Laboratory of General Biochemistry and Physical Pharmacy, Ghent University, Harelbekestraat 72, B-9000 Ghent, Belgium.
Biomaterials. 2011 Apr;32(11):3072-84. doi: 10.1016/j.biomaterials.2010.12.045. Epub 2011 Jan 22.
Extensive research is currently performed on designing safe and efficient non-viral carriers for gene delivery. To increase their efficiency, it is essential to have a thorough understanding of the mechanisms involved in cellular attachment, internalization and intracellular processing in target cells. In this work, we studied in vitro the cellular dynamics of polyplexes, composed of a newly developed bioreducible poly(amido amine) carrier, formed by polyaddition of N,N-cystamine bisacrylamide and 1-amino-4-butanol (p(CBA-ABOL)) on retinal pigment epithelium (RPE) cells, which are attractive targets for ocular gene therapy. We show that these net cationic p(CBA-ABOL)/DNA polyplexes require a charge-mediated attachment to the sulfate groups of cell surface heparan sulfate proteoglycans in order to be efficiently internalized. Secondly, we assessed the involvement of defined endocytic pathways in the internalization of the polyplexes in ARPE-19 cells by using a combination of endocytic inhibitors, RNAi depletion of endocytic proteins and live cell fluorescence colocalization microscopy. We found that the p(CBA-ABOL) polyplexes enter RPE cells both via flotillin-dependent endocytosis and a PAK1 dependent phagocytosis-like mechanism. The capacity of polyplexes to transfect cells was, however, primarily dependent on a flotillin-1-dependent endocytosis pathway.
目前，人们正在广泛研究设计安全有效的非病毒载体用于基因传递。为了提高它们的效率，深入了解目标细胞中细胞附着、内化和细胞内加工的机制至关重要。在这项工作中，我们研究了由新开发的生物还原型聚（酰胺胺）载体形成的复合物的细胞动力学，该载体通过 N，N-胱胺二丙烯酰胺和 1-氨基-4-丁醇（p（CBA-ABOL））的加成聚合作用在视网膜色素上皮（RPE）细胞上形成，这是眼部基因治疗的有吸引力的靶标。我们表明，这些带正电荷的 p（CBA-ABOL）/DNA 复合物需要通过电荷介导与细胞表面硫酸乙酰肝素蛋白聚糖的硫酸盐基团附着，以便有效地内化。其次，我们通过使用一系列内吞抑制剂、内吞蛋白的 RNAi 耗尽和活细胞荧光共定位显微镜，评估了定义的内吞途径在 ARPE-19 细胞中复合物内化的参与情况。我们发现 p（CBA-ABOL）复合物通过依赖于浮球素的内吞作用和依赖于 PAK1 的吞噬样机制进入 RPE 细胞。然而，复合物转染细胞的能力主要依赖于依赖于浮球素 1 的内吞作用途径。
