藻酸盐/PEI/DNA复合载体作为一种新型基因递送系统

目的克服多聚乙烯亚胺(PEI，polyethlenimine)/DNA载体对细胞的毒性以及在含血清培养基里对癌细胞基因的转移率低的问题。方法利用具有水溶性、可生物降解的、并带有负电的藻酸盐(alginate)对PEI/DNA载体进行包衣，制备出复合载体，并在体外含50%血清培养基里，与PEI/DNA载体比较对C3癌细胞转染率。结果 在含50%血清的培养基里，藻酸盐包衣制备的复合体载体［alginate:DNA，0.15 (w/w);PEI:DNA，N:P=10］与PEI/DNA载体相比，对C3癌细胞基因转染率高出10～30倍，而且其表面正电荷数比PEI/DNA载体减少了一半，颗粒较小，并降低对细胞毒性和红血球集聚反应。结论作为新型的藻酸盐包衣制备的复合载体能提高在体外含高浓度血清培养基里对C3癌细胞的转染率，并能减少其对细胞毒性。     

聚乙烯亚胺的修饰及其作为基因载体的研究

目的:考察聚氧乙烯硬脂酸酯(POES)修饰聚乙烯亚胺(PEI)后聚合物的细胞毒性及其与DNA形成复合物后的载体性质。方法:使用琥珀酰亚胺碳酸脂法将POES连接在PEI上,通过1H-NMR确定接枝聚合物的结构,将接枝聚合物与DNA形成复合物后考察其琼脂糖凝胶电泳行为及测定其粒径、Zeta电位;MTT法考察接枝聚合物的细胞毒性,使用质粒pGL3-lus作为报告基因,测定虫荧光素酶活性以评价复合物对Hela细胞的转染效率。结果:1H-NMR结果表明接枝聚合物具有较高的纯度。凝胶电泳表明接枝聚合物对DNA的包裹能力随着N/P值的增加而升高,随着POES接枝数目的增大而降低。复合物的粒径小于300nm,Zeta电位随N/P值的增加而升高。接枝聚合物细胞毒性显著低于PEI,POES接枝数目低的聚合物转染效率较高。结论:POES修饰的PEI可以作为一种有效的非病毒基因载体。     

不同相对分子质量聚乙烯亚胺体外介导基因传递的研究

目的研究4种不同相对分子质量聚乙烯亚胺（PEI）作为非病毒基因载体体外介导基因传递的能力。方法采用四甲基噻唑蓝法（MTT法）测定了PEI对Hela细胞的毒性,利用琼脂糖凝胶电泳阻滞试验考察PEI与DNA的结合能力,测定PEI—DNA复合物的粒径和Zeta电位,以及考察转染率。结果PEI的细胞毒性与相对分子质量呈正相关,高相对分子质量PEI的细胞毒性远大于低相对分子质量PEI;高相对分子质量PEI在较低的N／P比时就能对DNA起到完全阻滞作用;低相对分子质量PEI与DNA形成的复合物粒径明显大于高相对分子质量的PEI;Zeta电位随着PEI相对分子质量的增大而增大,复合物的粒径和Zeta电位都与组成中的N／P比有关;相对分子质量为2000的PEI（PEI2K）在Hela细胞中的转染率最低,而相对分子质量为25000的PEI（PEI25K）的转染率最高。结论PEI的相对分子质量对其各项性能指标以及介导基因传递的能力都有较大影响。     

鱼精蛋白／DNA复合物的构建与生物性能评价

目的研究非病毒基因载体鱼精蛋白／DNA复合物的制备方法及对其细胞毒性、不同量鱼精蛋白对pDNA的结合能力、体外细胞转染率。方法不同量鱼精蛋白与DNA在室温孵育后,得到鱼精蛋白／DNA复合物;用MTT法检测鱼精蛋白对HeLa子宫颈癌细胞的毒性作用,同时与PEI进行比较;利用琼脂糖电泳实验测定不同N／P比形成复合物时对DNA的阻滞情况;用结合沉淀试验比较不同量鱼精蛋白对包裹DNA的能力的影响;在荧光显微镜下观察比较它们对BEL-7402肝癌细胞转染率的大小。结果当鱼精蛋白／DNA复合物浓度升高时,对He—La子宫颈癌细胞的毒性均为0级,而聚乙烯亚胺（PEI）浓度升高时,对细胞的毒性明显增加;鱼精蛋白与质粒DNA形成复合物时所需的N／P比为1．5：1,较PEI／DNA复合物形成时所需的N／P比2：1要小;鱼精蛋白包裹DNA的能力随N／P比增大而增强,并且包裹DNA的能力要比PEI／DNA复合物转变得快;鱼精蛋白／DNA复合物对BEL-7402肝癌细胞的转染率较PEI／DNA复合物低,但毒性较低。结论鱼精蛋白／DNA复合物是一种制备工艺简单、细胞毒性小、对pDNA包裹能力高、转染率相对较高,具有一定应用潜力的非病毒基因载体。     

泊洛沙姆407修饰对聚乙烯亚胺的毒性和转染性质的影响

目的:考察泊洛沙姆407修饰对聚乙烯亚胺(PEI)的毒性和转染性质的影响.方法:使用琥珀酰亚胺碳酸脂法将P407连接在PEI的氨基上,得到新聚合物,通过1H-NMR确定新聚合物的结构,将该聚合物与DNA形成复合物,测定复合物的zeta电位,MTT法考察复合物的细胞毒性,使用质粒pGL3-lus作为报告基因,测定虫荧光素酶活性评价复合物对Hela细胞的转染效率.结果:1H-NMR结果表明合成的聚合物具有较高的纯度.复合物的Zeta电位随氮/磷比(N/P)值的增加而增高.复合物的细胞毒性随着N/P值的增加而增大,新聚合物其细胞毒性显著低于未修饰的PEI.新聚合物在高N/P值时仍能保持较高的转染效率.结论:泊洛沙姆407修饰的PEI可以作为一种有效的非病毒基因栽体.     

聚乙烯亚胺介导基因转染的影响因素研究

目的研究非病毒基因载体聚乙烯亚胺（PEI）介导基因转染的影响因素。方法采用透射电镜观察PEI与DNA形成复合物的形态,分别考察质粒量、复合物形成时间、复合物在细胞上作用时间和氮磷比（N/P）对转染效率的影响,以筛选较优的转染条件。结果PEI可有效地与DNA形成复合物,当质粒量为每孔2μg、复合物形成时间为30min、复合物在细胞上的作用时间为4h且N/P为20时转染效率最高。结论PEI可作为合成新型非病毒载体的骨架,但必须控制有关因素才能得到最佳的和可重复的转染结果。     

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。     

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。     

胎牛血清对聚乙烯亚胺基因传递系统的降毒作用研究

目的:研究胎牛血清(FBS)对聚乙烯亚胺(PEI)基因传递系统的降毒作用。运用Nanoparticles albumin bound技术的原理,制备以FBS修饰PEI/DNA的复合物(PEI/DNA/FBS)。采用MTT比色法计算人宫颈癌上皮Hela细胞存活率(CV),测定PEI质量浓度分别为0.5、1、2、5、10、15、20、30μg/ml时的细胞毒性,比较FBS含量分别为0、2.5%、5%、10%时PEI/FBS复合物和N(PEI中氮的物质的量)/P(DNA中磷的物质的量)比分别为10、20时的PEI/DNA/FBS复合物的细胞毒性差异,比较PEI和N/P比分别为10、20且FBS含量为0、5%、10%的PEI/DNA/FBS复合物作用4、8、12、16 h时的细胞毒性差异。结果:CV值与PEI质量浓度呈负相关,当PEI≥10μg/ml时,对细胞有明显的毒性;随FBS含量的增加CV值增加,当FBS含量为2.5%、5%、10%时PEI/DNA/FBS复合物CV值均在80%以上,且FBS含量为5%、10%时CV值均大于100%;且随作用时间的延长CV值基本维持稳定。结论:FBS可有效降低PEI基因传递系统的细胞毒性,经FBS修饰后的PEI基因传递系统不会随作用时间的延长而加大对细胞的损伤。     

自组装修饰在不同细胞系中对聚酰胺-胺介导的基因递送的影响

目的:通过自组装方法使用蛋白质分子修饰聚酰胺-胺树状大分子(PAMAM)与DNA形成的转染复合物,并考察其性质。方法:使用人血白蛋白(HSA)和表皮生长因子(EGF)修饰PAMAM-DNA转染复合物,测定复合物粒径及Zeta电位;通过DNA固缩程度测定试验和DNaseI消化试验考察修饰后复合物的稳定性;HEK 293T细胞与MCF-7细胞转染质粒,测定其荧光素酶表达水平;MTT检测修饰后复合物对细胞毒性的变化。结果:自组装修饰后的复合物的粒径无显著变化,Zeta电位显著降低,性质稳定;HSA修饰可显著提高复合物在HEK 293T和MCF-7细胞中的转染效率,EGF修饰仅显著提高复合物在MCF-7细胞中的转染效率;两种修饰都能降低PAMAM-DNA复合物对细胞的毒性。结论:自组装修饰方法快速、简便、有效,不影响PAMAM-DNA复合物的稳定性,并且能够实现提高转染效率、靶向递送、降低毒性等目的。     

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 biomimetic vectors with endosomal-escape agent enhancing gene transfection efficiency

Low cytotoxicity and high transfection efficiency are critical issues in designing current non-viral gene delivery vectors. In the present study, a novel biomimetic lipid-polycation copolymer, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-graft-poly(l-lysine)-block-poly(ethylene glycol) (DOPE-g-PLL-b-PEG) was first synthesized and the potential of this novel hybrid lipid-polycation as efficient gene vector was further evaluated. DOPE-g-PLL-b-PEG and DNA could self-assemble into lipid modified polyion complex micelles (LPCM) through electrostatic interactions. Compared with PEG-b-PLL/DNA polyion complex micelles (PIC), LPCM could protect DNA from plasma, nuclease degradation in vitro and showed lower cytotoxicity to HepG2 and HeLa cells (P<0.05). The results of transfection study in vitro indicated that LPCM exhibited higher gene expression than PIC. Especially, the corresponding LPCM displayed the highest transfection efficiency in HeLa cells (P<0.05) when DOPE grafting ratio reached up to 30%. These results suggested that LPCM could facilitate gene transfer in cultured cells and might alleviate the drawbacks of the conventional cationic vector/DNA complexes. As a novel hybrid lipid-polycation, DOPE-g-PLL-b-PEG was valuable to be evaluated for its further application as gene carrier in vivo.     

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.     

Low-molecular-weight polyethylenimine enhanced gene transfer by cationic cholesterol-based nanoparticle vector

Both polyethylenimine (PEI) polymers and cationic nanoparticles have been widely used for non-viral DNA transfection. Previously, we reported that cationic nanoparticles composed of cholesteryl-3beta-carboxyamidoethylene-N-hydroxyethylamine and Tween 80 (NP-OH) could deliver plasmid DNA (pDNA) with high transfection efficiency. To increase the transfection activity of NP-OH, we investigated the potential synergism of PEI and NP-OH for the transfection of DNA into human prostate tumor PC-3, human cervices tumor Hela, and human lung adenocarcinoma A549 cells. The transfection efficiency with low-molecular PEI (MW 600) was low, but that with a combination of NP-OH and PEI was higher than with NP-OH alone, being comparable to commercially available lipofectamine 2,000 and lipofectamine LTX, with very low cytotoxicity. Low-molecular weight PEI could not compact pDNA in size, but rather might help to dissociate pDNA from the complex and release pDNA from the endosome to cytoplasm by the proton sponge effect. Therefore, the combination of cationic cholesterol-based nanoparticles and a low-molecular PEI has potential as a non-viral DNA vector for gene delivery.     

Arginine-chitosan/DNA self-assemble nanoparticles for gene delivery: In vitro characteristics and transfection efficiency

Chitosan (Cs) is a natural cationic polysaccharide that has shown potential as non-viral vector for gene delivery because of its biocompatibility and low toxicity. However, chitosan used for gene delivery is limited due to its poor water solubility and low transfection efficiency. The purpose of this work was to prepare Arginine-chitosan (Arg-Cs)/DNA self-assemble nanoparticles (ACSNs), and determine their in vitro characteristics and transfection efficiency against HEK 293 and COS-7 cells. Our experimental results showed that the particle size and zeta potential of ACSNs prepared with different N/P ratios were 200-400nm and 0.23-12.25mV, respectively. The in vitro transfection efficiency of ACSNs showed dependence on pH of transfection medium, and the highest expression efficiency was obtained at pH 7.2. The transfection efficiency increased with the ratio of chitosan-amine/DNA phosphate (N/P ratio) from 1 to 5, and reached the highest level with the N/P ratio 5. Effect of plasmid dosage on the transfection efficiency showed the highest transfection efficiency was obtained at 4microg/well for HEK 293 cells and 6microg/well for COS-7 cells. The transfection efficiency of ACSNs was much higher than that of Cs/DNA self-assemble nanoparticles (CSNs). The average cell viability of ACSNs was over 90%. These results suggested that ACSNs could be a safe and effective non-viral vector for gene delivery.     

Novel cationic lipids possessing protonated cyclen and imidazolium salt for gene delivery

In this study, two novel protonated cyclen and imidazolium salt-containing cationic lipids differing only in their hydrophobic region (cholesterol or diosgenin) have been designed and synthesized for gene delivery. Cationic liposomes were easily prepared from each of these lipids individually or from the mixtures of each cationic lipid and dioleoylphosphatidyl ethanolamine (DOPE). Several studies including DLS, gel retardation assay, ethidium bromide intercalation assay, and TEM demonstrated that these amphiphilic molecules are able to bind and compact DNA into nanometer particles that could be used as non-viral gene delivery agents. Our results from in vitro transfection showed that in association with DOPE, two cationic lipids can induce effective gene transfection in HEK293 cells. Furthermore, the gene transfection efficiencies of two cationic lipids were dramatically increased in the presence of calcium ion (Ca²⁺). It is notable that the gene transfection abilities of two cationic lipids were maintained in the presence of 10% serum. Besides, different cytotoxicity was found for two lipoplexes. This study demonstrates that the title cationic lipids have large potential to be efficient non-viral gene vector.     

Chitosan-graft-spermine as a gene carrier in vitro and in vivo

Chitosan has been proposed as a non-viral gene carrier because of its biodegradable and biocompatible cationic polymeric properties. However, the transfection efficiency of chitosan-DNA complexes is still too low for clinical trials. To improve transfection efficiency, we prepared a chitosan-graft-spermine (CHI-g-SPE) copolymer by an imine reaction between periodate-oxidized chitosan and spermine. The CHI-g-SPE copolymer was complexed with plasmid DNA in various copolymer-DNA weight ratios, and the complexes were characterized. The CHI-g-SPE copolymer showed good DNA binding ability and high protection of DNA from nuclease attack. The CHI-g-SPE/DNA complexes had well-formed spherical shapes and a nanoscale size with homogenous size distribution. The CHI-g-SPE copolymer had low cytotoxicity and CHI-g-SPE/DNA complexes showed transfection efficiency that was enhanced over that of chitosan-DNA. Furthermore, aerosol delivery of CHI-g-SPE/GFP complexes showed higher GFP expression compared with chitosan/GFP complexes, without toxicity. Our results indicate that the CHI-g-SPE copolymer has potential as a gene carrier.     

Enhancing effects of galactosylated dendrimer/alpha-cyclodextrin conjugates on gene transfer efficiency

To improve in vitro gene transfer efficiency and/or achieve cell-specific gene delivery of polyamidoamine (PAMAM) starburst dendrimer (generation 2, G2) conjugate with alpha-cyclodextrin (alpha-CDE conjugate (G2)), we prepared alpha-CDE conjugate bearing galactose (Gal-alpha-CDE conjugates) with the various degrees of substitution of the galactose moiety (DSG) as a novel non-viral vector. The agarose gel electrophoretic studies revealed that Gal-alpha-CDE conjugates formed complexes with plasmid DNA (pDNA) and protected the degradation of pDNA by DNase I, but these effects impaired as the DSG value increased. Dendrimer and alpha-CDE conjugate exerted pDNA condensation through the complexation, but Gal-alpha-CDE conjugates did not. Gal-alpha-CDE conjugate (DSG 4) was found to have much higher gene transfer activity than dendrimer, alpha-CDE conjugate and Gal-alpha-CDE conjugates (DSG 8, 15) in HepG2, NIH3T3 and A549 cells, which are independent of the expression of the asialoglycoprotein receptor. Transfection activity of Gal-alpha-CDE conjugate (DSG 4) was insensitive to the existence of competitors (asialofetuin and galactose) and serum. In addition, no cytotoxicity after transfection of the complex of pDNA with Gal-alpha-CDE conjugate (DSG 4) was observed. These results suggest the potential use of Gal-alpha-CDE conjugate (DSG 4) as a non-viral vector in various cells.     

Cationic lipids containing cyclen and ammonium moieties as gene delivery vectors

In this study, two novel cationic lipids containing protonated cyclen and quaternary ammonium moieties were designed and synthesized as non-viral gene delivery vectors. The structures of the two lipids differ in their hydrophobic region (cholesterol or diosgenin). Cationic liposomes were easily prepared from the lipids individually or from the mixtures of each cationic lipid and dioleoylphosphatidylethanolamine. Several studies including DLS, gel retardation assay, and ethidium bromide intercalation assay suggest that these amphiphilic molecules are able to bind and compact DNA into nanometer particles which can be used as non-viral gene delivery agents. Our results from in vitro transfection show that in association with dioleoylphosphatidylethanolamine, two cationic lipids can induce effective gene transfection in human embryonic kidney 293 cells, although the gene transfection efficiencies of two cationic lipids were found to be lower than that of lipofectamine 2000(TM) . Besides, different cytotoxicity was found for two lipoplexes. This study demonstrates that the title cationic lipids have large potential to be efficient non-viral gene vectors.