半乳糖化脂质体-聚阳离子-DNA复合物的肝靶向性研究

目的 研究半乳糖苷修饰的脂质体-聚阳离子-DNA复合物(Gal-LPD)在体外的肝细胞靶向性.方法 合成胆甾五半乳糖苷(Gal-chol),并用薄膜-超声分散法制备空白阳离子脂质体,再与鱼精蛋白-DNA复合物形成Gal-LPD;用激光粒度仪及电位分析仪测定其粒径和电位;以lacZ质粒DNA作为报告基因,用HepG2肝癌细胞和A549肺癌细胞考察LPD的转染效率;用MTT法测定其细胞的毒性.结果 Gal-LPD的粒径为200 nm;Zeta电位随阳离子成分DDAB-DNA比例的不同而在20～55 mV间变化;与未用半乳糖修饰的LPD.相比,Gal-LPD在HepG2细胞中的转染率提高了2.4倍,但在A549细胞中的转染率却有所下降,而且半乳糖能竞争抑制Gal-LPD在HepG2细胞中的转基因效率;Gal-LPD无明显的细胞毒性.结论 Gal-LPD在HepG2细胞中有较高的转基因效率,具有体外的肝细胞靶向性.     

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。     

转铁蛋白修饰的载基因前阳离子脂质体制备及其表达研究

本文制备、优化转铁蛋白修饰的前阳离子脂质体，并研究其相关性质。通过薄膜分散膜挤压法制备空白前阳离子脂质体；以鱼精蛋白缩合质粒DNA与空白前阳离子脂质体作用形成载基因前阳离子脂质体(PLPD)；转铁蛋白(transferrin，Tf)再与PLPD作用形成转铁蛋白修饰的载基因前阳离子脂质体(Tf-PLPD)；中心组合设计优化制备工艺；以lacZ为报告基因转染人肝癌细胞株HepG2；测定形态、粒径、电位和转染效率。结果显示，PLPD形态近似于球体，平均粒径为(228.9±8.0) nm，多分散指数为0.122±0.020(n=3)；zeta电位为(-25.08±2.50) mV(n=3)，转染效率(12.18±3.80) mU·mg^-1(protein)。Tf-PLPD平均粒径为(240±12) nm，多分散指数为0.150±0.030(n=3)；zeta电位为(-24.10±2.50) mV(n=3)；转染效率(24.26±2.60) mU·mg^-1(protein)是裸质粒的20倍；实验结果也表明血清的存在不影响PLPD和Tf-PLPD的转染效率；PLPD和Tf-PLPD小于阳离子脂质体LPD对人肝癌细胞HepG2，SMMC7721和张氏正常肝细胞3种细胞株的毒性。由此可见，转铁蛋白修饰的前阳离子脂质体作为基因转运的非病毒载体具有良好的应用前景。     

葡聚糖-精胺阳离子聚合物基因载体体外基因转染的研究

本文研究了葡聚糖-精胺阳离子聚合物(DSP)基因载体的性能及其对体外细胞基因的转染效率。氧化葡聚糖与精胺通过还原胺化法反应制得DSP，所得DSP与质粒pEGFP通过静电吸附形成复合物；当DSP/DNA质量比在4∶1至20∶1，能形成稳定的复合物，复合物粒径为162.6～187.9 nm，zeta电位则从+8.45 mV增至+39.6 mV；DSP能有效保护DNA不受核酸酶I降解，同时在一定pH范围内载体具有较强的缓冲能力；复合物在质量比为8∶1时对SMMC-7721肝癌细胞、BHK-21细胞的转染率分别达到最高，其效果均与Lipofectamine 2000相当。该研究表明葡聚糖-精胺阳离子聚合物是一种高效的基因载体。     

脂质体-鱼精蛋白-DNA复合物的制备及对树突状细胞的成熟诱导

目的研究脂质体-鱼精蛋白-DNA复合物(LPD)的制备方法及对树突状细胞(DC2.4)的成熟诱导作用。方法薄膜-超声分散法制备空白的阳离子脂质体,再与鱼精蛋白-DNA复合物室温孵育形成LPD;测定其粒径和电位。流式细胞仪检测鼠源树突状细胞系DC2.4的甘露糖受体表达水平;用DC2.4表面标记分子CD80、CD40、CD86和MHC-2的表达水平,考查LPD对树突状细胞的成熟诱导作用。结果 LPD的最佳比例为DDAB-鱼精蛋白-DNA(2∶1.5∶1);LPD粒径为84.28±0.56 nm,Zeta电位为27.33±1.23 mV。通过FITC-ManBSA的结合作用检测到DC2.4表面有83%的甘露糖受体表达。LPD明显上调DC2.4表面标记分子的表达水平(P<0.05)。结论 LPD制备工艺简单,是一个良好的疫苗佐剂。     

Chol-PEG-GA修饰的马钱子碱脂质体体外肝癌细胞摄取特性

目的研究新型高分子材料Chol-PEG-GA修饰马钱子碱脂质体(CPGL)体外肝癌细胞摄取特性。方法采用硫酸铵梯度法制备马钱子碱普通脂质体(LP)和CPGL,测定其包封率,以人肝癌SMMC-7721细胞株为模型,研究孵化温度、给药剂量、Chol-PEG-GA及甘草次酸浓度等因素对LP和CPGL摄取的影响。结果 LP和CPGL的平均包封率分别为(81.5±1.2)%和(86.8±1.6)%,平均粒径分别为(131.3±15.8)nm和(147.2±16.6)nm。经过60 min孵育,肝癌细胞摄取CPGL的单位蛋白摄取量高于LP;在25～100 mg.L-1药物浓度范围内,肝癌细胞摄取CPGL的单位细胞摄取马钱子碱量随给药剂量的增加而增加,且明显高于LP(P<0.05)。4℃温度条件下,单位蛋白摄取量明显低于37℃,有显著差异(P<0.05)。结论CPGL可作为肝细胞靶向的载体,显著提高药物进入肝癌细胞的摄取量,为临床治疗肝脏疾病提供理论依据。     

人剪切修复基因XPD对肝癌细胞生长及ERG基因的影响

目的探讨人剪切修复基因XPD转染人肝癌细胞SMMC-7721后对细胞自身生长及癌基因ERG表达的影响。方法将XPD基因通过LipofectamineTM2000转染入人肝癌细胞SMMC-7721。实验分为4组,分别为重组质粒转染细胞SMMC-7721-pEGFP-N2-XPD组(XPD组)、空载质粒转染细胞SMMC-7721-pEGFP-N2组(N2组)、脂质体转染细胞SMMC-7721组(脂质体组)、肝癌细胞SMMC-7721无转染空白对照组(空白对照组)。分别用逆转录聚合酶链反应(RT-PCR)和Westernblot法检测各组细胞中XPD、ERG的mRNA和蛋白质的表达量,用四甲基偶氮唑盐比色法(MTT)检测各组细胞的增殖活力及流式细胞仪检测各组细胞的凋亡情况。结果与其他3组比较,XPD组中的ERGmRNA及蛋白表达量显著降低,而XPDmRNA及蛋白表达量明显升高(P<0.01)。转染了XPD的肝癌细胞SMMC-7721,其细胞增殖活性(0.455±0.009)显著降低,细胞凋亡率(42.06±0.01)%明显升高(P<0.001)。结论 XPD基因可以抑制癌基因ERG的表达,明显降低肝癌细胞的增殖活力并提高肝癌细胞的凋亡率。     

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。     

硬脂醇半乳糖苷修饰的阿西替尼脂质体的制备及体外活性研究

目的 制备硬脂醇半乳糖苷修饰的阿西替尼脂质体,并进行处方筛选及体外活性研究。方法 采用硫酸铵梯度法制备硬脂醇半乳糖苷修饰的阿西替尼脂质体,以包封率及粒径为评价指标,采用Box-Behnken响应面设计法优化制备工艺,并研究硬脂醇半乳糖苷修饰的阿西替尼脂质体对人肝癌SMMC-7721细胞株的增殖抑制及凋亡的诱导作用。采用CCK-8法检测硬脂醇半乳糖苷修饰的阿西替尼脂质体对人肝癌SMMC-7721细胞株的生长抑制情况,采用了Annexin V/PI流式细胞分析法检测细胞凋亡。结果 最佳工艺：药与磷脂比为1∶14.95,胆固醇与磷脂之比为1∶4.45,水浴温度为61.93 ℃,硫酸铵溶液的体积为4.31 mL。硬脂醇半乳糖苷修饰的阿西替尼脂质体的粒径为(252±6.4)nm,包封率为(68.50±0.85)%。CCK-8细胞毒性试验结果显示,在药物浓度相同时,抑制率随时间的延长而增加;作用时间相同时,抑制率随药物浓度的增大而增加。Annexin V/PI流式试验结果显示,硬脂醇半乳糖苷修饰的阿西替尼脂质体对人肝癌SMMC-7721细胞株的抑制率优于人肝癌A549细胞株。结论 硫酸铵梯度法制备硬脂醇半乳糖苷修饰的阿西替尼脂质体的处方合理,工艺可行,包封率高。硬脂醇半乳糖苷修饰的阿西替尼脂质体对人肝癌SMMC-7721细胞株有更高的细胞毒性,诱导SMMC-7721细胞株凋亡能力比A549的强。初步判定硬脂醇半乳糖苷修饰的阿西替尼脂质体具有主动肝靶向性。     

替诺福韦阳离子脂质体的制备及其对人体肝细胞SMMC-7721摄取和细胞毒性研究

目的:研究替诺福韦阳离子脂质体的制备及其促进肝实质细胞摄取的情况和细胞毒性作用。方法:采用叔丁醇冻干法制备替诺福韦阳离子脂质体,测定其包封率及理化性质;以SMMC-7721细胞为模型,研究脂质体对肝实质细胞摄取替诺福韦的促进作用,MTT法检测不同条件下载药脂质体对细胞的毒性情况。结果:制备的脂质体包封率为(88.3±1.6)%,粒径为(278.4±67.6)nm,Zeta电势为(31±5)mV;经半乳糖基及PEG修饰的脂质体较游离药物进入肝实质细胞的浓度明显升高,且时间延长;当替诺福韦脂质体、脂质浓度分别为7.5、30μg·mL-1时,细胞存活率在80%以上,毒性较小。结论:所制备的阳离子脂质体具有显著增加细胞摄取替诺福韦和保护替诺福韦的作用,有望成为抗病毒药物如替诺福韦等的高效传递系统。     

载TK基因聚丙交酯乙交酯纳米粒的性质及表达研究

目的载pEGFP-TKAFB重组质粒纳米粒的性质及其表达研究。方法以无毒的可生物降解的高分子材料聚丙交酯乙交酯作为载体材料,采用双乳化溶媒蒸发法制备了载pEGFP-TKAFB重组质粒纳米粒,考察其形态学及包封率,琼脂糖电泳分析抗核酸酶抗超声的能力,MTT法测定GCV对细胞的抑制率,流式细胞仪测定报告基因EGFP的表达。结果制得的纳米粒,形态圆整,大小均匀,平均粒径(72±12) nm,平均包封率91.25%,质粒制成纳米粒后提高了质粒对抗超声剪切及核酸酶降解的能力,细胞转染效率也显著优于裸质粒。结论质粒DNA制成纳米粒可进一步研究基因药物的给药系统。     

Targeted gene delivery to hepatoma cells using galactosylated liposome-polycation-DNA complexes (LPD)

A major goal for gene therapy is to obtain targeted vectors that transfer genes efficiently to specific cell types. The liver possesses a variety of characteristics that make this organ very attractive for gene therapy. In the present study, four cholesterylated thiogalactosides 1a approximately d with different spacer length were synthesized to formulate novel lipid-polycation-DNA (LPD) complexes, which were composed of galactosylated cationic liposomes, protamine sulfate and plasmid DNA. The galactosylated LPD1c significantly improved the levels of gene expression in cultured hepatoma cells HepG2 and SMMC-7721, while LPD1a and LPD1b did not significantly improve the levels compared with non-galactosylated LPD. Meanwhile, increased transfection activity was not observed in mouse fibroblasts L929 for galactosylated LPDs. Cytotoxicity of galactosylated LPDs assay showed they had no obvious toxicities to L929 cells and HepG2 cells. In summary, the length of the spacer between the anchor and galactose residues was important for the recognition of asialoglycoprotein receptor. The LPD1c described here, combining the condensing effect of protamine and the targeting capability of cholesterylated thiogalactosides, are potentially useful gene carriers to liver parenchymal cells.     

脂质体-聚乙烯亚胺-DNA三元复合物的构建及其对细胞的体外转染

目的研究非病毒基因载体脂质体-聚乙烯亚胺(PEI)-DNA三元复合物(TC)的制备方法,评价其体外细胞学性质。方法采用乙醇注入法制备空白阴离子脂质体,与PEI/DNA复合物37℃孵育30 min后,得到TC,考察其理化性质、抗核酸酶降解能力、血浆稳定性、细胞毒性及在卵巢癌细胞(Hela)中的转染效率。结果制备的TC呈类球形,大小较均匀,平均粒径为234.5 nm,Zeta电位为-20.72 mV;TC能在血浆中稳定存在4 h而不发生聚集;与核酸酶作用2 h后,其中的DNA几乎无降解;其细胞毒性较低,在无血清和含血清培养基中均能成功的转染Hela细胞,在含血清培养基中其转染效率明显高于PEI/DNA复合物。结论 TC是一种制备工艺简单、血浆稳定性好、转染率较高、极具应用潜力的非病毒纳米基因载体。     

Targeted gene delivery to hepatoma cells using galactosylated liposome-polycation-DNA complexes (LPD)

A major goal for gene therapy is to obtain targeted vectors that transfer genes efficiently to specific cell types. The liver possesses a variety of characteristics that make this organ very attractive for gene therapy. In the present study, four cholesterylated thiogalactosides 1a ～ d with different spacer length were synthesized to formulate novel lipid-polycation-DNA (LPD) complexes, which were composed of galactosylated cationic liposomes, protamine sulfate and plasmid DNA. The galactosylated LPD1c significantly improved the levels of gene expression in cultured hepatoma cells HepG2 and SMMC-7721, while LPD1a and LPD1b did not significantly improve the levels compared with non-galactosylated LPD. Meanwhile, increased transfection activity was not observed in mouse fibroblasts L929 for galactosylated LPDs. Cytotoxicity of galactosylated LPDs assay showed they had no obvious toxicities to L929 cells and HepG2 cells. In summary, the length of the spacer between the anchor and galactose residues was important for the recognition of asialoglycoprotein receptor. The LPD1c described here, combining the condensing effect of protamine and the targeting capability of cholesterylated thiogalactosides, are potentially useful gene carriers to liver parenchymal cells.     

Characterization of transferrin-modified procationic-liposome protamine-DNA complexes

We developed a novel transferrin modified non-viral gene delivery system, transferrin-modified procationic-liposome-protamine-DNA complexes (Tf-PLPD) and investigated its characteristics. Blank procationic liposomes were prepared using the film dispersion filter method. Protamine was used to condense plasmid DNA to form protamine-DNA complexes and the complexes were further incubated with blank procationic liposomes to form PLPD. Transferrin was adsorbed onto the surface of PLPD via an electrostatic interaction, and thus Tf-PLPD was produced. Characteristics such as stability in rat serum, morphology, average particle size, zeta potential, and transfection efficiency in HepG2 cells were further investigated. The results indicated that the procationic liposomes remained stable in rat serum for 24 h. Tf-PLPD protected plasmid DNA from enzymatic degradation even after lyophilization. The size distribution of Tf-PLPD was in the range of 240+/-12 nm and the zeta potential was -24.10+/-2.5 mV (n=3), respectively. The transfection efficiencies of Tf-PLPD were 24.26+/-2.6 mU beta-galactosidase/mg protein. Lyophilization and the presence of serum did not affect the transfectivity of Tf-PLPD and the procationic liposomes also had low cytotoxicity to cells.     

Galactosylated low molecular weight chitosan as DNA carrier for hepatocyte-targeting

Chitosan has the potential for DNA complexation and is useful as a non-viral vector for gene delivery. Highly purified low molecular weight chitosan (LMWC) was prepared. Lactobionic acid (LA) bearing galactose group was coupled with LMWC for liver-specificity. A series of galactosylated-LMWC (gal-LMWC) samples covering a range of galactose group contents were prepared. The chitosan/DNA complexes were obtained using a complex coacervation process. Gal-LMWCs were used to transfer pSV-beta-galactosidase reporter gene into human hepatocellular carcinoma cell (HepG2), L-02, SMMC-7721, and human cervix adenocarcinoma cell line (HeLa) cell lines in vitro. Transfection efficiency of gal-LMWCs was evaluated by beta-galactosidase assay and compared with those of lipofectin, calcium phosphate (CaP), high molecular weigh chitosan (HMWC) and LMWC. Gal-LMWC/DNA complex shows a very efficient cell selective transfection to hepatocyte. The transfection efficiency of gal-LMWCs increased with the improvement of the galactosylation degree. Cytotoxicity of gal-LMWC was determined by 3-(4,5-dimethylthiazd-2-yl)-2,5-diphenyltentrazolium bromide (MTT) assay and the results show that the modified chitosan has relatively low cytotoxicity, giving the evidence that the modified chitosan vector has the potential to be used as a safe gene-delivery system.     

Physical stability and in-vitro gene expression efficiency of nebulised lipid-peptide-DNA complexes

The lower respiratory tract provides a number of disease targets for gene therapy. Nebulisation is the most practical system for the aerosolisation of non-viral gene delivery systems. The aerosolisation process represents a significant challenge to the maintenance of the physical stability and biological activity of the gene vector. In this study we investigate the role of a condensing polycationic peptide on the stability and efficiency of nebulised lipid-DNA complexes. Complexes prepared from the cationic lipid 1, 2-dioleoyl-3-trimethylammonium propane (DOTAP) and plasmid DNA (pDNA) at mass (w/w) ratios of 12:1, 6:1 and 3:1, and complexes prepared from DOTAP, the polycationic peptide, protamine, and pDNA (LPD) at 3:2:1 w/w ratio were nebulised using a Pari LC Plus jet nebuliser. Samples from the nebuliser reservoir (pre- and post-nebulisation) and from the aerosol mist were collected and investigated for changes, including: particle diameter, retention of in-vitro transfection activity and the relative concentration and nature of the complexed pDNA remaining after the nebulisation procedure. The process of jet nebulisation adversely affected the physical stability of lipid:pDNA complexes with only those formulated at 12:1 w/w DOTAP:pDNA able to maintain their pre-nebulisation particle size distribution (145+/-3 nm pre-nebulisation vs. 142+/-2 nm aerosol mist) and preserve significant pDNA integrity in the reservoir (35% of pre-nebulisation pDNA band intensity). The LPD complexes were smaller (102+/-1 nm pre-nebulisation vs. 113+/-2 nm aerosol mist) with considerably greater retention of pDNA integrity in the reservoir (90% of pre-nebulisation pDNA band intensity). In contrast the concentration of pDNA in the aerosol mist for both the 12:1 w/w DOTAP:pDNA and LPD complexes were significantly reduced (10 and 12% of pre-nebulised values, respectively). Despite reduced pDNA concentration the transfection (% cells transfected) mediated by aerosol mist for the nebulised complexes was comparatively efficient (LPD aerosol mist 26 vs. 40% for pre-nebulised complex; the respective values for 12: 1 w/w DOTAP:pDNA were 12 vs. 28%). The physical stability and biological activity of nebulised lipid:pDNA complexes can be improved by inclusion of a condensing polycationic peptide such as protamine. The incorporation of the peptide precludes the use of potentially toxic excesses of lipid and charge and may act as a platform for the covalent attachment of peptide signals mediating sub-cellular targetting.     

Optimizing the novel formulation of liposome-polycation-dna complexes (lpd) by central composite design

LPD vectors are non-viral vehicles for gene delivery comprised of polycation-condensed plasmid DNA and liposomes. Here, we described a novel anionic LPD formulation containing protamine-DNA complexes and pH sensitive liposomes composed of DOPE and cholesteryl hemisuccinate (Chems). Central composite design (CCD) was employed to optimize stable LPD formulation with small particle size. A three factor, five-level CCD design was used for the optimization procedure, with the weight ratio of protamine/DNA (X1), the weight ratio of Chems/ DNA (X2) and the molar ratio of Chems/DOPE in the anionic liposomes (X3) as the independent variables. LPD size (Y1) and LPD protection efficiency against nuclease (Y2) were response variables. Zeta potential determination was utilized to define the experimental design region. Based on experimental design, responses for the 15 formulations were obtained. Mathematical equations and response surface plots were used to relate the dependent and independent variables. The mathematical model predicted optimized X1-X3 levels that achieve the desired particle size and the protection efficiency against nuclease. According to these levels, an optimized LPD formulation was prepared, resulting in a particle size of 185.3 nm and protection efficiency of 80.22%.