Polymers for DNA delivery

Nucleic acid delivery has many applications in basic science, biotechnology, agriculture, and medicine. One of the main applications is DNA or RNA delivery for gene therapy purposes. Gene therapy, an approach for treatment or prevention of diseases associated with defective gene expression, involves the insertion of a therapeutic gene into cells, followed by expression and production of the required proteins. This approach enables replacement of damaged genes or expression inhibition of undesired genes. Following two decades of research, there are two major methods for delivery of genes. The first method, considered the dominant approach, utilizes viral vectors and is generally an efficient tool of transfection. Attempts, however, to resolve drawbacks related with viral vectors (e.g., high risk of mutagenicity, immunogenicity, low production yield, limited gene size, etc.), led to the development of an alternative method, which makes use of non-viral vectors. This review describes non-viral gene delivery vectors, termed "self-assembled" systems, and are based on cationic molecules, which form spontaneous complexes with negatively charged nucleic acids. It introduces the most important cationic polymers used for gene delivery. A transition from in vitro to in vivo gene delivery is also presented, with an emphasis on the obstacles to achieve successful transfection in vivo.     

Recent progress in cationic polymeric gene carriers for cancer therapy

In recent years,various carriers for gene delivery nave been developed for biomedical applications.Among all kinds of gene carriers,cationic polymeric carriers for delivery therapeutic gene as non-viral carriers have received growing interests due to their improved high transfection efficiency with the relative safety.In particular,the advancement of novel polymeric gene carriers has gained much progress in the development of effective anticancer therapy.Herein,this review focused on the development of cationic polymeric carriers for cancer therapy,including polyethylenimine(PEI),polyamidoamine(PAMAM) dendrimers,polylysine(PLL),chitosan and modified cationic polymers.And recent progresses in the development of novel polymeric carriers for gene delivery,such as targeted gene carriers,responsive gene carriers and multifunctional gene carriers,were summarized.Finally,the future perspectives in the development of novel polymeric carriers for delivery gene were presented.     

Solid-phase synthesis of 89 polyamine-based cationic lipids for DNA delivery to mammalian cells

The ability of non-viral gene delivery systems to overcome extracellular and intracellular barriers is a critical issue for future clinical applications of gene therapy. In recent years much effort has been focused on the development of a variety of DNA carriers, and cationic liposomes have become the most common non-viral gene delivery system. Solid-phase synthesis was used to produce three libraries of polyamine-based cationic lipids with diverse hydrophobic tails. These were characterised, and structure-activity relationships were determined for DNA binding and transfection ability of these compounds when formulated as cationic liposomes. Two of the cationic lipids produced high-efficiency transfection of human cells. Surprisingly, these two compounds were from the library with two headgroups and one aliphatic tail, a compound class regarded as detergent-like and little investigated for transfection. These cationic lipids are promising reagents for gene delivery and illustrate the potential of solid-phase synthesis methods for lipoplex discovery.     

Novel cationic 6-lauroxyhexyl lysinate modified poly(lactic acid)-poly(ethylene glycol) nanoparticles enhance gene transfection

The leading principle of non-viral delivery systems for gene therapy is to mediate high levels of gene expression with low cytotoxicity. Nowadays, biodegradable nanoparticles formulated with poly(lactic acid)-poly(ethylene glycol) (PLA-PEG) were wildly developed. However, the relative lower gene transfection efficiency and higher cytotoxicity still remained critical problems. To address these limitations, PLA-PEG nanoparticles have been composited with other components in their formulation. Here, a novel cationic lipid, 6-lauroxyhexyl lysinate (LHLN), was fabricated onto PLA-PEG nanoparticles as a charge modifier to improve the transfection efficiency and cytotoxicity. The obtained cationic LHLN modified PLA-PEG nanoparticles (LHLN-PLA-PEG NPs) could condense pDNA thoroughly via electrostatic force, leading to the formation of the LHLN-PLA-PEG NPs/pDNA complexes (NPs/DNA complexes). The nanoparticles obtained have been characterized in relation to their physicochemical and biological properties, and the results are extremely promising in terms of low cell toxicity and high transfection efficiency. These results indicated that the novel cationic LHLN modified PLA-PEG nanoparticles could enhance gene transfection in vitro and hold the potential to be a promising non-viral nanodevice.     

Cationic lipid-DNA complexes-lipoplexes-for gene transfer and therapy

Cationic lipid-mediated gene transfer and delivery still attract great attention of many gene therapy laboratories. From the point of view of the most important characteristics of lipoplex particles, e.g. its charge and size, we reviewed recent studies available. In general, the paper deals with non-viral systems of gene transfer into eukaryotic cell based on various lipids. Having usually less efficiency in gene transfer, lipid-based gene transfer vehicles (lipoplexes/genosomes) are characterized with certain advantages even over viral ones: they are less toxic and immunogenic, could be targetable and are easy for large-scale production, a size of transferred DNA being quite high. Conditions of DNA condensation during interactions with lipids are described. Results of the studies of mechanism of DNA-lipid complex interactions with the cell membrane and their transport into the nucleus are summarized. Dependence of efficiency of gene transfer on lipoplex structure and physical-chemical properties is reviewed. Advantages and disadvantages of different macromolecule complexes from the point of view of transfection efficiency, possibility of use in vivo, cytotoxicity and targeted gene transfer in certain organs and tissues are also discussed. Results of transfection of different cells using neutral, anion and cation liposomes are reviewed. The conclusion reached was that efficiency and specificity of gene transfer may grow considerably when mixed macromolecule lipid systems including polycations and glycolipids are used.     

非病毒性基因载体的合成研究

基因治疗是一种有效的治疗先天性遗传性疾病以及后天获得性疾病的手段。它通过激发细胞的生物活性或者抑制细胞非正常的功能来治疗或者预防疾病的发生,例如细胞的基因紊乱,细胞的无序增殖。目前基因治疗所面临的问题是缺乏有效的基因递送载体。基因载体主要分为病毒性基因载体和非病毒性基因载体。与病毒性基因载体相比,非病毒性基因载体具有毒性小、安全性高、易于制备、能够荷载分子量大的DNA等优点。本文综述了非病毒性基因载体的合成研究进展。     

含氟高分子基因载体

阳离子高分子被广泛应用为非病毒类基因载体,但这类高分子材料的转染效率与细胞毒性之间通常存在"恶性"关联,即获得高转染效率时往往会伴随严重的细胞毒性.如何制备兼具高效、低毒特点的高分子载体是成功实施基因治疗的关键.含氟高分子是一类具有独特理化性质的高分子,能够在低电荷密度条件下与核酸形成稳定的复合物,从而实现高效、低毒的基因转染.含氟功能基团可帮助阳离子高分子改善复合物稳定性、细胞内吞、内涵体逃逸、胞内核酸释放等多个环节,从而赋予了含氟高分子在基因递送过程中的氟效应.该专论系统地总结了含氟高分子基因载体的研究,介绍了含氟高分子的基因递送性能、作用机理以及在基因治疗、基因编辑中的应用,并对含氟高分子载体的未来发展进行了展望.     

非病毒型DNA纳米微球层层组装构建多层膜用于原位基因传递

高效安全的基因传递体系是基因技术发展的关键问题. 基于聚阳离子的基因纳米微球是一种典型的非病毒型基因载体, 能够在体内外有效转染细胞. 本文通过层层组装方法构建装载基因纳米微球的可降解多层膜, 这种固相基因传递体系能实现材料表面的贴壁细胞的原位转染. 与装载裸DNA的多层膜相比, 基因纳米微球多层膜能更有效地原位转染贴壁细胞, 这主要是因为DNA在此多层膜中仍处于与聚阳离子缔合的状态. 构建于聚乳酸三维支架表面的基因纳米微球多层膜亦能实现支架表面贴壁细胞的原位转染. 这种结构可控、易制备的基因纳米微球多层膜为精确控制基因纳米微球传递提供了一种新方法, 也为基因治疗进一步应用于组织工程、介入治疗和医用植入体提供了一种可能的技术手段.     

Chemo-physical and biological evaluation of poly(L-lysine)-grafted chitosan copolymers used for highly efficient gene delivery

For the success of non-viral gene delivery, it is of great importance to develop gene vectors with high efficiency but low toxicity. We demonstrate that PLL-grafted chitosan copolymers combine the advantages of PLL with its good pDNA-binding ability and of chitosan with its good biocompatibility. The chemo-physical properties of the prepared Chi-g-PLL copolymers are thoroughly characterized. The in vitro transfection study shows that the copolymers have a much higher gene transfer ability than the starting materials chitosan and PLL. A positive correlation between PLL chain lengths and transfection efficiency of the copolymers is found. Our results suggest that these novel Chi-g-PLL copolymers are good candidates for gene delivery in vivo.     

A touch-and-go lipid wrapping technique in microfluidic channels for rapid fabrication of multifunctional envelope-type gene delivery nanodevices

Multifunctional envelope-type gene delivery nanodevices (MENDs) are promising non-viral vectors for gene therapy. Though MENDs remain strong in prolonged exposure to blood circulation, have low immunogenic response, and are suitable for gene targeting, their fabrication requires labor-intensive processes. In this work, a novel approach has been developed for rapid fabrication of MENDs by a touch-and-go lipid wrapping technique in a polydimethylsiloxane (PDMS)/glass microfluidic device. The MEND was fabricated on a glass substrate by introduction of a condensed plasmid DNA core into microfluidic channels that have multiple lipid bilayer films. The principle of the MEND fabrication in the microfluidic channels is based on electrostatic interaction between the condensed plasmid DNA cores and the coated lipid bilayer films. The constructed MEND was collected off-chip and characterized by dynamic light scattering. The MEND was constructed within 5 min with a narrow size distribution centered around 200 nm diameter particles. The size of the MEND showed strong dependence on flow velocity of the condensed plasmid DNA core in the microfluidic channels, and thus, could be controlled to provide the optimal size for medical applications. This approach was also proved possible for fabrication of a MEND in multiple channels at the same time. This on-chip fabrication of the MEND was very simple, rapid, convenient, and cost-effective compared with conventional methods. Our results strongly indicated that MENDs fabricated with our microfluidic device have a good potential for medical use. Moreover, MENDs fabricated by this microfluidic device have a great potential for clinical use because the devices are autoclavable and all the fabrication steps can be completed inside closed microfluidic channels without any external contamination.     

Effective gene delivery into primary dendritic cells using synthesized PDMAEMA-iron oxide nanocubes

Dendritic cells (DCs) have been a target of vaccine delivery, gene therapy, and cancer immunotherapy. However, gene delivery to primary DCs using traditional non-viral molecules has been a difficult challenge. Herein we have developed a gene delivery system to primary DCs using magnetic iron oxide nanocubes (MCs) coated with cationic polymer under the induction of a magnetic field. The MCs were coated with positively charged polymer, poly(2-dimethylamino) ethyl methacrylate (MCs-PD) before the plasmid gene (pMAX-GFP) was adsorbed on their surfaces. Three different sizes (15, 40 and 90 nm) of MCs were synthesized, and subsequently, PDMAEMA was assembled onto the MC surfaces (MCs-PD). MCs-PD exhibited zeta potentials of +23 to +26 mV, and the obtained particles showed superparamagnetic character with saturation magnetization of 17–66 emu/g. The MCs-PD of 10–100 μg/mL showed low toxicity on bone marrow-derived dendritic cells (BMDCs) in MTT assay, and they were well taken up by BMDCs under a magnetic field. Moreover, the particles with small size exhibited the enhanced plasmid transfection efficiency without the activation of BMDCs. The MCs-PD could be a promising non-viral gene delivery system that helps to manipulate primary DCs in vitro, which will be beneficial for cell-based immunotherapy.     

Delivery of surface-mediated non-viral gene nanoparticles from ultrathin layer-by-layer multilayers

An efficient and safe gene delivery system remains a challenge in the development of gene therapy.Polycation-based gene nanoparticles are a typical non-viral gene delivery system,which are able to transfect cells in vitro and in vivo.This paper reported a facile method for constructing biodegradable multilayers via layer-by-layer self-assembly,in which the polycation-based gene nanoparticles were loaded.Through this surface-mediated delivery system,adherent cells on the multilayer could be transfected in si...     

Function/structure correlation in novel pH-dependent cationic liposomes for glioma cells transfection in vitro

Cationic liposomes are good candidates as gene carriers in cell biology due to their ability to bind DNA through electrostatic interactions. These liposomes are used as non-viral delivery systems in the gene therapy of glioma. pH-dependency and transfection efficiency of seven novel lipids (MORF-1, MORF-2, MORF-3, PIPR-1, PIPR-2, MM54 and DC-Amy) were studied. Two of these molecules (PIPR-2 and MM54) show at specific charge ratios better transfection efficiency than that of some commercially available liposomes. To cite this article: C. Esposito et al., C. R. Chimie 6 (2003).     

Lipothiophosphoramidates for gene delivery: critical role of the cationic polar headgroup

When considering a family of cationic lipids designed for gene delivery, the nature of the cationic polar head probably has a great influence on both the transfection efficacy and toxicity. Starting from a cationic lipothiophosphoramidate bearing a trimethylammonium headgroup, we report herein the impact on gene transfection activity of the replacement of the trimethylammonium moiety by a trimethylphosphonium or a trimethylarsonium group. A series of three different human epithelial cell lines were used for the experimental transfection studies (HeLa, A549 and 16HBE14o(-)). The results basically showed that such structural modifications of the cationic headgroup can lead to a high transfection efficacy at low lipid/DNA charge ratios together with a low cytotoxicity. It thus appears that the use of a trimethylarsonium cationic headgroup for the design of efficient gene carriers, which was initially proposed in the lipophosphoramidate series, can be extended to other series of cationic lipids and might therefore have great potential for the development of novel non-viral vectors in general.     

Synthetic, self-assembly ABCD nanoparticles; a structural paradigm for viable synthetic non-viral vectors

Gene therapy research is still in trouble owing to a paucity of acceptable vector systems to deliver nucleic acids to patients for therapy. Viral vectors are efficient but may be too dangerous. Synthetic non-viral vectors are inherently safer but are currently not efficient enough to be clinically viable. The solution for gene therapy lies with improved synthetic non-viral vectors systems. This review is focused on synthetic cationic liposome/micelle-based non-viral vector systems and is a critical review written to illustrate the increasing importance of chemistry in gene therapy research. This review should be of primary interest to synthetic chemists and biomedical researchers keen to appreciate emerging technologies, but also to biological scientists who remain to be convinced about the relevance of chemistry to biology.     

Improved GFP gene transfection mediated by polyamidoamine dendrimer-functionalized multi-walled carbon nanotubes with high biocompatibility

The multi-walled carbon nanotubes (MWCNTs)-polyamidoamine (PAMAM) hybrid was prepared by covalent linkage approach, and characterized by transmission electron microscopy, Fourier transform infrared spectroscopy and ultraviolet-visible spectrometry. The PAMAM dendrimers were present on the surface of MWCNTs in high density, and the MWCNT-PAMAM hybrid exhibited good dispersibility and stability in aqueous solution. The interaction between MWCNT-PAMAM with plasmid DNA of enhanced green fluorescence protein (pEGFP-N1), intracellular trafficking of the hybrid, transfection performance and cytotoxicity to HeLa cells were evaluated in detail. We found that the MWCNT-PAMAM hybrid possessed good pEGFP-N1 immobilization ability and could efficiently delivery GFP gene into cultured HeLa cells. The surface modification of MWCNTs with PAMAM improved the transfection efficiency 2.4 and 0.9 times, and simultaneously decreased cytotoxicity by about 38%, as compared with mixed acid-treated MWCNTs and pure PAMAM dendrimers. The MWCNT-PAMAM hybrid can be considered as a new carrier for the delivery of biomolecules into mammalian cells. Therefore, this novel system may have good potential applications in biology and therapy, including gene delivery systems.     

纳米阳离子多聚物在基因载体系统的应用

阳离子多聚物能与DNA通过静电吸附作用而自组装成纳米微粒,防止DNA被核酸酶降解.阳离子多聚物由于具备合成简便、储存稳定、基因荷载率高、靶向性强、免疫原性低等优点而被用作基因载体.阳离子多聚物按特性可分为两类：合成型和天然型.经典的人工合成型阳离子多聚物基因载体主要有：多聚乙烯亚胺、多聚左旋赖氨酸和树状大分子等;天然生物型阳离子多聚物基因载体主要有壳聚糖及其衍生物和明胶等.本文详细论述了各种阳离子聚合物用作基因载体的性能特点、自身缺陷、介导基因进入细胞的机理和靶向性策略,并对非病毒基因载体的发展作出展望.     

脂质纳米颗粒的制备及在基因治疗中的应用

基因治疗已经成为人类治疗疾病的一种重要手段.然而,为了将基因药物用于临床,需要更加复杂的递送系统.脂质纳米颗粒(LNPs)系统是目前领先的非病毒递送系统,在治疗诊断学方面取得了许多令人鼓舞的进展,其具有实现基因药物临床治疗应用的潜力.由于LNPs纳米尺寸的优势及类脂化合物的生物相容性和生物降解性,LNPs能够克服阻碍基...     

Alkylated chitosans of low molecular weight as non-viral transfection vectors for gene therapy

Low molecular weight chitosans (5 kDa) hydrophobically modified with 3, 10, and 18 mol % of tetradecenoyl (TDC) groups have been synthesized. Their good solubility at neutral pH, their surface activity and micelle-forming properties as well as their ability to interact with negatively charged phospholipid vesicles mimicking the internal layer of cell plasma membranes, allow us to consider them as potential non-viral transfection vectors for gene therapy.