聚乙烯亚胺衍生物作为非病毒基因载体的研究进展

综述了通过结构修饰获得的各类聚乙烯亚胺（PEI）衍生物，如PEG修饰、小分子PEI共聚、连接配体或其它高分子骨架物等，及其在基因转染和细胞毒性方面的研究进展。     

PEG化聚乙烯亚胺作为非病毒基因给药载体的研究

采用不同比例的单甲氧基聚乙二醇-琥珀酰亚胺基丙酸盐（mPEG-SPA）对支链聚乙烯亚胺（PEI）进行修饰，考察了不同接枝率的mPEG-PEI与DNA复合物的粒径、ζ‘电位、包封率，及其对A549细胞的转染效率和细胞毒性。体外转染试验表明，低PEG接枝率的PEI转染效果与PEI相当，且细胞毒性大大降低。     

聚乙烯亚胺与壳聚糖在基因载体中的应用

基因治疗是攻克肿瘤最有潜力的方法之一,其关键是寻找可靠而有效的转基因载体。经过各种生物或化学修饰的聚乙烯亚胺和壳聚糖基因载体因转染效率高、细胞毒性低、靶向性强而成为研究热点。针对聚乙烯亚胺、壳聚糖及其衍生物应用于基因载体中存在的问题,将高转染效率的聚乙烯亚胺与高生物相容性的壳聚糖结合,可能得到兼具两者优点的新型基因载体。     

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.     

新型非病毒载体聚乙烯亚胺体内应用的研究进展

聚乙烯亚胺是1995年发现的重要的真核细胞基因转染载体，在非病毒载体的研究中处于重要的地位。聚乙烯亚胺体外研究已取得显著的进展，但在体内基因治疗的应用面临着一系列局限。本文综述了非病毒载体聚丙烯亚胺体内应用的障碍和克服办法，包括聚乙烯亚胺-DNA复合物的脂质体包裹或聚乙二醇化修饰等。     

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.     

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。     

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。     

基于聚乙烯亚胺的非病毒基因载体研究进展

如今,越来越多的非病毒基因载体被应用于基因传递中,聚乙烯亚胺（PEI）作为一种重要的阳离子聚合非病毒基因传递载体受到广泛的关注。针对如何克服其生物不可降解性和细胞毒性并进一步提高转染效率的问题,笔者通过文献综合介绍了一些新的策略,并具体对以PEI为基础的新型载体的合成、新型体外转染方法的建立以及体内应用等方面的研究进展进行综述。     

聚乙烯亚胺-胆固醇结合脂质微泡介导的基因转染系统

本文研究了聚乙烯亚胺-胆固醇阳离子聚合物(PEI-Chol)基因载体的性能及其结合中性脂质、聚乙二醇修饰脂质微泡介导的基因传递系统(微泡/PEI-Chol/DNA)的体外细胞基因转染效率。采用胆固醇甲酰氯与聚乙烯亚胺(Mw1800)通过酰胺化制得PEI-Chol,并运用IR、1H NMR和MADI-TOF-MS(基质辅助激光解吸附电离串联飞行时间质谱)对其结构和分子量进行测定,平均相对分子质量约为2000。采用反相挥发法制备DPPC、DSPE-PEG2000和全氟丙烷构建的粒径为2～8μm的脂质微泡,并运用自组装技术将PEI-Chol/DNA复合物与微泡融合,构建微泡/PEI-Chol/DNA(bubble/PEI-Chol/DNA)基因转染系统;以pEGFP-Cl(enhanced green fluorescent protein,增强型绿色荧光蛋白)为报告基因,用凝胶滞留试验考察PEI-Chol的DNA压缩能力,同时在A549和MCF-7两种细胞中比较以PEI-Chol为核心构建的各种载体系统的细胞毒性、抗血清沉淀作用以及细胞转染效率。结果发现,当N/P比值为4以上时,PEI-Chol能有效压缩DNA;...     

Comprehensive comparison of two new biodegradable gene carriers

Safety and high transfection efficiency are the prerequisites for an ideal gene vector. Polyethylenimine (PEI), especially PEI 25k (25 kDa), is a well-known cationic gene carrier with high transfection efficiency. However, the high toxicity, depended on its molecular weight, has limited its use as a potential gene carrier. In our research, for the purpose of reducing the toxicity and increasing the transfection efficiency and further to inspect where the degradation of these biodegradable polymers take place would be more beneficial, in cytoplasm or in endocytic vesicles, two kinds of degradable polymers were synthesized. One is an acid-liable PEI derivate (PEI-GA) which was cross-linked by PEI 2k with glutadialdehyde (GA) through imine linkages and the other one (PEI-TEG) was cross-linked PEI 2k with modified triethyleneglycol (TEG) through biscarbamate linkages and can be degraded at neutral environment. By the use of a series of assay methods both in vitro and in vivo, the results showed that PEI-TEG was found to be biodegradable at neutral environment and exhibit high transfection ability with low toxicity, which indicated its potential as a candidate carrier for gene therapy.     

Enhancing polysaccharide-mediated delivery of nucleic acids through functionalization with secondary and tertiary amines

Chitosan is a polysaccharide that has generated significant interest as a non-viral gene delivery vehicle due to its cationic and biocompatible characteristics. However, transfection efficiency of chitosan is significantly lower compared to other cationic gene delivery agents, e.g. polyethyleneimine (PEI), dendrimers or cationic lipids. This is primarily attributed to its minimal solubility and low buffering capacity at physiological pH leading to poor endosomal escape of the gene carrier and inefficient cytoplasmic decoupling of the complexed nucleic acid. Here we have developed an imidazole acetic acid (IAA)-modified chitosan to introduce secondary and tertiary amines to the polymer in order to improve its endosomal buffering and solubility. The modified polymer was characterized by ninhydrin and (1)H NMR assays for degree of modification, while buffering and solubility were analyzed by acid titration. Nanocomplex formation, studied at various polymer-nucleic acid ratios, showed an increase in particle zeta potential for chitosan-IAA, as well as an increase in the effective diameter. Up to 100-fold increase in transfection efficiency of pDNA was seen for chitosan-IAA as compared to native chitosan, nearly matching that of PEI. In addition, transfection of siRNA by the modified polymers showed efficient gene knockdown equivalent to commercially available siPORT Amines. Collectively, these results demonstrate the potential of the imidazole-grafted chitosan as a biocompatible and effective delivery vehicle for both pDNA and siRNA.     

Cross-linked polyethylenimine-hexametaphosphate nanoparticles to deliver nucleic acids therapeutics

Branched polyethylenimine (PEI; 25 kDa) as a nonviral vector exhibits high transfection efficiency and is a potential candidate for efficient gene delivery. However, the cytotoxicity of PEI limits its application in vivo. PEI was ionically interacted with hexametaphosphate, a compact molecule with high anionic charge density, to obtain nanoparticles (PEI-HMP). Nanoparticles were assessed for their efficacy in protecting complexed DNA against nucleases. The intracellular trafficking of nanoparticles was monitored by confocal microscopy. The cytotoxicity and transfection efficiency of PEI-HMP nanoparticles were evaluated in vitro. In vitro transfection efficiency of PEI-HMP (7.7%) was ～1.3- to 6.4-folds higher than that of the commercial reagents GenePORTER 2^TM, Fugene^TM, and Superfect^TM. Also, PEI-HMP (7.7%) delivered green fluorescent protein (GFP)-specific small interfering ribonucleic acid (siRNA) in culture cells leading to >80% suppression in GFP gene expression. PEI-HMP nanoparticles protected complexed DNA against DNase for at least 2 hours. A time-course uptake of PEI-HMP (7.7%) nanoparticles showed the internalization of nanoparticles inside the cell nucleus in 2 hours. Thus, PEI-HMP nanoparticles efficiently transfect cells with negligible cytotoxicity and show great promise as nonviral vectors for gene delivery.From the Clinical EditorBranched polyethylenimine (PEI) as a non-viral vector exhibits high transfection efficiency for gene delivery, but its cytotoxicity limits its applications. PEI hexametaphosphate nanoparticles (PEI-HMP) demonstrated a 1.3-6.4 folds higher transfection rate compared to commercial reagents. Overall, PEI-HMP nanoparticles efficiently transfect cells with negligible cytotoxicity and show great promise as non-viral vectors for gene delivery.     

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。     

Enhanced gene transfection efficiency by low-dose 25 kDa polyethylenimine by the assistance of 1.8 kDa polyethylenimine

Gene therapy is a promising strategy for treatments of various diseases. Efficient and safe introduction of therapeutic genes into targeted cells is essential to realize functions of the genes. High-molecular-weight polyethylenimines (HMW PEIs) including 25?kDa branched PEI and 22?kDa linear PEI are widely used for in vitro gene transfection. However, high-gene transfection efficiency is usually accompanied with high cytotoxicity, which hampers their further clinical study. On the contrary, low-molecular-weight polyethylenimines (LMW PEIs) such as 1.8?kDa PEI and 800?Da PEI show good biocompatibility but their applications are limited by the poor DNA condensation capability. In this study, we find that 1.8?kDa PEI, but not 800?Da PEI combined with low-dose 25?kDa PEI could significantly promote gene transfection with low cytotoxicity. Plasmids encoding enhanced green fluorescence protein (EGFP) were delivered by the combined PEI and gene transfection efficiency was evaluated by microscopic observation and flow cytometry. Parameters including concentrations of 25?kDa PEI and 1.8?kDa PEI and preparation ways were further optimized. This study presents an efficient and safe combined PEI-based non-viral gene delivery strategy with potential for in vivo applications.     

Poly(L-lactic acid)/polyethylenimine nanoparticles as plasmid DNA carriers

Non-viral vectors such as liposomes, polycations, and nanoparticles have been used as gene delivery systems. In this study, we prepared and characterized biodegradable poly(L-lactic acid) (PLA)/polyethylenimine (PEI) nanoparticles as gene carriers. pCMV/β-gal and pEGFP-C1 were utilized as model plasmid DNAs (pDNA). Nanoparticles were prepared using a double emulsion-solvent evaporation technique, and their pDNA binding capacity was assessed by agarose gel electrophoresis. Transfection was studied in HEK 293 and HeLa cell lines, and the transfection efficiencies were determined by β-galactosidase assay or flow cytometry. Three kinds of PLA/PEI systems were studied by varying the molecular weight of PEI. The PLA/PEI 25K system had a higher transfection efficiency than the PLA/PEI 0.8K or PLA/PEI 750K systems. The transfection efficiency was found to be dependent on the ratio of PLA/PEI nanoparticles to pDNA with an optimum ratio of 60:1 (w/w). The cytotoxicity was dependent on the quantity of PLA/PEI nanoparticles used, but it was comparable to that of commercial Lipofectin™. These results demonstrate the potential of PLA/PEI nanoparticles as gene carriers.     

Preparation and gene delivery of alkaline amino acids-based cationic liposomes

Cationic lipids 1, 2, and 3, based on hydrophobic cholesterol linked to L-lysine, L-histidine or L-arginine, respectively, were designed and tested as gene delivery vectors. Physicochemical and biological properties of all liposomes and lipoplexes were evaluated, including lipid-DNA interactions, size, morphology, zeta potential, acid-base buffering capability, protection of DNA from DNase I digestion, and cytotoxity. The efficiency of luciferase gene transfection of lipoplexes 1-3 was compared with that of commercial dioleoyl-trimethylammonium propane (DOTAP) and polyethyleneimine (PEI) in 293T cells and HepG2 cells with or without poly(ethylene glycol) PEG stabilizer. The complexation and protection of DNA of liposome 3 was the strongest among the three liposomes. The efficiency of gene transfection of liposomes 1-3 was two-to threefold higher than that of PEI and/or DOTAP in 293T cells. Liposomes 1 and 3 in PEG as stabilizer showed sixfold higher transfection efficiency than that of PEI and/or DOTAP, whereas liposome 2 showed very low transfection efficiency. In HepG2 cells, the transfection efficiency of all the cationic liposomes was much lower than that of DOTAP. In conclusion, lipids 1-3 were efficient and non-toxic gene vectors; the headgroup of cationic lipids and the stabilizer of liposome formulation had an important influence on gene transfection.     

Transfection efficiency and toxicity of polyethylenimine in differentiated Calu-3 and nondifferentiated COS-1 cell cultures

In the present study, we evaluated polyethylenimine (PEI) of different molecular weights (MWs) as a DNA complexing agent for its efficiency in transfecting nondifferentiated COS-1 (green monkey fibroblasts) and well-differentiated human submucosal airway epithelial cells (Calu-3). Studying the effect of particle size, zeta potential, presence of serum proteins or chloroquine, it appeared that transfection efficiency depends on the experimental conditions and not on the MW of the PEI used. Comparing transfection efficiencies in both cell lines, we found that PEI was 3 orders of magnitude more effective in COS-1 than in Calu-3 cells, because Calu-3 cells are differentiated and secrete mucins, which impose an additional barrier to gene delivery. Transfection efficiency was strongly correlated to PEI cytotoxicity. Also, some evidence for PEI-induced apoptosis in both cell lines was found. In conclusion, our results indicate that PEI is a useful vector for nonviral transfection in undifferentiated cell lines. However, results from studies in differentiated bronchial epithelial cells suggest that PEI has yet to be optimized for successful gene therapy of cystic fibrosis (CF).     

A novel non-viral gene transfer system, polycation liposomes.

To develop a novel non-viral gene transfer system, liposome was modified with cetylated polyethylenimine (PEI). This polycation liposome (PCL) showed remarkable transfection efficiency to COS-1 cells in vitro, in comparison with conventional cationic liposome preparations. Cytotoxicity against COS-1 cells and hemolytic activity of PCL or PCL-DNA complex were quite low in comparison with conventional cationic liposomes. Most conventional cationic liposomes require phosphatidylethanolamine or cholesterol as a component, though PCL did not. Egg yolk phosphatidylcholine- and dipalmitoylphosphatidylcholine-based PCL were as effective as dioleoylphosphatidylethanolamine-based PCL for gene transfer. Furthermore, the transfection efficacy of PCL was enhanced, instead of being diminished, in the presence of serum. Effective gene transfer was observed in all eight malignant and two normal line cells tested as well as in COS-1 cells. The effect of the molecular weight of PEI on PCL-mediated gene transfer was examined, and observed that PEIs with a molecular weight (Mr. Wt.) of 600 and 1800 Da were quite effective but PEI of 25,000 was far less effective. Effectiveness of gene transfer by using PCL was also observed in vivo: GFP and Luciferase genes were effectively expressed in mouse. We also discussed the mechanism of gene transfer by PCL. Taken together, PCL represents a new system useful for transfection and gene therapy.