用于转基因的阳离子脂质体

通过直接导入外源基因来治疗人类疾病的方法需要一种有效、安全并且可重复进行的载体,阳离子脂质体是基本满足这些条件的有限几种载体中的一员. 目前已经有十几种阳离子脂质体. 这些脂质体通过外周的电荷与DNA相结合,静电吸引形成复合物在与细胞膜相互作用后,通过细胞的内吞或融合作用使复合物进入细胞内,从而将外源基因导入细胞,这种DNA传递技术的有效性和安全性已经确立. 二例利用阳离子脂质体的人体基因治疗临床试验也已开始实施,将来会有更多的临床试验得到开展,阳离子脂质体在基因治疗领域有较好的前景.     

新型非病毒三元复合DNA载体的研究

基因治疗是未来临床医学最具潜力的治疗方式,目前阻碍临床基因治疗发展的主要因素是缺乏安全和高效的基因载体,因此研究理想的非病毒转基因载体具有重要的意义.构建了由质粒DNA(D)-抗DNA抗体(A)-阳离子脂质体(C)组成的三元复合纳米基因载体(DAC),研究表明,三组分在磷酸缓冲液中可通过分子组装形成复合纳米胶束,DAC在细胞培养中表现出显著高效的基因表达,DAC在血管平滑肌细胞中的基因转染效率比不含抗DNA抗体的二元组合(DC)高4倍,比不含阳离子脂质体的二元组合(DA)约高11倍.激光共聚焦荧光显微观察证明,DAC细胞摄取量和DNA进入细胞核的量均明显高于对照组,而DC二元组合(不含抗DNA抗体)的DNA很少进入细胞核,细胞在DAC存在下生长正常.未发现细胞毒性.研究结果提示,DAC的作用机理主要是三元复合胶束中DNA的装载量比二元载体大得多,抗DNA抗体与阳离子脂质体的协同作用明显有利于DNA被细胞摄取和胞吞,从而提高了基因的转染和表达.     

阳离子脂质体的转染机制及转染效率影响因素

阳离子脂质体是一种非常具有发展前景的基因载体。简要介绍了阳离子脂质体的结构特点;着重讨论了阳离子脂质体作为基因载体时介导基因转移的机制以及在转染过程中对基因转染效率产生影响的主要因素。     

阳离子脂质体的转染机制及转染效率影响因素

阳离子脂质体是一种非常具有发展前景的基因载体。简要介绍了阳离子脂质体的结构特点;着重讨论了阳离子脂质体作为基因载体时介导基因转移的机制以及在转染过程中对基因转染效率产生影响的主要因素。     

阳离子脂质体介导RNA干扰的效果评价

考察自制的肽型阳离子脂质体CDO14作为RNA转染载体的细胞毒性及其运载si RNA进行RNA干扰的效果。通过MTT法检测脂质体对稳定表达荧光素酶的肺癌A549(Luc-A549)细胞的毒性。以脂质体为载体将荧光素酶si RNA(Luc-si RNA)转染至Luc-A549细胞内,用发光仪检测转染细胞内荧光素酶含量,BCA法检测细胞内总蛋白含量。在裸鼠腋下接种Luc-A549细胞,成瘤后尾静脉注射Luc-si RNA和脂质体的复合物,利用活体成像系统检测模型小鼠体内荧光素酶的表达量。细胞毒性实验表明,自制脂质体的毒性与商品脂质体DOTAP相近,低于商品脂质体Lipo2000;细胞转染实验表明自制脂质体作为基因转染载体的转染效率高于DOTAP;体内转染实验表明CDO14作为载体转染效果优于DOTAP。结果表明,肽型阳离子脂质体CDO14具有毒性小、转染效率高等优点,有望作为转染载体用于基因治疗。     

表面电荷对阳离子基因载体的影响

阳离子基因载体表面带有大量正电荷,由于许多DNA和细胞膜表面带负电荷,因此阳离子基因载体表面电荷有利于提高结合DNA的效率,纳米粒子与细胞膜的吸附也受粒子表面电荷的影响。同时,其表面电荷也是产生细胞毒性的主要原因之一,因此揭示细胞毒性及其作用机制有利于开发出更安全高效的基因递送载体。本文综述了阳离子基因载体表面电荷对DNA结合能力、细胞摄入、转染效率以及细胞毒性及其作用机制的影响。     

蔗糖脂肪酸酯型阳离子脂质体的制备及基因转运性能研究

该文采用蔗糖脂肪酸酯(sucrose fatty acid esters,SEs)作为助脂质与季铵盐型阳离子脂质1,2-双-[N-十四烷氧酰胺乙基-N,N-二甲基碘化铵](CTA14)制备阳离子脂质体,测定了脂质体的粒径及Zeta电位,脂质体的平均粒径为210~230 nm,Zeta电位为50~65 mV。DNA延滞实验表明,蔗糖脂肪酸酯型脂质体能够有效压缩DNA。阳离子脂质体与绿色荧光蛋白基因(plasmid green fluorescent protein-N2,p GFP-N2)结合,形成脂质体/DNA复合物,通过载入人喉癌细胞(Hep-2)和人宫颈癌细胞(Hela),观察其转染效率和细胞毒性。结果表明,阳离子脂质与SEs以质量比1:1、2:1混合制备的脂质体均能高效转染Hep-2和Hela细胞。毒性实验显示,SEs对两种细胞的毒性很小,阳离子脂质单独存在时对癌细胞具有一定的细胞毒性,随着SEs加入量的增加,脂质体对的细胞毒性也明显减小。该文进一步证实了SEs能够作为助脂质用于基因载体系统进行基因转运。     

用作基因传递系统的阳离子纳米载体的 细胞毒性及其机制研究进展

作为基因治疗中的非病毒基因载体,阳离子纳米载体可通过电荷作用与核酸类药物相结合,具有广阔的应用前景。然而,其细胞毒 性,主要表现为诱导细胞凋亡,限制了其临床开发与应用,也成为阳离子纳米载体研究所关注的重点。揭示和准确评价阳离子纳米载体的 细胞毒性及其机制,将有助于设计和开发更安全、更高效地用于基因传递的阳离子纳米载体。综述常用作基因传递系统的阳离子纳米载体 材料阳离子脂质体、聚乙烯亚胺、多聚赖氨酸、聚苯乙烯纳米粒以及其他阳离子聚合物的细胞毒性及其机制研究进展。     

阳离子脂质体及其在体内基因转染中的应用

阳离子脂质体已经成为基因转移使用最广泛的载体之一。本文从阳离子脂质体的理化特性、质粒／阳性子脂质复合体与生物大分子的相互作用、质粒／脂质复合体的基因转移机制等方面,对阳离子脂质体及其在体内基因转染中的应用进行了综述。     

基于CRISPR/Cas9系统构建精准调控Wnt信号通路的表达载体及其效果的验证

目的:构建基于CRISPR/cas9系统调控Wnt信号通路的载体,并在细胞水平验证其调控基因表达的效率。方法:选取Wnt信号中负性调控分子,设计并合成能够表达靶向上述分子gRNA的互补DNA克隆序列,BsmBI限制性内切酶酶切载体后,采用分子克隆的方法将上述序列克隆至目的载体lenti-sgRNA-Ms2-zeo,测序正确的克隆通过Lipofectamine2000与lenti-Ms2-P65-HSF1-Hygro和lenti-dcas9-VP64-blastine共同转染入293细胞;转染24h后收集细胞,qRt-PCR检测目的基因的表达。结果:筛选了Wnt信号通路中已知的19个负性调控基因;针对每个基因设计了两对gRNA序列,并构建了能够表达gRNA和MS2融合序列的载体,测序结果显示重组质粒的DNA序列与预期完全相符。随机挑选了4个表达载体与lenti-Ms2-P65-HSF1-Hygro和lenti-dcas9-VP64-blastine共转进入细胞,qPCR结果显示构建的目的载体联合lenti-Ms2-P65-HSF1-Hygro和lenti-dcas9-VP64-blastine载体可以协同促进靶分子表达。结论:本研究成功构建了基于CRISPR/cas9基因编辑系统调控Wnt信号的载体。     

Nature as a source of inspiration for cationic lipid synthesis

Synthetic gene delivery systems represent an attractive alternative to viral vectors for DNA transfection. Cationic lipids are one of the most widely used non-viral vectors for the delivery of DNA into cultured cells and are easily synthesized, leading to a large variety of well-characterized molecules. This review discusses strategies for the design of efficient cationic lipids that overcome the critical barriers of in vitro transfection. A particular focus is placed on natural hydrophilic headgroups and lipophilic tails that have been used to synthesize biocompatible and non-toxic cationic lipids. We also present chemical features that have been investigated to enhance the transfection efficiency of cationic lipids by promoting the escape of lipoplexes from the endosomal compartment and DNA release from DNA-liposome complexes. Transfection efficiency studies using these strategies are likely to improve the understanding of the mechanism of cationic lipid-mediated gene delivery and to help the rational design of novel cationic lipids.     

Role of intracellular cationic liposome-DNA complex dissociation in transfection mediated by cationic lipids

The cationic lipid-mediated gene transfer process involves sequential steps: internalization of the cationic lipid-DNA complexes inside the cells via an endocytosis-like mechanism, escape from endosomes, dissociation of the complex, and finally entry of free DNA into the nucleus. However, cationic lipid-DNA complex dissociation in the cytoplasm and the ability of the subsequently released DNA to enter the nucleus have not yet been demonstrated. In this report we showed, using confocal laser scanning analysis, that microinjection of a double fluorescent-labeled cationic lipid-pCMV-LacZ plasmid complex into the cytoplasm of HeLa cells results in efficient complex dissociation. However, the released DNA did not enter the nucleus, and no significant transfection could be detected. In contrast, nuclear microinjection of the cationic lipid-pCMV-LacZ plasmid complex resulted in efficient complex dissociation and transfection of all the cells. Taken together, the data suggest that intracellular dissociation of the cationic lipid-DNA complex is not a limiting step for transfection as previously thought.     

A composite gene delivery system consisting of polyethylenimine and an amphipathic peptide KALA

BACKGROUND: Animal viruses such as enveloped virus carry multi-functional proteins in the virion that can mediate more than two distinct steps of a gene delivery process during the transfer of viral genome into host cells. We tested if the aspects of the viral gene delivery mechanism could be mimicked by forming composite formulae from multi-functional synthetic gene carriers having complementary action modes. METHODS: Polyethylenimine (PEI) was chosen as the component responsible for endosome escape and DNA condensation and KALA for cellular entry and DNA condensation. Compact DNA-carrier particles consisting of the core part where DNA chains were tightly condensed by PEI and the outer layer lined with KALA were formulated, characterized and compared with monolithic cationic formulae in terms of gene delivery efficiency and mechanism. RESULTS: High-level gene expression was observed when C2C12 cells were transfected with DNA that was first partially condensed with PEI and, then, fully with KALA. In these formulae KALA mediated enhanced cellular entry of DNA by facilitating endocytic vesicle formation, while PEI provided an effective endosomolytic capacity. An optimal PEI/KALA formula showed transfection efficiencies better than or comparable to the commercial cationic liposome in various cell types in culture and in vivo. CONCLUSIONS: Gene delivery by combining the membrane-active property of KALA with the endosomolytic activity of PEI can be more efficient than that by either of the properties alone. It appears that, in these formulae, the predominant role of KALA is to facilitate cellular entry of DNA by providing a fusogenic capability, rather than an endosomolytic activity.     

Plasmid DNA size does not affect the physicochemical properties of lipoplexes but modulates gene transfer efficiency.

Clinical applications of gene therapy mainly depend on the development of efficient gene transfer vectors. Large DNA molecules can only be transfected into cells by using synthetic vectors such as cationic lipids and polymers. The present investigation was therefore designed to explore the physicochemical properties of cationic lipid-DNA particles, with plasmids ranging from 900 to 52 500 bp. The colloidal stability of the lipoplexes formed by complexing lipopolyamine micelles with plasmid DNA of various lengths, depending on the charge ratio, resulted in the formation of three domains, respectively corresponding to negatively, neutrally and positively charged lipoplexes. Lipoplex morphology and structure were determined by the physicochemical characteristics of the DNA and of the cationic lipid. Thus, the lamellar spacing of the structure was determined by the cationic lipid and its spherical morphology by the DNA. The main result of this study was that the morphological and structural features of the lipopolyamine-DNA complexes did not depend on plasmid DNA length. On the other hand, their gene transfer capacity was affected by the size of plasmid DNA molecules which were sandwiched between the lipid bilayers. The most effective lipopolyamine-DNA complexes for gene transfer were those containing the shortest plasmid DNA.     

DNA internalized via caveolae requires microtubule-dependent, Rab7-independent transport to the late endocytic pathway for delivery to the nucleus

Using cationic liposomes to mediate gene delivery by transfection has the advantages of improved safety and simplicity of use over viral gene therapy. Understanding the mechanism by which cationic liposome:DNA complexes are internalized and delivered to the nucleus should help identify which transport steps might be manipulated in order to improve transfection efficiencies. We therefore examined the endocytosis and trafficking of two cationic liposomes, DMRIE-C and Lipofectamine LTX, in CHO cells. We found that DMRIE-C-transfected DNA is internalized via caveolae, while LTX-transfected DNA is internalized by clathrin-mediated endocytosis, with both pathways converging at the late endosome or lysosome. Inhibition of microtubule-dependent transport with nocodazole revealed that DMRIE-C:DNA complexes cannot enter the cytosol directly from caveosomes. Lysosomal degradation of transfected DNA has been proposed to be a major reason for poor transfection efficiency. However, in our system dominant negatives of both Rab7 and its effector RILP inhibited late endosome to lysosome transport of DNA complexes and LDL, but did not affect DNA delivery to the nucleus. This suggests that DNA is able to escape from late endosomes without traversing lysosomes and that caveosome to late endosome transport does not require Rab7 function. Lysosomal inhibition with chloroquine likewise had no effect on transfection product titers. These data suggest that DMRIE-C and LTX transfection complexes are endocytosed by separate pathways that converge at the late endosome or lysosome, but that blocking lysosomal traffic does not improve transfection product yields, identifying late endosome/lysosome to nuclear delivery as a step for future study.     

Plasmid DNA activates murine macrophages to induce inflammatory cytokines in a CpG motif-independent manner by complex formation with cationic liposomes

Plasmid DNA (pDNA) is very important in non-viral gene therapy and DNA vaccination. Unmethylated CpG motifs in bacterial DNA, but not in vertebrate DNA, are known to trigger an inflammatory response, which inhibits gene expression while improving immunological consequences. In this report, we investigated the cytokine secretion induced by pDNA/cationic liposome complexes using murine macrophages. Naked CpG DNA induced tumor necrosis factor-alpha (TNF-alpha) secretion from the macrophages, but DNA without CpG motif did not, demonstrating that the cytokine induction was mediated by CpG motifs. pDNA complexed with cationic liposomes, but not the cationic liposomes alone, produced a significant amount of TNF-alpha from the macrophages. Surprisingly, methylated pDNA and calf thymus DNA complexed with the cationic liposomes were also able to induce TNF-alpha production, indicating that these responses were not dependent on CpG motifs. Taken together, the present study demonstrated that for the first time DNA can stimulate murine macrophages in a CpG motif-independent manner when it is complexed with the cationic liposomes.     

Cationic lipids and cationic ligands induce DNA helix denaturation: detection of single stranded regions by KMnO4 probing

Cationic lipids and cationic polymers are widely used in gene delivery. Using 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) as a cationic lipid, we have investigated the stability of the DNA in DOTAP:DNA complexes by probing with potassium permanganate (KMnO4). Interestingly, thymidines followed by a purine showed higher susceptibility to cationic ligand-mediated melting. Similar studies performed with other water-soluble cationic ligands such as polylysine, protamine sulfate and polyethyleneimine also demonstrated melting of the DNA but with variations. Small cations such as spermine and spermidine and a cationic detergent, cetyl trimethylammonium bromide, also rendered the DNA susceptible to modification by KMnO4. The data presented here provide direct proof for melting of DNA upon interaction with cationic lipids. Structural changes subsequent to binding of cationic lipids/ligands to DNA may lead to instability and formation of DNA bubbles in double-stranded DNA.     

Multilamellar structures of DNA complexes with cationic liposomes.

Studies of DNA complexes with cationic liposomes are prompted by the search for nonviral DNA carriers for gene therapy. Recent experiments have identified a stable multilamellar phase in which ordered smectic layers of DNA alternate with cationic bilayers. In this paper we identify the forces governing DNA adsorption on cationic lamellae, including a membrane-induced attraction between the adsorbed DNA. Calculating the DNA interhelical spacing as a function of system composition, the model successfully explains recent surprising observations.     

Evaluation of cationic assemblies constructed with amino acid based lipids for plasmid DNA delivery

We synthesized cationic lipids bearing lysine, histidine, or arginine as a cationic headgroup for use in gene transfer studies. The cationic assemblies formed from lysine- or arginine-type lipids gave unilamellar vesicles (approximately 100 nm diameter), whereas the morphology of the histidine-type lipids was tube-like. The competences of the cationic assemblies were sufficient to form lipoplexes, and the resulting lipoplexes were evaluated in terms of gene expression efficiencies with COS-7 cells. The lysine- or arginine-type lipids exhibited higher gene expression efficiencies than that of Lipofectamine2000, a conventional transgenic reagent, indicating that stable lipoplexes could be prepared between spherical cationic assemblies and plasmid DNA. The gene expression efficiency in relation to the cationic headgroup of the lipids was as follows: lysine > or = arginine > histidine. In addition, gene expression efficiency was enhanced by decreasing the length of the alkyl chain of the hydrophobic moiety. Unlike Lipofectamine2000, no reduction in transfection efficiency in the presence of fetal bovine serum was observed for the lipoplexes formed using synthetic cationic lipids. Moreover, the synthetic cationic lipids revealed remarkably low cytotoxicity compared with Lipofectamine2000. In conclusion, cationic assemblies formed from 1,5-ditetradecyl-N-lysyl-L-glutamate or 1,5-ditetradecyl-N-arginyl-L-glutamate can be used as an effective plasmid DNA delivery system.     

On the Mechanism of Cationic Amphiphile-mediated Transfection. To Fuse or not to Fuse: Is that the Question?

Due to charge interaction, cationic lipids spontaneously associate with nucleic acids, resulting in the formation of so-called lipoplexes. Lipoplexes are membranous structures that are capable of transducing genes into cells, eventually leading to expression of the genes (transfection). The mechanism involved in the cellular uptake of lipoplexes is most likely endocytosis, which occurs after nonspecific charge-mediated binding to cellular receptors. An important step in the transfection process following the actual internalization of lipoplexes is the release of the lipoplex and/or its DNA into the cytoplasm in order to evade lysosomal degradation. Here, the membranous nature of the lipoplex seems to be crucial in that it allows the exchange of lipids between the endosomal membrane and the lipoplex, which results in membrane perturbations that are a prerequisite in the endosomal escape of DNA. Interestingly, a hexagonal phase of the lipoplexes has been correlated with efficient transfection and it can be envisaged that such a phase could be instrumental in the creation of membrane perturbations. Subsequent to its release into the cytoplasm, the DNA has to be transferred into the nucleus. The nuclear import of DNA is most likely a protein-mediated process. In addition, the nuclear uptake of DNA may be facilitated at the time of nuclear envelope disassembly during mitosis. Currently, cationic liposomes are widely used as gene carrier system to deliver nucleic acids into cells in culture to study the cell-biological functioning of genes plus accompanying proteins. Ultimately, cationic lipids may be used in gene therapeutic protocols.