阳离子膜融合脂质体的细胞转染机制及其保护作用

Study on the transfection mechanism of cationic fusogenic liposomes for antisense oligonucleotides and its role in protection against DNase Ⅰ

目的探讨阳离子膜融合脂质体(CFL)介导反义寡核苷酸(ASON)的细胞转染机制和抗核酸酶的作用。方法用流式细胞仪检测不同转染条件下,阳离子膜融合脂质体(CFL)介导反义寡核苷酸(ASON)细胞摄取的情况,变性聚丙烯酰胺凝胶电泳检测阳离子膜融合脂质体对反义寡核苷酸的保护作用。结果在转染4h后,细胞内总荧光强度已达峰,此时CFL/ASON细胞摄取效率约为游离ASON的30倍。在低能量(4℃)或有溶酶体酶抑制剂(氯喹)的条件下,细胞内的荧光强度未发生明显改变,可推测阳离子膜融合脂质体介导的细胞转染机制主要是融合而非内吞途径。变性聚丙烯酰胺凝胶电泳证实阳离子膜融合脂质体具有保护ASON的抵抗核酸酶降解的作用。结论阳离子膜融合脂质体有望成为ASON理想的传递系统。

OBJECTIVE To investigate the mechanism of cellular uptake based on cationic fusogenic liposomes(CFL) and the stability of CFL/ASON complexes in the presense of DNase Ⅰ. METHODS Transfection efficiency was estimated using 6-carboxy-fluorescence (FAM) labeled antisense oligonucleotides(ASON) by flow cytometric analysis and the mechanism of cellular uptake was investigated using temperature shifts and lysosomotropic agent. The ability of CFL in protecting ASON against DNase I in vitro was analyzed by 20% denaturing polyacryamide gel electrophoresis (PAGE). RESULTS The maximum fluorescence intensity was achieved after 4 hours, which was nearly 30 folds of that of free ASON. Cellular uptake efficiency was changed significantly under low energy and in the presense of lysosomotropic agent compared to that under normal transfection condition. These results indicated that CFL delivered ASON into cytoplasmic through membrane fusion, not via endocytosis.The DNase Ⅰ protection assay of CFL/ASON complexes showed that ASON loaded into CFL was hardly degraded within 4 hours while the naked ASON was obviously degraded within 10 minutes.CONCLUSION The preliminary experiment showed CFL is a promising formulation for ASON delivery.