Chitosan-mediated orally delivered nucleic acids: A gutful of gene therapy

Gene therapy is rapidly gaining its hold in preclinical drug evaluation. However, upfront, ease of administration and greater patient compliance should strictly drive research efforts for developing modes of gene delivery. It has been a decade since plasmid DNA was first introduced orally in animals. Since then, two main modes of such delivery for potentially therapeutic nucleic acids, chitosan-based systems and non-chitosan-based systems, have been developed, at a steady though slow pace. This slow pace is partly due to the various hurdles faced with oral delivery, especially for labile molecules like nucleic acids. The real challenge is to enhance delivery systems that can traverse the gut and gain entry into the bloodstream in sufficient quantities for efficacy in diseased tissues at a distance. This review examines some of the current chitosan-based vehicles used for oral administration of potentially therapeutic nucleic acids, and explores novel ways to better deliver such molecules in the future for non-vaccination applications.     

Cationic Lipid-Based Gene Delivery Systems: Pharmaceutical Perspectives

Gene delivery systems are designed to control the location of administered therapeutic genes within a patient's body. Successful in vivo gene transfer may require (i) the condensation of plasmid and its protection from nuclease degradation, (ii) cellular interaction and internalization of condensed plasmid, (iii) escape of plasmid from endosomes (if endocytosis is involved), and (iv) plasmid entry into cell nuclei. Expression plasmids encoding a therapeutic protein can be, for instance, complexed with cationic liposomes or micelles in order to achieve effective in vivo gene transfer. A thorough knowledge of pharmaceutics and drug delivery, bio-engineering, as well as cell and molecular biology is required to design optimal systems for gene therapy. This mini-review provides a critical discussion on cationic lipid-based gene delivery systems and their possible uses as pharmaceuticals.     

Design of lipid-based nanoparticles for delivery of therapeutic nucleic acids

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Novel Pentablock Copolymers for Selective Gene Delivery to Cancer Cells

Purpose  In this study, the novel poly(diethylaminoethylmethacrylate) (PDEAEM)/Pluronic F127 pentablock copolymers were found to be able to mediate high-efficiency transfection of human epithelial ovarian carcinoma (SKOV3) cell line while showing significantly lower efficacy in human epithelial retinal (ARPE-19) cell line and Swiss Mouse Fibroblast (3T3) cell line. Methods  The intracellular routes of polyplexes were investigated by confocal microscopy after appropriately labeling the polymer and DNA. Results  It was found that lesser nuclear entry in the ARPE-19 cells may result in the lower efficiency of transfection. Since the SKOV3 proliferation rate was found to be much higher than that of the ARPE-19 cells, the nuclear entry of polyplexes was assumed to be correlated with the proliferation rate, and it was hypothesized that the novel pentablock copolymers could mediate gene delivery selectively in fast growing cells. The different intracellular barriers to gene transfer may also account for the observed difference of transfection efficacy. Conclusions  Although the validity of the hypothesis that our pentablock copolymer could selectively transfect hyperproliferative cells needs further examination, this present work provides a new perspective to design targeting vectors for cancer therapies based on different characteristics among specific cell types. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Quantitative Analysis of Correlation Between Number of Nuclear Plasmids and Gene Expression Activity After Transfection with Cationic Liposomes

Purpose. A quantitative understanding of the intracellular trafficking of plasmids delivered by nonviral vectors is essential for optimizing vector functions to increase their transfection efficiency. In this study, quantitative methods were developed to measure plasmids delivered to the nucleus, and the relationship between transfection activity and the number of plasmids in the nucleus were analyzed. Methods. AH130 cells were transfected with plasmids in cationic liposomes at various doses. The nuclear fraction was isolated after NP-40 lysis, and the unincorporated plasmids were enzymatically degraded and washed away. Intranuclear plasmids were amplified by quantitative PCR, and the number of plasmids was determined. Plasmid amounts in the nucleus were also measured by Southern analysis to confirm the quantification. Results. Both methods led to similar results in measuring the nuclear plasmids within the same order of magnitude. A remarkable saturation was found for transfection activity vs. number of plasmids in the nucleus, whereas no saturation was observed in nuclear-delivered plasmids vs. dose. Conclusions. These results clearly demonstrate the importance of the quantitative measurement of intracellular trafficking of plasmids after transfection. The findings herein described suggest that efficient transgene expression as well as enhanced nuclear delivery is required in order to achieve the maximal transfection activity of nonviral vectors.     

The biological routes of gene delivery mediated by lipid-based non-viral vectors

Cationic lipid/DNA complexes (lipoplexes) represent an attractive alternative to viral vectors for cell transfection in vitro and in vivo but still suffer from relatively low efficiency. Comprehension of the interactions between vectors and DNA as well as cellular pathways and mechanisms in DNA entry into cells and ultimately nuclei will lead to the design of better adapted non-viral vectors for gene therapy applications. Here, some recent developments in the field on the pathways and mechanisms involved in lipoplex-mediated transfection are discussed. The techniques that are widely used to study the mechanism of gene delivery are also discussed.     

Pulmonary delivery of nucleic acids

The lung is an appropriate present and future target for gene therapy approaches designed to treat inherited monogenic diseases, eradicate bronchial tumours, transfer pharmacologically active products to the general circulation, express enzymes to catabolise toxins, manage pulmonary hypertension and lung injury and vaccinate against infection. Despite 35 years of gene therapy research and some significant milestones in molecular biology, the clinical potential of gene therapy has yet to be realised. In pulmonary gene therapy the nucleic acid cargo needs to be delivered to cells in the ?target region of the lung, and even in cases when these targets are well defined this is severely limited by the pulmonary architecture, clearance mechanisms, immune activation, the presence of respiratory mucus and the availability of a truly representative biological model. The challenge from a drug delivery perspective is to consider the suitability of conventional nebulisers and inhalers for delivering DNA to the lung and design and apply integrated formulation and device solutions specific to nucleic acid delivery.     

脂质-鱼精蛋白-DNA复合物的构建及其对细胞的体外转染

目的研究新型非病毒载体脂质-聚阳离子-DNA（LPD）复合物的制备方法及其对体外细胞的转染率。方法用薄膜-挤压法制备空白阳离子脂质体，与鱼精蛋白-DNA复合物在室温孵育后，得到LPD；用透射电镜观察其形态，用激光粒度仪测定其粒径和zeta电位；LPD与DNA酶I溶液在37 ℃下孵育不同时间后，用琼脂糖凝胶电泳观察其降解情况；用荧光法测定其包封率；用X-gal染色法考察了LPD对张氏（Chang）肝细胞，HepG2肝癌细胞和SMMC-7721肝癌细胞的转染率。结果LPD的形态近似于球体，平均粒径为143.5 nm，平均zeta电位为+32.6 mV；37 ℃下核酸酶作用2 h后，LPD中的DNA几乎无降解；平均包封率为93.42%；LPD对张氏（Chang）肝细胞、HepG2肝癌细胞和SMMC-7721肝癌细胞的转染率分别为(69±6)%，(43±7)%和(96.2±1.8)%。结论LPD是一种制备工艺简单、体外稳定性好、转染率高，具有应用潜力的非病毒载体系统。     

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

Abstract

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.     

Cancer,chitosan nanoparticles and catalytic nucleic acids

Objectives The aim of this review was to examine gene therapy involving DNAzyme and siRNA encapsulation into chitosan nanoparticles, discussing the current and future status of this drug delivery system in enhancing drug delivery and cancer therapy. Key findings Cancer is a disease state in which the cells in our body undergo mutations at the genetic level and are transformed, acquiring the ability to replicate limitlessly. Conventional cancer treatment involves the use of surgery and cytotoxic chemotherapy and/or radiotherapy, which have the potential of harming normal, otherwise healthy, non‐neoplastic cells. Newer forms of therapy such as immunotherapy and gene therapy have shown initial promise, but still require better ways to limit exposure to cancerous lesions in the body. As a result drug delivery systems have been developed in attempts to deliver therapeutics specifically to the target lesion site. One recent drug delivery system has revolved around the use of chitosan nanoparticle technology, where therapeutics are encapsulated into nanoparticles and targeted to tumours. Summary Though few, attempts at encapsulating therapeutics such as deoxyribozymes and small or short interfering RNA have been optimistic and encouraging.     

DQAsomes: A Novel Potential Drug and Gene Delivery System Made from Dequalinium™

Purpose. Dequalinium, a drug known for over 30 years, is a dicationic amphiphile compound resembling bolaform electrolytes. The purpose of our work was to determine the state of aggregation of dequalinium in aqueous medium and to investigate both, its ability to bind DNA and its potential to serve as a novel non-viral transfection vector. Methods. The form of aggregation was determined employing electron microscopic techniques. The DNA binding capacity of dequalinium was assayed using SYBR Green I stain. For in vitro cell transfection experiments plasmid DNA encoding for firefly luciferase was used. Results. Dequalinium forms in aqueous medium liposome-like aggregates, which we term DQAsomes. These dequalinium vesicles bind DNA and they are able to transfect cells in vitro with an efficiency comparable to Lipofectin. Conclusions. Based on the intrinsic properties of dequalinium such as the in vivo selectivity for carcinoma cells and selective accumulation in mitochondria we propose DQAsomes as a novel and unique drug and gene delivery system.     

Recent developments in cationic lipid-mediated gene delivery and gene therapy

Gene therapy will revolutionize medicine, helping us to cure and prevent diseases at their core level. Until becoming a widespread reality, the problem of efficient gene transfer and expression (transfection) must be solved. Cationic lipids represent a safer alternative than viral vectors, which, although more efficient, have the drawback of immunogenicity and propagation risks. Additionally, cationic lipids and cationic liposomes allow the delivery of larger plasmids and may be GMP manufactured and stored in bulk quantities. However, their specific transfection efficiency must be improved in order to reach the performance of biological vectors. In recent years, new structures have been released and tested, with designs adapted to recent findings in lipid-mediated transfection mechanisms. Another trend is the increased use of natural, biodegradable, building blocks in the backbone of these compounds. Here we review the very recent developments in the field of cationic lipids, both from industry and academia. Physicochemical characteristics, insights of transfection mechanisms, as well as therapeutic applications are also presented. Finally, some future prospects and trends are proposed.     

精蛋白在改进非病毒载体基因转染中的应用

非病毒基因载体的出现,为基因治疗提供了低毒、易于大规模制备的载体。但与病毒载体相比,非病毒基因载体的转染效率仍然偏低,阻碍了非病毒基因载体的临床应用。本文旨在探讨精蛋白在改进非病毒基因载体方面的应用,希望通过合理的载体设计与优化,制备出一种高效、低毒的基因载体。     

Cell-penetrating peptides for the delivery of nucleic acids

Introduction: Different gene therapy approaches have gained extensive interest lately and, after many initial hurdles, several promising approaches have reached to the clinics. Successful implementation of gene therapy is heavily relying on finding efficient measures to deliver genetic material to cells. Recently, non-viral delivery of nucleic acids and their analogs has gained significant interest. Among non-viral vectors, cell-penetrating peptides (CPPs) have been extensively used for the delivery of nucleic acids both in vitro and in vivo.

Areas covered: In this review we will discuss recent advances of CPP-mediated delivery of nucleic acid-based cargo, concentrating on the delivery of plasmid DNA, splice-correcting ONs, and small-interfering RNAs.

Expert opinion: CPPs have proved their potential as carriers for nucleic acids. However, similarly to other non-viral vectors, CPPs require further development, as efficient systemic delivery is still seldom achieved. To achieve this, CPPs should be modified with entities that would allow better endosomal escape, targeting of specific tissues and cells, and shielding agents that increase the half-life of the vehicles. Finally, to understand the clinical potential of CPPs, they require more thorough investigations in clinically relevant disease models and in pre-clinical and clinical studies.     

脂质体-聚乙烯亚胺-DNA三元复合物的构建及其对细胞的体外转染

目的研究非病毒基因载体脂质体-聚乙烯亚胺(PEI)-DNA三元复合物(TC)的制备方法,评价其体外细胞学性质。方法采用乙醇注入法制备空白阴离子脂质体,与PEI/DNA复合物37℃孵育30 min后,得到TC,考察其理化性质、抗核酸酶降解能力、血浆稳定性、细胞毒性及在卵巢癌细胞(Hela)中的转染效率。结果制备的TC呈类球形,大小较均匀,平均粒径为234.5 nm,Zeta电位为-20.72 mV;TC能在血浆中稳定存在4 h而不发生聚集;与核酸酶作用2 h后,其中的DNA几乎无降解;其细胞毒性较低,在无血清和含血清培养基中均能成功的转染Hela细胞,在含血清培养基中其转染效率明显高于PEI/DNA复合物。结论 TC是一种制备工艺简单、血浆稳定性好、转染率较高、极具应用潜力的非病毒纳米基因载体。     

Dendrosome-dendriplex inside liposomes: as a gene delivery system

Dendrosomes are lipid vesicular entities containing entrapped dendrimer-DNA complexes and possessing low toxicity, acceptable transfection efficiency, and good in vivo tolerance. Herein, an attempt was made to explore the potential of dendrosomes as a gene delivery system combining the advantages of both polyamidoamine (PAMAM) dendrimer (nucleic acid condensation, facilitated endosomal release) and of non-cationic liposomes (increased cellular uptake, low cytotoxicity), and at the same time overcoming the drawbacks of these system (low encapsulation efficiency of non-cationic liposome and toxicity of dendrimers). Dendrosomes were assembled by loading optimized DNA-dendrimer complexes into liposomes prepared by solvating of dried lipid films made of DOPE/EggPC/Cholesterol (4.74:4.75:1.5 mole ratio). Dendrosomes were characterized in terms of size, zeta, encapsulation efficiency and the ability to protect the system from DNA degradation. The transfection efficiency and toxicity of the preparations were evaluated in HeLa cells using flow cytometry and CellTiter-Blue^® methods. The efficient transfection and low toxicity makes them an appealing alternative to be further explored for gene delivery in vivo.     

Polyvinyl Derivatives as Novel Interactive Polymers for Controlled Gene Delivery to Muscle

Purpose. DNA plasmids (pDNA) can be taken up by and expressed in striated muscle after direct intramuscular injection. We have developed interactive polymeric gene delivery systems that increase pDNA bioavailability to muscle cells by both protecting pDNA from nucleases and controlling the dispersion and retention of pDNA in muscle tissue. Methods. A DNA plasmid, containing a CMV promoter and a -galactosidase reporter gene (CMV--gal), was injected either in saline or formulated in polyvinyl pyrrolidone (PVP) and polyvinyl alcohol (PVA) solutions. Interactions between PVP and pDNA were assessed by dynamic dialysis, Isothermal Titration Calorimetry (ITC), and Fourier-Transformed Infra Red (FT-IR) spectroscopy. Formulations (50 µl) were injected into rat tibialis muscles after surgical exposure. Immuno-histochemistry for -gal was used to visualize the sites of expression in muscle. Results. -gal expression using pDNA in saline reached a plateau while -gal expression using PVP formulations increased linearly in the dose range studied (12.5–150 µg pDNA injected) and resulted in an increase in the number and distribution of cells expressing -gal. The interaction between PVP and pDNA was found to be an endothermic process governed largely by hydrogen-bonding and results in protection of pDNA from extracellular nucleases. Conclusions. Significant enhancement of gene expression using interactive polyvinyl-based delivery systems has been observed. The improved tissue dispersion and cellular uptake of pDNA using polyvinyl-based systems after direct injection into muscle is possibly due to osmotic effects.     

非病毒基因递送载体的研究进展

目前,基因药物的递送成为药学研究的热点,基因递送载体主要包括病毒载体和非病毒载体。非病毒基因载体的毒性低,生物相容性好,转染效率高,具有潜在的临床应用价值。本文就靶向递送基因载体、多功能基因载体、同时载基因与化疗药物的载体、智能基因载体和脂质体等非病毒基因递送载体的研究进展做一综述。