Endocytic internalization mechanism of bioactive antibacterial nanoparticles by fibroblasts

Studying the uptake mechanism of photosensitizers is an important step in developing an ideal photosensitizer for use in photodynamic therapy (PDT). Understanding the uptake mechanism can help design novel photosensitizers that are selectively accumulated in the target tissue, with improved pharmacokinetics, and are dosed optimally to maximize the efficacy of the treatment. In our previous studies we synthesized and characterized the use of chitosan nanoparticles functionalized with rose bengal (CSRBnp) as a photosensitizer against dental biofilm. The aim of this study is to analyze the internalization mechanism and cellular proinflammatory activities of CSRBnps on fibroblasts. Fibroblasts (NIH 3T3) were incubated with chlorpromazine (5 µg/ml), nystatin (5 µg/ml), wortmannin (100 ng/ml) and at 4 °C for 30 min followed by CSRBnp (0.3 mg/ml). Cell viability (MTS assay), intracellular adenosine triphosphate content (Luminescence assay), cytokine expression (TNF-α) using ELISA and nitric oxide (NO) production by Griess reaction system were conducted at different time intervals (30 min, 1, 4, and 12 h). The internalization of CSRBnps was analyzed using live cell imaging confocal microscope with excitation wavelengths of 405 and 568 to detect nuclei (Hoechst 33,342) and CSRBnps respectively. CSRBnps and inhibitors at the applied concentrations were not cytotoxic. ATP content in chlorpromazine and without inhibitors groups were significantly lower than the control group at 12 h. All inhibitors showed significantly lower CSRBnps uptake compared to the control group at 30 min, 1 h, and 4 h time. Wortmannin resulted in the most significant inhibition of CSRBnps uptake as compared to chlorpromazine and nystatin (P < 0.05). TNF-α expression and NO production were not significant during the entire CSRBnps uptake. The results showed macropinocytosis was a dominant CSRBnps uptake mechanism by fibroblasts in the early stages and non-specific uptake pathways were activated after prolonged incubation time. CSRBnps uptake by fibroblasts was energy dependent and did not cause any proinflammatory response.