小分子聚乙烯亚胺与腺病毒结合增强基因转染效率的研究

目的 提高重组腺病毒在细胞中,特别是缺乏柯萨奇腺病毒受体的细胞中的转染效率.方法 将阳离子材料聚乙烯亚胺PEI与重组腺病毒通过电荷作用形成复合物,用激光粒度仪及电位分析仪测定其粒径和电位;以表达β半乳糖苷酶的重组腺病毒作为模型病毒,研究其在富含(A549)或者缺乏(MDCK、LLC)柯萨奇腺病毒受体细胞中的转染效率和毒性,并用荧光标记复合物,观察其进入细胞的能力.结果 与聚乙烯亚胺结合形成复合物可明显促进腺病毒进入细胞的能力,从而提高腺病毒在各种细胞中的转染效率,同时发现小分子的PEI-2 kD由于其较好的生物相容性,对腺病毒进入细胞后的传递影响较小,所以最终的基因传递效率要显著优于PEI-25 kD;PEI-2 kD在本复合物中表现出的细胞毒性要小于PEI-25 kD.结论 PEI-2 kD可以用于病毒载体和非病毒载体结合的基因传递研究.     

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.     

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.     

溶酶体酸性微环境敏感型粒径可变纳米粒的构建及评价

目的 构建一种以功能性支化聚乙烯亚胺(bPEI)为基本骨架材料的粒径可变纳米粒,用于递送药物至黑色素瘤的深部区域.方法 通过酰胺键修饰特定量的琥珀酸酐至bPEI骨架上,得到具有电荷翻转功能的材料NSP,再引入聚N-异丙基丙烯酰胺(PNIPAM)制备粒径可变纳米粒(PSP),分别考察其粒径、Zeta电位和释药行为;考察B16F10细胞对PSP的摄取及胞内内涵-溶酶体对PSP的影响;通过构建体外三维肿瘤球模型来考察纳米粒的穿透能力.结果 载阿霉素粒径可变纳米粒(D/PSP)在pH7.4缓冲液中的粒径、PDI和Zeta电位分别为31.2±4.8 nm、0.106 ±0.015、-2.26-±0.06 mV.在内涵-溶酶体所对应的pH5.0条件下,D/PSP的粒径可膨胀至891.6±26.3 nm,PDI为0.256±0.123,Zeta电位为15.61±0.19 mV.B16F10细胞对D/PSP的摄取在一定范围内呈孵育浓度依赖性及时间依赖性,其主要的入胞机制为网格蛋白介导和能量依赖型的内吞途径.结论 成功制备了具有较好的溶酶体酸性微环境响应性的PSP,D/PSP具有较强的肿瘤球穿透能力.     

腺病毒-阴离子脂质体复合物的制备和体外表征

为了制备腺病毒-阴离子脂质体复合物 (AL-Ad5), 首先利用HEK 293细胞大量扩增了带有绿色荧光蛋白报告基因的重组腺病毒, 并以氯化铯两步密度梯度离心法进行纯化, 再通过细胞病变效应 (CPE) 法、壳蛋白免疫法和定量聚合酶链反应 (Q-PCR) 法分别测定腺病毒滴度。以中心组合设计法优化处方和制备条件, 通过钙离子融合法制备目标复合物AL-Ad5, 以透射电镜和动态光散射法分别对其外观形态、粒径和zeta电位进行考察; 并制备双色荧光标记的复合物, 通过激光共聚焦技术观察该复合物被MDCK细胞摄取后的胞内定位, 以考察阴离子脂质体对腺病毒的包合情况。结果表明, 目标复合物分布均匀, 粒径和zeta电位分别为 (211 ± 10) nm和 (−41.2 ± 2.2) mV。激光共聚焦实验结果表明, 红色荧光标记的腺病毒和绿色荧光标记的脂质体在MDCK细胞内的定位一致。由此说明, 阴离子脂质体能够较好地包裹腺病毒以形成腺病毒-阴离子脂质体复合物。     

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。     

利福平固体脂质纳米粒的体外细胞摄取研究

目的 研究利福平固体脂质纳米粒的制备方法和体外细胞摄取.方法 采用薄膜分散法制备利福平固体脂质纳米粒(RFP-SLNs);用扫描电镜观察其形态,激光粒度仪测定其粒径大小和Zeta电位;用MTT法测定其对RAW 264.7、A549细胞的毒性,HPLC法测定其在这两种细胞中的摄取量.结果 RFP-SLNs的外观形态圆整,...     

兰索拉唑脂质体的制备及其药剂学性质考察

目的:研究兰索拉唑阳离子脂质体的制备方法并考察其药剂学性质。方法:采用正交设计筛选处方,乙醇注入法制备兰索拉唑脂质体;超滤法测定其包封率;用透射电镜观察脂质体的外观形态,并用粒径分析仪和Zeta电位仪分别测定脂质体的粒径和Zeta电位;进一步考察脂质体的释放规律。结果:所得脂质体包封率约为(80±1.23)%;形态为粒径均匀的球形和类球形,粒径为(184±21)nm,Zeta电位为(36.1±5)mV;脂质体的体外释放符合一级方程;具有较好的稳定性。结论:优选得到的脂质体处方和制备工艺合理、稳定,其体外释放具有缓释特点。     

PEG-b-PLL的合成及其自组装纳米基因载体的研究

目的合成两嵌段聚乙二醇-b-聚赖氨酸共聚物(PEG-b-PLL),并评价PEG-b-PLL载基因纳米复合物。方法以端氨基PEG引发Lys(z)-NCA,首先得到两嵌段共聚物PEG-b-PZLL,然后酸解去除苄氧羰基保护基团,得到了两嵌段共聚物PEG-b-PLL。通过正负电荷吸附作用自组装形成PEG-b-PLL载基因纳米复合物,考察其性质。结果制备的PEG-b-PLL载基因纳米复合物外观圆整,呈类球形,大小均匀,平均粒径为(150.3±5.5)nm,其Zeta电位为(-15.82±2.34)mV。该复合物在血浆中稳定,具有一定的抗核酸酶降解能力,且能成功的转染HepG2细胞。结论该复合物是一种制备工艺简单,性能良好,极富潜力的非病毒基因载体。     

Novel biotinylated chitosan-graft-polyethyleneimine copolymer as a targeted non-viral vector for anti-EGF receptor siRNA delivery in cancer cells

The major impediments to develop an efficient non-viral siRNA-mediated gene silencing method, as a therapeutic approach, are the low cellular uptake and intracellular delivery and release of non-viral vectors. To overcome these problems, designing a proper vector with high transfection efficiency is obviously under scrutiny of various studies. The present study, evaluate a novel biotinylated chitosan-graft-polyethyleneimine (Bio-Chi-g-PEI) copolymer as an appropriate non-viral vector for targeted delivery of siRNA to cancer cells. The composition of the synthesized Bio-Chi-g-PEI copolymer was thoroughly characterized using ¹H NMR and FTIR spectroscopy, besides the hydroxyazobenzene-2-carboxylic acid (HABA) assay. In vitro cytotoxicity assay of the Bio-Chi-g-PEI copolymers was performed by MTT assay. Cytotoxicity evaluations indicated that the new copolymer was markedly less toxic than PEI 25KD. Physicochemical properties of the Bio-Chi-g-PEI/siRNA complexes such as complex stability, size, zeta potential, and their morphology at various weight ratios, investigated by appropriate methods, revealed the suitability of the complexes for the transfection. The efficient cellular internalization of the complexes for HeLa and OVCAR-3 cells in culture media was confirmed by intracellular tracking of the prepared complexes using confocal laser scanning microscopy and Cy3-labeled anti-epidermal growth factor receptor siRNA. Finally, evaluation of the transfection efficiency and gene silencing by flow cytometry and real-time polymerase chain reaction highlighted the significantly higher efficiency of transfection and silencing for biotinylated copolymer compared with the PEI 25KD and non-biotinylated copolymer.     

Effect of DC/mDC iontophoresis and terpenes on transdermal permeation of methotrexate: in vitro study

We have reported previously that a basic peptide, arginine peptide, can be used as an efficient system for delivery of foreign genes. In this work, to better understand the mechanism of arginine peptide-mediated gene delivery, we further evaluated the process of cellular uptake and nuclear localization of the peptide/DNA complex. To investigate the effect of cellular proteoglycans on arginine peptide/DNA complexes, interactions between polyanionic glycosaminoglycans (GAGs) and peptide/DNA complexes were examined by the ethidium bromide interaction assay. Sulfated GAGs were found to relax the complexed DNA at low peptide/DNA charge ratios. Condensed peptide/DNA complexes facilitate cellular uptake, but their mechanism of uptake is poorly understood. Studies of various endocytosis inhibitors suggested that the peptide/DNA complex internalization involved the caveolar-related endocytosis pathway. A critical step in the gene delivery is the cytosol-to-nucleus transport of exogenous DNA following initial complex uptake. Nuclear localization of peptide/DNA complex was confirmed by confocal laser scanning microscopic observation. Further, we show that transfections with peptides result in an early accumulation of plasmid DNA in the nucleus of growth-arrested cells, which suggest nuclear transport. To assess the potential for arginine peptide as an agent for therapeutic gene delivery, in vivo complexed DNA transduction studies were performed. Mice were injected subcutaneously with the reporter gene beta-galactosidase, resulting in high levels of gene expression in dermal tissue.     

Vitreous Is a Barrier in Nonviral Gene Transfer by Cationic Lipids and Polymers

Purpose. To investigate the role of vitreous in nonviral gene delivery into retinal pigment epithelial (RPE) cells. Methods. Human RPE cell line D407 was cultured in six-well plates. Bovine vitreous, hyaluronan, or DMEM was added on the cells. Complexes of DNA and cationic carriers (polyethyleneimine, poly-L-lysine, DOTAP liposomes) were pipetted onto the vitreous, hyaluronan, or DMEM. Cellular uptake of DNA was studied with ethidium monoazide DNA and gene expression with GFP-plasmid complexes. FITC-dextrans and FITC-polylysines were used to probe the effects of the size and cationic charge on permeation in the vitreous in a similar experimental setup. Fluorescent cells were analyzed by flow cytometry. Results. Vitreous decreased the cellular uptake of DNA complexes 2-30 times, and GFP expression was also impaired. In hyaluronan solutions the cellular uptake of the complexes was also decreased significantly in most cases. In vitreous, cellular uptake of all FITC-dextrans decreased slightly, and uptake of poly-L-lysines was decreased substantially, whereas in hyaluronan solutions the effects were mild or nonexistent. Conclusions. Polymeric and liposomal gene delivery is substantially limited by the vitreous. This is probably because of the size and charge of the retinal gene delivery after intravitreal injections.     

Gene transfer efficacies of serum-resistant amino acids-based cationic lipids: dependence on headgroup, lipoplex stability and cellular uptake

Serum is a major obstacle to efficient cationic liposome-mediated gene transfection. In this paper, three alkaline amino acids based cationic lipids including lysinylated cholesterol (lipid 1), histidinylated cholesterol (lipid 2) and argininylated cholesterol (lipid 3) were used as non-viral gene vectors. The physicochemical properties such as size, Zeta potential, stability and cellular uptake of the lipoplexes formed from lipids 1-3 as well as the transfection efficacies with or without serum were investigated. The results demonstrated that lipid 1 and lipid 3 showed good properties in lipoplex stability and cellular uptake. Interestingly, lipid 3-based liposome showed serum-enhanced effect on the gene transfection. The transfection efficiency of lipid 1 and lipid 3 was remarkably higher than that of lipid 2. Moreover, they exhibited 10-20-fold more efficaciously than the control, 1,2-dioleoyloxy-3-(trimethylammonio)-propane (DOTAP) liposome in serum-containing media. The data suggested the strong effect of the type of the headgroup on gene transfection. The lysine/arginine derivative cationic lipids could be promising nonviral vectors for gene delivery in vivo.     

Hydrotropic Polymeric Mixed Micelles Based on Functional Hyperbranched Polyglycerol Copolymers as Hepatoma-Targeting Drug Delivery System

Mixed copolymer nanoparticles (NPs) self-assembled from β-cyclodextrin-grafted hyperbranched polyglycerol (HPG-g-CD) and lactobionic acid (LA)-grafted hyperbranched polyglycerol (HPG-g-LA) were applied as carriers for a hydrophobic antitumor drug, paclitaxel (PTX), achieving hepatocellular carcinoma-targeted delivery. The resulting NPs exhibited high drug loading capacity and substantial stability in aqueous solution. In vitro drug release studies demonstrated a controlled drug release profile with increased release at acidic pH. Remarkably, tumor proliferation assays showed that PTX-loaded mixed copolymer NPs inhibited asialoglycoprotein (ASGP) receptor positive HepG2 cell proliferation in a concentration-dependent manner in comparison with ASGP receptor negative BGC-823 cells. Moreover, the competition assay demonstrated that the small molecular LA inhibited the cellular uptake of the PTX-loaded mixed copolymer NPs, indicating the ASGP receptor-mediated endocytosis in HepG2 cells. In addition, the intracellular uptake tests by confocal laser scanning microscopy showed that the mixed copolymer NPs were more efficiently taken up by HepG2 cells compared with HPG-g-CD NPs. These results suggest a feasible application of the mixed copolymer NPs as nanocarriers for hepatoma-targeted delivery of potent antitumor drugs.     

Peptide-based cationic molecules for the production of positive charged liposomes and micelles

This paper describes the synthesis and the physico-chemical characterization of cationic peptides (CPs) for possible application as non-viral gene delivery systems. Particularly, the production of cationic liposomes and micelle solutions was considered. Liposomes were prepared by REV-phase and extrusion presenting an average diameter reflecting the pore size of the membrane used for the extrusion. After DNA complexation the mean diameter of complexes decreased by increasing the number of positive charges. The non-complexed liposome preparations showed a net positive zeta potential comprised between 17.8–30 mV. After adding Defibrotide (DFT) to liposomes (at a 1:4?±?molar ratio) the zeta potential fell down to a net negative value indicating the formation of the ionic complex. Concerning micelles, before complexation it was not possible to measure their size by PCS. However, after DFT complexation the size of complexes highly increased. In addition, as previously seen for liposomes, before complexation, the five CPs solutions showed a positive zeta potential ranging from 10–17.8 mV, while after addition of DFT the zeta potential fell to negative values. Concerning toxicity studies, in general CP-liposomes displayed a lower toxicity towards K562 cells as compared to the corresponding CP-solution. Taking into account these results, the studied CPs could be efficiently used to obtain both cationic liposomes and micelles. Moreover they are able to complex DNA with different interaction strength, depending on the type of peptide-based cationic molecule used.     

Specific Gene Transfer Based on Biotinylated and Gluconoylated Polylysine/Plasmid Complexes

Complexes formed between plasmids and polylysine derivatives bearing recognition signals are the basis of nonviral vehicles suitable for gene delivery into eukaryotic cells by a receptor-mediated endocytosis process. We used an alternative procedure with the aim of achieving noncovalent attachment of recognition signals to plasmids. Biotinylated polylysine/DNA complexes were made between a plasmid and biotinylated polylysine by electrostatic interactions, and then the DNA complex was lactosylated via streptavidin bridges in order to target the galactose-specific membrane lectin of human hepatoma (HepG2) cells. HepG2 cells were efficiently transfected in a sugar-dependent manner with a polymer/DNA complex lactosylated with either biotinylated and lactosylated bovine serum albumin or lactosylated streptavidin. In addition, reduction of the number of remaining cationic charges, by partial gluconoylation of biotinylated polylysine, increased the transfection efficiency of HepG2 cells compared with that of lactosylated polymer/DNA complexes. These results indicate that complexes between a plasmid and a partially gluconoylated and biotinylated polylysine are suitable DNA complexes for the binding of any biotinylated recognition signals for in vitro and ex vivo receptor-mediated gene delivery.     

Lipoplexes versus nanoparticles: pDNA/siRNA delivery

Abstract

Small interfering RNA (siRNA) has been widely used as potential therapeutic for treatment of various genetic disorders. However, rapid degradation, poor cellular uptake and limited stability in blood limit the effectiveness of the systemic delivery of siRNA. Therefore, an efficient delivery system is required to enhance its transfection and duration of therapeutics. In the present study, plasmid DNA (pEGFPN3) expressing green fluorescent protein (GFP) was used as a reporter gene. Chitosan nanoparticles/polyplexes and cationic liposomes/lipoplexes were developed and compared for their transfectivity and therapeutic activity in mammalian cell line (HEK 293). The nanoparticulates were first characterized by assessing the surface charge (zeta potential), size (dynamic light scattering) and morphology (transmission electron microscope) followed by evaluation for their DNA retardation ability, transfection efficiency and cytotoxicity on HEK 293 cell line. The chitosan nanoparticles/plasmid DNA (pDNA) complex and liposomes/pDNA complex were co-transfected with GFP-specific siRNA into HEK 293 cells and it was found that both are efficient delivery vehicles for siRNA transfection, resulting in ～57% and ～70% suppression of the targeted gene (GFP), respectively, as compared with the mock control (cells transfected with nanocarrier/pDNA complexes alone). This strong inhibition of GFP expression indicated that cationic liposomes are better than chitosan nanoparticles and can be used as an effective carrier of siRNA in mammalian cells.