新型非病毒载体聚乙烯亚胺介导基因转染参数的研究

聚乙烯亚胺是一种新型阳离子多聚物基因释放载体,可结合浓缩DNA,通过粘附内吞,进入细胞,使携带的质粒表达。本研究目的通过对聚乙烯亚胺各种转染参数的测定,为合成以聚乙烯亚胺为骨架的人工载体积累数据。方法:本研究利用聚乙烯亚胺分别结合含β半乳糖甙酶报告基因的pSVβ表达质粒、含绿色荧光蛋白报告基因的pEGFP质粒转染Cos-7细胞,通过组织化学法测定细胞抽提产物中β半乳糖甙酶的表达量、流式细胞仪法测定绿色荧光蛋白阳性细胞的表达比例,来测定影响转基因效率的各种参数。结果:在培养液中,6μg/ml聚乙烯亚胺作用24h,NIH 3T3细胞生存率为64.2%,7μg/ml聚乙烯亚胺细胞生存率为54.4%。电泳阻滞试验,聚乙烯亚胺在N/P比在3.0以上方可完全结合DNA。溶酶体抑制剂氯喹可增加聚乙烯亚胺的转染效率。培养液中的白蛋白、血清可降低转染效率。作为配制聚乙烯亚胺/DNA复合物的溶媒,HEPES缓冲液优于生理盐水,生理盐水优于5%葡萄糖。配制聚乙烯亚胺/DNA复合物的溶媒中加入Mg2+降低转染效率。聚乙烯亚胺转染效率优于SuperFectTM(断裂型树突状多聚物),而毒性低于SuperFectTM。结论:本研究首次报道了聚乙烯亚胺与DNA结合配伍的N/P比计算公式,N/P=7.75×b/c,这里b是PEI的质量(μg),c是质粒的质量(μg);PEI的工作终浓度应≤6μg/ml。通过体外细胞试验证明,聚乙烯亚胺是一种有效的真核细胞转染剂和人工合成基因载体的骨架。     

聚乙烯亚胺基因转染载体的研究进展

安全有效的基因载体是实现基因治疗的必要条件,由于聚乙烯亚胺易于合成和改性,可以方便地与DNA形成紧密的超分子复合物,保护DNA免受核酸酶的降解,并促进其进入细胞,从而成为非病毒基因载体研发热点。本文从提高聚乙烯亚胺作为基因载体的转染效率及降低其毒性方面综述了聚乙烯亚胺基因载体的研究进展。     

非病毒载体聚乙烯亚胺介导DNA转染的研究

目的研究新型非病毒类载体聚乙烯亚胺(PEI)的最适转染条件。方法分析各种因素对25 ku线性PEI转染效率的影响,优化实验条件。结果PEI与DNA的质量比≥2能保证DNA与PEI完全结合,最佳混合时间为10 min,但血清的存在将降低其结合效率。在一定范围内提高PEI与DNA的质量比有利于提高转染效率。293T细胞最适转染密度为培养面积的80%。结论确定了PEI转染细胞的最优条件。     

新型阳离子脂质体制备及其体外转基因特征

背景:阳离子脂质体及胶束、纳米粒等转基因载体是目前非病毒转基因载体领域研究热点;而目前市售转基因试剂盒价格昂贵,并且基本上都是进口商品,如果能够开发一种价廉,转基因效率高,具有自主知识产权的转基因试剂盒将展现广阔市场前景。目的:合成一种新型阳离子脂质复合物,并制备相应阳离子脂质体,对其体外转基因效率进行考察。方法:以1-乙基-3-(3-二甲胺丙基)碳二亚胺、胆固醇、丁二酸酐为原料合成阳离子脂质复合物,采用薄层色谱、红外光谱等方法对产物进行结构表征。然后用薄膜分散法,将适当比例的二油酰磷脂酰乙醇胺和阳离子脂质复合物混合制备成阳离子脂质体,并用其包封含绿色荧光蛋白报告基因的真核表达质粒pEGFPC1,对人宫颈癌Hela细胞株进行体外转染。结果与结论:通过两步法合成了一种全新的阳离子脂质复合物——胆固醇-丁二酸酯-1-乙基-3-(3-二甲胺丙基)碳二亚胺,薄层色谱及红外光谱结果表明合成了预期产物。薄膜分散法制得阳离子脂质体,其中以这种阳离子脂质复合物和二油酰磷脂酰乙醇胺摩尔比为1:1左右所制备的脂质体颗粒较为均匀、细小,形态基本呈圆形。体外对质粒包裹实验结果表明,阳离子脂质体与质粒比为5:3,可完全包封质粒。用该阳离子脂质体对Hela细胞进行转染,24h后,瞬时转染效率达35.6%。从初步实验结果看,所合成的阳离子脂质复合物结构总体与市售阳性成分结构类似,合成方法简单、廉价,体外对质粒DNA有很好包裹性能,细胞毒性低,然而体外对Hela细胞的转染效率还明显偏低。     

N-亚甲基磷酸化壳聚糖基因纳米粒子的体外细胞毒性及基因转染实验研究

目的 考察N-亚甲基磷酸化壳聚糖( NMPCS)基因纳米粒子的体外细胞毒性及基因转染效率.方法 采用均相反应法制备了NMPCS,用复凝聚法制备了NMPCS/DNA纳米粒子;通过MTT实验考察了NMPCS及其与DNA复合物对HeLa细胞的细胞毒性,以荧光索酶质粒为报告基因考察了NMPCS及NMPCS-CaZ+载体介导的体外基因转染效率.结果 NMPCS及其与DNA的复合物在体外表现出很小的细胞毒性,远远低于同等浓度时聚乙烯亚胺(PEI)的毒性.通过对壳聚糖进行亚甲基磷酸化修饰后,可大幅提高载体的基因转染效率.结论 N-亚甲基磷酸化壳聚糖有望成为一种新型、安全、高效的非病毒基因载体.     

聚乙烯亚胺-壳聚糖/DNA纳米粒的制备及介导体外转染关节软骨细胞

背景：壳聚糖对软骨细胞具有良好的生物相容性和可降解性,但存在基因转染效率偏低的缺陷。 目的：构建负载增强型绿色荧光蛋白基因的聚乙烯亚胺-壳聚糖/DNA纳米粒,检测其理化性能,以及体外对关节软骨细胞的基因转染效率。 方法：将聚乙烯亚胺共价连接于壳聚糖骨架上构建聚乙烯亚胺-壳聚糖复合物,再将聚乙烯亚胺-壳聚糖与负载增强型绿色荧光蛋白基因的质粒DNA以复凝聚法制成纳米粒,以扫描电镜检测纳米粒形态,Zeta电位粒度分析仪测定其粒径、表面电位;凝胶电泳阻滞实验观察聚乙烯亚胺-壳聚糖和质粒DNA的结合力。以聚乙烯亚胺-壳聚糖/DNA纳米粒、裸质粒DNA、脂质体2000及壳聚糖/DNA纳米粒转染体外培养的兔关节软骨细胞,流式细胞仪及荧光显微镜检测基因转染率;激光共聚焦显微镜检测DNA的入核情况。 结果与结论：聚乙烯亚胺-壳聚糖/DNA纳米粒多呈球形,粒径为(154.6±18.6) nm,表面Zeta电位为(24.68± 6.82) mV,可有效保护质粒DNA免受 DNaseⅠ的降解。体外转染实验证明聚乙烯亚胺-壳聚糖/DNA纳米粒能介导增强型绿色荧光蛋白基因转染关节软骨细胞并在细胞内表达绿色荧光蛋白,转染率达(23.80±1.74)%,转染率高于裸质粒DNA组及壳聚糖/DNA纳米粒组(P < 0.05),与脂质体2000组无显著差别(P=0.522)。表明聚乙烯亚胺-壳聚糖/DNA纳米粒能有效保护质粒DNA免受核酸酶降解,对关节软骨细胞有良好的基因转染能力。     

肝靶向DNA载体乳糖化多聚乙烯基亚胺及体外转染试验

目的 研制肝靶向的DNA载体,用于干扰素的肝细胞内表达或抗病毒反义核酸的肝靶问给药。方法 以还原胺化法制备了肝靶向基因载体乳糖化多聚乙烯基亚胺（L－PEI）,并进行了体外转染试验和稳定性试验。结果 靶向载体L－PEI与DNA形成的复合物,对受体阳性的Huh－7细胞具有较高的转染效率;靶向载体乳糖化白蛋白能够竞争拮抗这种转染。靶向载体／DNA复合物在大鼠血清中具有较好的稳定性。结论 乳糖化多聚乙烯基亚胺能将DNA选择的投放于肝细胞,具有较好的应用前景。     

长链非编码RNA-1700020I14Rik对高糖培养下小鼠肾系膜细胞纤维化的影响

目的探索长链非编码RNA表达质粒构建的方法,研究lncRNA-1700020I14Rik对肾系膜细胞纤维化的影响。方法从小鼠肾系膜细胞中提取RNA并反转录为c DNA作为模板,PCR法扩增目的片段,构建入载体pc DNA3.1(+)中。通过脂质体3000转染方法,将载体转染至高低糖培养的小鼠肾系膜细胞中。RT-q PCR法检测1700020I14Rik的表达水平;Western blot检测肾脏纤维化标记蛋白Col-4、FN及TGF-β1表达水平。结果1700020I14Rik在高糖培养的肾系膜细胞中显著性下调(P0.01)。与转染空质粒组相比,转染表达质粒的肾系膜细胞中1700020I14Rik表达水平升高(P0.01),且促使Col-4、FN以及TGF-β的表达水平下降(P0.05)。结论pc DNA3.1(+)-1700020I14Rik表达质粒能高表达1700020I14Rik,长链非编码RNA-1700020I14Rik可以缓解高糖培养下肾系膜细胞的纤维化发展。     

PSMA-CAR慢病毒表达载体的包装研究

目的前列腺特异性膜抗原-嵌合抗原受体(PSMA-CAR)慢病毒表达载体的包装方法研究。方法采用慢病毒三质粒病毒包装系统(Del8.9+VSVG、PsAX2+OG)及包装细胞(HEK-293T、HEK-293FT)在转染试剂[聚乙烯亚胺(PEI)、Lipofectamine 2000、磷酸钙]的介导下,包装慢病毒颗粒。通过检测转染效率筛选最佳包装细胞、最佳包装质粒组合、最佳转染试剂和剂量。结果 HEK-293FT细胞的转染效率高于HEK-293T细胞的转染效率。PsAX2+OG组合的转染效率高于Del8.9+VSVG组合的转染效率。PEI方法转染时,包装质粒相同,空载体的转染效率明显要高很多;目的质粒相同,采用包装质粒为PsAX2+OG组合时,转染效率更高;数据统计表明,质粒总量∶PEI的比例在1∶9时转染效率更高。Lipofectamine 2000方法转染时,包装质粒和包膜质粒相同,空载体的转染效率更高;目的质粒和包装、包膜质粒相同,质粒总量∶Lipofectamine 2000的比例在1.0∶2.0和1.0∶2.5时转染效率显著高于其他两组比例。磷酸钙转染法时,随着加入的磷酸钙的增多,转染效率呈递减趋势。结论采用包装质粒PsAX2和包膜质粒OG时转染效率相对更高。质粒总量和加入的PEI量比例在1∶9时转染效率更高;质粒总量和加入的Lipofectamine 2000量比例在1.0∶2.0和1.0∶2.5时转染效率更高。PEI包装法和Lipofectamine 2000包装法转染效率相当,Lipofectamine 2000包装法相对高一些,但在考虑包装成本的前提下后续大量包装PSMA-CAR慢病毒颗粒时,应选择PEI包装法。     

聚乙烯亚胺用作基因转染的研究进展

 基因载体问题以及与载体相关的免疫反应、细胞毒性和安全性等问题，是基因治疗领域亟待解决的关键问题之一。聚乙烯亚胺（PEI）是阳离子聚合物非病毒载体的典型代表^[1]，是一种很早便为人所知并予以应用的有机大分子。目前，以 PEI 阳离子聚合物与 DNA 形成的 PEI/DNA 复合物已成为非病毒基因载体的研究热点。本文就近年来这方面的研究进展作简要综述。 1 PEI的特性 PEI 每 3 个原子中有 1 个胺基原子，使其具有较高正电荷密度。根据 pH 与质子作用之间的对应关系可得出：自由 PEI 的结构在生理条件下有 1/6 至 1/5 胺基发生质子化反应，从而使溶酶体肿胀破裂，从而起到“质子海绵”作用，使 PEI/DNA 复合物得以释放入胞质，很大程度上减少了 DNA 在吞噬泡内富集并进而被降解的作用，因而可以提高转染效率^[2]。     

A low-toxic and efficient gene vector: carboxymethyl dextran-graft-polyethylenimine

In this study, the low molecular weight branched polyethylenimine (PEI) (800 Da PEI) was grafted to the biodegradable and biocompatible carboxymethyl dextran (CMD) to obtain CMD-g-PEI, and the plasmid DNA was complexed with CMD-g-PEI polycation to form the polyion complex. The acid-base titration profile showed that the CMD-g-PEI had endosomal disruption capacity, and the agarose gel electrophoresis suggested that the CMD-g-PEI could condense DNA efficiently. The transfection efficiency of CMD-g-PEI/DNA complexes was measured by luciferase and green fluorescent protein assay in HEK293 cells. The results revealed that the transfection efficiency of CMD-g-PEI at the N/P ratios over 30-70 was higher than or comparable to that of the 25 kDa PEI at optimal ratio (N/P = 10). The cytotoxicity of CMD-g-PEI as well as 25 kDa PEI was evaluated in NIH3T3 and HEK293 cells, respectively, and it was also found that the cytotoxicity of CMD-g-PEI was much lower than that of 25 kDa PEI. The resulted CMD-g-PEI with high transfection efficiency and low cytotoxicity have promising applications when used as gene vector.     

Transfection efficiency and intracellular fate of polycation liposomes combined with protamine

Endosomal escape and nuclear entry are the two main barriers for successful non-viral gene delivery. To overcome these barriers, polyethylenimine (PEI) with a molecular weight of 800, conjugated to cholesterol (PEI 800-Chol) was synthesized to prepare polycation liposomes (PCLs). The effect of cationic polymers on transfection was investigated by pre-condensing DNA with these before using PCLs. The complexes of PCLs and protamine/DNA nanoparticles (PLPD) were introduced as efficient gene transfer vectors, and displayed obviously higher transfection efficiency (approximately 39-fold) than PCLs/DNA complexes. Kinetics of transgene expression indicated PLPD complexes could be maintained at a relatively high level over 72?h. The order of protamine addition affected the transfection of PLPD complexes. Pre-mixed and post-mixed PLPD complexes improved transfection, although the former was preferred. Distribution of FAM-labeled oligonucleotides (FAM-ODN) in cells mediated by PCLs were throughout the whole cell, while most FAM-ODN were nuclear when transfected with PLPD. These results suggest that the protonation of PEI and membrane destabilization of 1, 2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) increases the endosomal escape ability of vectors. The addition of protamine, containing nuclear localization signals, improved nuclear entry of DNA. The internalization pathways for PCLs involved multiple processes and were possibly dependent on cell lines.     

Combinational therapy of ischemic brain stroke by delivery of heme oxygenase-1 gene and dexamethasone

Combinational therapies using genes and drugs are promising therapeutic strategies for various diseases. In this research, a co-delivery carrier of dexamethasone and plasmid DNA (pDNA) was developed by conjugation of dexamethasone to polyethylenimine (2 kDa, PEI2k) for combinational therapy of ischemic brain. Dynamic light scattering, atomic force microscopy and flow cytometry studies showed that the pDNA/dexamethasone-conjugated PEI2k (PEI2k-Dexa) complex was 150 nm in size and was taken up by cells more easily than PEI2k-Dexa only. The tumor necrosis factor-α (TNF-α) level was decreased more efficiently by pDNA/PEI2k-Dexa complex than dexamethasone only in hypoxia activated Raw 264.7 macrophage cells, suggesting that pDNA/PEI2k-Dexa complex increased the delivery efficiency and therapeutic effect of dexamethasone. In in vitro transfection assay, PEI2k-Dexa had higher transfection efficiency than PEI2k and lipofectamine. However, the simple mixture of PEI2k and dexamethasone did not show this effect, suggesting that the conjugation of dexamethasone to polyethylenimine increased DNA delivery efficiency of PEI2k. To evaluate the effects of combinational therapy in vivo, pDNA/PEI2k-Dexa complex was applied to a transient focal ischemia animal model. At 24 h after the injection, mean infarction volume and the TNF-α level were reduced more efficiently in the pDNA/PEI2k-Dexa injection group, compared with the control, pDNA/PEI2k, or dexamethasone injection group. The infarction volume and inflammatory cytokines were further decreased by delivery of pSV-HO-1 using PEI2k-Dexa. Magnetic resonance imaging and microPET studies confirmed the therapeutic effect of pSV-HO-1/PEI2k-Dexa complex at 10 days after the injection. Therefore, pSV-HO-1/PEI2k-Dexa complexes may be useful in combinational therapy for ischemic diseases such as stroke.     

Characterization of lactoferrin as a targeting ligand for nonviral gene delivery to airway epithelial cells

In this study lactoferrin (Lf) was investigated as a targeting ligand for receptor-mediated gene delivery to human bronchial epithelial cells. A high number of lactoferrin receptors (LfRs) were detected on bronchial epithelial (BEAS-2B), but not on alveolar epithelial (A549) cells by fluorescence microscopy and FACS measurements, suggesting potential targeting selectivity for bronchial epithelial cells. Molecular conjugates with ratios of Lf to branched polyethylenimine 25 kDa (PEI) ranging from 4:1 to 1:40 (mol/mol) were synthesized and analyzed for complexation of plasmid DNA (pDNA), transfection efficiency, and cytotoxicity. Whereas particle size increased with the degree of Lf coupling from 45 to 225 nm, surface charge was not significantly influenced. Transfection studies on BEAS-2B cells revealed that Lf-PEI 1:20 exhibited the highest luciferase gene expression which was 5-fold higher at an N/P ratio (molar ratio of PEI nitrogen to pDNA phosphate) of 4 than PEI and could be inhibited by an excess of free Lf. With A549 cells, no significant enhancement in transfection efficiency between Lf-PEI/pDNA and PEI/pDNA complexes could be observed. Increasing the degree of Lf coupling to PEI resulted in reduced transfection efficiency in both alveolar and bronchial epithelial cells. Cell viability assays resulted in significantly lower cellular toxicity of Lf-PEI/pDNA compared with PEI/pDNA complexes. We suggest that Lf represents a potent targeting ligand for receptor-mediated gene delivery to bronchial epithelial cells and might be a promising candidate for lung gene transfer in vivo.     

A biodegradable poly(ester amine) based on polycaprolactone and polyethylenimine as a gene carrier

The aim of research was to develop and optimize delivery systems for plasmid DNA (pDNA) based on biodegradable polymers, in particular, poly(ester amine)s (PEAs), suitable for non-viral gene therapy. Poly(ester amine)s were successfully synthesized by Michael addition reaction between polycaprolactone (PCL) diacrylate and low molecular weight polyethylenimine (PEI). PEA/DNA complexes showed effective and stable DNA condensation with the particle sizes below 200nm, implicating its potential for intracellular delivery. PEAs showed controlled degradation and were essentially non-toxic in all three cells (293T: Human kidney carcinoma, HepG2: Human hepatoblastoma and HeLa: Human cervix epithelial carcinoma cell lines) at higher doses in contrast to PEI 25K. PEAs also revealed much higher transfection efficiencies in three cell lines as compared to PEI 25K. The highest reporter gene expression was observed for PCL/PEI-1.2 (MW 1200) complex having transfection efficiency 15-25 folds higher than PEI 25K in vitro. Also PEA/DNA complexes successfully transfected cells in vivo after aerosol administration than PEI 25K. These PEAs can be used as most efficient polymeric vectors which provide a versatile platform for further investigation of structure property relationship along with the controlled degradation, significant low cytotoxicity and high transfection efficiency.     

Amphiphilic and biodegradable methoxy polyethylene glycol-block-(polycaprolactone-graft-poly(2-(dimethylamino)ethyl methacrylate)) as an effective gene carrier

A group of amphiphilic cationic polymers, methoxy polyethylene glycol-block-(polycaprolactone-graft-poly(2-(dimethylamino)ethyl methacrylate)) (PECD), were synthesized by combining ring-opening polymerization (ROP) and atom transfer radical polymerization (ATRP) methods to form nanoparticles (NPs). The structures of these amphiphilic cationic polymers were characterized by (1)H NMR measurement. The PECD NPs have hydrophobic cores covered with hydrophilic PEG and cationic PDMAEMA chains. These self-assembly nanoparticles were characterized by dynamic light scattering (DLS) technique. PECD NPs can effectively condense DNA to form compact complexes of the size 65-160 nm suitable for gene delivery. The in vitro gene transfection studies of HeLa and HepG2 cells show that PECD NPs have better transfection efficiency compared to polyethylenimine (PEI) and Lipofectamine 2000 at low dose (N/P = 5). The cytotoxicity result shows that PECD NPs/DNA complexes at the optimal N/P ratio for transfection have comparable toxicity with PEI and Lipofectamine. These results indicate that PECD NPs have a great potential to be used as efficient polymeric carriers for gene transfection.     

α,β-Poly(l-aspartate-graft-PEI): A pseudo-branched PEI as a gene carrier with low toxicity and high transfection efficiency

The aim of this research is to develop a novel branched polyethylenimine (PEI)-like polycation as a potential gene carrier with high gene transfection efficiency and low toxicity. In particular, α,β-poly(l-aspartate-graft-PEI) (Asp-g-PEI), a pseudo-branched PEI, was synthesized by the ring-opening reaction of poly(l-succinimide) (PSI) with low molecular weight branched PEI (LMW PEI, MW = 600 and 1200). Good plasmid condensation and protection ability of Asp-g-PEI were confirmed by agarose gel electrophoresis assay. Asp-g-PEI/DNA complexes showed high positive zeta potential, narrow size distribution, good dispersity and a compact spherical shape with size below 250 nm when the N/P ratio was above 5, suggesting that they can be endocytosed. Cytotoxicity of Asp-g-PEI/DNA complexes was rather lower than that of PEI25K/DNA complexes, especially at high N/P ratio. The most efficient gene transfection of Asp-g-PEI/DNA complexes was similar or a little higher than that of PEI25K in 293T, HeLa and HepG2 cell lines, while almost 4 and 6 times higher than that of parent PEI1200 and PEI600, respectively, in HeLa cell line; as the molecular weight of parent PEI in Asp-g-PEI was increased from 600 to 1200, the transfection efficiency showed a tendency to decrease. The mechanism of Asp-g-PEI-mediated gene transfection was attributed to the “proton sponge effect” due to PEI in the copolymer.     

Gene transfection of hyperbranched PEI grafted by hydrophobic amino acid segment PBLG

The complex copolymer of hyperbranched polyethylenimine (PEI) with hydrophobic poly(gamma-benzyl L-glutamate) segment (PBLG) at their chain ends was synthesized. This water-soluble copolymer PEI-PBLG (PP) was characterized for DNA complexation (gel retardation assay, particle size, DNA release and DNase I protection), cell viability and in vitro transfection efficiency. The experiments showed that PP can effectively condense pDNA into particles. Size measurement of the complexes particles indicated that PP/DNA tended to form smaller nanoparticles than those of PEI/DNA, which was caused by the hydrophobic PBLG segments compressing the PP/DNA complex particles in aqueous solution. The representative average size of PP/DNA complex prepared using plasmid DNA (pEGFP-N1, pDNA) was about 96 nm. The condensed pDNA in the PP/pDNA complexes was significantly protected from enzymatic degradation by DNase I. Cytotoxicity studies by MTT colorimetric assays suggested that the PP had much lower toxicity than PEI. The in vitro transfection efficiency of PP/pDNA complexes improved a lot in HeLa cells, Vero cells and 293T cells as compared to that of PEI-25K by the expression of Green Fluorescent Protein (GFP) as determined by flow cytometry. Thus, the water-soluble PP copolymer showed considerable potential as carriers for gene delivery.     

Starburst low-molecular weight polyethylenimine for efficient gene delivery

Low-molecular weight polyethylenimine (LMW PEI) shows the advantage of low-cytotoxicity, but has been inefficient in gene delivery as a consequence of the low-charge density. A number of previous studies employed the approach of crosslinking to solve this problem. In this study, a starburst LMW PEI gene vector has been developed. It has a polyamidoamine (PAMAM) core conjugated with a shell composed of LWM PEI and polyethylene glycol (PEG), that is PAMAM-PEI-PEG. Plasmid DNA (pEGFP-N1) and human cervix epithelial carcinoma (HeLa) cells were used in the study. The results showed that the starburst LMW PEI could effectively condense DNA at N/P above 5. The polyplexes had a size of about 500 nm and a nearly neutral surface because of the PEG shielding effect. This novel gene vector is able to maintain the low-cytotoxicity of LMW PEI, whereas its transfection efficiency was significantly improved.     

Cationic star polymers consisting of alpha-cyclodextrin core and oligoethylenimine arms as nonviral gene delivery vectors

A series of novel cationic star polymers were synthesized by conjugating multiple oligoethylenimine (OEI) arms onto an alpha-cyclodextrin (alpha-CD) core as nonviral gene delivery vectors. The molecular structures of the alpha-CD-OEI star polymers, which contained linear or branched OEI arms with different chain lengths ranging from 1 to 14 ethylenimine units, were characterized by using size exclusion chromatography, 13C and 1H NMR, and elemental analysis. The alpha-CD-OEI star polymers were studied in terms of their DNA binding capability, formation of nanoparticles with plasmid DNA (pDNA), cytotoxicity, and gene transfection in cultured cells. All the alpha-CD-OEI star polymers could inhibit the migration of pDNA on agarose gel through formation of complexes with pDNA, and the complexes formed nanoparticles with sizes ranging from 100 to 200 nm at N/P ratios of 8 or higher. The star polymers displayed much lower in vitro cytotoxicity than that of branched polyethylenimine (PEI) of molecular weight 25K. The alpha-CD-OEI star polymers showed excellent gene transfection efficiency in HEK293 and Cos7 cells. Generally, the transfection efficiency increased with an increase in the OEI arm length. The star polymers with longer and branched OEI arms showed higher transfection efficiency. The best one of the star polymers for gene delivery showed excellent in vitro transfection efficiency that was comparable to or even higher than that of branched PEI (25K). The novel alpha-CD-OEI star polymers with OEI arms of different chain lengths and chain architectures can be promising new nonviral gene delivery vectors with low cytotoxicity and high gene transfection efficiency for future gene therapy applications.