A preliminary study on the interaction between Asn-Gly-Arg (NGR)-modified multifunctional nanoparticles and vascular epithelial cells

Liu, Chunxi; Liu, Tingxian; Yu, Xiaoyue; Gu, Yizhu

Nanoparticles enter cells through endocytic pathways, and selective inhibition of these various pathways was initiated to identify the relevant intracellular track adopted by TPIC for cell internalization. Hence, HUVEC were treated separately with each inhibitor against different internalization routes including CME, CvME, and macropinocytosis before cell incubation with the TPIC. Chlorpromazine is effective to inhibit CME. Treatment with chlorpromazine (10 μg/mL) for 30 min resulted in a 67.19% inhibition of TPIC uptake into HUVEC (as shown in Fig. 8 ), which indicated that CME was involved. Although the involvement of CME was previously mentioned 34 , other types of transcellular mechanism were also implicated in the previously reported intracellular traffic experiment of TPIC in HUVEC. Inhibition of the CvME was tested using genistein, a tyrosine kinase inhibitor, which was reported to inhibit the CvME in some virus 42 . After treated by genistein (1 μg/mL) for 30 min, the cell uptake of TPIC was significantly decreased by 74.2% (Fig. 8) , suggesting the internalization of TPIC was mainly through CvME. Cytochalasin D was reported to inhibit actin polymerization and membrane ruffling involved in macropinocytosis 43 . The cell uptake of TPIC into HUVEC was reduced by 13.86% (Fig. 8) after cytochalasin D treatment, implying that macropinocytosis was not a major cell uptake mechanism for TPIC under normal condition. Taken together, the endocytic inhibition study presented herein indicated that the internalization of TPIC in HUVEC was a combined process of CME and CvME. The relative ratio between CME and CvME was about 1:1.1. Nevertheless, the unique mechanism of caveolae-mediated endocytosis was indeed mainly involved in the internalization which was consistent with our original hypothesis.