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.

Investigation of Cytotoxicity, Oxidative Stress, and Inflammatory Responses of Tantalum Nanoparticles in THP-1-Derived Macrophages

“…Further, TiO 2 internalization is also mediated, to a lesser extent, by clathrin-dependent endocytosis and micropinocytosis. These results are supported by previous studies, which have shown that TiO 2 particle internalization is mediated by phagocytosis and endocytosis in glial cells [28], H9c2 rat cardiomyoblasts [27], and rodent macrophages [41,42]. The multiple uptake pathways of TiO 2 NPs may partially explain why they were internalized more efficiently and why they induce more robust cytotoxicity, oxidative stress, and inflammatory cytokines compared to Ta NPs under the same conditions.…”

“…Phagocytosis and endocytosis are responsible for the uptake and clearance of particles [27,40]. However, to date, the mechanistic nature of TiO 2 and Ta NP internalization 10 Mediators of Inflammation in human macrophages remains obscure.…”

“…THP-1 cells were seeded into 24-well cell culture plates at 2 × 10 5 cells per well with PMA overnight. Then, THP-1 cells were preincubated for 30min with the following uptake inhibitors: 25 μM cytochalasin D (CytD), used to disrupt actin-dependent phagocytosis [27]; 100 μM amiloride (Ame), applied as an inhibitor of micropinocytosis [28]; and 25μM genistein (Gen) and 25 μM chlorpromazine hydrochloride (Cpz), used to inhibit caveolae-and clathrinmediated endocytosis, respectively [29]. Cells were subsequently exposed to TiO 2 or Ta NPs at concentrations of 100 and 500μg/mL (0.5mL/well) for 1 and 6h.…”

Endocytosis of abiotic nanomaterials and nanobiovectors: Inhibition of membrane trafficking

“…Receptor ligands that are internalized through receptor-mediated CME include low-density lipoprotein, transferrin, growth factor, G-protein, tyrosine kinase receptors and insulin [36] . Nanomaterials such as Ag and TiO 2 nanoparticles are also internalized by the endocytic pathways mediated by clathrin [37] , [38] .…”

Immunomodulatory Effects of Lycium barbarum Polysaccharide Extract and Its Uptake Behaviors at the Cellular Level

“…The clathrin-dependent pathway includes three key steps: cell membrane infolding that depends on clathrin, vesicle formation that relies on dynamin, and endosome maturation. To investigate the endocytic pathway of LBP, four specific inhibitors for receptor-mediated cellular endocytosis pathway were used, including Chloropromazine (preventing the assembly and disassembly of clathrin lattices on cell membrane and endosomes) [40], Dynasore (inhibiting dynamin activity) [41], Cytochalasin D (disrupting actin filaments and inhibiting actin polymerization) [42], Amiloride (Epidermal sodium channel blocker) [43]. The flow cytometry assay (Figure 4d) showed that compared with the cellular uptake in the control group, all the inhibitors showed different degrees of inhibitory effect on the internalization of LBP, and the order of inhibition intensity was Chloropromazine > Cytochalasin D > Dynasore > Amiloride.…”