阳离子脂质体在基因转染载体中的研究进展

目前基因治疗面临的首要技术问题是基因药物的载体,用于基因治疗的载体主要分为两大类:病毒载体和非病毒载体.病毒载体可能在体内发生基因的重组或互补,因此具有较大的潜在危险,限制了它在临床基因治疗上的应用[1].非病毒载体中发展最为成熟的是阳离子脂质体(cationic liposomes,CL)载体,它具有可自然降解、无免疫原性、可重复转染等优点,迄今已有数十种阳离子脂质体被用于基因转染[2].该项技术目前已成为基因治疗的研究热点之一,现就其进展综述如下.     

新型基因转染阳离子脂质体研究进展

阳离子脂质体是继病毒基因转染载体之后,近几年倍受国内外研究者关注的新一类基因转染载体。本文就用于基因的阳离子脂质体载体的阳离子脂质、载体－ＤＮＡ复合物、载体理化性质及载体肺部基因转染等方面的最新研究进展作一综述。     

用作基因载体的阳离子脂质体及其相关材料与制备技术

综述近年来阳离子脂质体在基因转染中的应用及其相关材料和制备技术。基因疗法面临的技术问题之一是寻找合适的基因载体,阳离子脂质体作为一种非病毒载体,具有无免疫原性、可自然降解等优点,在肺部、眼部疾病以及癌症的治疗中极具应用价值。随着研究的不断深入,新型脂质体材料相继出现、制备工艺也不断更新,阳离子脂质体的理化性质和转染效率均得到了极大的改善,推动了基因疗法和基因转染研究的发展。     

新型阳离子核酸载体的细胞转染效率研究

新型阳离子核酸载体ＬＷ１３ ２进行研究,分析其细胞毒性和基因转染效率,探讨其作为核酸载体的可能性。方法　使用ＭＴＴ比色法分析ＬＷ１３ ２与ＤＮＡ在不同比例下的细胞毒性。选用绿色荧光蛋白表达质粒ＥＧＦＰ为报告基因,以人胚胎肾ＨＥＫ２９３细胞为工具细胞,在荧光显微镜下观察计数,计算转染效率。结果　当ＬＷ１３ ２与ＤＮＡ的质量比为（１～３）∶１时,细胞毒性最低。ＬＷ１３ ２与ＤＮＡ的质量比为３∶１时,转染作用４ｈ,在无血清培养基中继续培养４８ｈ,得到ＬＷ１３ ２的最大转染效率为８７６％。结论　通过与市售成熟转染试剂Ｌｉｐｏｆｅｃｔａｍｉｎｅ２０００的对比,新型阳离子核酸载体在大幅提高基因转染效率的同时并没有增加其细胞毒性,有望成为核酸转移的有效载体,为相关研究及基因的靶向治疗研究提供基础。     

阳离子脂质在基因传递中构效关系的研究进展

阳离子脂质是非病毒载体中应用最为广泛的一种基因传递载体,其结构中的4个部分各自发挥着重要作用。构效关系研究表明,阳离子脂质的疏水基团、骨架链、连接键以及阳离子头基等结构的微小变化均会不同程度的影响阳离子脂质的细胞毒性、基因结合能力以及转染效率。本文综述了近几年有关阳离子脂质的文献,介绍了阳离子脂质各部分结构组成,归纳了阳离子脂质构效关系研究中的各种作用规律,希望为未来新型阳离子脂质的合成开发及应用提供参考。     

两种阳离子脂质体介导基因转染的比较研究

In order to study the important factors involved in cationic liposome-mediated gene transfer,   Lipofectamine 2000 or DOTAP was evaluated using three types of cells (Hep-2, MCF-7 and SW-480) in vitro transfection efficiencies.  Different properties of the two reagents were analyzed and compared by DNA     arrearage assay and MTT assay.  Both Lipofectamine 2000 and DOTAP had strong capability to combine with DNA; Lipofectamine 2000 can get higher transfection efficiency of the three cells by using GFP as report gene, meanwhile, DOTAP can also get higher transfection efficiency against Hep-2 cell.  However, DOTAP showed lower transfection efficiency against MCF-7 and SW-480 cell.  On the other hand, the cytotoxicity assay showed that over 85% cell viability of MCF-7 cell could be achieved both by Lipofectamine 2000 and DOTAP under the optimal transfection condition.  Relatively speaking, Lipofectamine 2000 has very high transfection efficiency in a broad range of cell lines, but because of the special selectivity of cell type on liposome, DOTAP also has a broad application prospect.     

阳离子聚合物在基因传递系统中的应用阳离子聚合物在基因传递系统中的应用

随着近代生物技术的发展与人类基因库的不断完善 ,各种与人类疾病相关基因逐步被揭晓 ,使人类从分子水平上认识某些疾病根源已逐步成为可能 ,为基因治疗提供了理论基础。基因治疗即ＤＮＡ给药系统 ,属于靶向给药系统 ,它不同于一般靶向药物作用于机体的某器官或某组织 ,它是细胞与分子水平上的靶向作用。基因治疗从 1980年第 1篇哺乳类基因转移公开报告[1]到 1994年 30 0余人接受临床基因治疗试验 ,整整花了 15年时间 ,1994年美国重组ＤＮＡ咨询委员会(ＲＡＣ)批准了人基因治疗方案。此后 ,许多分子疾病 (ｍｏｌｅｃｕｌａｒｄｉｓｅａｓｅ)…     

阴离子脂质体－阳离子脂质体复合物介导的基因转染

目的评价阴离子脂质体-阳离子脂质体复合物介导质粒转移至HepG2肝癌细胞中及其毒副作用。方法制备携载表达绿色荧光蛋白质粒的阳离子脂质体,与阴离子脂质体形成复合物。测定脂质体复合物的zeta电位,凝胶阻滞实验考察质粒包封情况,流式细胞仪测量各阴离子脂质体-阳离子脂质体复合物的转染效率,MTT法检测细胞毒性。结果复合物能完全包裹质粒,其zeta电位低于阳离子脂质体zeta电位;脂质体复合物介导的转染效率略低于阳离子脂质体,其细胞生存率高于阳离子脂质体。结论阴离子脂质体-阳离子脂质体复合物在降低细胞毒性的同时,可实现对HepG2细胞较高的转染效率。     

阳离子脂质材料和聚乙二醇对脂质体细胞转染率及膜流动性的影响阳离子脂质材料和聚乙二醇对脂质体细胞转染率及膜流动性的影响

目的制备包封荧光素钠（FS）的脂质体，考察阳离子脂质材料（DC-chol）和聚乙二醇（PEG）对脂质体包封率、细胞转染率及膜流动性的影响。方法以FS作为模型物质，制备并分离脂质体，测定脂质体包封率；通过观察荧光光谱的变化考察FS与脂质体膜之间的相互作用；以HepG₂ 2.2.15为细胞模型观察脂质体对FS细胞转染率的影响；通过荧光偏振技术考察阳离子脂质材料和PEG对脂质体膜流动性的影响。结果阳离子脂质材料和PEG能提高脂质体包封率（0.64％～86.57％）、细胞转染率（2.18％～48.46％）及脂质体膜流动性，PEG分子质量的增大有利于包封率、转染率的提高，并增加脂质体膜的流动性。结论在脂质体处方中加入阳离子脂质材料和高分子量的PEG有利于提高包封率、细胞转染率及增加脂质体膜的流动性。     

阳离子脂质体用做基因传递载体的研究进展

 基因治疗的难题之一在于研制安全有效的基因传递载体。常用的基因传递载体分为病毒载体和非病毒载体两类,其中,非病毒载体中的阳离子脂质体因具有低毒性与免疫原性、生物相容性好、易于制备等优点而受到广泛关注,具有良好的应用前景。近年来对阳离子脂质体载体的研究主要集中在对其传递基因机制的考察、各种影响其转基因效率的因素的探求、应用各种方法研制安全性和转染活性更佳的新型阳离子脂质体等方面。文中从转基因特点、传递机制、常用的制备材料、影响转基因效率的因素、近年来出现的新型阳离子脂质体等方面综述了此类基因传递载体的研究进展。     

Cationic niosomes composed of spermine-based cationic lipids mediate high gene transfection efficiency

Non-ionic surfactant-based vesicles (niosomes) composed of non-ionic surfactants (i.e., Tween and Span) and cholesterol were formulated, and their turbidity and particle size assayed. The most appropriate niosomes formulation was mixed with novel synthesized spermine-based cationic lipids to prepare cationic niosomes that could act as gene carriers. Factors affecting gene transfection and cell viability including differences in the acyl chain length (C14, C16 and C18) of cationic lipids and the weight ratio of niosomes to DNA were evaluated on a human cervical carcinoma cell line (HeLa cells) using pDNA encoding green fluorescent protein (pEGFP-C2). The morphology, size and charge of the niosomes were also characterized, and a gel retardation assay to determine complex formation was performed. The results revealed that the transfection efficiency of the Span 20-niosomes was the highest for the spermine-C14 formulation and decreased as follows: spermine-C14 > spermine-C16 > spermine-C18. In addition to the highest transfection efficiency, there was also no serum effect on transfection efficiency of the spermine-C14 niosomes at a weight ratio of 10. This formulation was safe in vitro and had good physical stability for at least 1 month at 4°C. In conclusion, the cationic niosomes may constitute a good alternative carrier for gene transfection.     

Structure-function relationship research of glycerol backbone-based cationic lipids for gene delivery

Transfection activities of two series of synthetic glycerol backbone-based cationic lipids were studied as gene delivery carriers. The variable length of hydrocarbon chains, diverse quaternary ammonium heads, different linkage, as well as alternative anion combined with them allowed to find how these factors affect cationic lipids on their gene delivery performance. The structure-function relationship of the synthetic glycerol backbone-based cationic lipids was discussed, and the transfection efficiency of some of the cationic liposomes was superior or parallel to that of two commercial transfection agents.     

Synthetic diether-linked cationic lipids for gene delivery

Quaternary ammonium lipids 2a-p, with diether linkages between hydrocarbon chains and their ammonium headgroups, were synthesized as potential vectors for cationic liposome-mediated gene delivery. Varying the length of carbon chains and quaternary ammonium heads as well as different anionic complexes will enable the study of the structure-function relationships of these cationic lipids in terms of gene delivery properties.     

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.     

Cationic lipids for gene delivery in vitro and in vivo

Certain disease states can be corrected by using nucleic acids as therapeutic agents. To achieve this, nucleic acids must be delivered into the affected cells efficiently. At the core of a successful gene therapy protocol is the design of the nucleic acid carrier. Cationic lipids, as one of the gene delivery systems, have a wide potential in delivering nucleic acids both in vivo and in vitro. They are synthetic in origin and, hence, can be produced in required quantities and are biologically safe. Significant inputs from synthetic chemists in the recent past have resulted in the exploration of cationic lipids with very interesting functionalities. Transfection efficiencies of cationic lipids are comparable to viral-mediated transfection in vitro. However, viral-based methods for gene delivery in vivo are comparatively more efficient. Current understanding of lipid-mediated transfection is partially due to incomplete characterisation of the lipoplex, poor understanding of cell biology of transfection and cell type variations in transfection efficiencies. The published patents and research demonstrates the need for incorporation of the biological information in the design of the gene delivery formulations. In this review, the cell biological aspects critical for lipid-mediated transfection are emphasised. The parameters that influence the colloidal stability of the lipoplexes, cell biological processes relevant to gene delivery, such as cell association/uptake, cytoplasmic stability of the DNA and nuclear import, are discussed. The main focus of this review is patents published in the last 5 years.     

Anionic liposomal delivery system for DNA transfection

The present study investigates the use of novel anionic lipoplexes composed of physiological components for plasmid DNA delivery into mammalian cells in vitro. Liposomes were prepared from mixtures of endogenously occurring anionic and zwitterionic lipids, 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (sodium salt) (DOPG) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), respectively, at a molar ratio of 17:83 (DOPG:DOPE). Anionic lipoplexes were formed by complexation between anionic liposomes and plasmid DNA molecules encoding green fluorescence protein (GFP) using Ca2+ ions. Transfection and toxicity were evaluated in CHO-K1 cells using flow cytometry and propidium iodide staining, respectively. Controls included Ca2+-DNA complexes (without lipids), anionic liposomes (no Ca2+), and a cationic liposomal formulation. Efficient delivery of plasmid DNA and subsequent GFP expression was achieved using anionic lipoplexes. Transfection efficiency increased with Ca2+ concentration up to 14 mM Ca2+, where transfection efficiency was 7-fold higher than in untreated cells, with minimum toxicity. Further increase in Ca2+ decreased transfection. Transfection efficiency of anionic lipoplexes was similar to that of cationic liposomes (lipofectAmine), whereas their toxicity was significantly lower. Ca2+-DNA complexes exhibited minimal and irregular transfection with relatively high cytotoxicity. A model was developed to explain the basis of anionic lipoplex uptake and transfection efficacy. Effective transfection is explained on the formation of nonbilayer hexagonal lipid phases. Efficient and relatively safe DNA transfection using anionic lipoplexes makes them an appealing alternative to be explored for gene delivery.     

Cationic lipids containing cyclen and ammonium moieties as gene delivery vectors

In this study, two novel cationic lipids containing protonated cyclen and quaternary ammonium moieties were designed and synthesized as non-viral gene delivery vectors. The structures of the two lipids differ in their hydrophobic region (cholesterol or diosgenin). Cationic liposomes were easily prepared from the lipids individually or from the mixtures of each cationic lipid and dioleoylphosphatidylethanolamine. Several studies including DLS, gel retardation assay, and ethidium bromide intercalation assay suggest that these amphiphilic molecules are able to bind and compact DNA into nanometer particles which can be used as non-viral gene delivery agents. Our results from in vitro transfection show that in association with dioleoylphosphatidylethanolamine, two cationic lipids can induce effective gene transfection in human embryonic kidney 293 cells, although the gene transfection efficiencies of two cationic lipids were found to be lower than that of lipofectamine 2000(TM) . Besides, different cytotoxicity was found for two lipoplexes. This study demonstrates that the title cationic lipids have large potential to be efficient non-viral gene vectors.