Oba Makoto, Fukushima Shigeto, Kanayama Naoki, Aoyagi Kazuhiro, Nishiyama Nobuhiro, Koyama Hiroyuki, Kataoka Kazunori
Department of Clinical Vascular Regeneration, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-8655, Japan.
Bioconjug Chem. 2007 Sep-Oct;18(5):1415-23. doi: 10.1021/bc0700133. Epub 2007 Jun 27.
A cyclic RGD peptide-conjugated block copolymer, cyclo[RGDfK(CX-)]-poly(ethylene glycol)-polylysine (c(RGDfK)-PEG-PLys), was synthesized from acetal-PEG-PLys under mild acidic conditions and spontaneously associated with plasmid DNA (pDNA) to form a polyplex micelle in aqueous solution. The cyclic RGD peptide recognizes alphavbeta3 and alphavbeta5 integrin receptors, which play a pivotal role in angiogenesis, vascular intima thickening, and the proliferation of malignant tumors. The c(RGDfK)-PEG-PLys/pDNA polyplex micelle showed a remarkably increased transfection efficiency (TE) compared to the PEG-PLys/pDNA polyplex micelle for the cultured HeLa cells possessing alphavbeta3 and alphavbeta5 integrins. On the other hand, in the transfection against the 293T cells possessing no alphavbeta3 and a few alphavbeta5 integrins, the TE of the c(RGDfK)-PEG-PLys/pDNA micelle showed no increase compared to the TE of the PEG-PLys/pDNA micelle. Flow cytometric analysis revealed a higher uptake of the c(RGDfK)-PEG-PLys/pDNA micelle than the PEG-PLys/pDNA micelle against HeLa cells, consistent with the transfection results. Furthermore, a confocal laser scanning microscopic observation revealed that the pDNA in the c(RGDfK)-PEG-PLys micelle preferentially accumulated in the perinuclear region of the HeLa cells within 3 h of incubation. No such fast and directed accumulation of pDNA to the perinuclear region was observed for the micelles without c(RGDfK) ligands. These results indicate that the increase in the TE induced by the introduction of the c(RGDfK) peptide ligand was due to an increase in cellular uptake as well as facilitated intracellular trafficking of micelles toward the perinuclear region via alphavbeta3 and alphavbeta5 integrin receptor-mediated endocytosis, suggesting that the cyclic RGD peptide-conjugated polyplex micelle has promising feasibility as a site-specifically targetable gene delivery system.
一种环状RGD肽共轭嵌段共聚物，环[RGDfK(CX-)]-聚乙二醇-聚赖氨酸（c(RGDfK)-PEG-PLys），在温和酸性条件下由缩醛-PEG-PLys合成，并在水溶液中与质粒DNA（pDNA）自发缔合形成多聚体胶束。环状RGD肽识别αvβ3和αvβ5整合素受体，它们在血管生成、血管内膜增厚和恶性肿瘤增殖中起关键作用。对于具有αvβ3和αvβ5整合素的培养HeLa细胞，与PEG-PLys/pDNA多聚体胶束相比，c(RGDfK)-PEG-PLys/pDNA多聚体胶束的转染效率（TE）显著提高。另一方面，在对不具有αvβ3且仅有少量αvβ5整合素的293T细胞进行转染时，与PEG-PLys/pDNA胶束的TE相比，c(RGDfK)-PEG-PLys/pDNA胶束的TE没有增加。流式细胞术分析显示，与PEG-PLys/pDNA胶束相比，c(RGDfK)-PEG-PLys/pDNA胶束对HeLa细胞的摄取更高，这与转染结果一致。此外，共聚焦激光扫描显微镜观察显示，在孵育3小时内c(RGDfK)-PEG-PLys胶束中的pDNA优先积累在HeLa细胞的核周区域。对于没有c(RGDfK)配体的胶束，未观察到pDNA如此快速且定向地积累到核周区域。这些结果表明，引入c(RGDfK)肽配体诱导的TE增加是由于细胞摄取增加以及通过αvβ3和αvβ5整合素受体介导的内吞作用促进胶束向核周区域的细胞内运输，这表明环状RGD肽共轭多聚体胶束作为一种位点特异性靶向基因递送系统具有良好的可行性。
