The role of ganglioside GM1 in cellular internalization mechanisms of poly(amidoamine) dendrimers.
The role of ganglioside GM1 in cellular internalization mechanisms of poly(amidoamine) dendrimers.
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DOI：
10.1021/bc900029k
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发表时间：
2009-08-19
影响因子：
4.7
通讯作者：
Holl MM
中科院分区：
文献类型：
--
作者：
Hong S;Rattan R;Majoros IJ;Mullen DG;Peters JL;Shi X;Bielinska AU;Blanco L;Orr BG;Baker JR Jr;Holl MM
制备了具有胺、乙酰胺和羧酸酯端基的第7代（G7）聚（酰胺基胺）（PAMAM）树枝状聚合物，以研究聚合物/细胞膜的体外相互作用。在本研究中使用G7 PAMAM树枝状聚合物，因为与较小的较低代树枝状聚合物相比，较高代树枝状聚合物在细胞质膜的透化和在支撑的脂质双层中形成纳米级孔方面更有效。通过1H NMR、UV/维斯光谱、GPC、HPLC和CE表征基于树枝状聚合物的缀合物。观察到带正电荷的胺封端的G7树枝状聚合物（G7-NH 2）在200 nM浓度下内化到KB、Rat 2和C6细胞中。相反，带负电荷的G7羧酸盐封端的树枝状聚合物（G7-COOH）和中性乙酰胺封端的G7树枝状聚合物（G7-Ac）在类似条件下均不与细胞质膜缔合或内化。采用内吞标记物霍乱毒素亚基B（CT B）、转铁蛋白和GM 1-芘进行了一系列体外实验，以进一步研究树状聚合物内化到细胞中的机制。G7-NH 2树枝状聚合物与CTB共定位，然而，C6细胞的实验表明G7-NH 2的内化不是神经节苷脂GM 1依赖性的。因此，G7/CTB共定位归因于两个物种之间直接相互作用的人为因素。GM 1在膜中的存在也没有影响XTT测定细胞活力或乳酸脱氢酶（LDH）测定膜渗透性。
Generation 7 (G7) poly(amidoamine) (PAMAM) dendrimers with amine, acetamide, and carboxylate end groups were prepared to investigate polymer/cell membrane interactions in vitro. G7 PAMAM dendrimers were used in this study because higher generation of dendrimers are more effective in permeabilization of cell plasma membranes and in the formation of nanoscale holes in supported lipid bilayers than smaller, lower generation dendrimers. Dendrimer-based conjugates were characterized by 1H NMR, UV/Vis spectroscopy, GPC, HPLC, and CE. Positively charged amine-terminated G7 dendrimers (G7-NH2) were observed to internalize into KB, Rat2 and C6 cells at a 200 nM concentration. By way of contrast, neither negatively charged G7 carboxylate-terminated dendrimers (G7-COOH) nor neutral acetamide-terminated G7 dendrimers (G7-Ac) associated with the cell plasma membrane or internalized under similar conditions. A series of in vitro experiments employing endocytic markers cholera toxin subunit B (CTB), transferrin, and GM1-pyrene were performed to further investigate mechanisms of dendrimer internalization into cells. G7-NH2 dendrimers co-localized with CTB, however, experiments with C6 cells indicated that internalization of G7-NH2 was not ganglioside GM1 dependent. The G7/CTB co-localization was thus ascribed to an artifact of direct interaction between the two species. The presence of GM1 in the membrane also had no effect upon XTT assays of cell viability or lactate dehydrogenase (LDH) assays of membrane permeability.