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Published in Chad A. Mirkin, Spherical Nucleic Acids, 2020
Chung Hang J. Choi, Liangliang Hao, Suguna P. Narayan, Evelyn Auyeung, Chad A. Mirkin
Finally, we examined whether SNA endocytosis could be mediated by lipid-rafts. Based on confocal immunofluorescence data, Cy5–SNAs exhibit some colocalization with cholera toxin subunit B (CTX-B), caveolin-1 (CAV-1), and flotillin-1 (FLOT-1) (Fig. 19.3A), all resident proteins of lipid-rafts [8]. Pretreatment of C166 cells with pharmacological inhibitors that act according to various blocking mechanisms of cholesterol, including methyl-ß-cyclodextrin (which depletes and removes cholesterol) and filipin (which sequesters cholesterol) [9], decreased intracellular accumulation of Cy5–SNAs. Remarkably, for filipin-treated cells, Cy5–SNAs are only localized along the cell membrane (as evidenced by the rings of fluorescence) and rarely enter the cytosol (Fig. 19.3B). Based on ICP-MS data from independent cell samples, pretreatment with methyl-ß-cyclodextrin and filipin reduced the cellular association of SNAs by 49% and 70%, respectively. We also used TEM to monitor the interaction between SNAs and the cell membrane at the initial stage of endocytosis. After 5 min of incubation, the portion of the cell membrane that contains large amounts of SNAs reveals a darker intensity than portions without SNAs (Fig. 19.3C), possibly indicating the presence of lipid-raft microdomains that contain high concentrations of lipidated proteins or transmembrane proteins [10]. These data suggest that the pathway of endocytosis of SNAs is mediated by lipid-rafts and motivated us to further investigate the specific component of lipid-raft involved in the process. The apparent sensitivity of uptake to pretreatment of C166 cells by filipin implies the involvement of caveolae [11], characterized by flask-shaped 60–80 nm diameter pits that are abundantly found in the membrane of specific cell types, including endothelial cells [12]. Two common classes of endogenous cargo implicated for caveolae-mediated endocytosis are CTX-B (which colocalizes with SNAs in lipid-rafts) and glycosylphosphatidylinositol (GPI)-anchored proteins [13]. Indeed, pretreatment of cells with phosphatidylinositol–phospholipase C, which enzymatically cleaves GPI-anchored proteins from the cell membrane [14], reduced the uptake of SNAs by ~50%. By TEM imaging, after 5 min of incubation with C166 cells, individual SNAs are localized in flask-shaped invaginations of 50–100 nm in diameter at the cell membrane (Fig. 19.3D). To provide direct evidence of caveolae-mediated endocytosis, we generated stable T-cells with deficient expression levels of caveolin-1 (denoted “CAV1-DF”) by lentiviral transfection of C166 cells with pGIPZ plasmids that contain shRNA sequences for silencing the expression of the CAV1 gene. Compared with pGIPZ-control cells, CAV1-DF cells exhibited —60% reduction in endocytosis of SNAs (Fig. 19.3E). Thus, we conclude that lipid-rafts, or more specifically caveolae, significantly mediate the endocytosis of SNAs for C166 cells.
Transferrin/folate dual-targeting Pluronic F127/poly(lactic acid) polymersomes for effective anticancer drug delivery
Published in Journal of Biomaterials Science, Polymer Edition, 2022
Qing Xiao Wang, Xiang Chen, Zi Ling Li, Yan Chun Gong, Xiang Yuan Xiong
The cellular uptake levels showed a significant decrease after incubation with filipin, suggesting that the cellular uptake of Ps relied on caveolae-mediated endocytosis. The cholesterol-fixing properties of filipin could bind to 3β-hydroxysteroid and cholesterol on the caveolae, interfering with the structure and function of caveolae, inhibiting caveolae-protein-mediated endocytosis [54].