A novel non-viral vector for DNA delivery based on low molecular weight, branched polyethylenimine:: Effect of molecular weight on transfection efficiency and cytotoxicity
A novel non-viral vector for DNA delivery based on low molecular weight, branched polyethylenimine:: Effect of molecular weight on transfection efficiency and cytotoxicity
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DOI：
10.1023/a:1014861900478
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发表时间：
1999-08-01
影响因子：
3.7
通讯作者：
Kissel, T
中科院分区：
文献类型：
--
作者：
Fischer, D;Bieber, T;Kissel, T
目的.合成了低分子量支化聚乙烯亚胺（LMW-PEI），并研究了其作为基因载体的理化性质、细胞毒性和转染效率。采用分子排阻色谱结合激光散射和C-13-NMR光谱对氮丙啶酸催化开环聚合合成的LMW-PEI的结构进行了表征。体外细胞毒性作用通过LDH和MTT测定定量，并通过透射电子显微镜观察。以SV 40启动子驱动的荧光素酶为报告基因系统，在ECV 304细胞中监测转基因表达的潜力。合成了具有低支化度的LMW-PEI（Mw 11'900 D）作为用于基因递送的DNA载体。与高分子量聚乙烯亚胺（HMW-PEI; Mw 1 '616'000 D）相比，本文所述的聚合物显示出不同的支化程度，并且在宽浓度范围内具有较小的细胞毒性。如透射电子显微镜所示，LMW-PEI仅形成小的聚集体，其在血清存在下被不同细胞有效吸收，最可能是通过内吞途径。LMW-PEI产生通过报告基因荧光素酶的表达测量的转染效率，其比用HMW-PEI获得的转染效率高两个数量级。报告基因的表达是浓度依赖性的，但与脂质体转染不依赖于血清添加。本文所述的LMW-PEI是一种新型、高效、无细胞毒性的载体，具有良好的效率/毒性特征，可用于基因治疗应用。
Purpose. Low molecular weight branched polyethylenimine (LMW-PEI) was synthesized and studied as a DNA carrier for gene delivery with regard to physico-chemical properties, cytotoxicity, and transfection efficiency.Methods. The architecture of LMW-PEI, synthesized by acid catalyzed ring-opening polymerization of aziridine was characterized by size exclusion chromatography in combination with laser light scattering and C-13-NMR-spectroscopy. In vitro cytotoxic effects were quantified by LDH and MTT assay and visualized by transmission electron microscopy. The potential for transgene expression was monitored in ECV304 cells using luciferase driven by a SV40 promotor as reporter gene system.Results. LMW-PEI (Mw 11'900 D) with a low degree of branching was synthesized as a DNA carrier for gene delivery. In contrast to high molecular weight polyethylenimines (HMW-PEI; Mw 1'616'000 D), the polymer described here showed a different degree of branching and was less cytotoxic in a broad range of concentrations. As demonstrated by transmission electron microscopy the LMW-PEI formed only small aggregates which were efficiently taken up by different cells in the presence of serum, most likely by an endocytic pathway. LMW-PEI yielded transfection efficiencies measured via expression of the reporter gene luciferase which were up to two orders of magnitude higher than those obtained with HMW-PEI. The reporter gene expression was concentration dependent, but in contrast to lipofection independent of serum addition.Conclusions. The LMW-PEI described here is a new, highly efficient, and non-cytotoxic vector with a favorable efficiency/toxicity profile for gene therapeutic applications.