Internalization of Titanium Dioxide Nanoparticles Is Mediated by Actin-Dependent Reorganization and Clathrin- and Dynamin-Mediated Endocytosis in H9c2 Rat Cardiomyoblasts

Abstract

Titanium dioxide nanoparticles (TiO₂ NPs) are widely used for industrial and commercial applications. Once inside the body, they translocate into the bloodstream and reach different areas of the cardiovascular system including the heart, increasing the risk of developing cardiovascular diseases; consequently, the investigation of their interaction with cardiac cells is required. We previously showed that TiO₂ NPs are internalized by H9c2 rat cardiomyoblasts, and here, we examined the molecular mechanisms underlying this process. TiO₂ NPs internalization was evaluated by transmission electron microscopy, time-lapse microscopy, and flow cytometry. Changes in the actin cytoskeleton were studied by phalloidin staining. Endocytic uptake mechanisms for nanoparticles were probed with chemical inhibitors, whereas clathrin and dynamin expression was measured by Western blot. Cellular uptake of TiO₂ NPs occurred early after 30 min exposure, and large aggregates were observed after 1 h. Actin cytoskeleton reorganization included cell elongation plus lower density and stability of actin fibers. Cytochalasin-D inhibited TiO₂ NPs uptake, indicating actin-mediated internalization. Dynamin and clathrin levels increased early after TiO₂ NPs exposure, and their inhibition reduced nanoparticle uptake. Therefore, TiO₂ NPs internalization by H9c2 rat cardiomyoblasts involves actin cytoskeleton reorganization and clathrin/dynamin-mediated endocytosis.