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。     

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.     

阳离子脂质体转染效率的主要影响因素

基因治疗的关键问题是选择合适的载体将外源基因有效保护,并完成穿膜入胞-入核-表达这一完整传递过程。其中,传递载体的转染效率高低对基因治疗有重要意义。本文对影响阳离子脂质体这一重要非病毒基因表达载体转染效率的多种因素进行了综述。     

阳离子脂质体介导肽核酸转染K562细胞的研究

目的 优化阳离子脂质体介导肽核酸(PNA)转染K562细胞的条件.方法 以阳离子脂质体lipofectamine 2000为载体,将标记有FITC荧光基团的PNA转染K562细胞,在荧光显微镜下观察并计算其转染效率,采用Cell Counting Kit(CCK-8)检测其细胞毒性.应用正交试验筛选出最佳的PNA和脂质体的用量及比例,并优化稀释用培养基血清浓度,以获得最优的转染效果.结果 以2.5× 105/mL～3×105/mL的细胞密度接种,100 μL/孔的培养基中加入PNA 6.25 pmol,PNA与脂质体的体积比为1∶3.5,稀释用培养基未加胎牛血清时,细胞转染效率和细胞毒性最佳,转染效率为87.2％,细胞存活率(RGR)为94.1％.结论 PNA可通过阳离子脂质体转染进入K562细胞,优化条件后得到的细胞转染效率和细胞存活率能够满足基因表达研究的实验要求.     

腺病毒及紫杉醇阴离子脂质体共传递复合物的制备及体外细胞转染

目的 制备腺病毒及紫杉醇阴离子脂质体(AL-Ad5-PTX)共传递复合物,考察该复合物物理性质及体外细胞转染效率.方法 先后用薄膜超声法和钙离子融合法制得AL-Ad5-PTX;测定AL-Ad5-PTX中紫杉醇的含量;测定粒径及电位;用透射电镜观察其形态;以表达荧光素酶的重组腺病毒作为病毒载体,比较AL-Ad5-PTX和裸腺病毒(naked Ad5)在富含柯萨奇腺病毒受体(CAR)或缺乏CAR受体细胞中的转染效率.结果 AL-AdS-PTX中紫杉醇包封率为82.70％±2.47％,粒径为241.6 ±2.6 nm,粒径分布为0.196±0.006,电位为-50.4±5.4 mV.AL-Ad5-PTX在两种细胞中的转染效率高于naked Ad5(P＜0.05).结论 成功制备了能够同时转运腺病毒及紫杉醇的复合物AL-Ad5-PTX,能够提高naked Ad5在细胞中的转染效率;HPLC法检测AL-Ad5-PTX中紫杉醇含量,操作简便、准确、可靠.     

脂质体转染技术进展及其应用

细胞转染是将目的基因导入细胞中,是研究基因功能和基因治疗的重要技术之一。细胞转染的成功的关键在于设计一种安全、高效的载体,能够将目的基因高效、特异地导入细胞中,而且要使其对细胞的毒性减到最低程度。与使用病毒性载体相比,使用非感染载体－脂质复合体日益受到人们的关注。     

阳性脂质体介导基因转染及其研究进展*

基因治疗是一种很有前景的治疗模式,而阳性脂质体介导的基因转染是目前基因治疗的研究热点之一。现综述近5年来有关阳性脂质体的文献,介绍了阳性脂质体的基本组成,并从生物学、理化性质及制剂学等几个方面介绍了影响阳性脂质体/DNA复合物转染效率的主要因素,最后从新的阳性脂质成分及阳性脂质体或阳性脂质体/DNA复合物的表面修饰等方面介绍了近年来有关改善阳性脂质体介导基因转染的研究进展。     

三种阳离子转染试剂对小鼠血管瘤内皮细胞转染效率及细胞毒性的影响

目的 从三种常用的阳离子转染试剂中筛选出对小鼠血管瘤内皮细胞(EOMA)有较高转染效率和较低细胞毒性的转染试剂.方法 以含有增强型绿色荧光蛋白(EGFP)报告基因的真核表达载体pEGFP-N1为报告基因,以阳离子脂质体LipofectamineTM2000、LipofectamineTM PLUS和阳离子聚合物JetPEITM为转染试剂,按照转染试剂盒的说明优化其转染条件,分别转染EOMA细胞,24 h后在荧光显微镜下计数阳性细胞率、MTT法检测各转染条件下对EOMA的细胞毒性.结果 经优化转染条件,LipofectamineTM PLUS和JetPEITM转染的细胞中阳性细胞的最高比例分别为45%和47%,LipofectamineTM2000转染的细胞中荧光蛋白表达的比例最高(＞80%),Lipofecta-mineTM2000试剂在最高转染效率的剂量下仍然保证了80%的细胞存活率.结论 阳离子脂质体LipofectamineTM2000对小鼠EOMA细胞有较高的转染效率和较低的细胞毒作用.     

载基因阳离子脂质体的制备与包封率测定

目的:制备包封基因质粒的阳离子脂质体并考察其性质、测定包封率。方法:以DC-Chol和DOPE为材料,薄膜分散法制备阳离子脂质体,与可表达增强型绿色荧光蛋白的基因质粒结合并考察其转染性能,激光粒度分析仪测定阳离子脂质体和脂质复合物的粒径及Zeta电位;使用葡聚糖凝胶过滤法测定包封率,并对该法进行详细考察。结果:所制备的阳离子脂质体和脂质复合物的平均粒径分别为161.6和216.3 nm,Zeta电位分别为+22.2和+3.2 mV;基因质粒在0.1925～3.85μg.mL-1浓度范围内线性良好,精密度高,与葡聚糖无吸附作用,柱回收率高,测得脂质复合物的包封率为89.94%。结论:采用该处方和工艺可成功制备质量良好、能有效转染细胞的阳离子脂质体载体,葡聚糖凝胶过滤法可准确测定其包封率,该法快速、简便、有效。     

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。     

Development of magnetic anionic liposome/atelocollagen complexes for efficient magnetic drug targeting

Magnetic nanoparticle-incorporated liposomes (magnetic liposomes) are considered a promising site-specific drug delivery carrier vehicle. With regard to their surface charge, magnetic anionic liposomes (Mag-AL) demonstrate little toxicity in comparison with magnetic cationic liposomes (Mag-CL), whereas their cellular association and uptake efficiency are low. In the current study, we constructed complexes of Mag-AL and atelocollagen (ATCOL), which is a biocompatible and minimally immunogenic biomaterial, to improve the cellular uptake properties of Mag-AL in vitro and in vivo. The cellular association and/or uptake of Mag-AL in RAW264 cells, a murine macrophage-like cell line, under a magnetic field was significantly increased when Mag-AL was complexed with ATCOL, and the highest cellular association was observed with complexes constructed using 5?µg/mL of ATCOL. The complexes showed liposome concentration-dependent and time-dependent cellular association under a magnetic field, and their cellular uptake efficiency was comparable with that of Mag-CL. In addition, Mag-CL showed significant cytotoxicity in a liposome concentration-dependent manner, whereas Mag-AL/ATCOL complexes produced no cytotoxic effect against RAW264 cells. Furthermore, the efficient cellular association of Mag-AL/ATCOL complexes in RAW264 cells was observed even in the presence of serum, and their liver accumulation was significantly increased at a magnetic field-exposed region after intravenous injection in rats. These results indicate that Mag-AL/ATCOL complexes could be a safe and efficient magnetic responsive drug carrier.     

Biotin-directed assembly of targeted modular lipoplexes and their transfection of human hepatoma cells in vitro

The asialoglycoprotein receptor, which is abundantly and near exclusively expressed on hepatocytes, has received much attention in the design of non-viral hepatotropic DNA delivery systems. Thus, asialoglycoproteins and hexopyranosyl ligands have been coupled to DNA-binding cationic polymers and liposomes in the assembly of complexes intended for uptake by liver parenchymal cells. The aim of the study was to construct a hepatocyte-targeted multimodular liposome-based transfecting complex, in which the biotin-streptavidin interaction provides the cohesive force between the ligand asialorosomucoid and the liposome bilayer, and to evaluate its transfection capabilities in the hepatocyte-derived human transformed cell line HepG2. Dibiotinylated asialoorosomucoid was attached to cationic liposomes constructed from 3β[N-(N’,N’-dimethylaminopropane)-carbamoyl] cholesterol (Chol-T):dioleoylphosphatidylethanolamine:biotinylcholesterylformylhydrazide (MSB1) (48:50:2 mole ratio) through streptavidin interposition. Liposome-pGL3 DNA interactions were studied by gel band shift and ethidium displacement assays. The cytotoxicity of assemblies was evaluated in the HepG2 cell line and transfection capabilities determined by measuring the activity of the transgene luciferase. Binding assays showed that all DNA was liposome associated at a DNA (negative):liposome (positive) charge ratio of 1:1. Accommodation of a streptavidin dibiotinylated asialoorosomucoid assembly was achieved at a DNA:liposome:streptavidin dibiotinylated asialoorosomucoid ratio of 1:4:9 (weight basis). Complexes showed optimal transfection activity at this ratio, which was reduced 10-fold by the presence of the competing ligand asialofetuin. The streptavidin-biotin interaction has been applied for the first time to the assembly of hepatocyte-targeted lipoplexes that display asialoorosomucoid and that are well tolerated by a human hepatoma cell line in which transfection is demonstrably achieved by receptor mediation. Favorable size and charge ratio characteristics suggest that this system may be suitable for in vivo application.     

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。     

兰索拉唑脂质体包封率的测定

目的:建立兰索拉唑脂质体包封率的测定方法。方法:利用透析法分离脂质体和游离药物,采用紫外分光光度法测定兰索拉唑脂质体的包封率,在284 nm 处测定兰索拉唑脂质体中的含药量。结果:透析平衡时间为10 h,兰索拉唑浓度在2.1～10.5μg·mL~(-1)范围内线性关系良好(r=0.9998);该方法的平均游离药物回收率(98.5%～99.9%)符合要求;高、中、低3种浓度的日内和日间精密度试验的 RSD 均小于1%;透析24 h 内无渗漏,重现性好。结论:本法便捷、准确,适用于脂溶性药物兰索拉唑脂质体包封率的测定。     

脂质体中药物包封率的测定方法

本文综述了各种脂质体包封率的测定方法,包括需要在分析前进行游离药物与脂质体分离的各种常规包封率测定方法和不需要进行分离的新型包封率测定方法,并对各种方法的适用范围、优点和局限性进行了归纳总结并进行了举例说明。     

盐酸莫西沙星脂质体的制备及包封率测定方法研究

目的：制备盐酸莫西沙星脂质体,建立盐酸莫西沙星脂质体包封率测定方法。方法：乙醇注入法制备盐酸莫西沙星脂质体,正交试验优选脂质体的最佳处方。采用葡聚糖凝胶柱法、超滤离心法分离脂质体与游离药物,并进行方法学考察,优选出测定盐酸莫西沙星脂质体包封率的方法。结果：盐酸莫西沙星脂质体的最佳处方为：卵磷脂与胆固醇比为 3：1,药脂比为 1：7,水合介质中聚山梨酯20的用量为1%,优化后的包封率为90.73%。葡聚糖凝胶柱法和超滤离心法都能将脂质体与游离药物分离,葡聚糖凝胶柱法的平均柱加样回收率为85.54%~88.15%,超滤离心法的平均加样回收率为94.40%~97.35%。结论：盐酸莫西沙星脂质体的制备工艺稳定,包封率较高。葡聚糖凝胶柱法不适于盐酸莫西沙星脂质体的包封率测定,超滤离心法可高效、准确、方便地测定盐酸莫西沙星脂质体包封率。