered and characterized a receptor fusion protein, mIL-6-RFP-Fc, for the inhibition of interleukin-6 (IL-6), a well-established target in anti-cytokine therapy. Upon application in mice mIL-6-RFP-Fc inhibited IL-6-induced activation of the transcription factor STAT3 and ERK1/2 kinases in liver and kidney. mIL-6-RFP-Fc is encoded by a single gene and therefore most relevant for gene transfer approaches. Gene transfer through hydrodynamic plasmid delivery in mice resulted in hepatic production and secretion of mIL-6-RFP-Fc into the blood in considerable amounts, blocked hepatic acute phase protein synthesis and improved kidney function in an ischemia and reperfusion injury model. Our study establishes receptor fusion proteins as promising agents in anti-cytokine therapies through gene therapeutic approaches for future targeted and cost-effective treatments. The strategy described here is applicable for many cytokines involved in inflammatory and other diseases. PMID:26423228

  5. Anti-interleukin-6 therapy through application of a monogenic protein inhibitor via gene delivery.

    PubMed

    Görtz, Dieter; Braun, Gerald S; Maruta, Yuichi; Djudjaj, Sonja; van Roeyen, Claudia R; Martin, Ina V; Küster, Andrea; Schmitz-Van de Leur, Hildegard; Scheller, Jürgen; Ostendorf, Tammo; Floege, Jürgen; Müller-Newen, Gerhard

    2015-01-01

    Anti-cytokine therapies have substantially improved the treatment of inflammatory and autoimmune diseases. Cytokine-targeting drugs are usually biologics such as antibodies or other engineered proteins. Production of biologics, however, is complex and intricate and therefore expensive which might limit therapeutic application. To overcome this limitation we developed a strategy that involves the design of an optimized, monogenic cytokine inhibitor and the protein producing capacity of the host. Here, we engineered and characterized a receptor fusion protein, mIL-6-RFP-Fc, for the inhibition of interleukin-6 (IL-6), a well-established target in anti-cytokine therapy. Upon application in mice mIL-6-RFP-Fc inhibited IL-6-induced activation of the transcription factor STAT3 and ERK1/2 kinases in liver and kidney. mIL-6-RFP-Fc is encoded by a single gene and therefore most relevant for gene transfer approaches. Gene transfer through hydrodynamic plasmid delivery in mice resulted in hepatic production and secretion of mIL-6-RFP-Fc into the blood in considerable amounts, blocked hepatic acute phase protein synthesis and improved kidney function in an ischemia and reperfusion injury model. Our study establishes receptor fusion proteins as promising agents in anti-cytokine therapies through gene therapeutic approaches for future targeted and cost-effective treatments. The strategy described here is applicable for many cytokines involved in inflammatory and other diseases.

  6. Global brain delivery of neprilysin gene by intravascular administration of AAV vector in mice

    PubMed Central

    Iwata, Nobuhisa; Sekiguchi, Misaki; Hattori, Yoshino; Takahashi, Akane; Asai, Masashi; Ji, Bin; Higuchi, Makoto; Staufenbiel, Matthias; Muramatsu, Shin-ichi; Saido, Takaomi C.

    2013-01-01

    Accumulation of amyloid-β peptide (Aβ) in the brain is closely associated with cognitive decline in Alzheimer's disease (AD). Stereotaxic infusion of neprilysin-encoding viral vectors into the hippocampus has been shown to decrease Aβ in AD-model mice, but more efficient and global delivery is necessary to treat the broadly distributed burden in AD. Here we developed an adeno-associated virus (AAV) vector capable of providing neuronal gene expression throughout the brains after peripheral administration. A single intracardiac administration of the vector carrying neprilysin gene in AD-model mice elevated neprilysin activity broadly in the brain, and reduced Aβ oligomers, with concurrent alleviation of abnormal learning and memory function and improvement of amyloid burden. The exogenous neprilysin was localized mainly in endosomes, thereby effectively excluding Aβ oligomers from the brain. AAV vector-mediated gene transfer may provide a therapeutic strategy for neurodegenerative diseases, where global transduction of a therapeutic gene into the brain is necessary. PMID:23503602

  7. Enhanced gene delivery using Bubble liposomes and ultrasound for folate-PEG liposomes.

    PubMed

    Omata, Daiki; Negishi, Yoichi; Hagiwara, Shoko; Yamamura, Sho; Endo-Takahashi, Yoko; Suzuki, Ryo; Maruyama, Kazuo; Aramaki, Yukihiko

    2012-05-01

    We have previously reported that the transfection efficiency of laminin-derived AG73-peptide labeled polyethyleneglycol-modified liposomes (AG73-PEG liposomes) was enhanced by echo-contrast gas entrapping PEG liposomes (Bubble liposomes, BLs) and ultrasound (US) exposure by improving endosomal escape. However, it has not been well understood whether BLs and US exposure can enhance the transfection efficiency of other carriers except AG73-PEG liposomes. In this study, to evaluate whether BLs and US exposure can be generally applied to gene delivery carriers, we focused on folate as a model ligand and examined whether BLs and US exposure could enhance the transfection efficiency of folate-PEG liposomes. Folate-PEG liposomes could internalize into cells efficiently, whereas they could not deliver genes into cytosol from endosomes sufficiently. BLs and US exposure could enhance the transfection efficiency of folate-PEG liposomes compared with folate-PEG liposomes alone without their direct induction into cells. These results suggested that BLs and US exposure could enhance the transfection efficiency of folate-PEG liposomes in the same manner as AG73-PEG liposomes. Thus, BLs and US exposure may be a promising tool to achieve efficient gene transfection into various gene carriers in general.

  8. Sleeping Beauty Transposon Vectors in Liver-directed Gene Delivery of LDLR and VLDLR for Gene Therapy of Familial Hypercholesterolemia.

    PubMed

    Turunen, Tytteli A K; Kurkipuro, Jere; Heikura, Tommi; Vuorio, Taina; Hytönen, Elisa; Izsvák, Zsuzsanna; Ylä-Herttuala, Seppo

    2016-03-01

    Plasmid-based Sleeping Beauty (SB) transposon vectors were developed and used to deliver genes for low-density lipoprotein and very-low-density lipoprotein receptors (LDLR and VLDLR, respectively) or lacZ reporter into liver of an LDLR-deficient mouse model of familial hypercholesterolemia (FH). SB transposase, SB100x, was used to integrate the therapeutic transposons into mice livers for evaluating the feasibility of the vectors in reducing high blood cholesterol and the progression of atherosclerosis. Hydrodynamic gene delivery of transposon-VLDLR into the livers of the mice resulted in initial 17-19% reductions in plasma cholesterol, and at the later time points, in a significant stabilization of the cholesterol level for the 6.5-month duration of the study compared to the control mice. Transposon-LDLR-treated animals also demonstrated a trend of stabilization in the cholesterol levels in the long term. Vector-treated mice had slightly less lipid accumulation in the liver and reduced aortic atherosclerosis. Clinical chemistry and histological analyses revealed normal liver function and morphology comparable to that of the controls during the follow-up with no safety issues regarding the vector type, transgenes, or the gene transfer method. The study demonstrates the safety and potential benefits of the SB transposon vectors in the treatment of FH.

  9. Sleeping Beauty Transposon Vectors in Liver-directed Gene Delivery of LDLR and VLDLR for Gene Therapy of Familial Hypercholesterolemia.

    PubMed

    Turunen, Tytteli A K; Kurkipuro, Jere; Heikura, Tommi; Vuorio, Taina; Hytönen, Elisa; Izsvák, Zsuzsanna; Ylä-Herttuala, Seppo

    2016-03-01

    Plasmid-based Sleeping Beauty (SB) transposon vectors were developed and used to deliver genes for low-density lipoprotein and very-low-density lipoprotein receptors (LDLR and VLDLR, respectively) or lacZ reporter into liver of an LDLR-deficient mouse model of familial hypercholesterolemia (FH). SB transposase, SB100x, was used to integrate the therapeutic transposons into mice livers for evaluating the feasibility of the vectors in reducing high blood cholesterol and the progression of atherosclerosis. Hydrodynamic gene delivery of transposon-VLDLR into the livers of the mice resulted in initial 17-19% reductions in plasma cholesterol, and at the later time points, in a significant stabilization of the cholesterol level for the 6.5-month duration of the study compared to the control mice. Transposon-LDLR-treated animals also demonstrated a trend of stabilization in the cholesterol levels in the long term. Vector-treated mice had slightly less lipid accumulation in the liver and reduced aortic atherosclerosis. Clinical chemistry and histological analyses revealed normal liver function and morphology comparable to that of the controls during the follow-up with no safety issues regarding the vector type, transgenes, or the gene transfer method. The study demonstrates the safety and potential benefits of the SB transposon vectors in the treatment of FH. PMID:26670130

  10. The structure of AAVrh32.33, a Novel Gene Delivery Vector

    PubMed Central

    Mikals, Kyle; Nam, Hyun-Joo; Vliet, Kim Van; Vandenberghe, Luk H.; Mays, Lauren E.; McKenna, Robert; Wilson, James M.; Agbandje-McKenna, Mavis

    2014-01-01

    The Adeno-Associated viruses (AAVs) are being developed as gene delivery vectors for therapeutic clinical applications. However, the host antibody immune response directed against their capsid, prevalent in ~40–70% of the general population, depending on serotype, negatively impacts efficacy. AAVrh32.33, a novel vector developed from rhesus macaques isolates, has significantly lower seroprevalence in human populations compared to AAV2 and AAV8, which are both in clinical use. To better understand the capsid determinants of this differential immune response to AAVrh32.33, its structure was determined by X-ray crystallography to 3.5 Å resolution. The capsid viral protein (VP) structure conserves the eight-stranded β-barrel core and αA helix reported for other parvoviruses and the distinct capsid surface topology of the AAVs: a depression at the icosahedral two-fold axis, three protrusions surrounding the three-fold axis, and a depression surround a cylindrical channel at the five-fold axis. A comparison to AAV2, AAV4, and AAV8, to which AAVrh32.33 shares ~61%, ~81%, and ~63% identity, respectively, identified differences in previously defined AAV VP structurally variable regions (VR-1 to VR-IX) which function as receptor attachment, transduction efficiency, and/or antigenic determinants. This structure thus provides a 3D platform for capsid engineering in ongoing efforts to develop AAVrh32.33, as well as other AAV serotypes, for tissue targeted gene-therapy applications with vectors that can evade pre-existing antibody responses against the capsid. These features are required for full clinical realization of the promising AAV gene delivery system. PMID:24704217

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

    PubMed

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

    2011-05-30

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

  12. Robust Lentiviral Gene Delivery But Limited Transduction Capacity of Commonly Used Adeno-Associated Viral Serotypes in Xenotransplanted Human Skin.

    PubMed

    Jakobsen, Maria; Askou, Anne Louise; Stenderup, Karin; Rosada, Cecilia; Dagnæs-Hansen, Frederik; Jensen, Thomas G; Corydon, Thomas J; Mikkelsen, Jacob Giehm; Aagaard, Lars

    2015-08-01

    Skin is an easily accessible organ, and therapeutic gene transfer to skin remains an attractive alternative for the treatment of skin diseases. Although we have previously documented potent lentiviral gene delivery to human skin, vectors based on adeno-associated virus (AAV) rank among the most promising gene delivery tools for in vivo purposes. Thus, we compared the potential usefulness of various serotypes of recombinant AAV vectors and lentiviral vectors for gene transfer to human skin in a xenotransplanted mouse model. Vector constructs encoding firefly luciferase were packaged in AAV capsids of serotype 1, 2, 5, 6, 8, and 9 and separately administered by intradermal injection in human skin transplants. For all serotypes, live bioimaging demonstrated low levels of transgene expression in the human skin graft, and firefly luciferase expression was observed primarily in neighboring tissue outside of the graft. In contrast, gene delivery by intradermally injected lentiviral vectors was efficient and led to extensive and persistent firefly luciferase expression within the human skin graft only. The study demonstrates the limited capacity of single-stranded AAV vectors of six commonly used serotypes for gene delivery to human skin in vivo. PMID:26204415

  13. Gene Therapy Delivery Systems for Enhancing Viral and Nonviral Vectors for Cardiac Diseases: Current Concepts and Future Applications

    PubMed Central

    Katz, Michael G.; Fargnoli, Anthony S.; Williams, Richard D.

    2013-01-01

    Abstract Gene therapy is one of the most promising fields for developing new treatments for the advanced stages of ischemic and monogenetic, particularly autosomal or X-linked recessive, cardiomyopathies. The remarkable ongoing efforts in advancing various targets have largely been inspired by the results that have been achieved in several notable gene therapy trials, such as the hemophilia B and Leber's congenital amaurosis. Rate-limiting problems preventing successful clinical application in the cardiac disease area, however, are primarily attributable to inefficient gene transfer, host responses, and the lack of sustainable therapeutic transgene expression. It is arguable that these problems are directly correlated with the choice of vector, dose level, and associated cardiac delivery approach as a whole treatment system. Essentially, a delicate balance exists in maximizing gene transfer required for efficacy while remaining within safety limits. Therefore, the development of safe, effective, and clinically applicable gene delivery techniques for selected nonviral and viral vectors will certainly be invaluable in obtaining future regulatory approvals. The choice of gene transfer vector, dose level, and the delivery system are likely to be critical determinants of therapeutic efficacy. It is here that the interactions between vector uptake and trafficking, delivery route means, and the host's physical limits must be considered synergistically for a successful treatment course. PMID:24164239

  14. Gene therapy delivery systems for enhancing viral and nonviral vectors for cardiac diseases: current concepts and future applications.

    PubMed

    Katz, Michael G; Fargnoli, Anthony S; Williams, Richard D; Bridges, Charles R

    2013-11-01

    Gene therapy is one of the most promising fields for developing new treatments for the advanced stages of ischemic and monogenetic, particularly autosomal or X-linked recessive, cardiomyopathies. The remarkable ongoing efforts in advancing various targets have largely been inspired by the results that have been achieved in several notable gene therapy trials, such as the hemophilia B and Leber's congenital amaurosis. Rate-limiting problems preventing successful clinical application in the cardiac disease area, however, are primarily attributable to inefficient gene transfer, host responses, and the lack of sustainable therapeutic transgene expression. It is arguable that these problems are directly correlated with the choice of vector, dose level, and associated cardiac delivery approach as a whole treatment system. Essentially, a delicate balance exists in maximizing gene transfer required for efficacy while remaining within safety limits. Therefore, the development of safe, effective, and clinically applicable gene delivery techniques for selected nonviral and viral vectors will certainly be invaluable in obtaining future regulatory approvals. The choice of gene transfer vector, dose level, and the delivery system are likely to be critical determinants of therapeutic efficacy. It is here that the interactions between vector uptake and trafficking, delivery route means, and the host's physical limits must be considered synergistically for a successful treatment course.

  15. Enhanced thermogenic program by non-viral delivery of combinatory browning genes to treat diet-induced obesity in mice.

    PubMed

    Park, Hongsuk; Cho, Sungpil; Janat-Amsbury, Margit M; Bae, You Han

    2015-12-01

    Thermogenic program (also known as browning) is a promising and attractive anti-obesity approach. Islet amyloid polypeptide (IAPP) and irisin have emerged as potential browning hormones that hold high potential to treat obesity. Here, we have constructed a dual browning gene system containing both IAPP and irisin (derived from fibronectin type III domain containing 5; FNDC5) combined with 2A and furin self-cleavage sites. Intraperitoneal administration of the construct complexed with a linear polyethylenimine into diet-induced obese mice demonstrated the elevation of anti-obesogenic effects characterized as the decreased body weight, adiposity, and levels of glucose and insulin. In addition, the construct delivery increased energy expenditure and the expression of core molecular determinants associated with browning. The additional advantages of the dual browning gene construct delivery compared to both single gene construct delivery and dual peptide delivery can be emphasized on efficacy and practicability. Hence, we have concluded that dual browning gene delivery makes it therapeutically attractive for diet-induced obesity treatment.

  16. Association with Amino Acids Does Not Enhance Efficacy of Polymerized Liposomes As a System for Lung Gene Delivery.

    PubMed

    Bandeira, Elga; Lopes-Pacheco, Miquéias; Chiaramoni, Nadia; Ferreira, Débora; Fernandez-Ruocco, Maria J; Prieto, Maria J; Maron-Gutierrez, Tatiana; Perrotta, Ramiro M; de Castro-Faria-Neto, Hugo C; Rocco, Patricia R M; Alonso, Silvia Del Valle; Morales, Marcelo M

    2016-01-01

    Development of improved drug and gene delivery systems directly into the lungs is highly desirable given the important burden of respiratory diseases. We aimed to evaluate the safety and efficacy of liposomes composed of photopolymerized lipids [1,2-bis-(tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine] associated with amino acids as vectors for gene delivery into the lungs of healthy animals. Lipopolymer vesicles, in particular, are more stable than other types of liposomes. In this study, lipopolymers were associated with l-arginine, l-tryptophan, or l-cysteine. We hypothesized that the addition of these amino acids would enhance the efficacy of gene delivery to the lungs by the lipopolymers. l-Arginine showed the highest association efficiency due to its positive charge and better surface interactions. None of the formulations caused inflammation or altered lung mechanics, suggesting that these lipopolymers can be safely administered as aerosols. All formulations were able to induce eGFP mRNA expression in lung tissue, but the addition of amino acids reduced delivery efficacy when compared with the simple lipopolymer particle. These results indicate that this system could be further explored for gene or drug delivery targeting lung diseases. PMID:27199766

  17. The transition from linear to highly branched poly(β-amino ester)s: Branching matters for gene delivery.

    PubMed

    Zhou, Dezhong; Cutlar, Lara; Gao, Yongsheng; Wang, Wei; O'Keeffe-Ahern, Jonathan; McMahon, Sean; Duarte, Blanca; Larcher, Fernando; Rodriguez, Brian J; Greiser, Udo; Wang, Wenxin

    2016-06-01

    Nonviral gene therapy holds great promise but has not delivered treatments for clinical application to date. Lack of safe and efficient gene delivery vectors is the major hurdle. Among nonviral gene delivery vectors, poly(β-amino ester)s are one of the most versatile candidates because of their wide monomer availability, high polymer flexibility, and superior gene transfection performance both in vitro and in vivo. However, to date, all research has been focused on vectors with a linear structure. A well-accepted view is that dendritic or branched polymers have greater potential as gene delivery vectors because of their three-dimensional structure and multiple terminal groups. Nevertheless, to date, the synthesis of dendritic or branched polymers has been proven to be a well-known challenge. We report the design and synthesis of highly branched poly(β-amino ester)s (HPAEs) via a one-pot "A2 + B3 + C2"-type Michael addition approach and evaluate their potential as gene delivery vectors. We find that the branched structure can significantly enhance the transfection efficiency of poly(β-amino ester)s: Up to an 8521-fold enhancement in transfection efficiency was observed across 12 cell types ranging from cell lines, primary cells, to stem cells, over their corresponding linear poly(β-amino ester)s (LPAEs) and the commercial transfection reagents polyethyleneimine, SuperFect, and Lipofectamine 2000. Moreover, we further demonstrate that HPAEs can correct genetic defects in vivo using a recessive dystrophic epidermolysis bullosa graft mouse model. Our findings prove that the A2 + B3 + C2 approach is highly generalizable and flexible for the design and synthesis of HPAEs, which cannot be achieved by the conventional polymerization approach; HPAEs are more efficient vectors in gene transfection than the corresponding LPAEs. This provides valuable insight into the development and applications of nonviral gene delivery and demonstrates great prospect for their

  18. Systemic gene delivery following intravenous administration of AAV9 to fetal and neonatal mice and late-gestation nonhuman primates.

    PubMed

    Mattar, Citra N; Wong, Andrew M S; Hoefer, Klemens; Alonso-Ferrero, Maria E; Buckley, Suzanne M K; Howe, Steven J; Cooper, Jonathan D; Waddington, Simon N; Chan, Jerry K Y; Rahim, Ahad A

    2015-09-01

    Several acute monogenic diseases affect multiple body systems, causing death in childhood. The development of novel therapies for such conditions is challenging. However, improvements in gene delivery technology mean that gene therapy has the potential to treat such disorders. We evaluated the ability of the AAV9 vector to mediate systemic gene delivery after intravenous administration to perinatal mice and late-gestation nonhuman primates (NHPs). Titer-matched single-stranded (ss) and self-complementary (sc) AAV9 carrying the green fluorescent protein (GFP) reporter gene were intravenously administered to fetal and neonatal mice, with noninjected age-matched mice used as the control. Extensive GFP expression was observed in organs throughout the body, with the epithelial and muscle cells being particularly well transduced. ssAAV9 carrying the WPRE sequence mediated significantly more gene expression than its sc counterpart, which lacked the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) sequence. To examine a realistic scale-up to larger models or potentially patients for such an approach, AAV9 was intravenously administered to late-gestation NHPs by using a clinically relevant protocol. Widespread systemic gene expression was measured throughout the body, with cellular tropisms similar to those observed in the mouse studies and no observable adverse events. This study confirms that AAV9 can safely mediate systemic gene delivery in small and large animal models and supports its potential use in clinical systemic gene therapy protocols. PMID:26062602

  19. Anionic Lipid, pH-Sensitive Liposome-Gold Nanoparticle Hybrids for Gene Delivery - Quantitative Research of the Mechanism.

    PubMed

    Du, Baoji; Tian, Li; Gu, Xiaoxiao; Li, Dan; Wang, Erkang; Wang, Jin

    2015-05-20

    Gene therapy is a potential method for treating a large range of diseases. Gene vectors are widely used in gene therapy for promoting the gene delivery efficiency to the target cells. Here, gold nanoparticles (AuNPs) coated with dimethyldioctadecylammonium bromide (DODAB)/dioleoylphosphatidylethanolamine (DOPE) are synthesized using a facile method for a new gene vector (DODAB/DOPE-AuNPs), which possess 3- and 1.5-fold higher transfection efficiency than those of DODAB-AuNPs and a commercial transfection agent, respectively. Meanwhile, it is nontoxic with concentrations required for effective gene delivery. Imaging and quantification studies of cellular uptake reveal that DOPE increases gene copies in cells, which may be attributed to the smaller size of AuNPs/DNA complexes. The dissociation efficiency of DNA from the endocytic pathway is quantified by incubating with different buffers and investigated directly in the cells. The results suggest that DOPE increases the internalization of AuNPs/DNA complexes and promotes DNA release from early endosomes for the vector is sensitive to the anionic lipid membrane and the decreasing pH along the endocytic pathway. The new vector contains the potential to be the new alternative as gene delivery vector for biomedical applications. PMID:25594807

  20. Preparation and testing of quaternized chitosan nanoparticles as gene delivery vehicles.

    PubMed

    Li, Guang-Feng; Wang, Jing-Cheng; Feng, Xin-Min; Liu, Zhen-Dong; Jiang, Chao-Yong; Yang, Jian-Dong

    2015-04-01

    The aim of this study was to synthesize a chitosan (CS) derivative, a quaternary ammonium salt crystal called N-2-hydroxypropyl trimethyl ammonium chloride chitosan (HACC), and test a series of HACC and pEGFP-DNA complexes at different weight ratios for their efficiency of gene delivery into human cells. CS was modified with cationic etherifying agent to obtain the CS derivative. Fourier transform infrared spectra were recorded on KBr pellets with a spectrometer. (1)H nuclear magnetic resonance (NMR) spectra of HACC were obtained using a spectrometer. HACC was subsequently used to prepare HACC/DNA complexes at different weight ratios by coacervation method. The resulting particle size and surface charge were assessed by laser light scattering using a zeta potential analyzer. The HACC/DNA complex formation and DNA protection in the nanoparticle complex was investigated by gel mobility shift assay and DNase I protection assay, respectively. The cytotoxicity of HACC and HACC/DNA nanoparticles was evaluated by MTT assay using (mesenchymal stem cell) MSC lines. The nanoscale structure of the particles was obtained by transmission electron microscope (TEM). The FTIR spectrum of HACC showed the characteristic quaternary ammonium group absorption band at 1475 cm(-1), which indicated the presence of quaternary ammonium group. The successful synthesis of HACC was also confirmed by (1)H NMR spectrum. HACC showed good solubility in water and was electropositive. HACC efficiently packed and protected pEGFP-DNA at a weight ratio of 10. With increased weight ratios, the surface charge of the composite particle increased from negative to positive, the average particle size increased, and HACC nanoparticle had a higher carrying efficiency. The nanoparticles released DNA in two distinct phases, and 55 % was released within the first 20 h of solubilization. The nanoparticles under TEM showed circular or oval shapes. The particles exhibited no cytotoxicity against human cells. No

  1. Preparation and testing of quaternized chitosan nanoparticles as gene delivery vehicles.

    PubMed

    Li, Guang-Feng; Wang, Jing-Cheng; Feng, Xin-Min; Liu, Zhen-Dong; Jiang, Chao-Yong; Yang, Jian-Dong

    2015-04-01

    The aim of this study was to synthesize a chitosan (CS) derivative, a quaternary ammonium salt crystal called N-2-hydroxypropyl trimethyl ammonium chloride chitosan (HACC), and test a series of HACC and pEGFP-DNA complexes at different weight ratios for their efficiency of gene delivery into human cells. CS was modified with cationic etherifying agent to obtain the CS derivative. Fourier transform infrared spectra were recorded on KBr pellets with a spectrometer. (1)H nuclear magnetic resonance (NMR) spectra of HACC were obtained using a spectrometer. HACC was subsequently used to prepare HACC/DNA complexes at different weight ratios by coacervation method. The resulting particle size and surface charge were assessed by laser light scattering using a zeta potential analyzer. The HACC/DNA complex formation and DNA protection in the nanoparticle complex was investigated by gel mobility shift assay and DNase I protection assay, respectively. The cytotoxicity of HACC and HACC/DNA nanoparticles was evaluated by MTT assay using (mesenchymal stem cell) MSC lines. The nanoscale structure of the particles was obtained by transmission electron microscope (TEM). The FTIR spectrum of HACC showed the characteristic quaternary ammonium group absorption band at 1475 cm(-1), which indicated the presence of quaternary ammonium group. The successful synthesis of HACC was also confirmed by (1)H NMR spectrum. HACC showed good solubility in water and was electropositive. HACC efficiently packed and protected pEGFP-DNA at a weight ratio of 10. With increased weight ratios, the surface charge of the composite particle increased from negative to positive, the average particle size increased, and HACC nanoparticle had a higher carrying efficiency. The nanoparticles released DNA in two distinct phases, and 55 % was released within the first 20 h of solubilization. The nanoparticles under TEM showed circular or oval shapes. The particles exhibited no cytotoxicity against human cells. No

  2. Regulated Non-Viral Gene Delivery from Coaxial Electrospun Fiber Mesh Scaffolds

    PubMed Central

    Saraf, Anita; Baggett, L. Scott; Raphael, Robert M.; Kasper, F. Kurtis; Mikos, Antonios G.

    2009-01-01

    In an effort to add to the versatility of three-dimensional scaffolds for tissue engineering applications, recent experimental designs are incorporating biological molecules such as plasmids and proteins within the scaffold structure. Such scaffolds act as reservoirs for the biological molecules of interest while regulating their release over various durations of time. Here, we describe the use of coaxial electrospinning as a means for the fabrication of fiber mesh scaffolds and the encapsulation and subsequent release of a non-viral gene delivery vector over a period of up to 60 days. Various fiber mesh scaffolds containing plasmid DNA (pDNA) within the core and the non-viral gene delivery vector poly(ethylenimine)-hyaluronic acid (PEI-HA) within the sheath of coaxial fibers were fabricated based on a fractional factorial design that investigated the effects of four processing parameters at two levels. Poly(ε-caprolactone) sheath polymer concentration, poly(ethylene glycol) core polymer molecular weight and concentration, and the concentration of pDNA were investigated for their effects on average fiber diameter, release kinetics of PEI-HA, and transfection efficiency. It was determined that increasing the values of each of the investigated parameters caused an increase in the average diameter of the fibers. The release kinetics of PEI-HA from the fibers were affected by the loading concentration of pDNA (with PEI-HA concentration adjusted accordingly to maintain a constant nitrogen to phosphorous (N:P) ratio within the complexes). Two-dimensional cell culture experiments with model fibroblast-like cells demonstrated that complexes of pDNA with PEI-HA released from fiber mesh scaffolds could successfully transfect cells and induce expression of enhanced green fluorescent protein (EGFP). Peak EGFP expression varied with the investigated processing parameters, and the average transfection observed was a function of poly(ethylene glycol) (core) molecular weight and

  3. Functionalized nanomaterials: are they effective to perform gene delivery to difficult-to-transfect cells with no cytotoxicity?

    NASA Astrophysics Data System (ADS)

    Tonelli, F. M. P.; Lacerda, S. M. S. N.; Paiva, N. C. O.; Pacheco, F. G.; Scalzo Junior, S. R. A.; de Macedo, F. H. P.; Cruz, J. S.; Pinto, M. C. X.; Junior, J. D. Corrêa; Ladeira, L. O.; França, L. R.; Guatimosim, S.; Resende, R. R.

    2015-10-01

    Nanodiamonds (NDs), multiwalled carbon nanotubes (MWCNTs) and gold nanorods (NRs) can be functionalized to promote gene delivery to hard-to-transfect cells with higher transfection efficiency than cationic lipids, and inducing less cell death.Nanodiamonds (NDs), multiwalled carbon nanotubes (MWCNTs) and gold nanorods (NRs) can be functionalized to promote gene delivery to hard-to-transfect cells with higher transfection efficiency than cationic lipids, and inducing less cell death. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr04173b

  4. Enhanced gene delivery to the lung using biodegradable polyunsaturated cationic phosphatidylcholine-detergent conjugates.

    PubMed

    Pierrat, Philippe; Kereselidze, Dimitri; Lux, Marie; Lebeau, Luc; Pons, Françoise

    2016-09-10

    Lung diseases are among the more representative causes of mortality and morbidity worldwide and gene therapy is considered as a promising therapeutic approach for their treatment. However the design of efficient nucleic acid carriers for airway administration still is a challenge and there is a pressing need for new developments in this field. Herein, new synthetic DNA carriers based on the conjugation of a phospholipid and C12E4, a nonionic detergent, are developed. DNA complexes with phosphatidylcholine-detergent conjugates are administered in mouse airways, and transgene expression and inflammatory activity as an index of toxicity are investigated as a function of time, DNA dose, and presence of helper and stealth lipids. Introduction of a biodegradable linker between the phosphatidylcholine and detergent moieties significantly attenuates the severity of inflammatory response that characterizes cationic lipid-mediated gene transfer. Concurrent introduction of polyunsaturated fatty acid chains in the carrier scaffold improves transgene expression and further reduces airway inflammation. Finally, the biodegradable phosphatidylcholine-detergent conjugates favorably compare to GL67A, the gold standard for DNA delivery to the airway that is currently under clinical evaluation. Our findings indicate that the lipid formulations described herein may have great potential as nucleic acid carriers for gene therapy. PMID:27418568

  5. Targeted gene delivery mediated by folate-polyethylenimine-block-poly(ethylene glycol) with receptor selectivity.

    PubMed

    Cheng, Han; Zhu, Jing-Ling; Zeng, Xuan; Jing, Yue; Zhang, Xian-Zheng; Zhuo, Ren-Xi

    2009-03-18

    The folate receptor (FR) is a tumor marker overexpressed in large numbers of cancer cells. Folic acid has high affinity to the FR and retains its binding affinity upon derivatization via its gamma-carboxyl. Therefore, in this article, folate-polyethylenimine-block-poly(ethylene glycol) (FOL-PEI-b-PEG) was designed for specific receptor targeted gene delivery. Physicochemical characterizations of resulting FOL-PEI-b-PEG/DNA complexes in terms of agarose gel electrophoresis, particle size, and zeta potential measurements were investigated. The results indicated that FOL-PEI-b-PEG was able to condense plasmid DNA tightly with a suitable particle size. The cytotoxicity study indicated that the copolymer exhibited less toxicity in comparison with that of 25 kDa PEI. Luciferase assay and green fluorescent protein (GFP) detections were also used to confirm that FOL-PEI-b-PEG could be an effective gene vector. Importantly, transfection efficiency of FOL-PEI-b-PEG with free folic acid was much lower than that of the copolymer without free folic acid on FR-positive HeLa cells, suggesting that FOL-PEI-b-PEG has great potential as a targeting gene vector.

  6. Needle-free gene delivery through the skin: an overview of recent strategies.

    PubMed

    Elsabahy, Mahmoud; Foldvari, Marianna

    2013-01-01

    Topical administration is attractive and non-invasive gene delivery approach. It is simple and allows repeated administration. In addition, the skin is active immune surveillance site. Topical gene therapy, although promising for treatment of cancer, dermatological disorders, vaccination and autoimmune disease, has not progressed yet to clinical trials. The inability of nucleic acids to survive the extraand intracellular environment and to permeate through the outermost layer of the skin, the stratum corneum, compromise the therapeutic outcomes of nucleic acids-based therapies. Nanostructured vehicles (e.g. transfersomes, niosomes, nanoemulsions, gemini-lipid nanoparticles and biphasic vesicles) have the ability to partially disrupt and perturb lipids that are found in the skin layers and deliver their nucleic acid cargos to their targeted subcellular compartments. However, the efficiency of these carriers is still inferior to other invasive methods (e.g. epidermal and intradermal injections). The goal of this review is to examine the critical parameters required to enhance the efficiency of the currently available nanostructured vehicles, for example, by combining them with minimally invasive techniques, such as, electroporation, iontophoresis, microneedles, ultrasound, gene gun and femtosecond laser. The recent advances in engineering these nanovectors will be discussed with a focus on their future prospects. PMID:23489207

  7. Low molecular weight PEI-appended polyesters as non-viral gene delivery vectors.

    PubMed

    Xun, Miao-Miao; Liu, Yan-Hong; Guo, Qian; Zhang, Ji; Zhang, Qin-Fang; Wu, Wan-Xia; Yu, Xiao-Qi

    2014-05-01

    Routine clinical implementation of human gene therapy requires safe and efficient gene delivery methods. Linear biodegradable polyesters with carbon-carbon double bonds are prepared from unsaturated diacids and diols. Subsequent appending of low molecular weight PEI by Michael addition gives target cationic polymers efficiently. Agarose gel retardation and fluorescence quenching assays show that these materials have good DNA binding ability and can completely retard plasmid DNA at weight ratio of 0.8. The formed polyplexes have appropriate sizes around 275 nm and zeta-potential values about +20-35 mV. The cytotoxicities of these polymers assayed by MTT are much lower than that of 25 kDa PEI. In vitro transfection toward 7402, HEK293 and U-2OS cells show that polymer P1 may give dramatically higher transfection efficiency (TE) than 25 kDa PEI, especially in U-2OS cells, suggesting that such polymer might be promising non-viral gene vectors.

  8. Multi-functionalized carbon dots as theranostic nanoagent for gene delivery in lung cancer therapy

    PubMed Central

    Wu, Yu-Fen; Wu, Hsi-Chin; Kuan, Chen-Hsiang; Lin, Chun-Jui; Wang, Li-Wen; Chang, Chien-Wen; Wang, Tzu-Wei

    2016-01-01

    Theranostics, an integrated therapeutic and diagnostic system, can simultaneously monitor the real-time response of therapy. Different imaging modalities can combine with a variety of therapeutic moieties in theranostic nanoagents. In this study, a multi-functionalized, integrated theranostic nanoagent based on folate-conjugated reducible polyethylenimine passivated carbon dots (fc-rPEI-Cdots) is developed and characterized. These nanoagents emit visible blue photoluminescence under 360 nm excitation and can encapsulate multiple siRNAs (EGFR and cyclin B1) followed by releasing them in intracellular reductive environment. In vitro cell culture study demonstrates that fc-rPEI-Cdots is a highly biocompatible material and a good siRNA gene delivery carrier for targeted lung cancer treatment. Moreover, fc-rPEI-Cdots/pooled siRNAs can be selectively accumulated in lung cancer cells through receptor mediated endocytosis, resulting in better gene silencing and anti-cancer effect. Combining bioimaging of carbon dots, stimulus responsive property, gene silencing strategy, and active targeting motif, this multi-functionalized, integrated theranostic nanoagent may provide a useful tool and platform to benefit clinicians adjusting therapeutic strategy and administered drug dosage in real time response by monitoring the effect and tracking the development of carcinomatous tissues in diagnostic and therapeutic aspects. PMID:26880047

  9. Polyamidoamine (PAMAM) dendrimers modified with short oligopeptides for early endosomal escape and enhanced gene delivery.

    PubMed

    Thuy, Le Thi; Mallick, Sudipta; Choi, Joon Sig

    2015-08-15

    Recently, non-viral vectors have become a popular research topic in the field of gene therapy. In this study, we conjugated short oligopeptides to polyamidoamine-generation 4 (PAMAM G4) to achieve higher transfection efficiency. Previous reports have shown that the PAMAM G4-histidine (H)-arginine (R) dendrimer enhances gene delivery by improving cell penetration and internalization mechanisms. Therefore, we synthesized PAMAM G4-H phenylalanine (F) R, PAMAM G4-FHR and PAMAM G4-FR derivatives to determine the best gene carrier with the lowest toxicity. Physicochemical studies were performed to determine mean diameters and surface charge of PAMAM derivatives/pDNA polyplexes. DNA condensation was confirmed using a gel retardation assay. Cytotoxicity and transfection efficiency were analyzed using human cervical carcinoma (HeLa) and human liver carcinoma (HepG2) cells. Similar levels of transfection were achieved in both cell lines by using gold standard transfection reagent PEI 25 kD. Therefore, our results show that these carriers are promising and may help achieve higher transfection with negligible cytotoxicity. PMID:26187169

  10. Chitosan/TPP Nanoparticles as a Gene Delivery Agent For Tumor Suppressant P53

    NASA Astrophysics Data System (ADS)

    Liu, Gaojun

    In the last decade, non-viral polymeric vectors have become more attractive than their viral counterparts due to their nontoxicity and good biocompatibility. However, one of the major drawbacks is the low transfection efficiency when compared to viruses. In this work, a naturally cationic polysaccharide, chitosan, was cross-linked with negatively charged tripolyphosphate (TPP) to synthesize chitosan/TPP nanoparticles (CNPs) for delivery of plasmid DNA (pDNA). Particle size and zeta potential were characterized for CNPs with chitosan-TPP mass ratios of 4:1 and 6:1 (w/w) using benchtop dynamic light scattering. And both potentiometric titration method and FTIR spectrometer were applied to measure the degree of deacetylation of chitosan. Release kinetics of a model protein (bovine serum albumin, BSA) showed a steady release that reached 7% after 6 days. Besides that, we also assessed the in vitro transfection efficiency of the CNP-pDNA system using fluorescence microscopy, as well as the effect of tumor suppressant p53. Later the release kinetics and encapsulation efficiency of plasmid DNA bound to the CNPs will be investigated. Additionally, we will try to improve the gene transfection efficiency in both MC3T3-E1 and osteosarcoma cells by applying Sonicator 740 therapeutic ultrasound. Key words: gene therapy, non-viral gene vector, chitosan/TPP nanoparticles, ionic gelation, p53.

  11. Efficient Gene Delivery of Primary Human Cells Using Peptide Linked Polyethylenimine Polymer Hybrid

    PubMed Central

    Dey, Devaveena; Inayathullah, Mohammed; Lee, Andrew S; Limiuex, Melbes; Zhang, Xuexiang; Wu, Yi; Nag, Divya; De Almeida, Patricia Eliza; Han, Leng; Rajadas, Jayakumar; Wu, Joseph C

    2011-01-01

    Polyethylenimine (PEI) based polymers are efficient agents for cell transfection. However, their use has been hampered due to high cell death associated with transfection thereby resulting in low efficiency of gene delivery within the cells. To circumvent the problem of cellular toxicity, metal binding peptides were linked to PEI. Eight peptide-PEI derivatives were synthesized to improve cell survival and transfection efficiency. TAT linked PEI was used as a control polymer. Peptides linked with PEI amines formed nano gels as shown by electron microscopy and atomic force microscopic measurements. Polymers were characterized by spectroscopic methods and their ability to form complexes with plasmids was tested using electrophoretic studies. These modifications improved polymer biocompatibility as well as cell survival markedly when compared to PEI alone. A subset of the modified peptide-polymers also showed significantly higher transfection efficiency in primary human cells with respect to the widely used transfection agent, lipofectamine. Study of the underlying mechanism of the observed phenomena revealed lower levels of ‘reactive oxygen species’ (ROS) in presence of the peptide-polymers when compared to PEI alone. This was further corroborated with global gene expression analysis which showed upregulation of multiple genes and pathways involved in regulating intracellular oxidative stress. PMID:21477858

  12. Recharging cationic DNA complexes with highly charged polyanions for in vitro and in vivo gene delivery.

    PubMed

    Trubetskoy, V S; Wong, S C; Subbotin, V; Budker, V G; Loomis, A; Hagstrom, J E; Wolff, J A

    2003-02-01

    The intravenous delivery of plasmid DNA complexed with either cationic lipids (CL) or polyethyleneimine (PEI) enables high levels of foreign gene expression in lung. However, these cationic DNA complexes cause substantial toxicity. The present study found that the inclusion of polyacrylic acid (pAA) with DNA/polycation and DNA/CL complexes prevented the serum inhibition of the transfection complexes in cultured cells. The mechanism mediating this increase seems to involve both particle size enlargement due to flocculation and electrostatic shielding from opsonizing serum proteins. The use of pAA also increased the levels of lung expression in mice in vivo substantially above the levels achieved with just binary complexes of DNA and linear PEI (lPEI) or CL and reduced their toxicity. Also, the use of a "chaser" injection of pAA 30 min after injection of the ternary DNA/lPEI/pAA complexes further aided this effort to reduce toxicity while not affecting foreign gene expression. By optimizing the amount of pAA, lPEI, and DNA within the ternary complexes and using the "chaser" injection, substantial levels of lung expression were obtained while avoiding adverse effects in lung or liver. These developments will aid the use of cationic DNA complexes in animals and for eventual human gene therapy.

  13. Quaternized chitosan/rectorite intercalative materials for a gene delivery system

    NASA Astrophysics Data System (ADS)

    Wang, Xiaoying; Pei, Xiaofeng; Du, Yumin; Li, Yan

    2008-09-01

    Non-viral vectors have gained increasing attention in gene therapy because of their safety, but with the shortcoming of low transfection efficiency. We have developed a hybrid material as a novel non-viral vector, which combines the advantages of both biopolymer and clay in a gene delivery system. Quaternized chitosan was intercalated into the interlayers of rectorite to obtain a new polymer/layered silicate nanocomposite. In vitro and in vivo toxicity studies revealed that the nanocomposites were biocompatible and non-toxic. At the nanocomposite:pDNA mass ratio of 8:1, they achieved 100% pDNA adsorption capacity. In vitro cell transfection revealed a transfection efficiency of 32.1% at 96 h as shown by a flow-cytometric study, and the intensive green fluorescence protein (GFP) expression could last for up to 120 h. Furthermore, an in vivo transfection study showed that the most prominent GFP expression was observed in the gastric and duodenum mucosa, and good transfection efficiency was also obtained when injected into the muscle. All the results suggest that quaternized chitosan/rectorite nanocomposite is a novel and potential non-viral gene carrier.

  14. Enhanced gene delivery to the lung using biodegradable polyunsaturated cationic phosphatidylcholine-detergent conjugates.

    PubMed

    Pierrat, Philippe; Kereselidze, Dimitri; Lux, Marie; Lebeau, Luc; Pons, Françoise

    2016-09-10

    Lung diseases are among the more representative causes of mortality and morbidity worldwide and gene therapy is considered as a promising therapeutic approach for their treatment. However the design of efficient nucleic acid carriers for airway administration still is a challenge and there is a pressing need for new developments in this field. Herein, new synthetic DNA carriers based on the conjugation of a phospholipid and C12E4, a nonionic detergent, are developed. DNA complexes with phosphatidylcholine-detergent conjugates are administered in mouse airways, and transgene expression and inflammatory activity as an index of toxicity are investigated as a function of time, DNA dose, and presence of helper and stealth lipids. Introduction of a biodegradable linker between the phosphatidylcholine and detergent moieties significantly attenuates the severity of inflammatory response that characterizes cationic lipid-mediated gene transfer. Concurrent introduction of polyunsaturated fatty acid chains in the carrier scaffold improves transgene expression and further reduces airway inflammation. Finally, the biodegradable phosphatidylcholine-detergent conjugates favorably compare to GL67A, the gold standard for DNA delivery to the airway that is currently under clinical evaluation. Our findings indicate that the lipid formulations described herein may have great potential as nucleic acid carriers for gene therapy.

  15. The Biology of the Sodium Iodide Symporter and its Potential for Targeted Gene Delivery

    PubMed Central

    Hingorani, M.; Spitzweg, C.; Vassaux, G.; Newbold, K.; Melcher, A.; Pandha, H.; Vile, R.; Harrington, K.

    2013-01-01

    The sodium iodide symporter (NIS) is responsible for thyroidal, salivary, gastric, intestinal and mammary iodide uptake. It was first cloned from the rat in 1996 and shortly thereafter from human and mouse tissue. In the intervening years, we have learned a great deal about the biology of NIS. Detailed knowledge of its genomic structure, transcriptional and post-transcriptional regulation and pharmacological modulation has underpinned the selection of NIS as an exciting approach for targeted gene delivery. A number of in vitro and in vivo studies have demonstrated the potential of using NIS gene therapy as a means of delivering highly conformal radiation doses selectively to tumours. This strategy is particularly attractive because it can be used with both diagnostic (99mTc, 125I, 124I) and therapeutic (131I, 186Re, 188Re, 211At) radioisotopes and it lends itself to incorporation with standard treatment modalities, such as radiotherapy or chemoradiotherapy. In this article, we review the biology of NIS and discuss its development for gene therapy. PMID:20201784

  16. Barriers to non-viral vector-mediated gene delivery in the nervous system.

    PubMed

    Pérez-Martínez, Francisco C; Guerra, Javier; Posadas, Inmaculada; Ceña, Valentín

    2011-08-01

    Efficient methods for cell line transfection are well described, but, for primary neurons, a high-yield method different from those relying on viral vectors is lacking. Viral transfection has several drawbacks, such as the complexity of vector preparation, safety concerns, and the generation of immune and inflammatory responses when used in vivo. However, one of the main problems for the use of non-viral gene vectors for neuronal transfection is their low efficiency when compared with viral vectors. Transgene expression, or siRNA delivery mediated by non-viral vectors, is the result of multiple processes related to cellular membrane crossing, intracellular traffic, and/or nuclear delivery of the genetic material cargo. This review will deal with the barriers that different nanoparticles (cationic lipids, polyethyleneimine, dendrimers and carbon nanotubes) must overcome to efficiently deliver their cargo to central nervous system cells, including internalization into the neurons, interaction with intracellular organelles such as lysosomes, and transport across the nuclear membrane of the neuron in the case of DNA transfection. Furthermore, when used in vivo, the nanoparticles should efficiently cross the blood-brain barrier to reach the target cells in the brain.

  17. Hyaluronan microspheres for sustained gene delivery and site-specific targeting.

    PubMed

    Yun, Yang H; Goetz, Douglas J; Yellen, Paige; Chen, Weiliam

    2004-01-01

    Hyaluronan is a naturally occurring polymer that has enjoyed wide successes in biomedical and cosmetic applications as coatings, matrices, and hydrogels. For controlled delivery applications, formulating native hyaluronan into microspheres could be advantageous but has been difficult to process unless organic solvents are used or hyaluronan has been modified by etherification. Therefore, we present a novel method of preparing hyaluronan microspheres using adipic dihydrazide mediated crosslinking chemistry. To evaluate their potential for medical applications, hyaluronan microspheres are incorporated with DNA for gene delivery or conjugated with an antigen for cell-specific targeting. The results show that our method, originally developed for preparing hyaluronan hydrogels, generates robust microspheres with a size distribution of 5-20mum. The release of the encapsulated plasmid DNA can be sustained for months and is capable of transfection in vitro and in vivo. Hyaluronan microspheres, conjugated with monoclonal antibodies to E- and P-selectin, demonstrate selective binding to cells expressing these receptors. In conclusion, we have developed a novel microsphere preparation using native hyaluronan that delivers DNA at a controlled rate and adaptable for site-specific targeting. PMID:14580918

  18. Influence of cationic lipid concentration on properties of lipid–polymer hybrid nanospheres for gene delivery

    PubMed Central

    Bose, Rajendran JC; Arai, Yoshie; Ahn, Jong Chan; Park, Hansoo; Lee, Soo-Hong

    2015-01-01

    Nanoparticles have been widely used for nonviral gene delivery. Recently, cationic hybrid nanoparticles consisting of two different materials were suggested as a promising delivery vehicle. In this study, nanospheres with a poly(d,l-lactic-co-glycolic acid) (PLGA) core and cationic lipid shell were prepared, and the effect of cationic lipid concentrations on the properties of lipid polymer hybrid nanocarriers investigated. Lipid–polymer hybrid nanospheres (LPHNSs) were fabricated by the emulsion-solvent evaporation method using different concentrations of cationic lipids and characterized for size, surface charge, stability, plasmid DNA-binding capacity, cytotoxicity, and transfection efficiency. All LPHNSs had narrow size distribution with positive surface charges (ζ-potential 52–60 mV), and showed excellent plasmid DNA-binding capacity. In vitro cytotoxicity measurements with HEK293T, HeLa, HaCaT, and HepG2 cells also showed that LPHNSs exhibited less cytotoxicity than conventional transfection agents, such as Lipofectamine and polyethyleneimine–PLGA. As cationic lipid concentrations increased, the particle size of LPHNSs decreased while their ζ-potential increased. In addition, the in vitro transfection efficiency of LPHNSs increased as lipid concentration increased. PMID:26379434

  19. A bacteria deriving peptide modified dendrigraft poly-l-lysines (DGL) self-assembling nanoplatform for targeted gene delivery.

    PubMed

    Liu, Yang; He, Xi; Kuang, Yuyang; An, Sai; Wang, Chenyu; Guo, Yubo; Ma, Haojun; Lou, Jinning; Jiang, Chen

    2014-10-01

    Achieving effective gene therapy for glioma depends on gene delivery systems. The gene delivery system should be able to cross the blood-brain barrier (BBB) and further target glioma at its early stage. Active brain tumor targeted delivery can be achieved using the "Trojan horse" technology, which involves either endogenous ligands or extraneous substances that can recognize and bind to specific receptors in target sites. This method facilitates receptor-mediated endocytosis to cross the BBB and enter into glioma cells. Dendrigraft poly-l-lysines (DGLs), which are novel nonviral gene vectors, are conjugated to a peptide (sequence: EPRNEEK) derived from Streptococcus pneumonia, a pathogen causing meningitis. This process yields peptide-modified nanoparticles (NPs) after DNA loading. Cellular uptake and in vivo imaging results indicate that EPRNEEK peptide-modified NPs have a better brain tumor targeted effect compared with a pentapeptide derived from endogenous laminin after intravenous injection. The mechanism of this effect is further explored in the present study. Besides, EPRNEEK peptide-modified NPs also exhibited a prolonged median survival time. In conclusion, the EPRNEEK peptide-modified DGL NPs exhibit potential as a nonviral platform for efficient, noninvasive, and safe brain glioma dual-targeted gene delivery.

  20. Crosslinked Polypropylenimine Dendrimers With Bioreducible Linkages for Gene Delivery Systems and Their Reductive Degradation Behaviors.

    PubMed

    Lee, Gyeong Jin; Ryu, Kitae; Kim, Kyunghwan; Choi, Ji-Yeong; Kim, Tae-Il

    2015-11-01

    Crosslinked bioreducible polypropylenimine-cystaminebisacrylamides (PPI-CBAs) were synthesized for gene delivery systems. They formed nano-sized polyplexes with high stability even in reducing condition probably due to the re-crosslinking. PPI-CBAs displayed high transfection efficiency comparable to PEI25k in serum condition. However, cytotoxicity increase, glutathione (GSH) decrease, and reactive oxygen species (ROS) increase were observed in cells with the elevation of PPI-CBAs concentration and crosslinking degree. These results suggest that the cytotoxicity of bioreducible polymers may be closely related with their structures and that reduction of GSH by degradation of re-crosslinked bioreducible polymers and the following increase of ROS may induce cytotoxicity by oxidative stress. PMID:26184085

  1. Effect of innate glutathione levels on activity of redox-responsive gene delivery vectors

    PubMed Central

    Manickam, Devika S.; Li, Jing; Putt, David A.; Zhou, Qing-Hui; Wu, Chao; Lash, Lawrence H.; Oupický, David

    2009-01-01

    Redox-responsive polyplexes represent a promising class of non-viral gene delivery vectors. The reducible disulfide bonds in the polyplexes undergo intracellular reduction owing to the presence of high concentrations of reduced glutathione (GSH). Available evidence suggests improved transfection activity of redox-sensitive polyplexes upon artificial modulation of intracellular GSH. This study investigates the effect of innate differences in GSH concentration in a panel of human pancreatic cancer cell lines on activity of reducible polyplexes of the four major classes of nucleic acid therapeutics: plasmid DNA (pDNA), messenger RNA (mRNA), antisense oligodeoxynucleotides (AON) and siRNA. In general, reducible polyplexes of linear poly(amido amines) (PAA) show improved activity compared to non-reducible polyplexes of PAA. Results demonstrate that increased GSH levels are associated with improved transfection of mRNA polyplexes but no clear trend is observed for pDNA, AON and siRNA polyplexes. PMID:19720098

  2. HPMA-oligolysine copolymers for gene delivery: optimization of peptide length and polymer molecular weight.

    PubMed

    Johnson, Russell N; Chu, David S H; Shi, Julie; Schellinger, Joan G; Carlson, Peter M; Pun, Suzie H

    2011-10-30

    Polycations are one of the most frequently used classes of materials for non-viral gene transfer in vivo. Several studies have demonstrated a sensitive relationship between polymer structure and delivery activity. In this work, we used reverse addition-fragmentation chain transfer (RAFT) polymerization to build a panel of N-(2-hydroxypropyl)methacrylamide (HPMA)-oligolysine copolymers with varying peptide length and polymer molecular weight. The panel was screened for optimal DNA-binding, colloidal stability in salt, high transfection efficiency, and low cytotoxicity. Increasing polyplex stability in PBS correlated with increasing polymer molecular weight and decreasing peptide length. Copolymers containing K(5) and K(10) oligocations transfected cultured cells with significantly higher efficiencies than copolymers of K(15). Four HPMA-oligolysine copolymers were identified that met the desired criteria. Polyplexes formed with these copolymers demonstrated both salt stability and transfection efficiencies on-par with poly(ethylenimine) PEI in cultured cells.

  3. Crosslinked Polypropylenimine Dendrimers With Bioreducible Linkages for Gene Delivery Systems and Their Reductive Degradation Behaviors.

    PubMed

    Lee, Gyeong Jin; Ryu, Kitae; Kim, Kyunghwan; Choi, Ji-Yeong; Kim, Tae-Il

    2015-11-01

    Crosslinked bioreducible polypropylenimine-cystaminebisacrylamides (PPI-CBAs) were synthesized for gene delivery systems. They formed nano-sized polyplexes with high stability even in reducing condition probably due to the re-crosslinking. PPI-CBAs displayed high transfection efficiency comparable to PEI25k in serum condition. However, cytotoxicity increase, glutathione (GSH) decrease, and reactive oxygen species (ROS) increase were observed in cells with the elevation of PPI-CBAs concentration and crosslinking degree. These results suggest that the cytotoxicity of bioreducible polymers may be closely related with their structures and that reduction of GSH by degradation of re-crosslinked bioreducible polymers and the following increase of ROS may induce cytotoxicity by oxidative stress.

  4. Transferrin protein nanospheres: a nanoplatform for receptor-mediated cancer cell labeling and gene delivery

    NASA Astrophysics Data System (ADS)

    McDonald, Michael A.; Spurlin, Tighe A.; Tona, Alessandro; Elliott, John T.; Halter, Michael; Plant, Anne L.

    2010-02-01

    This paper presents preliminary results on the use of transferrin protein nanospheres (TfpNS) for targeting cancer cells in vitro. Protein nanospheres represent an easily prepared and modifiable nanoplatform for receptor-specific targeting, molecular imaging and gene delivery. Rhodamine B isothiocyanate conjugated TfpNS (RBITC-TfpNS) show significantly enhanced uptake in vitro in SK-MEL-28 human malignant melanoma cells known to overexpress transferrin receptors compared to controls. RBITCTfpNS labeling of the cancer cells is due to transferrin receptor-mediated uptake, as demonstrated by competitive inhibition with native transferrin. Initial fluorescence microscopy studies indicate GFP plasmid can be transfected into melanoma cells via GFP plasmid encapsulated by TfpNS.

  5. Delivery and Specificity of CRISPR-Cas9 Genome Editing Technologies for Human Gene Therapy.

    PubMed

    Gori, Jennifer L; Hsu, Patrick D; Maeder, Morgan L; Shen, Shen; Welstead, G Grant; Bumcrot, David

    2015-07-01

    Genome editing using the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated 9 (Cas9) technology is revolutionizing the study of gene function and likely will give rise to an entire new class of therapeutics for a wide range of diseases. Achieving this goal requires not only characterization of the technology for efficacy and specificity but also optimization of its delivery to the target cells for each disease indication. In this review we survey the various methods by which the CRISPR-Cas9 components have been delivered to cells and highlight some of the more clinically relevant approaches. Additionally, we discuss the methods available for assessing the specificity of Cas9 editing; an important safety consideration for development of the technology.

  6. Inhibition of Experimental Liver Cirrhosis in Mice by Telomerase Gene Delivery

    NASA Astrophysics Data System (ADS)

    Rudolph, Karl Lenhard; Chang, Sandy; Millard, Melissa; Schreiber-Agus, Nicole; DePinho, Ronald A.

    2000-02-01

    Accelerated telomere loss has been proposed to be a factor leading to end-stage organ failure in chronic diseases of high cellular turnover such as liver cirrhosis. To test this hypothesis directly, telomerase-deficient mice, null for the essential telomerase RNA (mTR) gene, were subjected to genetic, surgical, and chemical ablation of the liver. Telomere dysfunction was associated with defects in liver regeneration and accelerated the development of liver cirrhosis in response to chronic liver injury. Adenoviral delivery of mTR into the livers of mTR-/- mice with short dysfunctional telomeres restored telomerase activity and telomere function, alleviated cirrhotic pathology, and improved liver function. These studies indicate that telomere dysfunction contributes to chronic diseases of continual cellular loss-replacement and encourage the evaluation of ``telomerase therapy'' for such diseases.

  7. Supercoiled Minivector DNA resists shear forces associated with gene therapy delivery

    PubMed Central

    Catanese, D J; Fogg, J M; Schrock, D E; Gilbert, B E; Zechiedrich, L

    2012-01-01

    Supercoiled DNAs varying from 281 to 5302 bp were subjected to shear forces generated by aerosolization or sonication. DNA shearing strongly correlated with length. Typical sized plasmids (⩾3000 bp) degraded rapidly. DNAs 2000–3000 bp persisted ∼10 min. Even in the absence of condensing agents, supercoiled DNA <1200 bp survived nebulization, and increased forces of sonication were necessary to shear it. Circular vectors were considerably more resistant to shearing than linear vectors of the same length. DNA supercoiling afforded additional protection. These results show the potential of shear-resistant Minivector DNAs to overcome one of the major challenges associated with gene therapy delivery. PMID:21633394

  8. Polyion complex stability and gene silencing efficiency with a siRNA-grafted polymer delivery system.

    PubMed

    Takemoto, Hiroyasu; Ishii, Atsushi; Miyata, Kanjiro; Nakanishi, Masataka; Oba, Makoto; Ishii, Takehiko; Yamasaki, Yuichi; Nishiyama, Nobuhiro; Kataoka, Kazunori

    2010-11-01

    An siRNA-grafted polymer through disulfide linkage was prepared to improve the physicochemical properties and transfection efficacies of the polyion complexes (PICs) as a nanocarrier of siRNA. The siRNA-grafted polymer formed stable PICs due to its larger numbers and higher density of anionic charges compared with monomeric siRNA, leading to effective internalization by cultured cells. Following the endosomal escape of the PIC, the disulfide linkage of the siRNA-grafted polymer allowed efficient siRNA release from the PIC under intracellular reductive conditions. Consequently, the PIC from the siRNA-grafted polymer showed a potent gene silencing effect without cytotoxicity or immunogenicity, demonstrating a promising feature of the siRNA-grafted polymer to construct the PIC-based nanocarrier for in vivo siRNA delivery.

  9. Validation of Mitochondrial Gene Delivery in Liver and Skeletal Muscle via Hydrodynamic Injection Using an Artificial Mitochondrial Reporter DNA Vector.

    PubMed

    Yasuzaki, Yukari; Yamada, Yuma; Ishikawa, Takuya; Harashima, Hideyoshi

    2015-12-01

    For successful mitochondrial transgene expression, two independent processes, i.e., developing a mitochondrial gene delivery system and construction of DNA vector to achieve mitochondrial gene expression, are required. To date, very few studies dealing with mitochondrial gene delivery have been reported and, in most cases, transgene expression was not validated, because the construction of a reporter DNA vector for mitochondrial gene expression is the bottleneck. In this study, mitochondrial transgene expression by the in vivo mitochondrial gene delivery of an artificial mitochondrial reporter DNA vector via hydrodynamic injection is demonstrated. In the procedure, a large volume of naked plasmid DNA (pDNA) is rapidly injected. We designed and constructed pHSP-mtLuc (CGG) as a mitochondrial reporter DNA vector that possesses a mitochondrial heavy strand promoter (HSP) and an artificial mitochondrial genome with the reporter NanoLuc (Nluc) luciferase gene that records adjustments to the mitochondrial codon system. We delivered the pDNA into mouse liver mitochondria by hydrodynamic injection, and detected exogenous mRNA in the liver using reverse transcription PCR analysis. The hydrodynamic injection of pHSP-mtLuc (CGG) resulted in the expression of the Nluc luciferase protein in liver and skeletal muscle. Our mitochondrial transgene expression reporter system would contribute to mitochondrial gene therapy and further studies directed at mitochondrial molecular biology.

  10. Ultrasound directs a transposase system for durable hepatic gene delivery in mice

    PubMed Central

    Anderson, Cynthia D; Urschitz, Johann; Khemmani, Mark; Owens, Jesse B; Moisyadi, Stefan

    2013-01-01

    Our aim was to evaluate the delivery of transposase-based vectors by Ultrasound Targeted Microbubble Destruction (UTMD) in mice. DNA vectors were attached to cationic lipid microbubbles (1-3 μm in diameter), injected intravenously, and delivered to the liver by destruction of the carrier bubbles with ultrasound in burst mode at a 1.0 MHz, 20 μs pulse duration, 10 Hz pulse repetition frequency, and acoustic peak negative pressure of ~1.3 MPa. We evaluated the expression and genomic integration of conventional (pcDNA3) and piggyBac transposase-based (pmGENIE) reporter vectors. In vivo, we observed UTMD-mediated liver-specific expression of pmGENIE for an average of 24 days, compared to 4 days with pcDNA3. Reporter expression was predominately located in proximity to blood vessels initially, while expression after three days was more evenly distributed through the parenchyma of the liver. We confirmed random genomic integration for pmGENIE in vitro, however, integration events for pmGENIE in vivo were targeted to specific areas of chromosome 14. Our results suggest that a combination of UTMD with nonviral DNA transposase vectors can mediate weeks of hepatic-specific gene transfer in vivo, and analyses performed by non-restrictive linear amplification-mediated (nrLAM) PCR, cloning, and sequencing identify an unexpected tropism for integration within a specific sequence on chromosome 14 in mice. UTMD delivery of transgenes may be useful for the treatment of hepatic gene deficiency disorders. PMID:24035623

  11. Mannosylated polyion complexes for in vivo gene delivery into CD11c(+) dendritic cells.

    PubMed

    Raviv, Lior; Jaron-Mendelson, Michal; David, Ayelet

    2015-02-01

    Dendritic cells (DCs) possess unique abilities in initiating primary immune responses and thus represent prime targets for DNA-based vaccinations. Here, we describe the design and synthesis of mannosylated polyion complexes (PICs) composed of cationic polyethylenimine (PEI) and hydrophilic polyethylene glycol (PEG) segments, and bearing mono- and trivalent mannose as a ligand for targeting mannose receptor (MR/CD206)-positive DCs. Amino-terminated mannose (Man)-containing ligands in mono- and trivalent presentations (Man- and Man3-, respectively) were prepared and conjugated to PEG via an N-hydroxysuccinimide (NHS)-activated terminal. Thiolated PEI was conjugated to the mannosylated PEG via the maleimide (MAL)-activated terminal. The resulting positively charged diblock copolymers bearing mannoses (Man-PEG-b-PEI and Man3-PEG-b-PEI) were self-assembled with DNA to form PICs with lower surface charge than did their PEI building block and mean hydrodynamic diameters in the range of 100-450 nm, depending on the N/P ratio. Man3-PEG-b-PEI demonstrated a 3-4-fold greater transfection efficiency in MR-positive dendritic cell lines (THP-1, DC2.4), relative to Man-PEG-b-PEI, exhibited low cytotoxicity when compared with PEI, and showed low transfection efficiency in nondendritic HeLa cells. In preliminary in vivo experiments, Man-PEG-b-PEI/DNA and Man3-PEG-b-PEI/DNA demonstrated 2-3-fold higher gene delivery efficiency into CD11c(+) DCs collected from inguinal lymph nodes of C57/BL6 mice, when compared to PEI/DNA complexes, as shown by GFP expression measurements, 24 h post subcutaneous injection. The results indicate that the mannosylated PICs are a safe and effective gene delivery system, showing in vivo specificity toward CD11c(+) DCs.

  12. Graphene oxide-cationic polymer conjugates: Synthesis and application as gene delivery vectors.

    PubMed

    Teimouri, Mohsen; Nia, Azadeh Hashem; Abnous, Khalil; Eshghi, Hossein; Ramezani, Mohammad

    2016-01-01

    Nanomedicine as the interface between nanotechnology and medical sciences is a new area that has attracted the attention of vast groups of researchers. Carbon nanomaterials are common platform for synthesis of nanoparticles for biomedical applications due to their low cytotoxicity and feasible internalization into mammalian cell lines (Yang et al., 2007; Arora et al., 2014; Oh and Park, 2014). Synthesis of vectors based on various cationic polymers polyethylenimine (PEI), polypropylenimine (PPI) and polyamidoamine (PAMAM) and their derivatives were considered as a strategy for transferring plasmid DNA and treatment of genetic diseases. Considering the low cytotoxicity of graphene, chemical modification of its surface has led to fabrication of novel gene delivery systems based on graphene and graphene oxide. Herein we report the synthesis of three groups of vectors based on conjugation of graphene oxide (GO) with alkylated derivatives of three different cationic polymers (polyethylenimine (PEI), polypropylenimine (PPI) and polyamidoamine (PAMAM)) through different linkers including surface carboxyl group, glycine and spermidine. Two main challenges in design of gene delivery vectors is decreasing cytotoxicity while improving the transfection efficiency. All synthesized vectors showed significantly lower cellular toxicity compared to bare polymer. A plasmid encoding green fluorescent protein (GFP) was used to evaluate the transfection efficiency of nanoparticles both qualitatively using live cell fluorescent imaging and quantitatively using flow cytometry and each vector was compared to its polymer base. Most successful conjugation strategy was observed in the case of PEI conjugates among which most efficient vector was PEI-GO conjugate bearing glycine linker. This vector was 9 fold more effective in terms of the percent of EGFP transfected cells. PMID:27072918

  13. Combined delivery of the adiponectin gene and rosiglitazone using cationic lipid emulsions.

    PubMed

    Davaa, Enkhzaya; Kang, Bong-Seok; Han, Joo-Hui; Lee, Sang-Eun; Ng, Choon Lian; Myung, Chang-Seon; Park, Jeong-Sook

    2015-04-10

    For the combined delivery of an insulin-sensitizing adipokine; i.e., the ADN gene, and the potent PPARγ agonist rosiglitazone, cationic lipid emulsions were formulated using the cationic lipid DOTAP, helper lipid DOPE, castor oil, Tween 20 and Tween 80. The effect of drug loading on the physicochemical characteristics of the cationic emulsion/DNA complexes was investigated. Complex formation between the cationic emulsion and negatively charged plasmid DNA was confirmed and protection from DNase was observed. The in vitro transfection efficiency and cytotoxicity were evaluated in HepG2 cells. The particle sizes of the cationic emulsion/DNA complex were in the range 230-540 nm and those of the rosiglitazone-loaded cationic emulsion/DNA complex were in the range 220-340 nm. Gel retardation of the complexes was observed when the complexation weight ratios of the cationic lipid to plasmid DNA exceeded 4:1 for both the drug-free and rosiglitazone-loaded complexes. Both complexes stabilized plasmid DNA against DNase. The ADN expression level increased dose-dependently when cells were transfected with the cationic emulsion/DNA complexes. The rosiglitazone-loaded cationic emulsion/DNA complexes showed higher cellular uptake in HepG2 cells depending on the rosiglitazone loading, but not depending on the type of plasmid DNA type such as pVAX/ADN, pCAG/ADN, or pVAX. The drug-loaded cationic emulsion/plasmid DNA complexes were less cytotoxic than free rosiglitazone. Therefore, a cationic emulsion could potentially serve as a co-delivery system for rosiglitazone and the adiponectin gene.

  14. The covalent bioconjugate of multiwalled carbon nanotube and amino-modified linearized plasmid DNA for gene delivery.

    PubMed

    Geyik, Caner; Evran, Serap; Timur, Suna; Telefoncu, Azmi

    2014-01-01

    Carbon nanotubes (CNTs) are allotropes of carbon, which have unique physical, mechanical, and electronic properties. Among various biomedical applications, CNTs also attract interest as nonviral gene delivery systems. Functionalization of CNTs with cationic groups enables delivery of negatively charged DNA into cells. In contrast to this well-known strategy for DNA delivery, our approach included the covalent attachment of linearized plasmid DNA to carboxylated multiwalled CNTs (MWCNTs). Carboxyl groups were introduced onto MWCNTs by oxidative treatment, and then the carboxyl groups were activated by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). The whole pQE-70 vector including the gene encoding green fluorescent protein (GFP) was subjected to polymerase chain reaction (PCR) using the modified nucleotide N6-(6-Amino)hexyl-2'-deoxyadenosine-5'-triphosphate. Hence, free amino groups were introduced onto the linearized plasmid. Covalent bonding between the amino-modified plasmid DNA and the carboxylated MWCNTs was achieved via EDC chemistry. The resulting bioconjugate was successfully transformed into chemically competent Escherichia coli cells, without necessity of a heat-shock step at 42°C. The presence of Ca(2+) in transformation medium was required to neutralize the electrostatic repulsion between DNA and negatively charged outer layer of E. coli. The transformants, which were able to express GFP were inspected manually on ampicillin agar plates. Our study represents a novelty with respect to other noncovalent CNT gene delivery systems. Considering the interest for delivery of linear DNA fragments, our study could give insights into further studies.

  15. Increased 1-aminocyclopropane-1-carboxylate deaminase activity enhances Agrobacterium tumefaciens-mediated gene delivery into plant cells

    PubMed Central

    Someya, Tatsuhiko; Nonaka, Satoko; Nakamura, Kouji; Ezura, Hiroshi

    2013-01-01

    Agrobacterium-mediated transformation is a useful tool for the genetic modification in plants, although its efficiency is low for several plant species. Agrobacterium-mediated transformation has three major steps in laboratory-controlled experiments: the delivery of T-DNA into plant cells, the selection of transformed plant cells, and the regeneration of whole plants from the selected cells. Each of these steps must be optimized to improve the efficiency of Agrobacterium-mediated plant transformation. It has been reported that increasing the number of cells transformed by T-DNA delivery can improve the frequency of stable transformation. Previously, we demonstrated that a reduction in ethylene production by plant cells during cocultivation with A. tumefaciens-expressing 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase resulted in increased T-DNA delivery into the plant cells. In this study, to further improve T-DNA delivery by A. tumefaciens, we modified the expression cassette of the ACC deaminase gene using vir gene promoter sequences. The ACC deaminase gene driven by the virD1 promoter was expressed at a higher level, resulting in a higher ACC deaminase activity in this A. tumefaciens strain than in the strain with the lac promoter used in a previous study. The newly developed A. tumefaciens strain improves the delivery of T-DNA into Solanum lycopersicum (tomato) and Erianthus ravennae plants and thus may be a powerful tool for the Agrobacterium-mediated genetic engineering of plants. PMID:24000136

  16. Exogenous DNA Loading into Extracellular Vesicles via Electroporation is Size-Dependent and Enables Limited Gene Delivery.

    PubMed

    Lamichhane, Tek N; Raiker, Rahul S; Jay, Steven M

    2015-10-01

    Extracellular vesicles (EVs) hold immense promise for utilization as biotherapeutics and drug delivery vehicles due to their nature as biological nanoparticles that facilitate intercellular molecular transport. Specifically, EVs have been identified as natural carriers of nucleic acids, sparking interest in their use for gene therapy and RNA interference applications. So far, small RNAs (siRNA and miRNA) have been successfully loaded into EVs for a variety of delivery applications, but the potential use of EVs for DNA delivery has scarcely been explored. Here, we report that exogenous linear DNA can be associated with EVs via electroporation in quantities sufficient to yield an average of hundreds of DNA molecules per vesicle. We determined that loading efficiency and capacity of DNA in EVs is dependent on DNA size, with linear DNA molecules less than 1000 bp in length being more efficiently associated with EVs compared to larger linear DNAs and plasmid DNAs using this approach. We further showed that EV size is also determinant with regard to DNA loading, as larger microvesicles encapsulated more linear and plasmid DNA than smaller, exosome-like EVs. Additionally, we confirmed the ability of EVs to transfer foreign DNA loaded via electroporation into recipient cells, although functional gene delivery was not observed. These results establish critical parameters that inform the potential use of EVs for gene therapy and, in agreement with other recent results, suggest that substantial barriers must be overcome to establish EVs as broadly applicable DNA delivery vehicles.

  17. A rapid pathway toward a superb gene delivery system: programming structural and functional diversity into a supramolecular nanoparticle library.

    PubMed

    Wang, Hao; Liu, Kan; Chen, Kuan-Ju; Lu, Yujie; Wang, Shutao; Lin, Wei-Yu; Guo, Feng; Kamei, Ken-ichiro; Chen, Yi-Chun; Ohashi, Minori; Wang, Mingwei; Garcia, Mitch André; Zhao, Xing-Zhong; Shen, Clifton K-F; Tseng, Hsian-Rong

    2010-10-26

    Nanoparticles are regarded as promising transfection reagents for effective and safe delivery of nucleic acids into a specific type of cells or tissues providing an alternative manipulation/therapy strategy to viral gene delivery. However, the current process of searching novel delivery materials is limited due to conventional low-throughput and time-consuming multistep synthetic approaches. Additionally, conventional approaches are frequently accompanied with unpredictability and continual optimization refinements, impeding flexible generation of material diversity creating a major obstacle to achieving high transfection performance. Here we have demonstrated a rapid developmental pathway toward highly efficient gene delivery systems by leveraging the powers of a supramolecular synthetic approach and a custom-designed digital microreactor. Using the digital microreactor, broad structural/functional diversity can be programmed into a library of DNA-encapsulated supramolecular nanoparticles (DNA⊂SNPs) by systematically altering the mixing ratios of molecular building blocks and a DNA plasmid. In vitro transfection studies with DNA⊂SNPs library identified the DNA⊂SNPs with the highest gene transfection efficiency, which can be attributed to cooperative effects of structures and surface chemistry of DNA⊂SNPs. We envision such a rapid developmental pathway can be adopted for generating nanoparticle-based vectors for delivery of a variety of loads.

  18. Mesoporous Silica Nanomaterials for Applications in Catalysis, Sensing, Drug Delivery and Gene Transfection

    SciTech Connect

    Radu, Daniela Rodica

    2004-01-01

    The central theme of this dissertation is represented by the versatility of mesoporous silica nanomaterials in various applications such as catalysis and bio-applications, with main focus on biological applications of Mesoporous Silica Nanospheres (MSN). The metamorphosis that we impose to these materials from catalysis to sensing and to drug and gene delivery is detailed in this dissertation. First, we developed a synthetic method that can fine tune the amount of chemically accessible organic functional groups on the pores surface of MSN by exploiting electrostatic and size matching between the cationic alkylammonium head group of the cetyltrimethylammonium bromide (CTAB) surfactant and various anionic organoalkoxysilane precursors at the micelle-water interface in a base-catalyzed condensation reaction of silicate. Aiming nature imitation, we demonstrated the catalytic abilities of the MSNs, We utilized an ethylenediamine functional group for chelating Cu2+ as a catalytic functional group anchored inside the mesopores. Thus, a polyalkynylene-based conducting polymer (molecular wire) was synthesized within the Cu-functionalized MSNs silica catalyst. For sensing applications, we have synthesized a poly(lactic acid) coated mesoporous silica nanosphere (PLA-MSN) material that serves as a fluorescence sensor system for detection of amino-containing neurotransmitters in neutral aqueous buffer. We exploited the mesoporosity of MSNs for encapsulating pharmaceutical drugs. We examined bio-friendly capping molecules such as polyamidoamine dendrimers of generations G2 to G4, to prevent the drug leaching. Next, the drug delivery system employed MSNs loaded with Doxorubicin, an anticancer drug. The results demonstrated that these nano-Trojan horses have ability to deliver Doxorubicin to cancer cells and induce their death. Finally, to demonstrate the potential of MSN as an universal cellular transmembrane nanovehicle, we anchored positively charged dendrimers on

  19. Coating nanocarriers with hyaluronic acid facilitates intravitreal drug delivery for retinal gene therapy.

    PubMed

    Martens, Thomas F; Remaut, Katrien; Deschout, Hendrik; Engbersen, Johan F J; Hennink, Wim E; van Steenbergen, Mies J; Demeester, Jo; De Smedt, Stefaan C; Braeckmans, Kevin

    2015-03-28

    Retinal gene therapy could potentially affect the lives of millions of people suffering from blinding disorders. Yet, one of the major hurdles remains the delivery of therapeutic nucleic acids to the retinal target cells. Due to the different barriers that need to be overcome in case of topical or systemic administration, intravitreal injection is an attractive alternative administration route for large macromolecular therapeutics. Here it is essential that the therapeutics do not aggregate and remain mobile in the vitreous humor in order to reach the retina. In this study, we have evaluated the use of hyaluronic acid (HA) as an electrostatic coating for nonviral polymeric gene nanomedicines, p(CBA-ABOL)/pDNA complexes, to provide them with an anionic hydrophilic surface for improved intravitreal mobility. Uncoated polyplexes had a Z-averaged diameter of 108nm and a zeta potential of +29mV. We evaluated polyplexes coated with HA of different molecular weights (22kDa, 137kDa and 2700kDa) in terms of size, surface charge and complexation efficiency and noticed their zeta potentials became anionic at 4-fold molar excess of HA-monomers compared to cationic monomers, resulting in submicron ternary polyplexes. Next, we used a previously optimized ex vivo model based on excised bovine eyes and fluorescence single particle tracking (fSPT) microscopy to evaluate mobility in intact vitreous humor. It was confirmed that HA-coated polyplexes had good mobility in bovine vitreous humor, similar to polyplexes functionalized with polyethylene glycol (PEG), except for those coated with high molecular weight HA (2700kDa). However, contrary to PEGylated polyplexes, HA-coated polyplexes were efficiently taken up in vitro in ARPE-19 cells, despite their negative charge, indicating uptake via CD44-receptor mediated endocytosis. Furthermore, the HA-polyplexes were able to induce GFP expression in this in vitro cell line without apparent cytotoxicity, where coating with low molecular

  20. Antitumor Effect of IP-10 by Using Two Different Approaches: Live Delivery System and Gene Therapy

    PubMed Central

    Taslimi, Yasaman; Zahedifard, Farnaz; Habibzadeh, Sima; Taheri, Tahereh; Abbaspour, Hossain; Sadeghipour, Alireza

    2016-01-01

    Purpose Immunotherapy is one of the treatment strategies for breast cancer, the most common cancer in women worldwide. In this approach, the patient's immune system is stimulated to attack microscopic tumors and control metastasis. Here, we used interferon γ-induced protein 10 (IP-10), which induces and strengthens antitumor immunity, as an immunotherapeutic agent. We employed Leishmania tarentolae, a nonpathogenic lizard parasite that lacks the ability to persist in mammalian macrophages, was used as a live delivery system for carrying the immunotherapeutic agent. It has been already shown that arginase activity, and consequently, polyamine production, are associated with tumor progression. Methods A live delivery system was constructed by stable transfection of pLEXSY plasmid containing the IP-10-enhanced green fluorescent protein (IP-10-egfp) fusion gene into L. tarentolae. Then, the presence of the IP-10-egfp gene and the accurate integration location into the parasite genome were confirmed. The therapeutic efficacy of IP-10 delivered via L. tarentolae and recombinant pcDNA-(IP-10-egfp) plasmid was compared by determining the arginase activity in a mouse 4T1 breast cancer model. Results The pcDNA-(IP-10-egfp) group showed a significant reduction in tumor weight and growth. Histological evaluation also revealed that only this group demonstrated inhibition of metastasis to the lung tissue. The arginase activity in the tissue of the pcDNA-(IP-10-egfp) mice significantly decreased in comparison with that in normal mice. No significant difference was observed in arginase activity in the sera of mice receiving other therapeutic strategies. Conclusion Our data indicates that IP-10 immunotherapy is a promising strategy for breast cancer treatment, as shown in the 4T1-implanted BALB/c mouse model. However, the L. tarentolae-(IP-10-EGFP) live delivery system requires dose modifications to achieve efficacy in the applied regimen (six injections in 3 weeks). Our results

  1. Metal-polybenzimidazole complexes as a nonviral gene carrier: effects of the DNA affinity on gene delivery.

    PubMed

    Huang, Xueying; Dong, Xiongwei; Li, Xue; Meng, Xianggao; Zhang, Dan; Liu, Changlin

    2013-12-01

    The metal complex-based carriers are emerging likely as a new type of gene-delivery systems prone to systematic structural alteration and chemical tailoring. In our work, the DNA affinity of metal complexes with polybenzimidazoles was found to be one of the determinants that can regulate expression of the transgenes. Here, the correlations between the DNA affinity and transfection efficacy were explored by characterizing gene-delivering properties of a series of Co(2+)- and Ca(2+)-polybenzimidazole complexes. The binding equilibrium constants (Kobs) of the divalent metal complexes to DNA, which is considered as a measure of the DNA affinity of metal complexes, were evaluated by isothermal titration calorimetry (ITC) and UV-visible absorption titration. The properties of DNA condensates formed with the metal complexes including sizes, ζ potential and morphology were observed to be altered with Kobs values. The monodispersed spherical condensates were found only for the Ca(2+) complexes whose DNA affinity is weaker than that of the Co(2+) complexes. However, the cell internalization examination indicated that cell uptake of the DNA condensates is independent of homogeneity in their sizes and morphology. The comparison of transgene expression showed that that the Ca(2+) complex-mediated transfection has higher efficiency than the Co(2+) complexes under the conditions tested, and the transfection efficacy cannot be correlated with the cell uptake of DNA condensates. Moreover, the Ca(2+) complexes and their DNA condensates had lower cytotoxicity than the Co(2+) complexes. Thus, the DNA affinity should be one of the factors to be capable of regulating the gene-delivering property of metal complexes. PMID:24099694

  2. Adenovirus-mediated gene delivery to cells of the magnocellular hypothalamo-neurohypophyseal system

    NASA Technical Reports Server (NTRS)

    Vasquez, E. C.; Beltz, T. G.; Haskell, R. E.; Johnson, R. F.; Meyrelles, S. S.; Davidson, B. L.; Johnson, A. K.

    2001-01-01

    The objective of the present study was to define the optimum conditions for using replication-defective adenovirus (Ad) to transfer the gene for the green fluorescent protein (GFP) to the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei and cells of the neurohypophysis (NH). As indicated by characterizing cell survival over 15 days in culture and in electrophysiological whole cell patch-clamp studies, viral concentrations up to 2 x 10(7) pfu/coverslip did not affect viability of transfected PVN and NH cultured cells from preweanling rats. At 2 x 10(7) pfu, GFP gene expression was higher (40% of GFP-positive cells) and more sustained (up to 15 days). Using a stereotaxic approach in adult rats, we were able to directly transduce the PVN, SON, and NH and visualize gene expression in coronal brain slices and in the pituitary 4 days after injection of Ad. In animals receiving NH injections of Ad, the virus was retrogradely transported to PVN and SON neurons as indicated by the appearance of GFP-positive neurons in cultures of dissociated cells from those brain nuclei and by polymerase chain reaction and Western blot analyses of PVN and SON tissues. Adenoviral concentrations of up to 8 x 10(6) pfu injected into the NH did not affect cell viability and did not cause inflammatory responses. Adenoviral injection into the pituitary enabled the selective delivery of genes to the soma of magnocellular neurons. The experimental approaches described here provide potentially useful strategies for the treatment of disordered expression of the hormones vasopressin or oxytocin. Copyright 2000 Academic Press.

  3. Injectable graphene oxide/hydrogel-based angiogenic gene delivery system for vasculogenesis and cardiac repair.

    PubMed

    Paul, Arghya; Hasan, Anwarul; Kindi, Hamood Al; Gaharwar, Akhilesh K; Rao, Vijayaraghava T S; Nikkhah, Mehdi; Shin, Su Ryon; Krafft, Dorothee; Dokmeci, Mehmet R; Shum-Tim, Dominique; Khademhosseini, Ali

    2014-08-26

    The objective of this study was to develop an injectable and biocompatible hydrogel which can efficiently deliver a nanocomplex of graphene oxide (GO) and vascular endothelial growth factor-165 (VEGF) pro-angiogenic gene for myocardial therapy. For the study, an efficient nonviral gene delivery system using polyethylenimine (PEI) functionalized GO nanosheets (fGO) complexed with DNAVEGF was formulated and incorporated in the low-modulus methacrylated gelatin (GelMA) hydrogel to promote controlled and localized gene therapy. It was hypothesized that the fGOVEGF/GelMA nanocomposite hydrogels can efficiently transfect myocardial tissues and induce favorable therapeutic effects without invoking cytotoxic effects. To evaluate this hypothesis, a rat model with acute myocardial infarction was used, and the therapeutic hydrogels were injected intramyocardially in the peri-infarct regions. The secreted VEGF from in vitro transfected cardiomyocytes demonstrated profound mitotic activities on endothelial cells. A significant increase in myocardial capillary density at the injected peri-infarct region and reduction in scar area were noted in the infarcted hearts with fGOVEGF/GelMA treatment compared to infarcted hearts treated with untreated sham, GelMA and DNAVEGF/GelMA groups. Furthermore, the fGOVEGF/GelMA group showed significantly higher (p < 0.05, n = 7) cardiac performance in echocardiography compared to other groups, 14 days postinjection. In addition, no significant differences were noticed between GO/GelMA and non-GO groups in the serum cytokine levels and quantitative PCR based inflammatory microRNA (miRNA) marker expressions at the injected sites. Collectively, the current findings suggest the feasibility of a combined hydrogel-based gene therapy system for ischemic heart diseases using nonviral hybrid complex of fGO and DNA.

  4. Injectable Graphene Oxide/Hydrogel-Based Angiogenic Gene Delivery System for Vasculogenesis and Cardiac Repair

    PubMed Central

    2015-01-01

    The objective of this study was to develop an injectable and biocompatible hydrogel which can efficiently deliver a nanocomplex of graphene oxide (GO) and vascular endothelial growth factor-165 (VEGF) pro-angiogenic gene for myocardial therapy. For the study, an efficient nonviral gene delivery system using polyethylenimine (PEI) functionalized GO nanosheets (fGO) complexed with DNAVEGF was formulated and incorporated in the low-modulus methacrylated gelatin (GelMA) hydrogel to promote controlled and localized gene therapy. It was hypothesized that the fGOVEGF/GelMA nanocomposite hydrogels can efficiently transfect myocardial tissues and induce favorable therapeutic effects without invoking cytotoxic effects. To evaluate this hypothesis, a rat model with acute myocardial infarction was used, and the therapeutic hydrogels were injected intramyocardially in the peri-infarct regions. The secreted VEGF from in vitro transfected cardiomyocytes demonstrated profound mitotic activities on endothelial cells. A significant increase in myocardial capillary density at the injected peri-infarct region and reduction in scar area were noted in the infarcted hearts with fGOVEGF/GelMA treatment compared to infarcted hearts treated with untreated sham, GelMA and DNAVEGF/GelMA groups. Furthermore, the fGOVEGF/GelMA group showed significantly higher (p < 0.05, n = 7) cardiac performance in echocardiography compared to other groups, 14 days postinjection. In addition, no significant differences were noticed between GO/GelMA and non-GO groups in the serum cytokine levels and quantitative PCR based inflammatory microRNA (miRNA) marker expressions at the injected sites. Collectively, the current findings suggest the feasibility of a combined hydrogel-based gene therapy system for ischemic heart diseases using nonviral hybrid complex of fGO and DNA. PMID:24988275

  5. Development of self-assembling peptide nanovesicle with bilayers for enhanced EGFR-targeted drug and gene delivery.

    PubMed

    Liang, Xiaofei; Shi, Bizhi; Wang, Kai; Fan, Mingliang; Jiao, Dejin; Ao, Junping; Song, Na; Wang, Chun; Gu, Jianren; Li, Zonghai

    2016-03-01

    Development of rational vectors for efficient drug and gene delivery is crucial for cancer treatment. In this study, epidermal growth factor receptor (EGFR)-binding peptide amphiphile (PA) were used as the primary bilayer skeleton material to construct ultra-stable self-assembling peptide nanovesicle (SPV). The resulted EGFR-targeted SPV (ESPV) could efficiently encapsulate therapeutic cargos (drugs or small interfering RNAs [siRNAs]) or labelled fluorescent cargo (quantum dots [QDs]) and exhibited excellent affinity for EGFR-positive cancer cells. Moreover, ESPV could deliver more drug or plasmid DNA to tumour sites and promote gene expression (a three-fold ratio of ESPVs vs cationic liposomes). Notably, the individual delivery or co-delivery of doxorubicin (DOX) and the acetylcholinesterase (AChE) gene via the ESPVs resulted in excellent drug/gene delivery both in vitro and in vivo and exerted a significant growth-suppressing effect on a liver cancer xenograft. This nanoscale, targeted cargo-packaging technology may provide a new strategy for the design of highly targeted cancer therapy vectors. PMID:26763734

  6. Quantitative measurement of delivery and gene silencing activities of siRNA polyplexes containing pyridylthiourea-grafted polyethylenimines.

    PubMed

    Pinel, Sophie; Aman, Emmanuel; Erblang, Felix; Dietrich, Jonathan; Frisch, Benoit; Sirman, Julien; Kichler, Antoine; Sibler, Annie-Paule; Dontenwill, Monique; Schaffner, Florence; Zuber, Guy

    2014-05-28

    The activity of synthetic interfering nucleic acids (siRNAs) relies on the capacity of delivery systems to efficiently transport nucleic acids into the cytosol of target cells. The pyridylthiourea-grafted 25KDa polyethylenimine (πPEI) is an excellent carrier for siRNA delivery into cells and it was extensively investigated in this report. Quantification of the siRNA-mediated gene silencing efficiency indicated that the πPEI specific delivery activity at the cell level may be measured and appears relatively constant in various cell lines. Delivery experiments assaying inhibitors of various entry pathways or concanamycin A, an inhibitor of the H(+)/ATPase vacuolar pump showed that the πPEI/siRNA polyplexes did not require any specific entry mode but strongly relied on vacuolar acidification for functional siRNA delivery. Next, πPEI polyplexes containing a siRNA targeting the transcription factor HIF-1α, known to be involved in tumor progression, were locally injected into mice xenografted with a human glioblastoma. A 55% reduction of the level of the target mRNA was observed at doses comparable to those used in vitro when the πPEI delivery activity was calculated per cell. Altogether, our study underscores the usefulness of "simple"/rough cationic polymers for siRNA delivery despite their intrinsic limitations. The study underscores as well as that bottom-up strategies make sense. The in vitro experiments can precede in vivo administration and be of high value for selection of the carrier with enhanced specific delivery activity and parallel other research aiming at improving synthetic delivery systems for resilience in the blood and for enhanced tissue-targeting capacity. PMID:24631860

  7. Polyglycerol-functionalized nanodiamond as a platform for gene delivery: Derivatization, characterization, and hybridization with DNA

    PubMed Central

    Zhao, Li; Nakae, Yuki; Qin, Hongmei; Ito, Tadamasa; Kimura, Takahide; Kojima, Hideto; Chan, Lawrence

    2014-01-01

    Summary A gene vector consisting of nanodiamond, polyglycerol, and basic polypeptide (ND-PG-BPP) has been designed, synthesized, and characterized. The ND-PG-BPP was synthesized by PG functionalization of ND through ring-opening polymerization of glycidol on the ND surface, multistep organic transformations (–OH → –OTs (tosylate) → –N3) in the PG layer, and click conjugation of the basic polypeptides (Arg8, Lys8 or His8) terminated with propargyl glycine. The ND-PG-BPP exhibited good dispersibility in water (>1.0 mg/mL) and positive zeta potential ranging from +14.2 mV to +44.1 mV at neutral pH in Milli-Q water. It was confirmed by gel retardation assay that ND-PG-Arg8 and ND-PG-Lys8 with higher zeta potential hybridized with plasmid DNA (pDNA) through electrostatic attraction, making them promising as nonviral vectors for gene delivery. PMID:24778723

  8. Guanidinylated bioresponsive poly(amido amine)s designed for intranuclear gene delivery

    PubMed Central

    Yu, Jiankun; Zhang, Jinmin; Xing, Haonan; Yang, Zhen; Cai, Cuifang; Zhang, Conglu; Zhao, Xiaoyun; Wei, Minjie; Yang, Li; Ding, Pingtian

    2016-01-01

    Guanidinylated poly(amido amine)s with multiple disulfide linkages (Gua-SS-PAAs) were designed and constructed as nonviral gene carriers. The main chains of these novel carriers were synthesized based on monomers containing guanidino groups (guanidine hydrochloride and chlorhexidine), which could avoid complicated side-chain-modification reactions while introducing the guanidino groups. The synthesized Gua-SS-PAAs polymers were characterized by 1H nuclear magnetic resonance, molecular weight, and polydispersity. Furthermore, Gua-SS-PAAs polymers were complexed with pDNA, and the properties of the complexes were determined, including entrapment efficiency, particle size, ζ-potential, atomic force microscopy images, stability, DNA complexation ability, reduction sensitivity, cytotoxicity, and transfection efficiency. The new Gua-SS-PAAs carriers exhibited higher transfection efficiency and lower cytotoxicity compared with two widely used gene delivery carriers, polyethylenimine and lipofectamine 2000. Furthermore, the relationship between the side-chain structure and morphological/biological properties was extrapolated, and the results showed that guanidine in the side chain aids in the improvement of transfection efficiency. In addition, the introduction of guanidino group might confer the new carriers with nuclear localization function compared to carriers without it. PMID:27574429

  9. Differential analysis of "protein corona" profile adsorbed onto different nonviral gene delivery systems.

    PubMed

    Capriotti, Anna Laura; Caracciolo, Giulio; Caruso, Giuseppe; Foglia, Patrizia; Pozzi, Daniela; Samperi, Roberto; Laganà, Aldo

    2011-12-15

    A shotgun proteomics approach was used to characterize and compare the proteins that lead to the formation of a rich "protein corona" adsorbed onto the surfaces of cationic liposomes (CLs), lipoplexes, and lipid/polycation/DNA (LPD) complexes, when they come into contact with plasma. After separation of the nanoparticle-protein complex from plasma, the protein mixture was digested, and peptides were analyzed by nanoliquid chromatography-Orbitrap LTQ-XL mass spectrometry. The number of proteins bound to lipoplexes was double that of those identified in the corona of CLs (208 vs 105), while 77 proteins were common to both coronas. The number of proteins bound to the surface of the LPD complexes (158, 133 of which are common to lipoplexes) is intermediate between those found in the protein corona of both CLs and lipoplexes. About half of them were found in the protein corona of CLs. By overlapping the three formulations, it can be seen that only 12 proteins are peculiar to LPD complexes. These results may help in designing gene delivery systems capable of binding the minimum possible quantity of proteins that influence transfection negatively, binding selectively proteins capable of helping in steering in vivo the vector toward the target, and obtaining more efficient and effective gene therapy.

  10. Self-assembled nanoparticles based on amphiphilic chitosan derivative and hyaluronic acid for gene delivery.

    PubMed

    Liu, Ya; Kong, Ming; Cheng, Xiao Jie; Wang, Qian Qian; Jiang, Li Ming; Chen, Xi Guang

    2013-04-15

    The present work described nanoparticles (NPs) made of oleoyl-carboxymethy-chitosan (OCMCS)/hyaluronic acid (HA) using coacervation process as novel potential carriers for gene delivery. An N/P ratio of 5 and OCMCS/HA weight ratio of 4 were the optimal conditions leading to the smallest (164.94 nm), positive charged (+14.2 mV) and monodispersed NPs. OCMCS-HA/DNA (OHD) NPs showed higher in vitro DNA release rates and increased cellular uptake by Caco-2 cells due to the HA involved in NPs. The MTT survival assay indicated no significant cytotoxicity. The transfection efficiency of OHD NPs was 5-fold higher than OCMCS/DNA (OD) NPs; however, it decreased significantly in the presence of excess free HA. The results indicated that OHD NPs internalized in Caco-2 cells were mediated by the hyaluronan receptor CD44. The data obtained in the present research gave evidence of the potential of OHD NPs for the targeting and further transfer of genes to the epithelial cells. PMID:23544543

  11. New Dendritic Lipids for Improved Gene Delivery by Cationic Liposome-DNA Complexes

    NASA Astrophysics Data System (ADS)

    Ewert, Kai

    2005-03-01

    Cationic Liposome-DNA (CL-DNA) complexes are widely used in non-viral gene delivery, including clinical trials, but their efficiency still requires optimization. Membrane charge density is a universal parameter for transfection with lamellar CL-DNA complexes (Lin AJ et al., Biophys. J. 2003; 84: 3307; Ahmad A et al., J. Gene Med., accepted). Newly synthesized lipids with dendritic headgroups, based on an ornithine scaffold, have headgroup charges of +4e to +16e. These lipids form lamellar complexes if the headgroup charge is small or the fraction of dendritic lipid in the membrane (in mixtures with DOPC) is low. Higher contents of highly charged lipids exhibit a novel phase of CL-DNA complexes, whose structure was determined by synchrotron x-ray diffraction. Cylindrical micelles of lipid are arranged on a hexagonal lattice, with DNA rods placed around them in the interstices. Complexes with this structure are highly transfecting, preventing the previously observed drop in transfection efficiency at very high membrane charge densities. Funded by NIH GM-59288.

  12. Caproic acid grafted chitosan cationic nanocomplexes for enhanced gene delivery: effect of degree of substitution.

    PubMed

    Layek, Buddhadev; Singh, Jagdish

    2013-04-15

    This work was designed to investigate the effect of the degree of substitutions of caproic acid on plasmid DNA (pDNA) binding, cellular uptake, biocompatibility, and transfection efficiency of caproic acid grafted chitosan (CGC). The CGC with three substitution degrees (CGC-5, CGC-15, and CGC-25) were synthesized by coupling caproic acid with chitosan. Chemical characterization of graft polymers was performed using FTIR, NMR, and elemental analysis. The CGC polymers showed good pDNA condensing capacity and efficient protection of pDNA from DNase I. The nanosized CGC/pDNA polyplexes exhibited well-defined spherical shapes and stability in serum. Isothermal titration calorimetry demonstrated reduction in CGC-pDNA binding constant with increase in the degree of caproic acid substitution. Caproic acid substitution resulted in 2-7-fold higher cellular uptake in HEK 293 cells mainly via the clathrin-mediated pathway without affecting biocompatibility. In vitro transfection study suggested a dependence of transfection efficiency on the variability of caproic acid substitution. The CGC-15 polymer exhibited 31-fold and 1.33-fold higher gene expression compared to chitosan and the marketed non-viral vector FuGENEHD, respectively. These finding suggests that the CGC-15 graft polymer is a promising non-viral gene delivery vector due to its superior transfection efficiency and biocompatibility.

  13. Linear cyclen-based polyamine as a novel and efficient reagent in gene delivery.

    PubMed

    Xiang, Yong-Zhe; Feng, Zhi-Hua; Zhang, Ji; Liao, Yi-Le; Yu, Chuan-Jiang; Yi, Wen-Jing; Zhu, Wen; Yu, Xiao-Qi

    2010-02-01

    Linear cyclen-based polyamine (LCPA, M(w) = 7392, M(w)/M(n) = 1.19) as a novel non-viral gene vector was designed and synthesized from 1,7-diprotected 1,4,7,10-tetraazacyclododecane (cyclen), bis(beta-hydroxylethyl)amine and epichlorohydrin. Agarose gel retardation and fluorescent titration using ethidium bromide showed the good DNA-binding ability of LCPA. It could retard pDNA at an N/P ratio of 4 and form polyplexes with sizes around 250-300 nm from an N/P ratio of 10 to 60 and relatively lower zeta-potential values (< +3 mV) even at the N/P ratio of 60. The cytotoxicity of LCPA assayed by MTT is much lower than that of 25 kDa PEI. In vitro transfection against A549 and 293 cells showed that the transfection efficiency of LCPA/DNA polyplexes is close to that of 25 kDa PEI at an N/P ratio of 10-15, indicating that the new material could be a promising non-viral polycationic reagent for gene delivery.

  14. Cationic Nanoemulsions as a Gene Delivery System: Proof of Concept in the Mucopolysaccharidosis I Murine Model.

    PubMed

    Fraga, Michelle; de Carvalho, Talita Giacomet; Diel, Dirnete da Silva; Kretzmann Filho, Nélson Alexandre; Teixeira, Helder Ferreira; Matte, Ursula

    2015-01-01

    Mucopolysaccharidosis I (MPS I) is an autosomal recessive lysosomal storage disease due to deficient a-L-iduronidase (IDUA) activity. It results in the accumulation of the glycosaminoglycans (GAGs) heparan and dermatan sulfate and leads to several clinical manifestations. This study describes the use of cationic nanoemulsions as a non-viral carrier for the plasmid named pIDUA, which has the gene that encodes for the IDUA enzyme. Cationic nanoemulsions, composed by a medium chain triglycerides oil core stabilized by DOTAP, DOPE and DSPE-PEG, were prepared by high pressure homogeneization. pIDUA was complexed with nanoemulsions in the end of manufacturing process. Physicochemical properties of complexes were influenced by the charge ratio used. From a charge ratio of +2/-, it was observed a total complexation of pIDUA with formulation as well as a protection of plasmid against DNAse I digestion. In vitro assay in fibroblasts of one MPS I patient presented greater and significant trasfection efficiency for pIDUA complexed to formulation in the +4/- charge ratio. This formulation was administered via the tail vein and the portal vein. Animals were compared to untreated MPS I mice. Transfection efficiency was measured as IDUA enzyme activity. After intravenous administration, IDUA activity was significantly higher in lungs and liver. The set of results shows the formulation obtained at the +4/- charge ratio as a promising non-viral gene delivery system, once showed increased enzyme activity both in vitro and in vivo. PMID:26328445

  15. Folic Acid Linked Chondroitin Sulfate-Polyethyleneimine Copolymer Based Gene Delivery System.

    PubMed

    Lo, Yu-Lun; Lo, Pei-Chi; Chiu, Chien-Chih; Wang, Li-Fang

    2015-08-01

    In our previous study, chondroitin sulfate-polyethylenimine copolymers (CP) have been synthesized and confirmed as potential gene delivery vectors. Efficient gene transfection is realized by chondroitin sulfate (ChS) that promotes CD44- mediated endocytosis and enhances the cellular uptake of CP/pDNA polyplexes besides clathrin-mediated endocytosis. In this study, the CP was functionalized with a folic acid (FA) molecule. This ancillary ligand allows polyplexes to bind with folate receptors (FR) in addition to the CD44 receptor. We conjugated FA-linked polyethylene glycol (FA-PEG) onto CP (FPCP) for tumor targeting and also synthesized mPEG-CP (MPCP) for comparison. The in vitro cell tests of polymer/pDNA polyplexes were done in FR-expressed U87 and FR-deficient A549 cells. The polymers exhibited less cytotoxicity than PEI-10K as well as PEI-25K against U87 and A549 cells. The transfection efficiency of FPCP/pDNA was higher than those of MPCP/pDNA and CP/pDNA. The cellular uptake pathways of FPCP/pDNA were tested in the cells in the presence of different endocytic chemical inhibitors. The CD44-, folate-, and caveolae-mediated pathways are involved in internalization of FPCP/pDNA. Recognition of FPCP to those receptors on the tumor surface is beneficial for enhanced cellular uptake of FPCP/pDNA, resulting in higher transgene expression than CP/pDNA and MPCP/pDNA.

  16. Multiscale Simulation as a Framework for the Enhanced Design of Nanodiamond-Polyethylenimine-based Gene Delivery

    PubMed Central

    Kim, Hansung; Man, Han Bin; Saha, Biswajit; Kopacz, Adrian M.; Lee, One-Sun; Schatz, George C.; Ho, Dean; Liu, Wing Kam

    2012-01-01

    Nanodiamonds (NDs) are emerging carbon platforms with promise as gene/drug delivery vectors for cancer therapy. Specifically, NDs functionalized with the polymer polyethylenimine (PEI) can transfect small interfering RNAs (siRNA) in vitro with high efficiency and low cytotoxicity. Here we present a modeling framework to accurately guide the design of ND-PEI gene platforms and elucidate binding mechanisms between ND, PEI, and siRNA. This is among the first ND simulations to comprehensively account for ND size, charge distribution, surface functionalization, and graphitization. The simulation results are compared with our experimental results both for PEI loading onto NDs and for siRNA (C-myc) loading onto ND-PEI for various mixing ratios. Remarkably, the model is able to predict loading trends and saturation limits for PEI and siRNA, while confirming the essential role of ND surface functionalization in mediating ND-PEI interactions. These results demonstrate that this robust framework can be a powerful tool in ND platform development, with the capacity to realistically treat other nanoparticle systems. PMID:23304428

  17. Guanidinylated bioresponsive poly(amido amine)s designed for intranuclear gene delivery.

    PubMed

    Yu, Jiankun; Zhang, Jinmin; Xing, Haonan; Yang, Zhen; Cai, Cuifang; Zhang, Conglu; Zhao, Xiaoyun; Wei, Minjie; Yang, Li; Ding, Pingtian

    2016-01-01

    Guanidinylated poly(amido amine)s with multiple disulfide linkages (Gua-SS-PAAs) were designed and constructed as nonviral gene carriers. The main chains of these novel carriers were synthesized based on monomers containing guanidino groups (guanidine hydrochloride and chlorhexidine), which could avoid complicated side-chain-modification reactions while introducing the guanidino groups. The synthesized Gua-SS-PAAs polymers were characterized by (1)H nuclear magnetic resonance, molecular weight, and polydispersity. Furthermore, Gua-SS-PAAs polymers were complexed with pDNA, and the properties of the complexes were determined, including entrapment efficiency, particle size, ζ-potential, atomic force microscopy images, stability, DNA complexation ability, reduction sensitivity, cytotoxicity, and transfection efficiency. The new Gua-SS-PAAs carriers exhibited higher transfection efficiency and lower cytotoxicity compared with two widely used gene delivery carriers, polyethylenimine and lipofectamine 2000. Furthermore, the relationship between the side-chain structure and morphological/biological properties was extrapolated, and the results showed that guanidine in the side chain aids in the improvement of transfection efficiency. In addition, the introduction of guanidino group might confer the new carriers with nuclear localization function compared to carriers without it. PMID:27574429

  18. PEG conjugation of a near-infrared fluorescent probe for noninvasive dual imaging of lung deposition and gene expression by pulmonary gene delivery.

    PubMed

    Okuda, Tomoyuki; Kobayashi, Yuko; Yanamoto, Shinya; Okamoto, Hirokazu

    2012-11-01

    Dual imaging of lung deposition and gene expression following the pulmonary delivery of a gene formulation is useful for a precise analysis of gene transfection efficiency in vivo. As a novel probe for evaluating lung deposition, in this study, a poly(ethylene glycol)-conjugated near-infrared fluorescent probe (PEG-NIRF) was newly synthesized, and compared with indocyanine green (ICG), for application to pDNA/polyethyleneimine (PEI) complex. PEG-NIRF had superior characteristics including a larger Stokes shift (absorption maximum, 662 nm; emission maximum, 772 nm) and relatively equivalent fluorescence intensity compared with ICG. ICG affected the physicochemical properties of pDNA/PEI complex with a loss of fluorescence intensity, while PEG-NIRF did not. Experiments in mice demonstrated that PEG-NIRF showed greater lung localization than ICG following pulmonary co-delivery with pDNA/PEI complex, indicating the possibility of accurately evaluating lung deposition. Moreover, it was clarified that the evaluation of lung deposition by PEG-NIRF even at 60 min could be significantly correlated with gene expression in each mouse following pulmonary co-delivery with pDNA/PEI complex. These results suggest that PEG-NIRF is widely applicable to the dual imaging of the lung deposition and gene expression of inhaled gene formulations.

  19. RGD peptide-modified dendrimer-entrapped gold nanoparticles enable highly efficient and specific gene delivery to stem cells.

    PubMed

    Kong, Lingdan; Alves, Carla S; Hou, Wenxiu; Qiu, Jieru; Möhwald, Helmuth; Tomás, Helena; Shi, Xiangyang

    2015-03-01

    We report the use of arginine-glycine-aspartic (Arg-Gly-Asp, RGD) peptide-modified dendrimer-entrapped gold nanoparticles (Au DENPs) for highly efficient and specific gene delivery to stem cells. In this study, generation 5 poly(amidoamine) dendrimers modified with RGD via a poly(ethylene glycol) (PEG) spacer and with PEG monomethyl ether were used as templates to entrap gold nanoparticles (AuNPs). The native and the RGD-modified PEGylated dendrimers and the respective well characterized Au DENPs were used as vectors to transfect human mesenchymal stem cells (hMSCs) with plasmid DNA (pDNA) carrying both the enhanced green fluorescent protein and the luciferase (pEGFPLuc) reporter genes, as well as pDNA encoding the human bone morphogenetic protein-2 (hBMP-2) gene. We show that all vectors are capable of transfecting the hMSCs with both pDNAs. Gene transfection using pEGFPLuc was demonstrated by quantitative Luc activity assay and qualitative evaluation by fluorescence microscopy. For the transfection with hBMP-2, the gene delivery efficiency was evaluated by monitoring the hBMP-2 concentration and the level of osteogenic differentiation of the hMSCs via alkaline phosphatase activity, osteocalcin secretion, calcium deposition, and von Kossa staining assays. Our results reveal that the stem cell gene delivery efficiency is largely dependent on the composition and the surface functionality of the dendrimer-based vectors. The coexistence of RGD and AuNPs rendered the designed dendrimeric vector with specific stem cell binding ability likely via binding of integrin receptor on the cell surface and improved three-dimensional conformation of dendrimers, which is beneficial for highly efficient and specific stem cell gene delivery applications. PMID:25658033

  20. Spontaneous Preterm Delivery, Particularly with Reduced Fetal Growth, is Associated with DNA Hypomethylation of Tumor Related Genes

    PubMed Central

    Chen, Xinhua; Bai, Guang; Scholl, Theresa O

    2016-01-01

    Background Preterm delivery and sub-optimal fetal growth are associated with each other and affect both mother and infant. Our aim was to determine (i) whether there are detectable differences in DNA methylation between early and late gestation and (ii) whether changes in DNA methylation from entry are associated with spontaneous preterm delivery with and without reduced fetal growth. Methods We conducted a case-control study nested within a large prospective cohort. Gene specific methylation was measured by Methyl-Profiler PCR Array in a Human Breast Cancer Signature Panel of 24 genes from maternal peripheral leukocytes genomic DNA at entry and 3rd trimester (sampled at 16 and 30 weeks of gestation, respectively). Clonal bisulfite DNA sequencing was performed to confirm the changes in selected genes (CYP1B1, GADD45A and CXCL12). Multivariable analysis was used for data analysis. Results There was significantly decrease in DNA methylation in 15 of 24 genes during the 3rd trimester in cases of spontaneous preterm delivery (n=23) as compared to the controls (n=19) (p<0.05–p<0.01 for each gene). Similar results were observed by bisulfite sequencing for 3 genes. The change in DNA methylation between late and early gestation was significantly different in cases (overall decrease in methylation was −4.0 ± 1.5%) compared to the controls (overall increase in methylation was 12.6 ± 2.19%, p<0.0001). A graded pattern of DNA methylation was observed in 15 genes. Cases who delivered preterm with reduced fetal growth had the lowest level of methylation, cases delivering preterm without reduced fetal growth were next and term controls were highest in methylation (p for trend <0.05 to p<0.01 for each gene). Cases of preterm delivery also had significantly lower dietary choline intake. Conclusions These data suggest that epigenetic modification is associated with an increased risk of spontaneous preterm delivery, spontaneous preterm delivery with reduced fetal growth in

  1. Self-assembled supramolecular nano vesicles for safe and highly efficient gene delivery to solid tumors

    PubMed Central

    Li, Wei; Li, Huafei; Li, Jinfeng; Wang, Huajing; Zhao, He; Zhang, Li; Xia, Yu; Ye, Zengwei; Gao, Jie; Dai, Jianxin; Wang, Hao; Guo, Yajun

    2012-01-01

    The main obstacles for cationic polyplexes in gene delivery are in vivo instability and low solid-tumor accumulation. Safe vectors with high transfection efficiency and in vivo tumor accumulation are therefore highly desirable. In this study, the amphiphilic block copolymer poly(n-butyl methacrylate)-b-poly(N-acryloylmorpholine) was synthesized by reversible addition–fragmentation chain-transfer (RAFT) radical polymerization. The corresponding well-defined vesicles with narrow size distribution were tailored by finely regulating the packing parameter (β) of copolymer (1/2 < β < 1). Compared with traditional “gold-standard” polycation (polyethylenimine, 25 kDa), plasmid DNA condensing efficiency, DNase I degradation protection, and cellular uptake were improved by the supramolecular nano vesicles. In addition, the plasmid DNA transferring efficiency in 10% fetal bovine serum medium was enlarged five times to that of polyethylenimine in renal tubular epithelial and human hepatocellular carcinoma cell lines. This improved in vitro transfection was mainly attributed to the densely packed bilayer. This stealth polyplex showed high serum stability via entropic repulsion, which further protected the polyplex from being destroyed during sterilization. As indicated by the IVIS® Lumina II Imaging System (Caliper Life Sciences, Hopkinton, MA) 24 hours post-intravenous administration, intra-tumor accumulation of the stealth polyplex was clearly promoted. This study successfully circumvented the traditional dilemma of efficient gene transfection at a high nitrogen-from-polyethylenimine to phosphate-from-DNA ratio that is accompanied with site cytotoxicity and low stability. As such, these simply tailored noncytotoxic nano vesicles show significant potential for use in practical gene therapy. PMID:22977303

  2. Agmatine-Containing Bioreducible Polymer for Gene Delivery Systems and Its Dual Degradation Behavior.

    PubMed

    Choi, Ji-Yeong; Ryu, Kitae; Lee, Gyeong Jin; Kim, Kyunghwan; Kim, Tae-Il

    2015-09-14

    Agmatine-containing bioreducible polymer, poly(cystaminebis(acrylamide)-agmatine) (poly(CBA-AG)) was synthesized for gene delivery systems. It could form 200-300 nm sized and positively charged polyplexes with pDNA, which could release pDNA in reducing the environment due to the internal disulfide bonds cleavage. Poly(CBA-AG) also showed a spontaneous degradation behavior in aqueous condition in contrast to the backbone polymer, poly(cystaminebis(acrylamide)-diaminobutane) (poly(CBA-DAB)) lacking guanidine moieties, probably due to the self-catalyzed hydrolysis of internal amide bonds by guanidine moieties. The cytotoxicity of poly(CBA-AG) was cell-dependent but minimal. Poly(CBA-AG) exhibited highly enhanced transfection efficiency in comparison with poly(CBA-DAB) and even higher transfection efficiency than PEI25k. However, cellular uptake efficiency of the polyplexes did not show positive correlation with the transfection efficiency. Confocal microscopy observation revealed that pDNA delivered by poly(CBA-AG) was strongly accumulated in cell nuclei. These results suggested that high transfection efficiency of poly(CBA-AG) may be derived from the efficient pDNA localization in cell nuclei by guanidine moieties and that the polyplexes dissociation via self-catalyzed hydrolysis as well as disulfide bonds cleavage in cytosol also may facilitate the transfection process. Finally, poly(CBA-AG)/pJDK-apoptin polyplex showed a high anticancer activity induced by apoptosis, demonstrating a potential of poly(CBA-AG) as a gene carrier for cancer gene therapy.

  3. Serum-resistant lipopolyplexes for gene delivery to liver tumour cells.

    PubMed

    García, Leire; Buñuales, Maria; Düzgüneş, Nejat; Tros de Ilarduya, Conchita

    2007-08-01

    In this study, an efficient non-viral gene transfer system has been developed by employing polyethylenimine (PEI 800, 25 and 22kDa) and DOTAP and cholesterol (Chol) as lipids (lipopolyplex), at three different lipid/DNA molar ratios (2/1, 5/1 and 17/1) by using five different protocols of formulation. Condensation assays revealed that PEI of 800, 25 and 22kDa were very effective in condensing plasmid DNA, leading to a complete condensation at N/P ratios above 4. Addition of DOTAP/Chol liposomes did not further condense DNA. Increasing the molar ratio lipid/DNA in the complex resulted in higher positive values of the zeta-potential, while the particle size increased in some protocols, but not in others. High molecular weight PEI (800kDa) used in the formulation of lipopolyplexes lead to a bigger particle size, compared to that obtained with smaller PEI species, whether branched (25kDa) or linear (22kDa). These vectors were also highly effective in protecting DNA from attack by DNAse I. Transfection activity was maximal by using protocols 3 and 4 and a lipid/DNA molar ratio of 17/1. These complexes showed high efficiency in gene delivery of DNA to liver cancer cells, even in the presence of high concentration of serum (60% FBS). On the other hand, complexes formed with linear PEI (22kDa) were more effective than lipopolyplexes containing branched PEI (800 or 25kDa). The complexes resulted to be much more efficient than conventional lipoplexes (cationic lipid and DNA) and polyplexes (cationic polymer and DNA). The same behaviour was observed for complexes prepared in the presence of the therapeutic gene pCMVIL-12. Toxicity assays revealed a viability higher than 80% in all cases, independently of the protocol, molar ratio (lipid/DNA), molecular weight and type of PEI.

  4. Efficient gene delivery to human umbilical cord mesenchymal stem cells by cationized Porphyra yezoensis polysaccharide nanoparticles

    PubMed Central

    Yu, Qingtong; Cao, Jin; Chen, Baoding; Deng, Wenwen; Cao, Xia; Chen, Jingjing; Wang, Yan; Wang, Shicheng; Yu, Jiangnan; Xu, Ximing; Gao, Xiangdong

    2015-01-01

    This study centered on an innovative application of Porphyra yezoensis polysaccharide (PPS) with cationic modification as a safe and efficient nonviral gene vector to deliver a plasmid encoding human Wnt3a (pWnt3a) into human umbilical cord mesenchymal stem cells (HUMSCs). After modification with branched low-molecular-weight (1,200 Da) polyethylenimine, the cationized PPS (CPPS) was combined with pWnt3a to form spherical nanoscale particles (CPPS-pWnt3a nanoparticles). Particle size and distribution indicated that the CPPS-pWnt3a nanoparticles at a CPPS:pWnt3a weight ratio of 40:1 might be a potential candidate for DNA plasmid transfection. A cytotoxicity assay demonstrated that the nanoparticles prepared at a CPPS:pWnt3a weight ratio of 40:1 were nontoxic to HUMSCs compared to those of Lipofectamine 2000 and polyethylenimine (25 kDa). These nanoparticles were further transfected to HUMSCs. Western blotting demonstrated that the nanoparticles (CPPS:pWnt3a weight ratio 40:1) had the greatest transfection efficiency in HUMSCs, which was significantly higher than that of Lipofectamine 2000; however, when the CPPS:pWnt3a weight ratio was increased to 80:1, the nanoparticle-treated group showed no obvious improvement in translation efficiency over Lipofectamine 2000. Therefore, CPPS, a novel cationic polysaccharide derived from P. yezoensis, could be developed into a safe, efficient, nonviral gene vector in a gene-delivery system. PMID:26604758

  5. Controllably local gene delivery mediated by polyelectrolyte multilayer films assembled from gene-loaded nanopolymersomes and hyaluronic acid

    PubMed Central

    Teng, Wei; Wang, Qinmei; Chen, Ying; Huang, Hongzhang

    2014-01-01

    To explore a spatiotemporally controllable gene delivery system with high efficiency and safety, polyelectrolyte multilayer (PEM) films were constructed on titanium or quartz substrates via layer-by-layer self-assembly technique by using plasmid deoxyribonucleic acid-loaded lipopolysaccharide–amine nanopolymersomes (pNPs) as polycations and hyaluronic acid (HA) as polyanions. pNPs were chosen because they have high transfection efficiency (>95%) in mesenchymal stem cells (MSCs) and induce significant angiogenesis in zebrafish in conventional bolus transfection. The assembly process of PEM films was confirmed by analyses of quartz crystal microbalance with dissipation, X-ray photoelectron spectroscopy, infrared, contact angle, and zeta potential along with atomic force microscopy observation. Quartz crystal microbalance with dissipation analysis reveals that this film grows in an exponential mode, pNPs are the main contributor to the film mass, and the film mass can be modulated in a relatively wide range (1.0–29 μg/cm2) by adjusting the deposition layer number. Atomic force microscopy observation shows that the assembly leads to the formation of a patterned film with three-dimensional tree-like nanostructure, where the branches are composed of beaded chains (pNP beads are strung on HA molecular chains), and the incorporated pNPs keep structure intact. In vitro release experiment shows that plasmid deoxyribonucleic acid can be gradually released from films over 14 days, and the released plasmid deoxyribonucleic acid exists in a complex form. In vitro cell experiments demonstrate that PEM films can enhance the adhesion and proliferation of MSCs and efficiently transfect MSCs in situ in vitro for at least 4 days. Our results suggest that a (pNPs/HA)n system can mediate efficient transfection in stem cells in a spatially and temporally controllable pattern, highlighting its huge potential in local gene therapy. PMID:25378927

  6. A bio-recognition device developed onto nano-crystals of carbonate apatite for cell-targeted gene delivery.

    PubMed

    Chowdhury, E H; Akaike, Toshihiro

    2005-05-20

    The DNA delivery to mammalian cells is an essential tool for analyzing gene structure, regulation, and function. The approach holds great promise for the further development of gene therapy techniques and DNA vaccination strategies to treat and control diseases. Here, we report on the establishment of a cell-specific gene delivery and expression system by physical adsorption of a cell-recognition molecule on the nano-crystal surface of carbonate apatite. As a model, DNA/nano-particles were successfully coated with asialofetuin to facilitate uptake by hepatocyte-derived cell lines through the asialoglycoprotein receptor (ASGPr) and albumin to prevent non-specific interactions of the particles with cell-surface. The resulting composite particles with dual surface properties could accelerate DNA uptake and enhance expression to a notable extent. Nano-particles coated with transferrin in the same manner dramatically enhanced transgene expression in the corresponding receptor-bearing cells and thus our newly developed strategy represents a universal phenomenon for anchoring a bio-recognition macromolecule on the apatite crystal surface for targeted gene delivery, having immediate applications in basic research laboratories and great promise for gene therapy.

  7. A novel target-specific gene delivery system combining baculovirus and sequence-specific long interspersed nuclear elements.

    PubMed

    Kawashima, Tomoko; Osanai, Mizuko; Futahashi, Ryo; Kojima, Tetsuya; Fujiwara, Haruhiko

    2007-07-01

    Transposable elements are valuable for somatic and germ-line transformation. However, long interspersed nuclear elements (LINEs) have not been used because of poor information on the transposition mechanism. We have developed a novel gene delivery system combining baculovirus AcNPV and two silkworm LINEs, SART1 and R1, which integrate into specific sequences of telomeric repeats and 28S ribosomal DNA, respectively. When two LINEs containing the enhanced green fluorescent protein gene recombined into AcNPV were infected into fifth instar larvae of the silkworm, we observed target-specific retrotransposition of LINEs at 72h post-infection, using polymerase chain reaction amplification and sequencing. Telomere- and 28S rDNA-specific transposition occurred in all nine tissues tested, including the ovary and testis. This is the first demonstration of site-specific gene delivery in living larvae. Insertion efficiencies were dependent on the virus titer for injection and the host strains of Bombyx mori. Using this system, we successfully detected the intergeneration transmission of retrotransposed sequences. In addition, AcNPV-mediated SART1 also transposed into telomere of another lepidopteran, Orgyia recens, suggesting that this system is useful for a wide variety of AcNPV-infectious insects. Site-specific gene delivery by virus-mediated LINE will be a potential gene therapy tool to avoid harmful unexpected insertions.

  8. Folate-associated lipoplexes mediate efficient gene delivery and potent antitumoral activity in vitro and in vivo.

    PubMed

    Duarte, Sónia; Faneca, Henrique; Lima, Maria C Pedroso de

    2012-02-28

    The lack of suitable vectors for efficient nucleic acid delivery into target cells represents a major hurdle for the successful application of gene therapy. Cationic liposomes exhibit attractive features for gene delivery, but their efficacy is still unsatisfactory, particularly for in vivo applications, which justifies the drive to further improve their performance by developing novel and efficient formulations. In the present study, we generated a new formulation of lipoplexes through electrostatic association of folate (FA) to 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (EPOPC):cholesterol (Chol) liposomes, prepared at different lipid/DNA charge ratios, and explored their potential to mediate gene delivery. The optimal FA-lipoplex formulation was evaluated for its efficacy to mediate antitumoral activity upon application of HSV-tk suicide gene therapy, both in vitro and in an animal model of oral cancer. Our results demonstrate that FA-EPOPC:Chol/DNA lipoplexes were able to promote a great enhancement of transfection and high in vitro antitumoral activity compared to plain lipoplexes in two different cancer cell lines. Most importantly, a considerable reduction of tumor growth was achieved with the developed FA-lipoplexes as compared to that observed for control FA-lipoplexes or plain lipoplexes. Overall, our study shows that FA-EPOPC:Chol/DNA lipoplexes constitute a promising system for the successful application of suicide gene therapy aiming at treating solid tumors. PMID:22209825

  9. Self-Assembling Micelle-like Nanoparticles Based on Phospholipid-Polyethyleneimine Conjugates for Systemic Gene Delivery

    PubMed Central

    Ko, Young Tag; Kale, Amit; Hartner, William C.; Papahadjopoulos-Sternberg, Brigitte; Torchilin, Vladimir P.

    2011-01-01

    With few exceptions, where local administration is feasible, progress towards broad clinical application of gene therapies requires the development of effective delivery systems. Here we report a novel non-viral gene delivery vector, ‘micelle-like nanoparticle’ (MNP) suitable for systemic application. MNP were engineered by condensing plasmid DNA with a chemical conjugate of phospholipid with polyethylenimine (PLPEI) and then coating the complexes with an envelope of lipid monolayer additionally containing polyethylene glycol-phosphatidyl ethanolamine (PEG-PE), resulting in spherical ‘hard-core’ nanoparticles loaded with DNA. MNP allowed for complete protection of the loaded DNA from enzymatic degradation, resistance to salt-induced aggregation, and reduced cytotoxicity. MNP also demonstrated prolonged blood circulation and low RES accumulation. Intravenous injection of MNP loaded with plasmid DNA encoding for the Green Fluorescence Protein (GFP) resulted in an effective transfection of a distal tumor. Thus, MNP provide a promising tool for systemic gene therapy. PMID:18929605

  10. In vivo targeted gene delivery to peripheral neurons mediated by neurotropic poly(ethylene imine)-based nanoparticles.

    PubMed

    Lopes, Cátia Df; Oliveira, Hugo; Estevão, Inês; Pires, Liliana Raquel; Pêgo, Ana Paula

    2016-01-01

    A major challenge in neuronal gene therapy is to achieve safe, efficient, and minimally invasive transgene delivery to neurons. In this study, we report the use of a nonviral neurotropic poly(ethylene imine)-based nanoparticle that is capable of mediating neuron-specific transfection upon a subcutaneous injection. Nanoparticles were targeted to peripheral neurons by using the nontoxic carboxylic fragment of tetanus toxin (HC), which, besides being neurotropic, is capable of being retrogradely transported from neuron terminals to the cell bodies. Nontargeted particles and naked plasmid DNA were used as control. Five days after treatment by subcutaneous injection in the footpad of Wistar rats, it was observed that 56% and 64% of L4 and L5 dorsal root ganglia neurons, respectively, were expressing the reporter protein. The delivery mediated by HC-functionalized nanoparticles spatially limited the transgene expression, in comparison with the controls. Histological examination revealed no significant adverse effects in the use of the proposed delivery system. These findings demonstrate the feasibility and safety of the developed neurotropic nanoparticles for the minimally invasive delivery of genes to the peripheral nervous system, opening new avenues for the application of gene therapy strategies in the treatment of peripheral neuropathies. PMID:27354797

  11. In vivo targeted gene delivery to peripheral neurons mediated by neurotropic poly(ethylene imine)-based nanoparticles

    PubMed Central

    Lopes, Cátia DF; Oliveira, Hugo; Estevão, Inês; Pires, Liliana Raquel; Pêgo, Ana Paula

    2016-01-01

    A major challenge in neuronal gene therapy is to achieve safe, efficient, and minimally invasive transgene delivery to neurons. In this study, we report the use of a nonviral neurotropic poly(ethylene imine)-based nanoparticle that is capable of mediating neuron-specific transfection upon a subcutaneous injection. Nanoparticles were targeted to peripheral neurons by using the nontoxic carboxylic fragment of tetanus toxin (HC), which, besides being neurotropic, is capable of being retrogradely transported from neuron terminals to the cell bodies. Nontargeted particles and naked plasmid DNA were used as control. Five days after treatment by subcutaneous injection in the footpad of Wistar rats, it was observed that 56% and 64% of L4 and L5 dorsal root ganglia neurons, respectively, were expressing the reporter protein. The delivery mediated by HC-functionalized nanoparticles spatially limited the transgene expression, in comparison with the controls. Histological examination revealed no significant adverse effects in the use of the proposed delivery system. These findings demonstrate the feasibility and safety of the developed neurotropic nanoparticles for the minimally invasive delivery of genes to the peripheral nervous system, opening new avenues for the application of gene therapy strategies in the treatment of peripheral neuropathies. PMID:27354797

  12. Synthesis and evaluation of cationic nanomicelles for in vitro and in vivo gene delivery

    NASA Astrophysics Data System (ADS)

    Mandke, Rhishikesh Subhash

    The goal of proposed study was to contribute towards the development of a nano size, high efficiency and low toxicity non-viral polymeric vector for gene delivery in vitro and in vivo. A series of fatty acid grafted low-molecular-weight chitosan (N-acyl LMWCs) were synthesized, purified and characterized for their physicochemical properties using various analytical techniques such as infrared spectroscopy, elemental analysis and dynamic light scattering. The formulation parameters including pH, sonication duration, and filtration altered the physicochemical characteristics of N-acyl LMWC nanomicelles. The acyl chain length and degree of unsaturation in fatty acids also had an impact on the physicochemical properties and the transfection efficiency of nanomicelles. N-acyl LMWC nanomicelles showed efficient in vitro transfection as visualized and quantified using a reporter plasmid (encoding green fluorescent protein), and therapeutic plasmids (encoding for interleukin-4 and interleukin-10), respectively. The in vitro transfection efficiencies of N-acyl LMWCs with 18:1 and 18:2 grafts (oleic and linoleic acids) were comparable with FuGENERTM HD (marketed non-viral vector) but were ˜8-fold and 35-fold higher as compared to LMWC and naked DNA, respectively. The in vivo transfection efficiency of N-acyl LMWC to deliver plasmids individually encoding IL-4 and IL-10 as well as a bicistronic plasmid encoding both IL-4 and IL-10 was studied in a multiple, low-dose streptozotocin induced diabetic mouse model. The transfection efficiency of pDNA/N-acyl LMWC polyplexes injected via intramuscular route showed significant improvement (p<0.05) over passive (naked DNA) or positive (FuGENE HD) controls. Additionally, a sustained and efficient expression of IL-4 and IL-10 was observed, accompanied by a reduction in interferon-gamma (INF-gamma), and tumor necrosis factor-alpha (TNF-alpha) levels. The pancreas of pDNA/N-acyl LMWC polyplex treated animals exhibited protection from

  13. Reiterated Targeting Peptides on the Nanoparticle Surface Significantly Promote Targeted Vascular Endothelial Growth Factor Gene Delivery to Stem Cells.

    PubMed

    Wang, Dong-Dong; Yang, Mingying; Zhu, Ye; Mao, Chuanbin

    2015-12-14

    Nonviral gene delivery vectors hold great promise for gene therapy due to the safety concerns with viral vectors. However, the application of nonviral vectors is hindered by their low transfection efficiency. Herein, in order to tackle this challenge, we developed a nonviral vector integrating lipids, sleeping beauty transposon system and 8-mer stem cell targeting peptides for safe and efficient gene delivery to hard-to-transfect mesenchymal stem cells (MSCs). The 8-mer MSC-targeting peptides, when synthetically reiterated in three folds and chemically presented on the surface, significantly promoted the resultant lipid-based nanoparticles (LBNs) to deliver VEGF gene into MSCs with a high transfection efficiency (∼52%) and long-lasting gene expression (for longer than 170 h) when compared to nonreiterated peptides. However, the reiterated stem cell targeting peptides do not enable the highly efficient gene transfer to other control cells. This work suggests that the surface presentation of the reiterated stem cell-targeting peptides on the nonviral vectors is a promising method for improving the efficiency of cell-specific nonviral gene transfection in stem cells. PMID:26588028

  14. RNAi-based therapeutic nanostrategy: IL-8 gene silencing in pancreatic cancer cells using gold nanorods delivery vehicles.

    PubMed

    Panwar, Nishtha; Yang, Chengbin; Yin, Feng; Yoon, Ho Sup; Chuan, Tjin Swee; Yong, Ken-Tye

    2015-09-11

    RNA interference (RNAi)-based gene silencing possesses great ability for therapeutic intervention in pancreatic cancer. Among various oncogene mutations, Interleukin-8 (IL-8) gene mutations are found to be overexpressed in many pancreatic cell lines. In this work, we demonstrate IL-8 gene silencing by employing an RNAi-based gene therapy approach and this is achieved by using gold nanorods (AuNRs) for efficient delivery of IL-8 small interfering RNA (siRNA) to the pancreatic cell lines of MiaPaCa-2 and Panc-1. Upon comparing to Panc-1 cells, we found that the dominant expression of the IL-8 gene in MiaPaCa-2 cells resulted in an aggressive behavior towards the processes of cell invasion and metastasis. We have hence investigated the suitability of using AuNRs as novel non-viral nanocarriers for the efficient uptake and delivery of IL-8 siRNA in realizing gene knockdown of both MiaPaCa-2 and Panc-1 cells. Flow cytometry and fluorescence imaging techniques have been applied to confirm transfection and release of IL-8 siRNA. The ratio of AuNRs and siRNA has been optimized and transfection efficiencies as high as 88.40 ± 2.14% have been achieved. Upon successful delivery of IL-8 siRNA into cancer cells, the effects of IL-8 gene knockdown are quantified in terms of gene expression, cell invasion, cell migration and cell apoptosis assays. Statistical comparative studies for both MiaPaCa-2 and Panc-1 cells are presented in this work. IL-8 gene silencing has been demonstrated with knockdown efficiencies of 81.02 ± 10.14% and 75.73 ± 6.41% in MiaPaCa-2 and Panc-1 cells, respectively. Our results are then compared with a commercial transfection reagent, Oligofectamine, serving as positive control. The gene knockdown results illustrate the potential role of AuNRs as non-viral gene delivery vehicles for RNAi-based targeted cancer therapy applications.

  15. RNAi-based therapeutic nanostrategy: IL-8 gene silencing in pancreatic cancer cells using gold nanorods delivery vehicles

    NASA Astrophysics Data System (ADS)

    Panwar, Nishtha; Yang, Chengbin; Yin, Feng; Yoon, Ho Sup; Swee Chuan, Tjin; Yong, Ken-Tye

    2015-09-01

    RNA interference (RNAi)-based gene silencing possesses great ability for therapeutic intervention in pancreatic cancer. Among various oncogene mutations, Interleukin-8 (IL-8) gene mutations are found to be overexpressed in many pancreatic cell lines. In this work, we demonstrate IL-8 gene silencing by employing an RNAi-based gene therapy approach and this is achieved by using gold nanorods (AuNRs) for efficient delivery of IL-8 small interfering RNA (siRNA) to the pancreatic cell lines of MiaPaCa-2 and Panc-1. Upon comparing to Panc-1 cells, we found that the dominant expression of the IL-8 gene in MiaPaCa-2 cells resulted in an aggressive behavior towards the processes of cell invasion and metastasis. We have hence investigated the suitability of using AuNRs as novel non-viral nanocarriers for the efficient uptake and delivery of IL-8 siRNA in realizing gene knockdown of both MiaPaCa-2 and Panc-1 cells. Flow cytometry and fluorescence imaging techniques have been applied to confirm transfection and release of IL-8 siRNA. The ratio of AuNRs and siRNA has been optimized and transfection efficiencies as high as 88.40 ± 2.14% have been achieved. Upon successful delivery of IL-8 siRNA into cancer cells, the effects of IL-8 gene knockdown are quantified in terms of gene expression, cell invasion, cell migration and cell apoptosis assays. Statistical comparative studies for both MiaPaCa-2 and Panc-1 cells are presented in this work. IL-8 gene silencing has been demonstrated with knockdown efficiencies of 81.02 ± 10.14% and 75.73 ± 6.41% in MiaPaCa-2 and Panc-1 cells, respectively. Our results are then compared with a commercial transfection reagent, Oligofectamine, serving as positive control. The gene knockdown results illustrate the potential role of AuNRs as non-viral gene delivery vehicles for RNAi-based targeted cancer therapy applications.

  16. RNAi-based therapeutic nanostrategy: IL-8 gene silencing in pancreatic cancer cells using gold nanorods delivery vehicles.

    PubMed

    Panwar, Nishtha; Yang, Chengbin; Yin, Feng; Yoon, Ho Sup; Chuan, Tjin Swee; Yong, Ken-Tye

    2015-09-11

    RNA interference (RNAi)-based gene silencing possesses great ability for therapeutic intervention in pancreatic cancer. Among various oncogene mutations, Interleukin-8 (IL-8) gene mutations are found to be overexpressed in many pancreatic cell lines. In this work, we demonstrate IL-8 gene silencing by employing an RNAi-based gene therapy approach and this is achieved by using gold nanorods (AuNRs) for efficient delivery of IL-8 small interfering RNA (siRNA) to the pancreatic cell lines of MiaPaCa-2 and Panc-1. Upon comparing to Panc-1 cells, we found that the dominant expression of the IL-8 gene in MiaPaCa-2 cells resulted in an aggressive behavior towards the processes of cell invasion and metastasis. We have hence investigated the suitability of using AuNRs as novel non-viral nanocarriers for the efficient uptake and delivery of IL-8 siRNA in realizing gene knockdown of both MiaPaCa-2 and Panc-1 cells. Flow cytometry and fluorescence imaging techniques have been applied to confirm transfection and release of IL-8 siRNA. The ratio of AuNRs and siRNA has been optimized and transfection efficiencies as high as 88.40 ± 2.14% have been achieved. Upon successful delivery of IL-8 siRNA into cancer cells, the effects of IL-8 gene knockdown are quantified in terms of gene expression, cell invasion, cell migration and cell apoptosis assays. Statistical comparative studies for both MiaPaCa-2 and Panc-1 cells are presented in this work. IL-8 gene silencing has been demonstrated with knockdown efficiencies of 81.02 ± 10.14% and 75.73 ± 6.41% in MiaPaCa-2 and Panc-1 cells, respectively. Our results are then compared with a commercial transfection reagent, Oligofectamine, serving as positive control. The gene knockdown results illustrate the potential role of AuNRs as non-viral gene delivery vehicles for RNAi-based targeted cancer therapy applications. PMID:26291710

  17. Impact of homeobox genes in gastrointestinal cancer

    PubMed Central

    Joo, Moon Kyung; Park, Jong-Jae; Chun, Hoon Jai

    2016-01-01

    Homeobox genes, including HOX and non-HOX genes, have been identified to be expressed aberrantly in solid tumors. In gastrointestinal (GI) cancers, most studies have focused on the function of non-HOX genes including caudal-related homeobox transcription factor 1 (CDX1) and CDX2. CDX2 is a crucial factor in the development of pre-cancerous lesions such as Barrett’s esophagus or intestinal metaplasia in the stomach, and its tumor suppressive role has been investigated in colorectal cancers. Recently, several HOX genes were reported to have specific roles in GI cancers; for example, HOXA13 in esophageal squamous cell cancer and HOXB7 in stomach and colorectal cancers. HOXD10 is upregulated in colorectal cancer while it is silenced epigenetically in gastric cancer. Thus, it is essential to examine the differential expression pattern of various homeobox genes in specific tumor types or cell lineages, and understand their underlying mechanisms. In this review, we summarize the available research on homeobox genes and present their potential value for the prediction of prognosis in GI cancers. PMID:27729732

  18. The transition from linear to highly branched poly(β-amino ester)s: Branching matters for gene delivery

    PubMed Central

    Zhou, Dezhong; Cutlar, Lara; Gao, Yongsheng; Wang, Wei; O’Keeffe-Ahern, Jonathan; McMahon, Sean; Duarte, Blanca; Larcher, Fernando; Rodriguez, Brian J.; Greiser, Udo; Wang, Wenxin

    2016-01-01

    Nonviral gene therapy holds great promise but has not delivered treatments for clinical application to date. Lack of safe and efficient gene delivery vectors is the major hurdle. Among nonviral gene delivery vectors, poly(β-amino ester)s are one of the most versatile candidates because of their wide monomer availability, high polymer flexibility, and superior gene transfection performance both in vitro and in vivo. However, to date, all research has been focused on vectors with a linear structure. A well-accepted view is that dendritic or branched polymers have greater potential as gene delivery vectors because of their three-dimensional structure and multiple terminal groups. Nevertheless, to date, the synthesis of dendritic or branched polymers has been proven to be a well-known challenge. We report the design and synthesis of highly branched poly(β-amino ester)s (HPAEs) via a one-pot “A2 + B3 + C2”–type Michael addition approach and evaluate their potential as gene delivery vectors. We find that the branched structure can significantly enhance the transfection efficiency of poly(β-amino ester)s: Up to an 8521-fold enhancement in transfection efficiency was observed across 12 cell types ranging from cell lines, primary cells, to stem cells, over their corresponding linear poly(β-amino ester)s (LPAEs) and the commercial transfection reagents polyethyleneimine, SuperFect, and Lipofectamine 2000. Moreover, we further demonstrate that HPAEs can correct genetic defects in vivo using a recessive dystrophic epidermolysis bullosa graft mouse model. Our findings prove that the A2 + B3 + C2 approach is highly generalizable and flexible for the design and synthesis of HPAEs, which cannot be achieved by the conventional polymerization approach; HPAEs are more efficient vectors in gene transfection than the corresponding LPAEs. This provides valuable insight into the development and applications of nonviral gene delivery and demonstrates great prospect for their

  19. Development of a successive targeting liposome with multi-ligand for efficient targeting gene delivery

    PubMed Central

    Ma, Kun; Shen, Haijun; Shen, Song; Xie, Men; Mao, Chuanbin; Qiu, Liyan; Jin, Yi

    2012-01-01

    Background A successful gene delivery system needs to breakthrough several barriers to allow efficient transgenic expression. In the present study, successive targeting liposomes (STL) were constructed by integrating various targeting groups into a nanoparticle to address this issue. Methods Polyethylenimine (PEI) 1800-triamcinolone acetonide (TA) with nuclear targeting capability was synthesized by a two-step reaction. Lactobionic acid was connected with cholesterol to obtain a compound of [(2-lactoylamido) ethylamino]formic acid cholesterol ester (CHEDLA) with hepatocyte-targeting capability. The liposome was modified with PEI 1800-TA and CHEDLA to prepare successive targeting liposome (STL). Its physicochemical properties and transfection efficiency were investigated both in vitro and in vivo. Results The diameter of STL was approximately 100 nm with 20 mV of potential. The confocal microscopy observation and potential assay verified that lipid bilayer of STL was decorated with PEI 1800-TA. Cytotoxicity of STL was significantly lower than that of PEI 1800-TA and PEI 25K. The transfection efficiency of 10% CHEDLA STL in HepG2 cells was the higher than of the latter two with serum. Its transfection efficiency was greatly reduced with excessive free galactose, indicating that STL was absorbed via galactose receptor-mediated endocytosis. The in vivo study in mice showed that 10% CHEDLA STL had better transgenic expression in liver than the other carriers. Conclusions STL with multi-ligand was able to overcome the various barriers to target nucleus and special cells and present distinctive transgenic expression. Therefore, it has a great potential for gene therapy as a nonviral carrier. PMID:21574214

  20. Tropism-Modification Strategies for Targeted Gene Delivery Using Adenoviral Vectors

    PubMed Central

    Coughlan, Lynda; Alba, Raul; Parker, Alan L.; Bradshaw, Angela C.; McNeish, Iain A.; Nicklin, Stuart A.; Baker, Andrew H.

    2010-01-01

    Achieving high efficiency, targeted gene delivery with adenoviral vectors is a long-standing goal in the field of clinical gene therapy. To achieve this, platform vectors must combine efficient retargeting strategies with detargeting modifications to ablate native receptor binding (i.e. CAR/integrins/heparan sulfate proteoglycans) and “bridging” interactions. “Bridging” interactions refer to coagulation factor binding, namely coagulation factor X (FX), which bridges hepatocyte transduction in vivo through engagement with surface expressed heparan sulfate proteoglycans (HSPGs). These interactions can contribute to the off-target sequestration of Ad5 in the liver and its characteristic dose-limiting hepatotoxicity, thereby significantly limiting the in vivo targeting efficiency and clinical potential of Ad5-based therapeutics. To date, various approaches to retargeting adenoviruses (Ad) have been described. These include genetic modification strategies to incorporate peptide ligands (within fiber knob domain, fiber shaft, penton base, pIX or hexon), pseudotyping of capsid proteins to include whole fiber substitutions or fiber knob chimeras, pseudotyping with non-human Ad species or with capsid proteins derived from other viral families, hexon hypervariable region (HVR) substitutions and adapter-based conjugation/crosslinking of scFv, growth factors or monoclonal antibodies directed against surface-expressed target antigens. In order to maximize retargeting, strategies which permit detargeting from undesirable interactions between the Ad capsid and components of the circulatory system (e.g. coagulation factors, erythrocytes, pre-existing neutralizing antibodies), can be employed simultaneously. Detargeting can be achieved by genetic ablation of native receptor-binding determinants, ablation of “bridging interactions” such as those which occur between the hexon of Ad5 and coagulation factor X (FX), or alternatively, through the use of polymer-coated

  1. Tropism-modification strategies for targeted gene delivery using adenoviral vectors.

    PubMed

    Coughlan, Lynda; Alba, Raul; Parker, Alan L; Bradshaw, Angela C; McNeish, Iain A; Nicklin, Stuart A; Baker, Andrew H

    2010-10-01

    Achieving high efficiency, targeted gene delivery with adenoviral vectors is a long-standing goal in the field of clinical gene therapy. To achieve this, platform vectors must combine efficient retargeting strategies with detargeting modifications to ablate native receptor binding (i.e. CAR/integrins/heparan sulfate proteoglycans) and "bridging" interactions. "Bridging" interactions refer to coagulation factor binding, namely coagulation factor X (FX), which bridges hepatocyte transduction in vivo through engagement with surface expressed heparan sulfate proteoglycans (HSPGs). These interactions can contribute to the off-target sequestration of Ad5 in the liver and its characteristic dose-limiting hepatotoxicity, thereby significantly limiting the in vivo targeting efficiency and clinical potential of Ad5-based therapeutics. To date, various approaches to retargeting adenoviruses (Ad) have been described. These include genetic modification strategies to incorporate peptide ligands (within fiber knob domain, fiber shaft, penton base, pIX or hexon), pseudotyping of capsid proteins to include whole fiber substitutions or fiber knob chimeras, pseudotyping with non-human Ad species or with capsid proteins derived from other viral families, hexon hypervariable region (HVR) substitutions and adapter-based conjugation/crosslinking of scFv, growth factors or monoclonal antibodies directed against surface-expressed target antigens. In order to maximize retargeting, strategies which permit detargeting from undesirable interactions between the Ad capsid and components of the circulatory system (e.g. coagulation factors, erythrocytes, pre-existing neutralizing antibodies), can be employed simultaneously. Detargeting can be achieved by genetic ablation of native receptor-binding determinants, ablation of "bridging interactions" such as those which occur between the hexon of Ad5 and coagulation factor X (FX), or alternatively, through the use of polymer-coated "stealth" vectors

  2. Enhancement of electric field-mediated gene delivery through pretreatment of tumors with a hyperosmotic mannitol solution.

    PubMed

    Henshaw, J; Mossop, B; Yuan, F

    2011-01-01

    Pulsed electric fields can enhance interstitial transport of plasmid DNA (pDNA) in solid tumors. However, the extent of enhancement is still limited. To this end, the effects of cellular resistance to electric field-mediated gene delivery were investigated. The investigation used two tumor cell lines (4T1 (a murine mammary carcinoma) and B16.F10 (a metastatic subline of B16 murine melanoma)) either in suspensions or implanted in two in vivo models (dorsal skin-fold chamber (DSC) and hind leg). The volume fraction of cells was altered by pretreatment with a hyperosmotic mannitol solution (1 M). It was observed that the pretreatment reduced the volumes of 4T1 and B16.F10 cells, suspended in an agarose gel, by 50 and 46%, respectively, over a 20-min period, but did not cause significant changes ex vivo in volumes of hind-leg tumor tissues grown from the same cells in mice. The mannitol pretreatment in vivo improved electric field-mediated gene delivery in the hind-leg tumor models, in terms of reporter gene expression, but resulted in minimal enhancement in pDNA electrophoresis over a few microns distance in the DSC tumor models. These data demonstrated that hyperosmotic mannitol solution could effectively improve electric field-mediated gene delivery around individual cells in vivo by increasing the extracellular space. PMID:20847751

  3. Minicircle microporation-based non-viral gene delivery improved the targeting of mesenchymal stem cells to an injury site.

    PubMed

    Mun, Ji-Young; Shin, Keun Koo; Kwon, Ohsuk; Lim, Yong Taik; Oh, Doo-Byoung

    2016-09-01

    Genetic engineering approaches to improve the therapeutic potential of mesenchymal stem cells (MSCs) have been made by viral and non-viral gene delivery methods. Viral methods have severe limitations in clinical application because of potential oncogenic, pathogenic, and immunogenic risks, while non-viral methods have suffered from low transfection efficiency and transient weak expression as MSCs are hard-to-transfect cells. In this study, minicircle, which is a minimal expression vector free of bacterial sequences, was employed for MSC transfection as a non-viral gene delivery method. The conventional cationic liposome method was not effective for MSC transfection as it resulted in very low transfection efficiency (less than 5%). Microporation, a new electroporation method, greatly improved the transfection efficiency of minicircles by up to 66% in MSCs without any significant loss of cell viability. Furthermore, minicircle microporation generated much stronger and prolonged transgene expression compared with plasmid microporation. When MSCs microporated with minicircle harboring firefly luciferase gene were subcutaneously injected to mice, the bioluminescence continued for more than a week, whereas the bioluminescence of the MSCs induced by plasmid microporation rapidly decreased and disappeared in mice within three days. By minicircle microporation as a non-viral gene delivery, MSCs engineered to overexpress CXCR4 showed greatly increased homing ability toward an injury site as confirmed through in vivo bioluminescence imaging in mice. In summary, the engineering of MSCs through minicircle microporation is expected to enhance the therapeutic potential of MSCs in clinical applications. PMID:27315214

  4. Application of pulsed-magnetic field enhances non-viral gene delivery in primary cells from different origins

    NASA Astrophysics Data System (ADS)

    Kamau Chapman, Sarah W.; Hassa, Paul O.; Koch-Schneidemann, Sabine; von Rechenberg, Brigitte; Hofmann-Amtenbrink, Margarethe; Steitz, Benedikt; Petri-Fink, Alke; Hofmann, Heinrich; Hottiger, Michael O.

    Primary cell lines are more difficult to transfect when compared to immortalized/transformed cell lines, and hence new techniques are required to enhance the transfection efficiency in these cells. We isolated and established primary cultures of synoviocytes, chondrocytes, osteoblasts, melanocytes, macrophages, lung fibroblasts, and embryonic fibroblasts. These cells differed in several properties, and hence were a good representative sample of cells that would be targeted for expression and delivery of therapeutic genes in vivo. The efficiency of gene delivery in all these cells was enhanced using polyethylenimine-coated polyMAG magnetic nanoparticles, and the rates (17-84.2%) surpassed those previously achieved using other methods, especially in cells that are difficult to transfect. The application of permanent and pulsating magnetic fields significantly enhanced the transfection efficiencies in synoviocytes, chondrocytes, osteoblasts, melanocytes and lung fibroblasts, within 5 min of exposure to these magnetic fields. This is an added advantage for future in vivo applications, where rapid gene delivery is required before systemic clearance or filtration of the gene vectors occurs.

  5. Role of Cell Membrane-Vector Interactions in Successful Gene Delivery.

    PubMed

    Vaidyanathan, Sriram; Orr, Bradford G; Banaszak Holl, Mark M

    2016-08-16

    Cationic polymers have been investigated as nonviral vectors for gene delivery due to their favorable safety profile when compared to viral vectors. However, nonviral vectors are limited by poor efficacy in inducing gene expression. The physicochemical properties of cationic polymers enabling successful gene expression have been investigated in order to improve expression efficiency and safety. Studies over the past several years have focused on five possible rate-limiting processes to explain the differences in gene expression: (1) endosomal release, (2) transport within specific intracellular pathways, (3) protection of DNA from nucleases, (4) transport into the nucleus, and (5) DNA release from vectors. However, determining the relative importance of these processes and the vector properties necessary for optimization remain a challenge to the field. In this Account, we describe over a decade of studies focused on understanding the interaction of cationic polymer and cationic polymer/oligonucleotide (polyplex) interactions with model lipid membranes, cell membranes, and cells in culture. In particular, we have been interested in how the interaction between cationic polymers and the membrane influences the intracellular transport of intact DNA to the nucleus. Recent advances in microfluidic patch clamp techniques enabled us to quantify polyplex cell membrane interactions at the cellular level with precise control over material concentrations and exposure times. In attempting to relate these findings to subsequent intracellular transport of DNA and expression of protein, we needed to develop an approach that could distinguish DNA that was intact and potentially functional for gene expression from the much larger pool of degraded, nonfunctional DNA within the cell. We addressed this need by developing a FRET oligonucleotide molecular beacon (OMB) to monitor intact DNA transport. The research highlighted in this Account builds to the conclusion that polyplex

  6. Role of Cell Membrane-Vector Interactions in Successful Gene Delivery.

    PubMed

    Vaidyanathan, Sriram; Orr, Bradford G; Banaszak Holl, Mark M

    2016-08-16

    Cationic polymers have been investigated as nonviral vectors for gene delivery due to their favorable safety profile when compared to viral vectors. However, nonviral vectors are limited by poor efficacy in inducing gene expression. The physicochemical properties of cationic polymers enabling successful gene expression have been investigated in order to improve expression efficiency and safety. Studies over the past several years have focused on five possible rate-limiting processes to explain the differences in gene expression: (1) endosomal release, (2) transport within specific intracellular pathways, (3) protection of DNA from nucleases, (4) transport into the nucleus, and (5) DNA release from vectors. However, determining the relative importance of these processes and the vector properties necessary for optimization remain a challenge to the field. In this Account, we describe over a decade of studies focused on understanding the interaction of cationic polymer and cationic polymer/oligonucleotide (polyplex) interactions with model lipid membranes, cell membranes, and cells in culture. In particular, we have been interested in how the interaction between cationic polymers and the membrane influences the intracellular transport of intact DNA to the nucleus. Recent advances in microfluidic patch clamp techniques enabled us to quantify polyplex cell membrane interactions at the cellular level with precise control over material concentrations and exposure times. In attempting to relate these findings to subsequent intracellular transport of DNA and expression of protein, we needed to develop an approach that could distinguish DNA that was intact and potentially functional for gene expression from the much larger pool of degraded, nonfunctional DNA within the cell. We addressed this need by developing a FRET oligonucleotide molecular beacon (OMB) to monitor intact DNA transport. The research highlighted in this Account builds to the conclusion that polyplex

  7. Bio-reducible polycations from ring-opening polymerization as potential gene delivery vehicles.

    PubMed

    Yu, Qing-Ying; Liu, Yan-Hong; Huang, Zheng; Zhang, Ji; Luan, Chao-Ran; Zhang, Qin-Fang; Yu, Xiao-Qi

    2016-07-01

    Synthetic polycations show great potential for the construction of ideal non-viral gene delivery systems. Several cationic polymers were synthesized by the epoxide ring-opening polymerization between diepoxide and various polyamines. Disulfide bonds were introduced to afford the polymers bio-reducibility, while the oxygen-rich structure might enhance the serum tolerance and biocompatibility. The polycations have much lower molecular weights than PEI 25 kDa, but still could well bind and condense DNA into nano-sized particles. DNA could be released from the polyplexes by addition of reductive DTT. Compared to PEI, the polycations have less cytotoxicity possibly due to their lower molecular weights and oxygen-rich structure. More significantly, these materials exhibit excellent serum tolerance than PEI, and up to 6 times higher transfection efficiency than PEI could be obtained in the presence of serum. The transfection mediated by was seldom affected even at a high concentration of serum. Much lower protein adsorption of polycations than PEI was proved by bovine serum albumin adsorption experiments. Flow cytometry also demonstrates their good serum resistance ability.

  8. Nanosized bioceramic particles could function as efficient gene delivery vehicles with target specificity for the spleen.

    PubMed

    Tan, K; Cheang, P; Ho, I A W; Lam, P Y P; Hui, K M

    2007-05-01

    We have compared the ability of several nanosized bioceramic particles including negatively charged silica (SiO(2)), neutrally charged hydroxyapatite (HA) and positively charged zirconia (ZrO(2)) nanoparticles as non-viral vectors for efficient in vivo gene delivery. A mixture of highly monodispersed aqueous suspension of HA or SiO(2) nanoparticles, coated with protamine sulfate (PS), complexed efficiently with plasmid DNA and significantly enhanced transgene expression in vitro. In comparison, ZrO(2) nanoparticles gave poor transfection efficiency under similar conditions tested. It was also determined that, under the same conditions, PS-SiO(2)-DNA, but not PS-HA-DNA-nanoplexes, were able to mediate efficient transgene expression in vitro in the presence of 50% serum. Intraperitoneal injections of PS-SiO(2)-luciferase DNA nanoplexes targeted the highest level of transgene expression in the spleen of recipient mice that lasted for more than 48 h. Injection of PS-SiO(2)-pNGVL-hFLex-MUC-1 nanoplexes was able to mediate the production of Flt-3L in the sera of recipient mice. Simultaneously, the production of Flt-3L was accompanied by the stimulation of IL-2 and interferon-gamma (IFN-gamma). Most importantly, the injection of PS-SiO(2)-pNGVL-hFLex-MUC-1 nanoplexes could mount potent anti-tumour specific immune responses that led to the subsequent regression of parental tumor cells containing the muc-1 determinant.

  9. Thermoresponsive polymers as gene delivery vectors: cell viability, DNA transport and transfection studies.

    PubMed

    Twaites, Beverley R; de Las Heras Alarcón, Carolina; Lavigne, Matthieu; Saulnier, Annabelle; Pennadam, Sivanand S; Cunliffe, David; Górecki, Dariusz C; Alexander, Cameron

    2005-11-28

    A range of gene delivery vectors containing the thermoresponsive polymer, poly(N-isopropylacrylamide) (PNIPAm) was evaluated for effects on cell viability, intracellular trafficking and transgene expression in C2C12 mouse muscle cells. Polymers were complexed with plasmid DNA at pH 7.4 and the ability of the resulting particles to transfect cells was assessed via confocal microscopy and protein expression studies in tissue culture. Cell viability assays indicated that these polymers were toxic at high concentrations when not complexed to DNA or at certain polymer:DNA ratios. Poly(ethyleneimine) co-polymers with side-chain grafted PNIPAm were shown to be less toxic than poly(ethyleneimine) alone or PNIPAm-co-(N,N'-dimethylaminoethylmethacrylate) linear co-polymers and the effects were concentration dependent. Confocal micrographs of labeled polymers and DNA indicated rapid cellular entry for all the complexes but expression of Green Fluorescent Protein was achieved only when the branched PEI-PNIPAm co-polymers were used as vectors. The results indicate that design of appropriate co-polymer components and overall polymer architecture can be used to mediate, and perhaps ultimately control, DNA transport and transgene expression. PMID:16214254

  10. Cationic Liposome-DNA Complexes: From supramolecular assembly toward gene delivery

    NASA Astrophysics Data System (ADS)

    Evans, Heather M.; Ahmad, A.; Ewert, K.; Martin, A.; Safinya, Cr

    2003-03-01

    Cationic liposomes (CL) present a viable alternative to viral delivery of therapeutic DNA and peptides to cells. We complex CL with DNA to deliver foreign DNA (genes) to cells. Typical self-assembly of CL-DNA shown by x-ray diffraction reveals multilamellar lipids with DNA intercalated between the lipid layers, having a DNA interaxial spacing d(DNA)[1]. The length d(DNA) can be tuned at the subnanometer level (from 35 down to 5 angstroms) by control of the membrane charge density and other parameters. Three distinct DNA-DNA interaction regimes were found due to repulsive long-range electrostatic forces, repulsive short-range hydration forces, and a polymer induced attractive depletion force [2-4]. We correlate d(DNA) to transfection in mammalian cells. These compact DNA structures suggest use for high density storage of genetic information, as well as for biological templates. Supported by NSF DMR-0203755, NIH GM59288. 1. J Radler et al, Science 275, 810 (1997). 2. AJ Lin et al, Biophys. J. (in press). 3. K Ewert, A Ahmad, H Evans et al, J. Med. Chem. 45, 5023 (2002). 4. A Martin et al, (submitted).

  11. Gene delivery using ternary lipopolyplexes incorporating branched cationic peptides: the role of Peptide sequence and branching.

    PubMed

    Welser, Katharina; Campbell, Frederick; Kudsiova, Laila; Mohammadi, Atefeh; Dawson, Natalie; Hart, Stephen L; Barlow, David J; Hailes, Helen C; Lawrence, M Jayne; Tabor, Alethea B

    2013-01-01

    Cationic peptide sequences, whether linear, branched, or dendritic, are widely used to condense and protect DNA in both polyplex and lipopolyplex gene delivery vectors. How these peptides behave within these particles and the consequences this has on transfection efficiency remain poorly understood. We have compared, in parallel, a complete series of cationic peptides, both branched and linear, coformulated with plasmid DNA to give polyplexes, or with plasmid DNA and the cationic lipid, DOTMA, mixed with 50% of the neutral helper lipid, DOPE, to give lipopolyplexes, and correlated the transfection efficiencies of these complexes to their biophysical properties. Lipopolyplexes formulated from branched Arg-rich peptides, or linear Lys-rich peptides, show the best transfection efficiencies in an alveolar epithelial cell line, with His-rich peptides being relatively ineffective. The majority of the biophysical studies (circular dichroism, dynamic light scattering, zeta potential, small angle neutron scattering, and gel band shift assay) indicated that all of the formulations were similar in size, surface charge, and lipid bilayer structure, and longer cationic sequences, in general, gave better transfection efficiencies. Whereas lipopolyplexes formulated from branched Arg-containing peptides were more effective than those formulated from linear Arg-containing sequences, the reverse was true for Lys-containing sequences, which may be related to differences in DNA condensation between Arg-rich and Lys-rich peptides observed in the CD studies.

  12. Triolein-based polycation lipid nanocarrier for efficient gene delivery: characteristics and mechanism.

    PubMed

    Zhang, Zhiwen; Fang, Xiaoling; Hao, Junguo; Li, Yajuan; Sha, Xianyi

    2011-01-01

    We proposed to develop a polycation lipid nanocarrier (PLN) with higher transfection efficiency than our previously described polycation nanostrucutred lipid nanocarrier (PNLC). PLN was composed of triolein, cetylated low-molecular-weight polyethylenimine, and dioleoyl phosphatidylethanolamine. The physicochemical properties of PLN and the PLN/DNA complexes (PDC) were characterized. The in vitro transfection was performed in human lung adenocarcinoma (SPC-A1) cells, and the intracellular mechanism was investigated as well. The measurements indicated that PLN and PDC are homogenous nanometer-sized particles with a positive charge. The transfection efficiency of PDC significantly increased with the content of triolein and was higher than that of PNLC and commercial Lipofectamine 2000. In particular, the transfection of PLN in the presence of 10% serum was more effective than that in its absence. With the help of specific inhibitors of chlorpromazine and filipin, the clathrin-dependent endocytosis pathway was determined to be the main contributor to the successful transfection mediated by PLN in SPC-A1 cells. The captured images verified that the fluorescent PDC was localized in the lysosomes and nuclei after endocytosis. Thus, PLN represents a novel efficient nonviral gene delivery vector. PMID:22114487

  13. Synthesis of water-based cationic polyurethane for antibacterial and gene delivery applications.

    PubMed

    Wu, Geng-Hsi; Hsu, Shan-Hui

    2016-10-01

    Cationic polymers are often used as antimicrobial materials and transfection reagents. Water-based process could reduce environmental pollution and prevent the risk of solvent residue in the final product. In this study, waterborne biodegradable cationic polyurethane (WCPU) was synthesized by reacting polycaprolactone (PCL diol), isophorone diisocyanate (IPDI), and N-methyldiethanolamine (N-MDEA) under 75°C. An aqueous dispersion of WCPU nanoparticles (NPs) could be acquired by vigorous stirring under acidic condition. The particles in the dispersion had an average size of ∼80nm and a zeta potential of ∼60mV. When cast into films, the contact angle of the film was ∼67° and the zeta potential was ∼16mV. WCPU NPs demonstrated excellent antibacterial activity against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) (100% inhibition with a contact time of 3h). Meanwhile, the antibacterial ratio of WCPU films to E. coli and S. aureus reached 100% after 24h of contact. Moreover, WCPU NPs could be used as a transfection reagent without significant toxicity for concentrations less than 1000μg/mL and showed the ability to condensate plasmid DNA. The transfection efficiency for HEK293T cells and hBMSCs was ∼60% and ∼30% at 48h, respectively, after the transfection. Therefore, the WCPU synthesized in this study has potential antibacterial and gene delivery applications. PMID:27451371

  14. Disulphide cross linked pullulan based cationic polymer for improved gene delivery and efflux pump inhibition.

    PubMed

    S, Priya S; R, Rekha M

    2016-10-01

    Multidrug resistance is a hurdle to successful cancer chemotherapy. Over expression of P-glycoprotein (P-gp) is a prime contributing factor for drug resistance. In this study, a disulphide cross-linked pullulan-based cationic polymer (PPSS) was synthesized to act simultaneously as gene delivery vehicle and efflux pump inhibitor. The PPSS nanoplexes were of size <200nm with the zeta potential of +15 to +20mV. The cytotoxicity studies using C6 and L929 cells showed that PPSS polymers are non-toxic even at high polymer concentrations. The PPSS/pDNA nanoplex showed superior uptake in confocal microscopy with 97% uptake by flow cytometry. The efficacy of efflux pump inhibition by the PPSS nanoplex was established by the enhanced intracellular retention of DOX. The enhanced cell death by p53/PPSS/DOX nanoplexes was attributed to the synergistic effect of P-gp inhibition and p53 transfection efficiency. Therefore, this multifunctional polymeric system may have significant promise for therapeutic application against cancer drug resistance. PMID:27459414

  15. DNA melting temperature assay for assessing the stability of DNA polyplexes intended for nonviral gene delivery.

    PubMed

    Schallon, Anja; Synatschke, Christopher V; Pergushov, Dmitry V; Jérôme, Valérie; Müller, Axel H E; Freitag, Ruth

    2011-10-01

    Many synthetic polycations have the ability to form complexes with the polyanion DNA, yet only a few, most notably poly(ethylene imine) (PEI), are efficient gene-delivery vehicles. Although a common explanation of this observation relies on the buffering capacity of the polycation, the intracellular stability of the complex may also play a role and should not be neglected. Assays typically used to follow complex formation, however, often do not provide the required information on stability. In this article, we propose the change in the DNA melting temperature observable after complex formation to be a significant indicator of complex stability. For a given DNA/polycation ratio, changes in the melting temperature are shown to depend on the polycation chemistry but not on the DNA topology or the polycation architecture. Effects of changes in the DNA/polycation ratio as well as the effect of polycation quaternization can be interpreted using the melting temperature assay. Finally, the assay was used to follow the displacement of DNA from the complexes by poly(methacrylic acid) or short single-stranded DNA sequences as competing polyanions.

  16. Ultrasound-mediated gene and drug delivery using a microbubble-liposome particle system.

    PubMed

    Yoon, Young Il; Kwon, Yong-Su; Cho, Hee-Sang; Heo, Sun-Hee; Park, Kyeong Soon; Park, Sang Gyu; Lee, Soo-Hong; Hwang, Seung Il; Kim, Young Il; Jae, Hwan Jun; Ahn, Gook-Jun; Cho, Young-Seok; Lee, Hakho; Lee, Hak Jong; Yoon, Tae-Jong

    2014-01-01

    Theranostic agents present a promising clinical approach for cancer detection and treatment. We herein introduce a microbubble and liposome complex (MB-Lipo) developed for ultrasound (US) imaging and activation. The MB-Lipo particles have a hybrid structure consisting of a MB complexed with multiple Lipos. The MB components are used to generate high echo signals in US imaging, while the Lipos serve as a versatile carrier of therapeutic materials. MB-Lipo allows high contrast US imaging of tumor sites. More importantly, the application of high acoustic pressure bursts MBs, which releases therapeutic Lipos and further enhances their intracellular delivery through sonoporation effect. Both imaging and drug release could thus be achieved by a single US modality, enabling in situ treatment guided by real-time imaging. The MB-Lipo system was applied to specifically deliver anti-cancer drug and genes to tumor cells, which showed enhanced therapeutic effect. We also demonstrate the clinical potential of MB-Lipo by imaging and treating tumor in vivo.

  17. Intracellular gene delivery is dependent on the type of non-viral carrier and defined by the cell surface glycosaminoglycans.

    PubMed

    Nomani, Alireza; Hyvönen, Zanna; Pulkkinen, Eveliina; Hiekkala, Marjo; Ruponen, Marika

    2014-08-10

    Intracellular limiting steps and molecules involved in internalization and intracellular routing of non-viral gene delivery systems are still poorly understood. In this study, the intracellular kinetics of three different gene delivery systems calcium phosphate precipitates (CaP), polyethyleneimine (PEI) and N-[1-(2,3-dioleyl)propyl]-N,N,N-trimethylammonium chloride (DOTAP)) were quantified at cellular, nuclear, transcriptional and translational levels by using qRT-PCR. Additionally, a role of cell surface glycosaminoglycans (GAGs) was evaluated by performing the aforementioned studies in cells devoid of GAGs (pgsB-618) and cells lacking heparan sulphate (HS). The obtained data showed that the intracellular kinetics was dependent on the type of gene carrier and the weakest intracellular step varied between the carriers; rapid elimination of cell-associated pDNA in CaP, nuclear uptake in DOTAP and transcriptional and translational events in PEI mediated transfections. Overall, neither the amount of cell- nor nuclear associated pDNA correlated with transgene expression but the mRNA expression of the transgene correlated well with the expression at protein level. The nuclear uptake of pDNA in all cases was rapid and efficient thus indicating that the post-nuclear processes including transcription and translation steps have a critical role in defining the efficiency of non-viral gene delivery systems. Our study demonstrated that cell-surface GAGs are not essential for cell surface binding and internalization of gene delivery complexes, but they are able to define the intracellular routing of the complexes by leading them to pathways with high pDNA elimination.

  18. The Ad5 [E1-, E2b-]-based vector: a new and versatile gene delivery platform

    NASA Astrophysics Data System (ADS)

    Jones, Frank R.; Gabitzsch, Elizabeth S.; Balint, Joseph P.

    2015-05-01

    Based upon advances in gene sequencing and construction, it is now possible to identify specific genes or sequences thereof for gene delivery applications. Recombinant adenovirus serotype-5 (Ad5) viral vectors have been utilized in the settings of gene therapy, vaccination, and immunotherapy but have encountered clinical challenges because they are recognized as foreign entities to the host. This recognition leads to an immunologic clearance of the vector that contains the inserted gene of interest and prevents effective immunization(s). We have reported on a new Ad5-based viral vector technology that can be utilized as an immunization modality to induce immune responses even in the presence of Ad5 vector immunity. We have reported successful immunization and immunotherapy results to infectious diseases and cancers. This improved recombinant viral platform (Ad5 [E1-, E2b-]) can now be utilized in the development of multiple vaccines and immunotherapies.

  19. Cationic Lipid-Nucleic Acid Complexes for Gene Delivery And Silencing: Pathways And Mechanisms for Plasmid Dna And Sirna

    SciTech Connect

    Ewert, K.K.; Zidovska, A.; Ahmad, A.; Bouxsein, N.F.; Evans, H.M.; McAllister, C.S.; Samuel, C.E.; Safinya, C.R.; /SLAC

    2012-07-17

    Motivated by the promises of gene therapy, there is great interest in developing non-viral lipid-based vectors for therapeutic applications due to their low immunogenicity, low toxicity, ease of production, and the potential of transferring large pieces of DNA into cells. In fact, cationic liposome (CL) based vectors are among the prevalent synthetic carriers of nucleic acids (NAs) currently used in gene therapy clinical trials worldwide. These vectors are studied both for gene delivery with CL-DNA complexes and gene silencing with CL-siRNA (short interfering RNA) complexes. However, their transfection efficiencies and silencing efficiencies remain low compared to those of engineered viral vectors. This reflects the currently poor understanding of transfection-related mechanisms at the molecular and self-assembled levels, including a lack of knowledge about interactions between membranes and double stranded NAs and between CL-NA complexes and cellular components. In this review we describe our recent efforts to improve the mechanistic understanding of transfection by CL-NA complexes, which will help to design optimal lipid-based carriers of DNA and siRNA for therapeutic gene delivery and gene silencing.

  20. Gene Silencing in Skin After Deposition of Self-Delivery siRNA With a Motorized Microneedle Array Device

    PubMed Central

    Hickerson, Robyn P; Wey, Winston C; Rimm, David L; Speaker, Tycho; Suh, Susie; Flores, Manuel A; Gonzalez-Gonzalez, Emilio; Leake, Devin; Contag, Christopher H; Kaspar, Roger L

    2013-01-01

    Despite the development of potent siRNAs that effectively target genes responsible for skin disorders, translation to the clinic has been hampered by inefficient delivery through the stratum corneum barrier and into the live cells of the epidermis. Although hypodermic needles can be used to transport siRNA through the stratum corneum, this approach is limited by pain caused by the injection and the small volume of tissue that can be accessed by each injection. The use of microneedle arrays is a less painful method for siRNA delivery, but restricted payload capacity limits this approach to highly potent molecules. To address these challenges, a commercially available motorized microneedle array skin delivery device was evaluated. This device combines the positive elements of both hypodermic needles and microneedle array technologies with little or no pain to the patient. Application of fluorescently tagged self-delivery (sd)-siRNA to both human and murine skin resulted in distribution throughout the treated skin. In addition, efficient silencing (78% average reduction) of reporter gene expression was achieved in a transgenic fluorescent reporter mouse skin model. These results indicate that this device effectively delivers functional sd-siRNA with an efficiency that predicts successful clinical translation. PMID:24150576

  1. Gene Silencing in Skin After Deposition of Self-Delivery siRNA With a Motorized Microneedle Array Device.

    PubMed

    Hickerson, Robyn P; Wey, Winston C; Rimm, David L; Speaker, Tycho; Suh, Susie; Flores, Manuel A; Gonzalez-Gonzalez, Emilio; Leake, Devin; Contag, Christopher H; Kaspar, Roger L

    2013-01-01

    Despite the development of potent siRNAs that effectively target genes responsible for skin disorders, translation to the clinic has been hampered by inefficient delivery through the stratum corneum barrier and into the live cells of the epidermis. Although hypodermic needles can be used to transport siRNA through the stratum corneum, this approach is limited by pain caused by the injection and the small volume of tissue that can be accessed by each injection. The use of microneedle arrays is a less painful method for siRNA delivery, but restricted payload capacity limits this approach to highly potent molecules. To address these challenges, a commercially available motorized microneedle array skin delivery device was evaluated. This device combines the positive elements of both hypodermic needles and microneedle array technologies with little or no pain to the patient. Application of fluorescently tagged self-delivery (sd)-siRNA to both human and murine skin resulted in distribution throughout the treated skin. In addition, efficient silencing (78% average reduction) of reporter gene expression was achieved in a transgenic fluorescent reporter mouse skin model. These results indicate that this device effectively delivers functional sd-siRNA with an efficiency that predicts successful clinical translation.Molecular Therapy-Nucleic Acids (2013) 2, e129; doi:10.1038/mtna.2013.56; published online 22 October 2013. PMID:24150576

  2. Brain-targeted co-delivery of therapeutic gene and peptide by multifunctional nanoparticles in Alzheimer's disease mice.

    PubMed

    Liu, Yang; An, Sai; Li, Jianfeng; Kuang, Yuyang; He, Xi; Guo, Yubo; Ma, Haojun; Zhang, Yu; Ji, Bin; Jiang, Chen

    2016-02-01

    Multifunctional nanocarriers are increasingly promising for disease treatment aimed to regulate multiple pathological dysfunctions and overcome barriers in drug delivery. Here we develop a multifunctional nanocarrier for Alzheimer's disease (AD) treatment by achieving therapeutic gene and peptide co-delivery to brain based on PEGylated dendrigraft poly-l-lysines (DGLs) via systemic administration. The dendritic amine-rich structure of DGLs provides plenty reaction sites and positive charge for drug loading. Successful co-delivery of drugs overcoming the blood-brain barrier by brain-targeted ligand modification was demonstrated both in vitro and in vivo. The pharmacodynamics study of the system following multiple-dosing treatment was verified in transgenic AD mice. Down-regulation of the key enzyme in amyloid-β formation was achieved by delivering non-coding RNA plasmid. Simultaneous delivery of the therapeutic peptide into brain leads to reduction of neurofibrillary tangles. Meanwhile, memory loss rescue in AD mice was also observed. Taken together, the multifunctional nanocarrier provides an excellent drug co-delivery platform for brain diseases.

  3. Lentiviral Delivery of RNAi for In Vivo Lineage-Specific Modulation of Gene Expression in Mouse Lung Macrophages

    PubMed Central

    Wilson, Andrew A; Kwok, Letty W; Porter, Emily L; Payne, Julie G; McElroy, Gregory S; Ohle, Sarah J; Greenhill, Sara R; Blahna, Matthew T; Yamamoto, Kazuko; Jean, Jyh C; Mizgerd, Joseph P; Kotton, Darrell N

    2013-01-01

    Although RNA interference (RNAi) has become a ubiquitous laboratory tool since its discovery 12 years ago, in vivo delivery to selected cell types remains a major technical challenge. Here, we report the use of lentiviral vectors for long-term in vivo delivery of RNAi selectively to resident alveolar macrophages (AMs), key immune effector cells in the lung. We demonstrate the therapeutic potential of this approach by RNAi-based downregulation of p65 (RelA), a component of the pro-inflammatory transcriptional regulator, nuclear factor κB (NF-κB) and a key participant in lung disease pathogenesis. In vivo RNAi delivery results in decreased induction of NF-κB and downstream neutrophilic chemokines in transduced AMs as well as attenuated lung neutrophilia following stimulation with lipopolysaccharide (LPS). Through concurrent delivery of a novel lentiviral reporter vector (lenti-NF-κB-luc-GFP) we track in vivo expression of NF-κB target genes in real time, a critical step towards extending RNAi-based therapy to longstanding lung diseases. Application of this system reveals that resident AMs persist in the airspaces of mice following the resolution of LPS-induced inflammation, thus allowing these localized cells to be used as effective vehicles for prolonged RNAi delivery in disease settings. PMID:23403494

  4. Production, purification and titration of a lentivirus-based vector for gene delivery purposes.

    PubMed

    Nasri, Masoud; Karimi, Ali; Allahbakhshian Farsani, Mehdi

    2014-12-01

    Viral vectors are valuable tools to deliver genetic materials into cells. Vectors derived from human immunodeficiency virus type 1 are being widely used for gene delivery, mainly because they are able to transduce both dividing and non-dividing cells which leads to stable and long term gene expression. In addition, these types of vectors are safe, with low toxicity, high stability and cell type specificity. Therefore, this work was aimed to produce lentivirus-based vector using a three-plasmid system. To produce this system, the eGFP marker gene was cloned into the plasmid pWPXLd. Subsequently, this vector plasmid, along with packaging plasmids, psPAX2 and envelope plasmid, pMD2.G, was co-transfected into packaging cell line (293T) using calcium phosphate method. 48 h post transfection, the constructed viral vector was harvested, purified and concentrated and stored at -80 °C for next experiments. The titration of the vector was carried out, using ELISA, flowcytometry, and fluorescent microscopy. Finally, transduction of HEK-293T, CHO, HepG2, MCF-7, MEFs and Jurkat cell lines was carried out with indicated cell numbers and multiplicities of infections of the vector in the presence of polybrene. Using this system, high titer lentivirus at titers of up to 2 × 10(8) transducing units/ml (TU/ml) was successfully generated and its transduction efficacy was improved by seven to over 20-fold in various cell types. We demonstrate the applicability of this vector for the efficient transduction of dividing and non-dividing cells, including HEK-293T, CHO, HepG2, MCF-7, MEFs and Jurkat cell line. Transduction efficiency yielded titers of (6.3 ± 1.2) 10(5) TU/ml. Furthermore, lentivirus transferred transgene was expressed at high level in the target cells and expression was followed until 90 days after transduction. Thus, the vector generated in this work, might be able to deliver the transgene into a wide range of mammalian cells.

  5. Magnetic resonance-guided regional gene delivery strategy using a tumor stroma-permeable nanocarrier for pancreatic cancer

    PubMed Central

    Wang, Qingbing; Li, Jianfeng; An, Sai; Chen, Yi; Jiang, Chen; Wang, Xiaolin

    2015-01-01

    Background Gene therapy is a very promising technology for treatment of pancreatic ductal adenocarcinoma (PDAC). However, its application has been limited by the abundant stromal response in the tumor microenvironment. The aim of this study was to prepare a dendrimer-based gene-free loading vector with high permeability in the tumor stroma and explore an imaging-guided local gene delivery strategy for PDAC to promote the efficiency of targeted gene delivery. Methods The experimental protocol was approved by the animal ethics committee of Zhongshan Hospital, Fudan University. Third-generation dendrigraft poly-L-lysines was selected as the nanocarrier scaffold, which was modified by cell-penetrating peptides and gadolinium (Gd) chelates. DNA plasmids were loaded with these nanocarriers via electrostatic interaction. The cellular uptake and loaded gene expression were examined in MIA PaCa-2 cell lines in vitro. Permeability of the nanoparticles in the tumor stroma and transfected gene distribution in vivo were studied using a magnetic resonance imaging-guided delivery strategy in an orthotopic nude mouse model of PDAC. Results The nanocarriers were synthesized with a dendri