胆固醇嵌入式阳离子脂质体运载基因研究

阳离子脂质体是一种有临床应用潜力的抗肿瘤药物递药系统,助类脂能起到稳定双层膜和降低阳性成分毒性的作用,同时提供阳性类脂的细胞渗透功能。为了进一步发掘助类脂的应用潜力,该文采用胆固醇(cholesterol)作为助类脂制备阳离子脂质体,测定了脂质体的粒径及Zeta电位,脂质体的平均粒径为100~140 nm,Zeta电位为45~60 mV。脂质体分别与绿色荧光蛋白基因(pGFP-N2)、荧光素酶基因(pGL3)结合,形成脂质体/DNA复合物,通过载入人喉癌细胞(Hep-2),考察了其转染效率和细胞毒性。结果表明,阳离子类脂与胆固醇以1:1、1:2和1:4摩尔比例混合制备脂质体均能高效转染Hep-2细胞。毒性实验显示,阳离子类脂单独存在时对癌细胞具有一定的细胞毒性,随着胆固醇的加入,脂质体对细胞的毒性明显减小,与商品试剂DOTAP和Lipofectamine 2000相当。     

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。     

脂质体介导法转染肿瘤细胞效率的优化

目的:研究优化影响脂质体转染效率的因素,以提高脂质体转染效率,为相关研究和应用提供参考.方法:以绿色荧光蛋白(GFP)作为报告基因,采用脂质体Lipofectamine 2000包裹pU6H1-GFP-FAK重组质粒转染Caco-2细胞,研究了细胞接种密度、DNA用量、脂质体与DNA的比例、脂质体-DNA复合物的形成时间、细胞与脂质体复合物的孵育时间、血清的有无及细胞的传代次数等因素对脂质体转染效率的影响.结果:2-5次细胞传代,2×105接种密度、4μg DNA用量、2.5:1的脂质体与DNA比例、30min脂质体-DNA复合物形成时间以及6h细胞与复合物孵育时间,转染效率最高.血清在本实验室条件下并不影响转染效率.结论:实验获得的优化条件可以明显提高脂质体对肿瘤细胞的转染效率,可作为有关研究或应用的参考.     

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。     

一种线性化聚乙烯亚胺介导的EGFR靶向siRNA转运系统在人肺腺癌细胞中的优化与评价研究

RNA干扰技术在靶基因验证和人类肿瘤治疗研究方面都已显示出良好的应用前景。目前小干扰RNA（siRNA）体内应用较为理想的转运工具是非病毒载体,其中聚乙烯亚胺（PEI）以其高效性和通用性而最具代表性。本研究以一种线性化PEI为载体构建靶向表皮生长因子受体的siRNA转运系统,采用琼脂糖凝胶电泳阻滞实验,和以Lipofectamine2000为对照的SPC-A1腺癌细胞转染实验,对决定LPEI／siRNA-EGFR复合物转染效率的N／P比条件进行优化,用RealtimeRT-PCR和流式细胞技术检测各组细胞的EGFR表达,最后以动态光散射技术和原子力显微镜对最优化条件下形成的LPEI／siRNA-EGFR复合物进行基本物理特性观察。结果显示,N／P≥5时siRNA可被LPEI充分聚合浓缩,且N／P=5时形成的LPEI／siRNA-EGFR复合物可引发最高效且特异的EGFR表达抑制,其效率与Lipofectamine2000转染结果相当。该最优化N／P比条件下所形成的复合物是一种大小较为均一的椭圆形颗粒,其粒径小于100nm,表面带有较低的正电荷,该特性可有利于细胞摄取和内吞。以上研究表明,这种LPEI介导的以EGFR为靶向的siRNA转运系统极可能成为人肺腺癌基因治疗的新策略,对此仍需进一步的体内验证。     

脂质体介导的人甲状腺过氧化物酶基因转染肺癌A549细胞及其蛋白表达

目的:利用基因转染技术,研究以脂质体Lipofectamine2000为载体介导的人甲状腺过氧化物酶(TPO)基因体外转染肺癌细胞,检测感染细胞内TPO蛋白的表达,为放射性碘治疗肺癌提供理论和实验依据.方法:将获得的含TPO基因的质粒pcDAN3.1-hTPO进行扩增、纯化,并经酶切鉴定和DNA测序.将肺癌A549细胞在体外复苏与培养并分为两组:转染质粒peDAN3.1-hTPO的为实验组,转染空质粒pcDAN3.1的为对照组.以脂质体Lipofectamine2000为栽体,介导TPO基因转染肺癌细胞.采用Western Blot免疫印迹法和免疫组化法分别检测肺癌细胞中TPO蛋白的表达.结果:①酶切鉴定和DNA测序结果表明质粒pcDAN3.1-hTPO中插入的基因为hTPO基因,其片段大小和方向正确.②体外培养的肺癌细胞活力及数量正常,细胞活力为96%,细胞生长密度为1× 106/ml,满足实验要求.③质粒转染A549细胞后,Western Blot免疫印迹法显示:在实验组中,肺癌A549细胞有TPO蛋白的表达,而在对照组中无表达.④免疫组化染色结果显示:在实验组的肺癌A549细胞中,TPO蛋白表达阳性且主要分布于细胞膜上,阳性表达率可达75%,而在对照组中TPO蛋白表达阴性,两组比较差异有显著性(P=0.000).结论:①获得的hTPO基因片段的核苷酸序列与GeneBank报道完全一致.②在脂质体Lipofectamine2000的介导下,TPO基因能够有效地转染肺癌细胞.③转染人甲状腺过氧化物酶基因的肺癌细胞能够在体外成功地表达TPO蛋白.     

猪肾细胞脂质磁转染系统的构建与表征

磁性纳米基因载体是一种非病毒基因载体,经过功能性基团修饰后能够连接阳离子转染剂构建细胞转染系统。本文将磁转染技术结合常用的脂质体转染,形成了一种新型动物体细胞转染方法,即称脂质磁转染(Liposomal magnetofection,LMF)。这将为体细胞克隆培育转基因动物提供稳定遗传的细胞系。为构建脂质磁性纳米基因载体复合物系统,本研究利用一种磁性纳米基因载体通过分子自组装与脂质阳离子转染剂结合,用于携带外源基因转染动物体细胞。通过原子力显微镜(AFM)观测、ζ电位-粒度等分析表征手段,研究磁性纳米基因载体的形貌、粒径分布、负载及浓缩DNA的方式。结果表明,通过猪肾(PK)细胞的LMF实验,与脂质体(Lipofectamine2000)介导的转染比较,具有较高的转染率,更重要的是克服了脂质体转染瞬时表达的缺陷。MTT细胞毒性试验结果也显示该方法具有较低的细胞毒性。因此LMF是一种切实可行的高效低毒性的细胞转染方法。     

腺病毒载体介导的H9C2心肌细胞外源基因表达系统的构建

本研究旨在探讨不同剂量的脂质体与腺病毒载体对转染H9C2细胞的效率的影响。应用常规剂量脂质体与减半剂量脂质体介导梯度剂量腺病毒载体转染H9C2细胞,并通过观察报告基因GFP表达计算各转染条件下的转染效率。结果发现应用常规剂量减半的脂质体反而能获得更高的转染效率;腺病毒载体与转染效率具有明显的量效关系,但是当载体量过大时转染效率有所下降。因此脂质体与腺病毒载体都具有一定的细胞毒性,细胞处于良好的状态可能更有利于外源基因的转入。     

磁性纳米颗粒作为基因转染载体的研究

通过扫描电子显微镜和Zeta电位仪对磁性纳米颗粒的形貌、粒径、表面电位等进行了表征。利用凝胶电泳阻滞试验分析磁性纳米颗粒与DNA的结合情况,研究磁性纳米颗粒对DNA的保护效果,运用MTT和流式细胞术分析磁性纳米颗粒对细胞的毒性。以绿色荧光蛋白基因为报告基因进行293T细胞的转染,研究磁性纳米颗粒与质粒DNA不同比例条件下对293T细胞的转染效率,并与脂质体(Lipofectamine2000)介导的转染进行比较分析。结果表明,磁性纳米颗粒与DNA可以稳定结合,可以保护DNA免受酶的消化作用,当磁性纳米颗粒与DNA比为1 1时,转染效率最高,优于脂质体(Lipotamine2000)介导的转染,且对细胞的毒害作用小于Lipotamine2000。     

新型“脂质HAPDNA复合体”的制备和用于真核细胞基因转染研究

为寻找一种简单、经济、有效的DNA递送系统用于基因转染和基因治疗,制备了表面电荷为正电的纳米HAP,与表面电荷为负电的DNA结合形成DNA-HAP复合物,采用逆向蒸发法,用卵磷脂、DOPE和胆固醇制备成脂质体包封DNA-HAP复合物形成脂质HAP-DNA复合体, 脂质体和HAP对照,对所形成的脂质HAP-DNA复合体（LHD）的特性、包封率、转染Hela细胞的效果进行初步检测研究。所获得的脂质HAP-DNA复合体呈球形、平均粒径为643nm;平均包封率达11.67%,为中性脂质体;能有效转染真核细胞。该方法可作为提高基因转染效果的简单、经济、有效的手段之一,也为进一步提高非病毒载体的转染效率提供了一个思路。     

Chitosan enhanced gene delivery of cationic liposome via non-covalent conjugation

Two new types of stable ternary complexes were formed by mixing chitosan with DOTAP/pDNA lipoplex and DOTAP with chitosan/pDNA polyplex via non-covalent conjugation for the efficient delivery of plasmid DNA. They were characterized by atomic force microscopy, gel retarding, and dynamic light scattering. The DOTAP/CTS/pDNA complexes were in compacted spheroids and irregular lump of larger aggregates in structure, while the short rod- and toroid-like and donut shapes were found in CTS/DOTAP/pDNA complexes. The transfection efficiency of the lipopolyplexes showed higher GFP gene expression than DOTAP/pDNA and CTS/pDNA controls in Hep-2 and Hela cells, and luciferase gene expression 2–3-fold than DOTAP/pDNA control and 70–120-fold than CTS/pDNA control in Hep-2 cells. The intracellular trafficking was examined by confocal laser scanning microscopy. Rapid pDNA delivery to the nucleus enchanced by chitosan was achieved after 4 h transfection.     

Synthesis and biological activity of carbamate-linked cationic lipids for gene delivery in vitro

We have introduced a convenient synthesis method for carbamate-linked cationic lipids. Two cationic lipids N-[1-(2,3-didodecylcarbamoyloxy)propyl]-N,N,N-trimethylammonium iodide (DDCTMA) and N-[1-(2,3-didodecyl carbamoyloxy)propyl]-N-ethyl-N,N-dimethylammonium iodide (DDCEDMA), with identical length of hydrocarbon chains, alternative quaternary ammonium heads, carbamate linkages between hydrocarbon chains and quaternary ammonium heads, were synthesized for liposome-mediated gene delivery. Liposomes composed of DDCEDMA and DOPE in 1:1 ratio exhibited a lower zeta potential as compared to those made of pure DDCEDMA alone, which influences their DNA-binding ability. pGFP-N2 plasmid was transferred by cationic liposomes formed from the above cationic lipids into Hela and Hep-2 cells, and the transfection efficiency of some of cationic liposomes was superior or parallel to that of two commercial transfection agents, Lipofectamine2000 and DOTAP. Combined with the results of the agarose gel electrophoresis and transfection experiment, the DNA-binding ability of cationic lipids was too strong to release DNA from complex in the transfection, which could lead to relative low transfection efficiency and high cytotoxicity.     

Transmembrane routes of cationic liposome-mediated gene delivery using human throat epidermis cancer cells

For studying the mechanism of cationic liposome-mediated transmembrane routes for gene delivery, various inhibitors of endocytosis were used to treat human throat epidermis cancer cells, Hep-2, before transfection with Lipofectamine 2000/pGFP-N2 or Lipofectamine 2000/pGL3. To eliminate the effect of inhibitor toxicity on transfection, the RLU/survival rate was used to represent the transfection efficiency. Chlorpromazine and wortmannin, clathrin inhibitors, decreased transfection efficiency by 44 % (100 μM) and 31 % (100 nM), respectively. At the same time, genistein, a caveolin inhibitor, decreased it by 30 % (200 μM). Thus combined transmembrane routes through the clathrin and caveolae-mediated pathways were major mechanisms of cell uptake for the cationic liposome-mediated gene delivery. After entering the cells, microtubules played an important role on gene delivery as vinblastine, a microtubulin inhibitor, could reduce transfection efficiency by 41 % (200 nM).     

Transient non-viral cutaneous gene delivery in burn wounds

BACKGROUND: Gene transfer to burn wounds could present an alternative to conventional and often insufficient topical and systemic application of therapeutic agents to aid in wound healing. The goals of this study were to assess and optimize the potential of transient non-viral gene delivery to burn wounds. METHODS: HaCaT cells were transfected with luciferase or beta-galactosidase transgene using either pure plasmid DNA (pDNA) or complexed with Lipofectamine 2000, FuGENE6, or DOTAP-Chol. Expression was determined by bioluminescence and fluorescence. Forty male Sprague-Dawley rats received naked pDNA, lipoplexes, or carrier control intradermally into either unburned skin, superficial, partial, or full-thickness scald burn. Animals were sacrificed after 24 h, 48 h, or 7 days, and transgene expression was assessed. RESULTS: Gene transfer to HaCaT cells showed the overall highest expression for DOTAP/Chol (77.85 ng luciferase/mg protein), followed by Lipofectamine 2000 (33.14 ng luciferase/mg protein). pDNA-derived gene transfer to superficial burn wounds showed the highest expression among burn groups (0.77 ng luciferase/mg protein). However, lipoplex-derived gene transfer to superficial burns and unburned skin failed to show higher expression. CONCLUSIONS: Lipofectamine 2000 and DOTAP/Chol lipoplex showed significantly enhanced gene transfer, whereas no transfection was detectable for naked DNA in vitro. In contrast to the in vitro study, naked DNA was the only agent with which gene delivery was successful in experimental burn wounds. These findings highlight the limited predictability of in vitro analysis for gene delivery as a therapeutic approach.     

Synthesis and optimization of cholesterol-based diquaternary ammonium Gemini Surfactant (Chol-GS) as a new gene delivery vector

Amongst a number of potential nonviral vectors, cationic liposomes have been actively researched, with both gemini surfactants and bola amphiphiles reported as being in possession of good structures in terms of cell viability and in vitro transfection. In this study, a cholesterol-based diquaternary ammonium gemini surfactant (Chol-GS) was synthesized and assessed as a novel nonviral gene vector. Chol-GS was synthesized from cholesterol by way of four reaction steps. The optimal efficiency was found to be at a weight ratio of 1:4 of lipid:DOPE (1,2-dioleoyl-L-alpha- glycero-3-phosphatidylethanolamine), and at a ratio of between 10:1~15:1 of liposome:DNA. The transfection efficiency was compared with commercial liposomes and with Lipofectamine, 1,2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide (DMRIE-C), and N-[1-(2,3-dioleoyloxy)propyl]- N,N,N-trimethylammonium chloride (DOTAP). The results indicate that the efficiency of Chol-GS is greater than that of all the tested commercial liposomes in COS7 and Huh7 cells, and higher than DOTAP and Lipofectamine in A549 cells. Confirmation of these findings was observed through the use of green fluorescent protein expression. Chol-GS exhibited a moderate level of cytotoxicity, at optimum concentrations for efficient transfection, indicating cell viability. Hence, the newly synthesized Chol-GS liposome has the potential of being an excellent nonviral vector for gene delivery.     

Proteolipidic vectors for gene transfer to the lung

In order to develop improved synthetic gene transfer vectors, we have synthesized bifunctional peptides composed of a DNA binding peptide (P2) and ligand peptides selected by the phage display technique on tracheal epithelial cells. We have evaluated the capacity of these peptides to enhance the gene transfer efficiency of the cationic lipid DOTAP to the mouse lung. To optimize the in vivo transfection efficiency, we first compared the efficiency of DOTAP to transfect the lung by either intravenous injection or aerosolization. We then tested DNA/Peptide/DOTAP complexes formed at different Peptide/DNA and DOTAP/DNA charge ratios. Under optimal conditions, precompaction of DNA by peptide P2 gave a higher expression in the mouse lung using the luciferase reporter gene than DOTAP/DNA complexes. A further increase of transfection efficiency was obtained with the bifunctional peptide P2-9. Experiments performed with the GFP reporter gene showed expression in the alveolar parenchyme.     

Novel carbamate-linked quaternary ammonium lipids containing unsaturated hydrophobic chains for gene delivery

In this paper, two novel carbamate-linked quaternary ammonium lipids (MU18: a lipid with a mono-ammonium head; GU18: a lipid with a Gemini-ammonium head) containing unsaturated hydrophobic chains were designed and synthesized. The chemical structures of the synthetic lipids were characterized by infrared spectrum, ESI-MS, ¹H NMR, ¹³C NMR, and HPLC. For investigating the effect of unsaturation on gene delivery, the previous reported saturated cationic liposomes (MS18 and GS18) were used as comparison. Cationic liposomes were prepared by using these cationic lipids and neutral lipid DOPE at the molar ratio of 1:1. Particle sizes and zeta potentials of the cationic liposomes were studied to show that they were suitable for gene transfection. The binding abilities of the cationic liposomes were investigated by gel electrophoresis at various N/P ratios from 0.5/1 to 8/1. The results indicated that the binding ability of GU18 was much better than MU18 and the saturated cationic liposomes (MS18 and GS18). DNA transfection of these liposomes comparable to commercially available reagent (DOTAP) was achieved in vitro against Hela, HepG-2 and NCI-H460 cell lines. GU18 showed higher transfection at the N/P ratio of 3/1 than other cationic liposomes and the positive control, DOTAP. All of the liposomes presented a relatively low cytotoxicity, which was measured by MTT. Therefore, the synthetic lipids bearing unsaturated hydrophobic chains and Gemini-head could be promising candidates for gene delivery.     

Low molecular weight polyethylenimine for efficient transfection of human hematopoietic and umbilical cord blood-derived CD34+ cells

With the emerging role of hematopoietic stem cells as potential gene and cell therapy vehicles, there is an increasing need for safe and effective nonviral gene delivery systems. Here, we report that gene transfer and transfection efficiency in human hematopoietic and cord blood CD34+ cells can be enhanced by the use of low molecular weight polyethylenimine (PEI). PEIs of various molecular weights (800-750,000) were tested, and our results showed that the uptake of plasmid DNA by hematopoietic TF-1 cells depended on the molecular weights and the N/P ratios. Treatment with PEI 2K (m.w. 2000) at an N/P ratio of 80/1 was most effective, increasing the uptake of plasmid DNA in TF-1 cells by 23-fold relative to Lipofectamine 2000. PEI 2K-enhanced transfection was similarly observed in hematopoietic K562, murine Sca-1+, and human cord blood CD34+ cells. Notably, in human CD34+ cells, a model gene transferred with PEI 2K showed 21,043- and 513-fold higher mRNA expression levels relative to the same construct transfected without PEI or with PEI 25 K, respectively. Moreover, PEI 2K-treated TF-1 and human CD34+ cells retained good viability. Collectively, these results indicate that PEI 2K at the optimal N/P ratio might be used to safely enhance gene delivery and transfection of hematopoietic and human CD34+ stem cells.     

Transfection property of a new cholesterol-based cationic lipid containing tri-2-hydroxyethylamine as gene delivery vehicle

A novel cholesterol-based cationic lipid containing a tri-2- hydroxyethylamine head group and ether linker (Chol- THEA) was synthesized and examined as a potent gene delivery vehicle. In the preparation of cationic liposome, the addition of DOPE as helper lipid significantly increased the transfection efficiency. To find the optimum transfection efficiency, we screened various weight ratios of DOPE and liposome/DNA (N/P). The best transfection efficiency was found at the Chol-THEA:DOPE weight ratio of 1:1 and N/P weight ratio of 10~15. Most of the plasmid DNA was retarded by this liposome at the optimum N/P weight ratio of 10. The transfection efficiency of Chol-THEA liposome was compared with DOTAP, Lipofectamine, and DMRIE-C using the luciferase assay and GFP expression. Chol-THEA liposome with low toxicity had better or similar potency of gene delivery compared with commercial liposomes in COS-7, Huh-7, and MCF-7 cells. Therefore, Chol-THEA could be a useful non-viral vector for gene delivery.