Virology Unit, Department of Biomedical Sciences, Institute of Tropical Medicine of Antwerp, Nationalestraat 155, Antwerp, Belgium.
Mol Pharm. 2012 Oct 1;9(10):2942-9. doi: 10.1021/mp3003336. Epub 2012 Aug 28.
Lipoplexes, composed of Lipofectamine and mRNA encoding HIV Gag protein, were shown to be internalized by dendritic cells (DCs) and promote antigen presentation to stimulate HIV-specific T cell responses. Using confocal microscopy, we showed that one-third of fluorescently labeled mRNA containing lipoplexes are colocalized with late endosomes. We further investigated the effect of inhibitors, blocking phagocytosis, macropinocytosis, and clathrin- and caveolae-mediated endocytosis, on both the internalization of the lipoplexes by DCs and the transfection efficiency. We observed that chloropromazine had no effect on the cellular uptake or transfection efficiency, excluding the involvement of clathrin-mediated endocytosis. Cytochalasin D, inhibiting macropinocytosis and phagocystosis, strongly reduced internalization (50%) of the lipoplexes as well as protein expression (70%). Amiloride, which should specifically block macropinocytosis, induced only a modest reduction of uptake and transfection. Genistein and dynasore induced a strong reduction of on the level of protein expression (>70%), but not the overall uptake. Our results indicate that transfection-effective mRNA lipoplex internalization by DCs, i.e., uptake that results in protein expression, preferentially proceeds by macropinocytosis and/or phagocytosis.
脂质体复合物由 Lipofectamine 和编码 HIV Gag 蛋白的 mRNA 组成，被证明可以被树突状细胞 (DC) 内吞，并促进抗原呈递，以刺激 HIV 特异性 T 细胞反应。通过共聚焦显微镜，我们发现三分之一荧光标记的含有脂质体复合物的 mRNA 与晚期内体共定位。我们进一步研究了抑制剂对脂质体复合物被 DC 内化和转染效率的影响，这些抑制剂分别阻断吞噬作用、巨胞饮作用以及网格蛋白和 caveolae 介导的内吞作用。我们观察到氯丙嗪对细胞摄取或转染效率没有影响，排除了网格蛋白介导的内吞作用的参与。细胞松弛素 D 抑制巨胞饮作用和吞噬作用，强烈降低脂质体复合物的内化（50%）以及蛋白表达（70%）。阿米洛利应该特异性阻断巨胞饮作用，仅引起摄取和转染的适度减少。染料木黄酮和 dynasore 强烈降低蛋白表达水平（>70%），但不影响总体摄取。我们的结果表明，转染有效的 mRNA 脂质体复合物被 DC 内化，即导致蛋白表达的摄取，优先通过巨胞饮作用和/或吞噬作用进行。
