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。     

Evaluation of multicomponent non-viral vectors for liver directed gene delivery

Multicomponent, non-viral gene delivery vehicles are designed to have as a minimum, a DNA binding component, and a cell recognition component for specific delivery to target cells. The DNA binding component cannot only bind, but also protect DNA from serum degradation, and tends to condense DNA to sizes that can be taken up by receptor-mediated processes of target cells. Generally, cationic peptides, single chained, e.g. poly-L-lysine or branched polymers or synthetic peptides with DNA binding properties are used for DNA binding components. Ligands for binding to receptors on cell surfaces can be covalently linked to the DNA binding component. Multicomponent, non-viral vectors have been successfully used to deliver genes into cells in vitro and in vivo. Improvements have been made to the non-viral carriers resulting in increased solubility of DNA/carrier complexes and longer survival in serum. Improvements have also been made by incorporating fusogenic/lysosomolytic components that enable DNA/carrier complexes to escape intracellular degradation and enhance the levels and duration of expression of genes in vitro and in vivo.     

Novel cationic solid-lipid nanoparticles as non-viral vectors for gene delivery

In this paper, the suitability of novel cationic solid-lipid nanoparticles (SLN) as a nonviral transfection agent for gene delivery was investigated. SLN were produced by using the microemulsion method and Compritol ATO 888 as matrix lipid, dimethyldioctadecylammonium bromide as charge carrier and Pluronic F68 as surfactant. Obtained nanoparticles were approximately 120 nm in size and positively charged, with a zeta potential value equal to +45 mV in twice-distilled water. Cationic SLN were able to form stable complexes with DNA and to protect DNA against DNase I digestion. The SLN–DNA complexes were characterized by mean diameter and zeta potential measurements. In vitro studies on human liver cancer cells demonstrated a very low degree of toxicity of both SLN and SLN–DNA complexes. Further, SLN–DNA complexes were able to promote transfection of liver cancer cells. These data suggest that our cationic SLN may be potentially useful for gene therapy.     

Novel cationic solid-lipid nanoparticles as non-viral vectors for gene delivery

In this paper, the suitability of novel cationic solid-lipid nanoparticles (SLN) as a nonviral transfection agent for gene delivery was investigated. SLN were produced by using the microemulsion method and Compritol ATO 888 as matrix lipid, dimethyldioctadecylammonium bromide as charge carrier and Pluronic F68 as surfactant. Obtained nanoparticles were approximately 120 nm in size and positively charged, with a zeta potential value equal to +45 mV in twice-distilled water. Cationic SLN were able to form stable complexes with DNA and to protect DNA against DNase I digestion. The SLN-DNA complexes were characterized by mean diameter and zeta potential measurements. In vitro studies on human liver cancer cells demonstrated a very low degree of toxicity of both SLN and SLN-DNA complexes. Further, SLN-DNA complexes were able to promote transfection of liver cancer cells. These data suggest that our cationic SLN may be potentially useful for gene therapy.     

Calcium phosphate nanoparticles as novel non-viral vectors for targeted gene delivery

Calcium phosphate nanoparticles present a unique class of non-viral vectors, which can serve as efficient and alternative DNA carriers for targeted delivery of genes. In this study we report the design and synthesis of ultra-low size, highly monodispersed DNA doped calcium phosphate nanoparticles of size around 80 nm in diameter. The DNA encapsulated inside the nanoparticle is protected from the external DNase environment and could be used safely to transfer the encapsulated DNA under in vitro and in vivo conditions. Moreover, the surface of these nanoparticles could be suitably modified by adsorbing a highly adhesive polymer like polyacrylic acid followed by conjugating the carboxylic groups of the polymer with a ligand such as p-amino-1-thio-beta-galactopyranoside using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride as a coupling agent. We have demonstrated in our studies that these surface modified calcium phosphate nanoparticles can be used in vivo to target genes specifically to the liver.     

siRNA非病毒递送载体的研究现状

RNA干扰(RNA interference,RNAi)是近年发展起来的一种新技术。RNAi是指通过外源性或内源性的双链RNA在体内诱导靶基因mRNA产生特异性降解,进而引起不同水平的基因沉默。RNAi已经用于肿瘤、病毒感染、乙型肝炎等多种疾病的治疗。小干扰RNA(siRNA)是RNAi的效应分子,可在体内诱导RNAi效应。但是裸siRNA在体内容易被核酶(RNase)降解,且半衰期短,转染效率低。因此,siRNA需要借助递送载体进入细胞发挥治疗作用。病毒载体在基因治疗中有潜在的免疫原性、致突变等副作用。所以,非病毒载体成为当前的研究热点。本文对siRNA非病毒递送载体的研究现状进行了综述。     

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.     

Novel hyaluronic acid-chitosan nanoparticles as non-viral gene delivery vectors targeting osteoarthritis

Gene therapy is a promising new treatment strategy for common joint-disorders such as osteoarthritis. The development of safe, effective, targeted non-viral gene carriers is important for the clinical success of gene therapy. The present work describes the use of hybrid hyaluronic acid (HA)/chitosan (CS) nanoparticles as novel non-viral gene delivery vectors capable of transferring exogenous genes into primary chondrocytes for the treatment of joint diseases. HA/CS plasmid-DNA nanoparticles were synthesized through the complex coacervation of the cationic polymers with pEGFP. Particle size and zeta potential were related to the weight ratio of CS to HA, where increases in nanoparticle size and decreases in surface charge were observed as HA content increased. The particle size and the zeta potential varied according to pH. Transfection of primary chondrocytes was performed under different conditions to examine variations in the pH of the transfection medium, different N/P ratios, different plasmid concentrations, and different molecular weights of chitosan. Transfection efficiency was maximized for a medium pH of approximately 6.8, an N/P ratio of 5, plasmid concentration of 4 μg/ml, and a chitosan molecular weight of 50 kDa. The transfection efficiency of HA/CS-plasmid nanoparticles was significantly higher than that of CS-plasmid nanoparticles under the same conditions. The average viability of cells transfected with HA/CS-plasmid nanoparticles was over 90%. These results suggest that HA/CS-plasmid nanoparticles could be an effective non-viral vector suitable for gene delivery to chondrocytes.     

Recent advances in aerosol gene delivery systems using non-viral vectors for lung cancer therapy

ABSTRACT

Introduction: Lung cancer commonly occurs at a high incidence worldwide. Application of aerosol gene delivery systems using various kinds of vectors can improve the patient’s quality of life by prolonging the survival rate.

Areas covered: This review provides a recent update on aerosol gene delivery strategies using various kinds of vectors and gene-modification technologies. Peptide-mediated gene therapy achieves specific targeting of cells and highly improves efficacy. Promoter-operating expression and the CRISPR/Cas9 system are novel gene therapy strategies for effective lung cancer treatment. Furthermore, hybrid systems with a combination of vectors or drugs have been recently applied as new trends in gene therapy.

Expert opinion: Although aerosol gene delivery has many advantages, physiological barriers in the lungs pose formidable challenges. Targeted gene delivery and gene-editing technology are promising strategies for lung cancer therapy. These strategies may allow the development of safety and high efficiency for clinical application. Recently, hybrid gene therapy combining novel and specific vectors has been developed as an advanced strategy. Although gene therapy for lung cancer is being actively researched, aerosol gene therapy strategies are currently lacking, and further studies on aerosol gene therapy are needed to treat lung cancer.     

pH-responsive shielding of non-viral gene vectors

PEG shielding of non-viral vectors reduces undesired interactions with the extracellular environment. Combination with cell-binding domains enables in vivo targeting via specific attachment to the target cells. Pegylation, however, also interferes with effective intracellular nucleic acid delivery. Consistently triggered removal of the PEG shield after reaching the target cell would make non-viral vectors more compatible with the intracellular delivery steps. Physiological triggers may include changes in pH, enzyme concentration or redox potential. This review focuses on pH-sensitive shielding strategies that exploit the endosomal acidification process after endocytosis for deshielding of the delivery system.     

pH-responsive shielding of non-viral gene vectors

PEG shielding of non-viral vectors reduces undesired interactions with the extracellular environment. Combination with cell-binding domains enables in vivo targeting via specific attachment to the target cells. Pegylation, however, also interferes with effective intracellular nucleic acid delivery. Consistently triggered removal of the PEG shield after reaching the target cell would make non-viral vectors more compatible with the intracellular delivery steps. Physiological triggers may include changes in pH, enzyme concentration or redox potential. This review focuses on pH-sensitive shielding strategies that exploit the endosomal acidification process after endocytosis for deshielding of the delivery system.     

脑靶向非病毒基因递释系统的研究进展

脑靶向非病毒基因递释系统可有效介导基因药物跨越血脑屏障,到达病变部位发挥疗效,已成为研究热点之一。多项研究结果显示,通过适当机制如配体-受体特异性结合作用可显著提高非病毒基因递释系统在脑部的蓄积量,从而提高所携带外源基因在脑部的表达量。本文主要从受体介导和吸附介导两种机制入手,综述脑靶向非病毒基因递释系统的最新研究进展。     

Key issues in non-viral gene delivery

The future of non-viral gene therapy depends on a detailed understanding of the barriers to delivery of polynucleotides. These include physicomechanical barriers, which limit the design of delivery devices, physicochemical barriers that influence self-assembly of colloidal particulate formulations, and biological barriers that compromise delivery of the DNA to its target site. It is important that realistic delivery strategies are adopted for early clinical trials in non-viral gene therapy. In the longer term, it should be possible to improve the efficiency of gene delivery by learning from the attributes which viruses have evolved; attributes that enable translocation of viral components across biological membranes. Assembly of stable, organized virus-like particles will require a higher level of control than current practice. Here, we summarize present knowledge of the biodistribution and cellular interactions of gene delivery systems and consider how improvements in gene delivery will be accomplished in the future.     

Polyethylenimine-based non-viral gene delivery systems.

Gene therapy has become a promising strategy for the treatment of many inheritable or acquired diseases that are currently considered incurable. Non-viral vectors have attracted great interest, as they are simple to prepare, rather stable, easy to modify and relatively safe, compared to viral vectors. Unfortunately, they also suffer from a lower transfection efficiency, requiring additional effort for their optimization. The cationic polymer polyethylenimine (PEI) has been widely used for non-viral transfection in vitro and in vivo and has an advantage over other polycations in that it combines strong DNA compaction capacity with an intrinsic endosomolytic activity. Here, we give some insight into strategies developed for PEI-based non-viral vectors to overcome intracellular obstacles, including the improvement of methods for polyplex preparation and the incorporation of endosomolytic agents or nuclear localization signals. In recent years, PEI-based non-viral vectors have been locally or systemically delivered, mostly to target gene delivery to tumor tissue, the lung or liver. This requires strategies to efficiently shield transfection polyplexes against non-specific interaction with blood components, extracellular matrix and untargeted cells and the attachment of targeting moieties, which allow for the directed gene delivery to the desired cell or tissue. In this context, materials, facilitating the design of novel PEI-based non-viral vectors are described.     

Endosomal disruptors in non-viral gene delivery

Importance of the field: Non-viral gene delivery for the treatment of genetic and non-genetic diseases has been under investigation for several decades, but there has been very little application in patients because of poor gene expression and toxicity.

Areas covered in this review: As gene delivery almost invariably involves endocytosis, many of its limitations are related to compartmentalisation of the transgene within the endosomes. Gene expression enhancers have become an essential part of manipulating endosomal release, as well as protecting transgene from intracellular degradation. However, disruption of the endosomes can also release proteases that have been shown to activate apoptotic pathways.

What the reader will gain: An understanding of the role that endosomal release plays in the toxicity of gene delivery vehicles will help identify new approaches to minimise adverse effects while enhancing non-viral gene expression.

Take home message: The future of non-viral gene therapy needs to identify new approaches that limit endosome-induced toxicity while enhancing expression so that a pharmacological response can be reliably observed in vivo.     

Self-amplifying vectors for gene delivery

Current methods of gene transfer have a rather low efficiency, especially in vivo. Therefore, one tries to achieve the highest possible levels of expression in the few cells that do take up foreign DNA. One approach is to use self-amplifying expression vectors. These vectors are based on the (+)-strand RNA viruses (alphaviruses) Sindbis virus and Semliki Forest virus. In these vectors, the viral capsid protein coding sequences are replaced with the gene of interest. After introduction into the target cells, the viral replication proteins will replicate the recombinant genome. The increased levels of mRNA generate very high transgene expression levels. Furthermore, spread throughout large cells (muscle, neurons) is much better compared to conventional expression cassettes. Self-amplifying vectors can be introduced into target cells as RNA, DNA or virions.     

Recent progress in microRNA delivery for cancer therapy by non-viral synthetic vectors

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression. Because of significant changes in their expression in cancer, miRNAs are believed to be key factors in cancer genetics and to have potential as anticancer drugs. However, the delivery of miRNAs is limited by many barriers, such as low cellular uptake, immunogenicity, renal clearance, degradation by nucleases, elimination by phagocytic immune cells, poor endosomal release, and untoward side effects. Nonviral delivery systems have been developed to overcome these obstacles. In this review, we provide insights into the development of non-viral synthetic miRNA vectors and the promise of miRNA-based anticancer therapies, including therapeutic applications of miRNAs, challenges of vector design to overcome the delivery obstacles, and the development of miRNA delivery systems for cancer therapy. Additionally, we highlight some representative examples that give a glimpse into the current trends into the design and application of efficient synthetic systems for miRNA delivery. Overall, a better understanding of the rational design of miRNA delivery systems will promote their translation into effective clinical treatments.     

Key issues in non-viral gene delivery

The future of non-viral gene therapy depends on a detailed understanding of the barriers to delivery of polynucleotides. These include physicomechanical barriers, which limit the design of delivery devices, physicochemical barriers that influence self-assembly of colloidal particulate formulations, and biological barriers that compromise delivery of the DNA to its target site. It is important that realistic delivery strategies are adopted for early clinical trials in non-viral gene therapy. In the longer term, it should be possible to improve the efficiency of gene delivery by learning from the attributes which viruses have evolved; attributes that enable translocation of viral components across biological membranes. Assembly of stable, organized virus-like particles will require a higher level of control than current practice. Here, we summarize present knowledge of the biodistribution and cellular interactions of gene delivery systems and consider how improvements in gene delivery will be accomplished in the future.     

Galactomannan-PEI based non-viral vectors for targeted delivery of plasmid to macrophages and hepatocytes

Intracellular nature and diversified locations of infectious and parasitic diseases such as leishmaniasis, trypanosomiasis, tuberculosis and hepatitis B and C pose a significant global burden and challenge to the scientists working in the area of drug discovery and drug delivery. The macrophages and hepatocytes are considered as potential target sites as they together play an important role in various infectious diseases. The present study scrutinizes the applicability of a natural biopolymer-based chemical vectors, capable of targeting both macrophages and hepatocytes, that can form a complex with plasmid and administer it into cells to produce a desired protein. The investigations were made to develop a novel series of gene carriers by conjugating depolymerized galactomannan (guar gum), a biocompatible polysaccharide with low molecular weight branched PEI (LMWP). A series of conjugates were developed and characterized using physicochemical techniques. All the GP/pDNA complexes showed significantly higher transfection efficiency with GP-3/pDNA, one of the best formulations, showed ∼2.0–7.7-folds higher transfection efficacy when compared with the standard transfection reagents. Further, GP-3/pDNA displayed significantly higher target specific transfection efficiency under both in vitro and in vivo conditions. The data demonstrate the potential of GP vectors to deliver nucleic acids simultaneously to macrophages and hepatocytes in gene delivery applications.