Mahato R I, Kim S W
Center for Controlled Chemical Delivery (CCCD), Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, Utah 84112, USA.
Bioconjug Chem. 2001 May-Jun;12(3):337-45. doi: 10.1021/bc000120w.
The use of biocompatible polymeric gene carriers may overcome the current problems associated with viral vectors in safety, immunogenicity, and mutagenesis. Nontoxic water-soluble lipopolymer (WSLP), poly(ethylenimine)-co-[N-(2-aminoethyl) ethyleneimin]-co-N-(N-cholesteryloxycarbonyl-(2-aminoethyl)ethylenimine) was synthesized using branched poly(ethylenimine) (PEI, mw 1800) and cholesteryl chloroformate. Following synthesis and purification, the structure and molecular weight of WSLP were confirmed by (1)H NMR and MADI-TOF mass spectrometry, respectively. The percentage of cholesterol conjugated to PEI was about 47%, and the average molecular weight of WSLP was approximately 2000 Da. WSLP/pDNA complexes were prepared at different N/P (nitrogen atoms of WSLP/phosphate of plasmid DNA) ratios and characterized in terms of particle size, zeta potential, osmolarity, surface morphology, and cytotoxicity. WSLP condensed plasmid DNA when N/P ratio reached 2.5/1 and no free DNA was detected at N/P ratio of 5/1 and above, as determined by agarose gel electrophoresis. The mean particle size was in the range of 25.9 to 148.5 nm and was dependent on N/P ratios. Atomic force microscopy (AFM) showed complete condensation of plasmid DNA with spherical particles of approximately 50 nm in diameter. WSLP/pDNA complexes or WSLP itself were nontoxic to CT-26 colon adenocarcinoma and 293 T human embryonic kidney transformed cells when formulated at the N/P ratio of 10/1 and below as determined by MTT assay. In contrast, PEI25000/pDNA complexes were toxic to these cells. Erythrocytes aggregated when incubated with PEI25000/pCMV-Luc complexes at high DNA concentrations, but there was little aggregation with WSLP/pCMV-Luc complexes. WSLP/pCMV-Luc complexes demonstrated higher transfection efficiency in both CT-26 and 293 T cells compared to PEI25000- or PEI1800-based formulations. WSLP/pCMV-Luc complexes are nontoxic and showed enhanced in vitro transfection. Thus, WSLP will be a suitable carrier for in vivo gene delivery.
使用生物相容性聚合物基因载体可以克服目前与病毒载体相关的安全性、免疫原性和诱变问题。使用支化聚乙烯亚胺（PEI，分子量1800）和胆固醇氯甲酸酯合成了无毒水溶性脂聚合物（WSLP），即聚（乙烯亚胺）-co-[N-（2-氨基乙基）乙烯亚胺]-co-N-（N-胆固醇氧基羰基-（2-氨基乙基）乙烯亚胺）。合成和纯化后，分别通过¹H NMR和基质辅助激光解吸电离飞行时间质谱法确认了WSLP的结构和分子量。与PEI共轭的胆固醇百分比约为47%，WSLP的平均分子量约为2000 Da。以不同的N/P（WSLP的氮原子/质粒DNA的磷酸根）比率制备WSLP/pDNA复合物，并从粒径、zeta电位、渗透压、表面形态和细胞毒性方面进行表征。通过琼脂糖凝胶电泳测定，当N/P比率达到2.5/1时，WSLP使质粒DNA凝聚，在N/P比率为5/1及以上时未检测到游离DNA。平均粒径在25.9至148.5 nm范围内，且取决于N/P比率。原子力显微镜（AFM）显示质粒DNA完全凝聚成直径约50 nm的球形颗粒。通过MTT测定法确定，当以10/1及以下的N/P比率配制时，WSLP/pDNA复合物或WSLP本身对CT-26结肠腺癌和293 T人胚胎肾转化细胞无毒。相比之下，PEI25000/pDNA复合物对这些细胞有毒。当在高DNA浓度下与PEI25000/pCMV-Luc复合物一起孵育时，红细胞会聚集，但与WSLP/pCMV-Luc复合物几乎没有聚集。与基于PEI25000或PEI1800的制剂相比，WSLP/pCMV-Luc复合物在CT-26和293 T细胞中均表现出更高的转染效率。WSLP/pCMV-Luc复合物无毒且在体外转染中表现出增强效果。因此，WSLP将是体内基因递送的合适载体。
