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。     

古细菌极性脂质片段的提取和四醚脂质体的制备及稳定性研究

脂质体作为药物口服载体,面临的主要障碍之一是其在肠道胆汁盐和肠道酶的作用下极不稳定,造成包裹药物的损失和破坏。研究中,应用丙酮沉淀、薄层制备层析以及甲醇沉淀相结合的方法,从培养的古细菌Sulfolobus acidocaldarius中分离和纯化极性脂质抽提片段(polarlipid fraction extract,PLFE),制备了由PLFE为组分的四醚脂质体;并着重评估其在模拟肠液中的稳定性。结果显示:改进的提取方法操作简单、有效;在模拟人肠道环境的体外稳定性实验中,四醚脂质体的稳定性明显优于普通磷脂脂质体。这些结论表明,具有独特结构的极性脂质组成的四醚脂质体,具备作为药物的口服输送载体的潜力。     

一种可用于SiRNA转染的可降解的新型脂质聚合物

合成一种兼具阳离子脂质体和阳离子聚合物优点的可用于SiRNA的新型脂质聚合物载体.以低分子量的支链聚乙烯亚胺(BPEI,分子量600D)为基础,通过交联剂将油酸与之交联,形成PEI-交联剂-油酸为单元的高分子脂质聚合物.以稳定表达EGFP基因的HeLa-EGFP细胞为模型,比较脂质聚合物、高分子聚合物BPEI(MW 25KD)及阳离子脂质体Lipofectamine 2000介导的SiRNA转染效率,以MTT法检测转染后细胞毒性.将聚合物放置于中性环境37℃保温,检测在温育不同时间后结合FITC标记的寡核苷酸能力.脂质聚合物介导转染EGFP-SiRNA的细胞中,绿色荧光蛋白的信号下降了72.3%,下降幅度高于Lipofectamine 2000介导转粢的细胞,而BPEI介导的转染细胞中的荧光强度仅比空白对照下降20%左右.MTT结果显示在最适条件下,脂质聚合物的介导的SiRNA转染48h后,细胞存活率为94.87%,高于BPEI 25K和Lipofectamine 2000对照组(80.68%和64.87%).降解实验证实,脂质聚合物和BPEI25K相似,在与FITC标记的寡核苷酸结合后,使其荧光水平下降65%左右;在保温后,脂质聚合物对荧光的抑制能力逐步下降,在8h后其荧光强度与BPEI600对照组相似;BPEI 25K实验组在保温前后荧光强度无显著的变化.脂质聚合物是一种可降解的化合物,具有与Lipofectamine 2000相似的SiRNA转染效率,细胞毒性明显低于BPEI和Lipofectamine 2000.提示脂质聚合物兼具阳离子脂质体和阳离子聚合物的优点.     

阳离子脂质体运载基因的跨膜机制

阳离子脂质体等非病毒载体以其制备简单、低毒性、低免疫原性、可生物降解等优点,成为近年来基因转运中的常用载体。理解阳离子脂质体运载基因的机制对阳离子脂质体的研究具有重要意义。从跨膜机制和信号调控的角度,介绍了脂质体/DNA复合体以特定构象避免细胞外基质中核酸酶的降解,跨越细胞膜进入细胞的过程;阐明了DNA在信号调控的作用下,逃离溶酶体并安全释放的机制;讨论了基因穿过核被膜进入到细胞核的方式,为进一步阐明阳离子脂质体运载基因的分子机制奠定基础。     

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。     

阳离子脂质体诱导的细胞氧化应激损伤

人工合成制备的阳离子脂质体作为一种重要的基因和药物载体,具有很多优点,但其对细胞的毒性作用机制尚不明确,严重影响其临床应用。该文对阳离子脂质体是否通过诱导细胞产生氧化应激反应,进而导致细胞毒性的相关作用机制进行了研究。实验使用人肺癌细胞(NCI-H460)和人胚肺正常细胞(MRC-5),采用CCK-8法、活性氧簇(ROS)荧光标记形态学观察、流式细胞术定量测定及活性氧簇自由基检测等方法,研究对比了三种头部结构不同,连接键和疏水尾部均相同的阳离子脂质体细胞毒性作用机制。这三种脂质体分别由三肽头部的N,N-双十四烷氧酰胺乙基三聚鸟氨酸酰胺(CDO14)、单季铵盐头部的N,N-双十四烷氧酰胺乙基-N,N-二甲基碘化铵(CDA14)和双季铵盐头部的1,2-双-[N-十四烷氧酰胺乙基-N,N-二甲基碘化铵]乙烷(CTA14)制备。研究结果表明,三种阳离子脂质体诱导的超氧化物、过氧化氢(H_2O_2)、羟自由基(?OH)、一氧化氮(NO)和丙二醛(MDA)等ROS均与作用剂量呈正相关,其中CDA14和CTA14高浓度处理组(0.10 μmol/L和0.20 μmol/L)与低浓度处理组(0.05 μmol/L常规转染浓度)及空白对照组相比差异极显著,而CDO14只在0.20 μmol/L处理组有显著差异,且三种脂质体对NCI-H460细胞的氧化应激损伤值均高于MRC-5细胞。因此,肽头部脂质体CDO14产生的氧化应激损伤远小于单季铵盐头部脂质体CDA14,而CDA14又小于双季铵盐头部脂质体CTA14。综上所述,阳离子脂质体诱导的细胞氧化应激损伤与其头部结构和剂量密切相关。该研究为阳离子脂质体的生物安全性及新型阳离子类脂结构设计提供科学参考。     

阳离子脂质体对HepG2.2.15细胞病毒分泌、毒性及凋亡的影响

目的:探讨阳离子脂质体作为基因治疗药物载体对HepG2.2.15细胞的病毒分泌、毒性及凋亡的研究.方法:以2.5、5.0、7.510.0 μL/mL浓度的阳离子脂质体转染HepG2.2.15细胞,共温育10 d,ELISA法检测细胞上清中HBsAg和HBeAg含量的变化.转染48 h后,MTT法检测阳离子脂质体对细胞的毒性、荧光显微镜检测细胞凋亡情况.结果:各个剂量组阳离子脂质体对HepG2.2.15细胞均有抑制作用,其中10 μL/mL组在第10 d对HBsAg、HBeAg抑制率分别达31.5%、29.9%,表现出较高的毒性作用.2.5、5.0、7.5 10.0 μL/mL阳离子脂质体转染HepG2.2.15细胞后,MTT检测结果显示其细胞存活率分别为82.14%、77.62%、77.4%、61.9%.荧光显微镜下可见各浓度组均有凋亡,且随脂质体剂量增加细胞凋亡数增加.结论:阳离子脂质体能抑制HepG2.2.15细胞分泌HBsAg和HBeAg,其毒性及诱导凋亡作用呈剂量和时间依赖性.作为基因治疗药物载体时,剂量宜小于7.5 μL/mL.     

胆固醇衍生物脂质体的物理稳定性和细胞相容性研究

目的：研究胆固醇衍生物（CTBBA）脂质体的物理稳定性,细胞相容性以及药物输送。方法：CTBBA组入脂质体并测定不同pH下的zeta电位。对长期保存的脂质体进行粒径分析和含磷量分析,评价脂质体的物理稳定性。通过测定包封在脂质体内的阿霉素的体外释放,来评价CTBBA对脂质体释放药物的影响。用MTT法检测CTBBA本身以及CTBBA脂质体的细胞相容性。评估了用流式细胞仪和荧光显微镜检测脂质体细胞内药物输送能力的可行性。结果：zeta电位数据表明CTBBA脂质体带有正电荷,可以改善脂质体在长期保存过程中的物理稳定性;作为胆固醇衍生物的CTBBA能有效的降低药物的渗漏;相比某些正电荷载体,CTBBA的细胞毒性较低;流式细胞仪在反映阿霉素脂质体的细胞定位上有局限,固定液的使用会改变阿霉素荧光的细胞内分布。结论：正电荷CTBBA脂质体具有改善脂质体长期保存稳定性,且细胞毒性低的特点。流式细胞仪不能完全反映载药的CTBBA脂质体在细胞内的分布。     

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。     

脂质体过氧化对DNA的损伤研究

研究了以脂质体为材料的脂质过氧化引起的ＥＮＡ损伤,同时检测了脂质过氧化程度与ＤＮＡ受损情况。结果表明：在脂质体过氧化程度中,ＤＮＡ的增色效应,对核酸酶的敏感程度,ＤＮＡ双链百分含量和ＤＮＡ－溴乙锭复合物的荧光强度随着氧化时间的增加而降低。在四种碱基中,鸟嘌呤损伤最严重。多种自由基清除列实验表明：脂质过氧化所产生的羟基自由基和单线态氧可能是引起ＤＮＡ氧化损伤的重要因素。     

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.     

Evaluation and optimization of DNA delivery into gliosarcoma 9L cells by a cholesterol-based cationic liposome

This paper reports results concerning the transfection of gliosarcoma cells 9L using an original cholesterol-based cationic liposome as carrier. This cationic liposome was prepared from triethyl aminopropane carbamoyl cholesterol (TEAPC-Chol) and a helper lipid, dioleoyl phosphatidyl ethanolamine (DOPE). The used concentration of liposome was not cytotoxic as revealed by the MTT test. TEAPC-Chol/DOPE liposomes allowed the plasmids encoding reporter genes to enter the nucleus as observed both by electron microscopy and functionality tests using fluorescence detection of green fluorescent protein (GFP) and luminometric measurements of luciferase activity. By changing the cationic lipid/DNA molar charge ratio, optimal conditions were determined. Further, improvement of the transfection level has been obtained by either precondensing plasmid DNA with poly-l-lysine or by adding polyethylene glycol (PEG) in the transfection medium. The optimal conditions determined are different depending on whether the transfection is made with cells in culture or with tumors induced by subcutaneous (s.c.) injection of cells in Nude mice. For in vivo assays, a simple method to overcome the interference of haemoglobin with the chemiluminescence intensity of luciferase has been used. These results would be useful for gaining knowledge about the potential for the cationic liposome TEAPC-Chol/DOPE to transfect brain tumors efficiently.     

Evaluation and optimization of DNA delivery into gliosarcoma 9L cells by a cholesterol-based cationic liposome

This paper reports results concerning the transfection of gliosarcoma cells 9L using an original cholesterol-based cationic liposome as carrier. This cationic liposome was prepared from triethyl aminopropane carbamoyl cholesterol (TEAPC-Chol) and a helper lipid, dioleoyl phosphatidyl ethanolamine (DOPE). The used concentration of liposome was not cytotoxic as revealed by the MTT test. TEAPC-Chol/DOPE liposomes allowed the plasmids encoding reporter genes to enter the nucleus as observed both by electron microscopy and functionality tests using fluorescence detection of green fluorescent protein (GFP) and luminometric measurements of luciferase activity. By changing the cationic lipid/DNA molar charge ratio, optimal conditions were determined. Further, improvement of the transfection level has been obtained by either precondensing plasmid DNA with poly-L-lysine or by adding polyethylene glycol (PEG) in the transfection medium. The optimal conditions determined are different depending on whether the transfection is made with cells in culture or with tumors induced by subcutaneous (s.c.) injection of cells in Nude mice. For in vivo assays, a simple method to overcome the interference of haemoglobin with the chemiluminescence intensity of luciferase has been used. These results would be useful for gaining knowledge about the potential for the cationic liposome TEAPC-Chol/DOPE to transfect brain tumors efficiently.     

In Vivo Gene Transfer to the Mouse Nasal Cavity Mucosa Using a Stable Cationic Lipid Emulsion

We evaluate a new cationic emulsion as a mucosal gene carrier and elucidate the relationship between the transfection efficiency and the stability of the carrier/DNA complex. A cationic lipid emulsion was formulated with soybean oil and 1,2-dioleoyl-sn-glycero-3-trimethylammonium-propane (DOTAP) as major components and was used to transfer genes to the epithelial cells of the mouse nasal cavity via intranasal instillation. Correlation between the transfection efficiency and the stability of the carrier/DNA complex was investigated by measuring the carrier size changes and by observing the degree of DNA protection against DNase I digestion in the presence of heparin. The cationic emulsion showed at least 3 times better transfection activity than the liposomal carriers in nasal mucosae. The cationic emulsion was stable in the presence of heparin whereas the liposomal carriers became very unstable. Unlike DNA in liposome/DNA complexes, DNA in the emulsion/DNA complex was resistant to heparin exchange and DNase I digestion. The cationic emulsion was more effective in delivering DNA to nasal mucosae than commercially available liposomal carriers. The transfection activities of the lipid carriers in nasal cavity mucosae are in agreement with the stability of the lipid carriers and their complexes with DNA.     

Analysis and optimization of the cationic lipid component of a lipid/peptide vector formulation for enhanced transfection in vitro and in vivo

We have previously described a lipopolyplex formulation comprising a mixture of a cationic peptide with an integrin-targeting motif (K16GACRRETAWACG) and Lipofectin, a liposome consisting of DOTMA and DOPE in a 1:1 ratio. The high transfection efficiency of the mixture involved a synergistic interaction between the lipid/peptide components. The aim of this study was to substitute the lipid component of the lipopolyplex to optimize transfection further and to seek information on the structure-activity relationship of the lipids in the lipopolyplex. Symmetrical cationic lipids with diether linkages that varied in alkyl chain length were formulated into liposomes and then incorporated into a lipopolyplex by mixing with an integrin-targeting peptide and plasmid DNA. Luciferase transfections were performed of airway epithelial cells and fibroblasts in vitro and murine lung airways in vivo. The biophysical properties of lipid structures and liposome formulations and their potential effects on bilayer membrane fluidity were determined by differential scanning calorimetry and calcein-release assays. Shortening the alkyl tail from C18 to C16 or C14 enhanced lipopolyplex and lipoplex transfection in vitro but with differing effects. The addition of DOPE enhanced transfection when formulated into liposomes with saturated lipids but was more variable in its effects with unsaturated lipids. A substantial improvement in transfection efficacy was seen in murine lung transfection with unsaturated lipids with 16 carbon alkyl tails. The optimal liposome components of lipopolyplex and lipoplex vary and represent a likely compromise between their differing structural and functional requirements for complex formation and endosomal membrane destabilization.     

How are Nucleic Acids Released in Cells from Cationic Lipid-Nucleic Acid Complexes?

Abstract

Cationic lipid-nucleic acid complexes are widely used to deliver oligonucleotides, RNA and DNA into cells. Although much has been learned about the structure and forces that hold the complex together, an understanding of the mechanism of release of the nucleic acids from the complex into cells has been lacking. Recent studies have shown that anionic liposomes with compositions similar to the cytoplasmic face of the endosomal membrane are potent agents for inducing the rapid release of oligonucleotides and DNA from cationic lipid-nucleic acid complexes. Based upon these results, we propose that after the cationic lipid/nucleic complex is internalized by endocytosis it destabilizes the endosomal membrane. This destabilization induces flip-flop of anionic lipids from the cytoplasmic facing monolayer, which laterally diffuse into the complex and form a charge neutral ion-pair with the cationic lipids. This results in displacement of the nucleic acid from the cationic lipid and subsequent release of the nucleic acid into cytoplasm of the cell. We review the data that show the proposed mechanism accounts for a variety of observations on cationic lipid/nucleic acid complex-cell interactions.     

Analysis and Optimization of the Cationic Lipid Component of a Lipid/Peptide Vector Formulation for Enhanced Transfection In Vitro and In Vivo

We have previously described a lipopolyplex formulation comprising a mixture of a cationic peptide with an integrin-targeting motif (K₁₆GACRRETAWACG) and Lipofectin^®, a liposome consisting of DOTMA and DOPE in a 1:1 ratio. The high transfection efficiency of the mixture involved a synergistic interaction between the lipid/peptide components. The aim of this study was to substitute the lipid component of the lipopolyplex to optimize transfection further and to seek information on the structure-activity relationship of the lipids in the lipopolyplex. Symmetrical cationic lipids with diether linkages that varied in alkyl chain length were formulated into liposomes and then incorporated into a lipopolyplex by mixing with an integrin-targeting peptide and plasmid DNA. Luciferase transfections were performed of airway epithelial cells and fibroblasts in vitro and murine lung airways in vivo. The biophysical properties of lipid structures and liposome formulations and their potential effects on bilayer membrane fluidity were determined by differential scanning calorimetry and calcein-release assays. Shortening the alkyl tail from C18 to C16 or C14 enhanced lipopolyplex and lipoplex transfection in vitro but with differing effects. The addition of DOPE enhanced transfection when formulated into liposomes with saturated lipids but was more variable in its effects with unsaturated lipids. A substantial improvement in transfection efficacy was seen in murine lung transfection with unsaturated lipids with 16 carbon alkyl tails. The optimal liposome components of lipopolyplex and lipoplex vary and represent a likely compromise between their differing structural and functional requirements for complex formation and endosomal membrane destabilization.     

Novel macrocyclic and acyclic cationic lipids for gene transfer: synthesis and in vitro evaluation

The synthesis and in vitro evaluation of four cationic lipid gene delivery vectors, characterized by acyclic or macrocyclic, and saturated or unsaturated hydrophobic regions, is described. The synthesis employed standard protocols, including ring-closing metathesis for macrocyclic lipid construction. All lipoplexes studied, formulated from plasmid DNA and a liposome composed of a synthesized lipid, 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (EPC), and either 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or cholesterol as co-lipid, exhibited plasmid DNA binding and protection from DNase I degradation, and concentration dependent cytotoxicity using Chinese hamster ovary-K1 cells. The transfection efficiency of formulations with cholesterol outperformed those with DOPE, and in many cases the EPC/cholesterol control, and formulations with a macrocyclic lipid (+/- 10:1) outperformed their acyclic counterparts (+/- 3:1).     

Structure Relationship of Cationic Lipids on Gene Transfection Mediated by Cationic Liposomes

The aim of this study was to investigate the transfection efficiency of cationic liposomes formulated with phosphatidylcholine (PC) and novel synthesized diethanolamine-based cationic lipids at a molar ratio of 5:1 in comparison with Lipofectamine™ 2000. Factors affecting transfection efficiency and cell viability, including the chemical structure of the cationic lipids, such as different amine head group (diamine and polyamine; and non-spermine and spermine) and acyl chain lengths (C14, C16, and C18) and the weight ratio of liposomes to DNA were evaluated on a human cervical carcinoma cell line (HeLa cells) using the pDNA encoding green fluorescent protein (pEGFP-C2). Characterizations of these lipoplexes in terms of size and charge measurement and agarose gel electrophoresis were performed. The results from this study revealed that almost no transfection was observed in the liposome formulations composed of cationic lipids with a non-spermine head group. In addition, the transfection efficiency of these cationic liposomes was in the following order: spermine-C14 > spermine-C16 > spermine-C18. The highest transfection efficiency was observed in the formulation of spermine-C14 liposomes at a weight ratio of 25; furthermore, this formulation was safe for use in vitro. In conclusion, cationic liposomes containing spermine head groups demonstrated promising potential as gene carriers.Key words: cationic lipids, cationic liposomes, gene transfection     

Fat metabolism in higher plants. Properties of a soluble stearyl-acyl carrier protein desaturase from maturing Carthamus tinctorius

A soluble extract from maturing safflower seeds (Carthamus tinctorius) synthesized [¹⁴C]oleic acid from [¹⁴C]malonate, or [¹⁴C]stearyl-acyl carrier protein. Stearyl-acyl carrier protein was generated from [¹⁴C]malonate by the seed extract. The desaturase had only a trace of activity when stearyl-CoA was the substrate. The stearyl-acyl carrier protein desaturase had a specific requirement for ferredoxin which was only partially replaced by flavodoxin. While NADPH was an effective reductant, NADH was ineffective. However, the most effective reductant was a system composed of ferredoxin, grana lamellae, ascorbic acid, dichlorophenolindophenol, and light. No NADPH requirement was observed when this reducing system was employed. Stearylacyl carrier protein desaturase activity was enhanced by dithiothreitol and reduced glutathione, but was partially inhibited by β-mercaptoethanol. The desaturase activity was inhibited by 1 mm potassium cyanide but insensitive to carbon monoxide. No lipid micelle requirement could be demonstrated.