Evaluating Endocytic Pathways of Nanoparticles

HeLa or MCF10A cells were seeded in a 48 well plate at a density of ~2×10⁴ cells/well 24 h prior to the experiment. On the following day, cells were washed one time with phosphate buffered saline (PBS) and incubated with following endocytic inhibitors in serum free media for 1 h at 37 °C: cytochalasin D (10 μg/mL), methyl-β-cyclodextrin (5 mg/mL), nocodazole (10 μg/mL), 3 mg/mL NaN₃/50 mM 2-deoxyglucose, wortmannin (100 ng/mL), pertussis toxin (100 ng/mL), U-73122 (4 μg/mL), polyinosinic acid (10 μg/mL), chlorpromazine (10 μg/mL) and dynasore (80 μM). The concentrations of the inhibitors were used as described in previous reports.^{[39 (link)]} After 1 h, media were replaced with fresh media containing the inhibitors with NPs (100 nM) and further incubated for 1 h at 37 °C. Untreated cells and cells treated with only NPs (no inhibitor) were used as negative and positive controls, respectively. After incubation, cells were washed three times with PBS and lysis buffer was added to each well. All lysed cell samples were then further processed for ICP-MS analysis (vide infra) to determine the intracellular amount of gold. All inhibitors were purchased from Sigma except for dynasore, chlorpromazine, and pertusis toxin that were obtained from Fisher Scientific. Particle uptake (%) was calculated based on the following equation:
$$\text{NP uptake}(\%)=(\text{NP uptake in presence of inhibitors}∕\text{NP uptake in absence of inhibitors})\times 100$$