van der Aa M A E M, Huth U S, Häfele S Y, Schubert R, Oosting R S, Mastrobattista E, Hennink W E, Peschka-Süss R, Koning G A, Crommelin D J A
Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht University, P.O. Box 80082, 3508 TB, Utrecht, The Netherlands.
Pharm Res. 2007 Aug;24(8):1590-8. doi: 10.1007/s11095-007-9287-3. Epub 2007 Mar 24.
Knowledge about the uptake mechanism and subsequent intracellular routing of non-viral gene delivery systems is important for the development of more efficient carriers. In this study we compared two established cationic polymers pDMAEMA and PEI with regard to their transfection efficiency and mechanism of cellular uptake.
The effects of several inhibitors of particular cellular uptake routes on the uptake of polyplexes and subsequent gene expression in COS-7 cells were investigated using FACS and transfection. Moreover, cellular localization of fluorescently labeled polyplexes was assessed by spectral fluorescence microscopy.
Both pDMAEMA- and PEI-complexed DNA showed colocalization with fluorescently-labeled transferrin and cholera toxin after internalization by COS-7 cells, which indicates uptake via the clathrin- and caveolae-dependent pathways. Blocking either routes of uptake with specific inhibitors only resulted in a marginal decrease in polyplex uptake, which may suggest that uptake routes of polyplexes are interchangeable. Despite the marginal effect of inhibitors on polyplex internalization, blocking the caveolae-mediated uptake route resulted in an almost complete loss of polyplex-mediated gene expression, whereas gene expression was not negatively affected by blocking the clathrin-dependent route of uptake.
These results show the importance of caveolae-mediated uptake for successful gene expression and have implications for the rational design of non-viral gene delivery systems.
了解非病毒基因递送系统的摄取机制及其随后的细胞内转运过程，对于开发更高效的载体至关重要。在本研究中，我们比较了两种成熟的阳离子聚合物聚甲基丙烯酸二甲氨基乙酯（pDMAEMA）和聚乙烯亚胺（PEI）在转染效率和细胞摄取机制方面的差异。
使用荧光激活细胞分选术（FACS）和转染技术，研究了几种特定细胞摄取途径的抑制剂对COS-7细胞中多聚体摄取及随后基因表达的影响。此外，通过光谱荧光显微镜评估了荧光标记多聚体的细胞定位。
COS-7细胞内化后，pDMAEMA和PEI复合的DNA均与荧光标记的转铁蛋白和霍乱毒素共定位，这表明通过网格蛋白和小窝蛋白依赖途径摄取。用特异性抑制剂阻断任何一种摄取途径仅导致多聚体摄取略有下降，这可能表明多聚体的摄取途径是可互换的。尽管抑制剂对多聚体内化的影响较小，但阻断小窝蛋白介导的摄取途径几乎导致多聚体介导的基因表达完全丧失，而阻断网格蛋白依赖的摄取途径对基因表达没有负面影响。
这些结果表明小窝蛋白介导的摄取对于成功的基因表达很重要，并对非病毒基因递送系统的合理设计具有启示意义。
