Cellular Uptake and Cytotoxicity of Cy5.5-Labeled Nanoparticles

The cellular uptake of Cy5.5-CNPs was investigated in three types of normal cells, such as breast cancer cells (4T1), rat cardiomyocytes (H9C2), mouse fibroblasts (L929) and macrophages (Raw264.7). Each cell was incubated with Cy5.5-CNPs (100, 225 or 900 μg/ml) for 4 or 24 h. After treatment, cells were fixed with 4% paraformaldehyde for 15 min, and stained with 4′,6-diamidino-2-phenylindole (DAPI) for 10 min. The cellular uptake was observed using a Leica TCS SP8 confocal laser-scanning microscope (CLSM; Leica Microsystems GmbH; Wetzlar, Germany). Quantitative analyses of the fluorescence images were performed using ImageJ software (NIH, Bethesda, MD, USA). The cytotoxicity was assessed by the Cell Counting Kit-8 (CCK-8) assay. Briefly, 5 × 10⁴ 4T1, H9C2, L929 and Raw264.7 cells were seeded into 96-well cell culture plates. Then, each cell was treated with different concentration of Cy5.5-CNPs ranging from 0 to 900 μg/ml. After 24 h of incubation, the cells were further incubated with cell culture medium containing 10% of CCK-8 solution for 20 min. Finally, the cell viability was analyzed using a microplate reader (VERSAmaxTM; Molecular Devices Corp., USA) with a wavelength of 450 nm.