Multiple Internalization Pathways of Polyelectrolyte Multilayer Capsules into Mammalian Cells
Multiple Internalization Pathways of Polyelectrolyte Multilayer Capsules into Mammalian Cells
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DOI：
10.1021/nn306032k
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发表时间：
2013-08-01
期刊：
影响因子：
17.1
通讯作者：
Rivera Gil, Pilar
中科院分区：
文献类型：
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作者：
Kastl, Lena;Sasse, Daniel;Rivera Gil, Pilar
聚电解质多层胶囊是一种具有巨大生物医学应用潜力的载体。为了设计生物相容性的、有效的治疗递送系统（如癌症），研究了PEM胶囊的内化途径（摄取和命运）。具体而言，进行了以下实验：(i)研究胶囊与已建立的内吞标记物的共定位，（ii）用药物/化学抑制剂关闭内吞途径，以及（iii）用共聚焦显微镜和电子显微镜表征和定量胶囊摄取。结果，胶囊与脂筏和嗜溶酶体共定位，但不与其他内吞小泡共定位。化学阻断剂对胞吞作用的化学干扰表明，PEM胶囊通过一种对静电相互作用微敏感的机制进入所研究的细胞系，不依赖于网格蛋白和小泡，强烈依赖于富含胆固醇的结构域和细胞器酸化。在胶囊摄取过程中，细胞的显微特征显示形成吞噬杯（囊泡）以吞噬胶囊，线粒体数量增加，并最终定位于核周细胞质。综合所有这些指标，我们得出结论，PEM胶囊内化通常是不同顺序机制的组合。最初，由于强静电相互作用的吸附机制决定了胶囊在细胞表面的稳定性。膜皱折和丝状足延伸是通过形成吞噬杯吞噬胶囊的原因。与脂筏结构域的共定位激活细胞启动脂筏介导的巨噬细胞增多症。内化囊泡是非常酸性的，只与吞噬溶酶体标记物共定位，排除了小窝蛋白介导的途径，这表明在吞噬作用后，胶囊被分类到异噬溶酶体。
Polyelectrolyte multilayer (PEM) capsules are carrier vehicles with great potential for biomedical applications. With the future aim of designing biocompatible, effective therapeutic delivery systems (e.g., for cancer), the pathway of internalization (uptake and fate) of PEM capsules was investigated. In particular the following experiments were performed: (i) the study of capsule co-localization with established endocytic markers, (ii) switching off endocytotic pathways with pharmaceutical/chemical inhibitors, and (iii) characterization and quantification of capsule uptake with confocal and electron microscopy. As result, capsules co-localized with lipid rafts and with phigolysosomes, but not with other endocytic Vesicles. Chemical interference of endocytosis with chemical blockers indicated that PEM capsules enter the investigated cell lines through a mechanism slightly sensitive to electrostatic interactions, independent of clathrin and caveolae, and strongly dependent on cholesterol-rich domains and organelle acidification. Microscopic characterization of cells during capsule uptake showed the formation of phagocytic cups (vesicles) to engulf the capsules, an increased number of mitochondria, and a final localization in the perinuclear cytoplasma. Combining all these indicators we conclude that PEM capsule internalization in general occurs as a combination of different sequential mechanisms. Initially, an adsorptive mechanism due to strong electrostatic interactions governs the stabilization of the capsules at the cell surface. Membrane ruffling and filopodia extensions are responsible for capsule engulfing through the formation of a phagocytic cup. Co-localization with lipid raft domains activates the cell to initiate a lipid-raft-mediated macropinocytosis. Internalization vesicles are very acidic and co-localize only with phagolysosome markers, excluding caveolin-mediated pathways and indicating that upon phagocytosis the capsules are sorted to heterophagolysosomes.