Mechanisms of Nanoparticle Internalization and Transport Across an Intestinal Epithelial Cell Model: Effect of Size and Surface Charge
Mechanisms of Nanoparticle Internalization and Transport Across an Intestinal Epithelial Cell Model: Effect of Size and Surface Charge
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DOI：
10.1021/mp500439c
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发表时间：
2014-12-01
影响因子：
4.9
通讯作者：
Stolnik, Snjezana
中科院分区：
文献类型：
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作者：
Bannunah, Azzah M.;Vllasaliu, Driton;Stolnik, Snjezana
本研究调查了纳米颗粒尺寸（50和100 nm）和表面电荷对它们与Caco-2单层相互作用的影响，作为肠上皮的模型，包括细胞内化途径和跨上皮转运水平。最初，毒性试验表明，细胞活力和细胞膜的完整性依赖于表面电荷和应用的质量，数量和总表面积的纳米粒子，在两个上皮细胞系，结肠癌Caco-2和气道Calu-3测试。这也确定了用于随后的细胞摄取实验的合适的纳米颗粒浓度。以低于半数最大有效浓度（EC 50）的剂量施用纳米颗粒揭示，尽管带正电荷的系统的内化水平更高，但带负电荷的纳米颗粒跨Caco-2细胞单层的转运效率（转运与细胞摄取的比率）与其带正电荷的对应物（相似大小）相比显著更高。因此，使用一组药理学抑制剂探测细胞内化途径，旨在确定转运效率的差异是否是由于不同的摄取和转运途径。通过抑制发动蛋白（通过dynasore）和微管网络（通过诺考达唑），证实了带正电荷和带负电荷的纳米颗粒的囊泡跨单层转运，这显著降低了两种纳米颗粒系统的转运。对于带正电荷的纳米颗粒，（分别为46%和37%）发生在网格蛋白途径抑制剂的存在下（氯丙嗪），巨胞饮抑制（42%;通过5-（N-乙基-N-异丙基）-阿米洛利实现），并且在胆固醇耗尽下（38%;通过甲基-β-环糊精），但仍然不受染料木素抑制脂筏相关摄取（小窝）的影响。相反，带负电荷的纳米颗粒的内化和转运的最显著的减少（分别为51%和48%）遵循脂筏相关途径的抑制（染料木素抑制小窝），但不受网格蛋白途径的抑制的显著影响。
This study investigated the effect of nanoparticle size (50 and 100 nm) and surface charge on their interaction with Caco-2 monolayers as a model of the intestinal epithelium, including cell internalization pathways and the level of transepithelial transport. Initially, toxicity assays showed that cell viability and cell membrane integrity were dependent on the surface charge and applied mass, number, and total surface area of nanoparticles, as tested in two epithelial cell lines, colon carcinoma Caco-2 and airway Calu-3. This also identified suitable nanoparticle concentrations for subsequent cell uptake experiments. Nanoparticle application at doses below half maximal effective concentration (EC50) revealed that the transport efficiency (ratio of transport to cell uptake) across Caco-2 cell monolayers is significantly higher for negatively charged nanoparticles compared to their positively charged counterparts (of similar size), despite the higher level of internalization of positively charged systems. Cell internalization pathways were hence probed using a panel of pharmacological inhibitors aiming to establish whether the discrepancy in transport efficiency is due to different uptake and transport pathways. Vesicular trans-monolayer transport for both positively and negatively charged nanoparticles was confirmed via inhibition of dynamin (by dynasore) and microtubule network (via nocodazole), which significantly reduced the transport of both nanoparticle systems. For positively charged nanoparticles a significant decrease in internalization and transport (46% and 37%, respectively) occurred in the presence of a clathrin pathway inhibitor (chlorpromazine), macropinocytosis inhibition (42%; achieved by 5-(N-ethyl-N-isopropyi)-amiloride), and under cholesterol depletion (38%; via methyl-beta-cyclodextrin), but remained unaffected by the inhibition of lipid raft associated uptake (caveolae) by genistein. On the contrary, the most prominent reduction in internalization and transport of negatively charged nanoparticles (51% and 48%, respectively) followed the inhibition of lipid raft-associated pathway (caveolae inhibition by genistein) but was not significantly affected by the inhibition of clathrin pathway.