Cytotoxicity Evaluation of HHC-Loaded Nanoparticles

The cytotoxicity of the blank NPs without HHC (CS-NPs), the unencapsulated HHC, and the HHC-CS-NPs against MDA-MB-231 breast cancer cells (ATCC, Manassas, VA, USA) was determined by the MTT assay, as previously reported by Muangnoi et al. [36 (link)], with modifications. Briefly, the breast cancer cells were cultured in a complete medium (CM) comprising DMEM supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) and 1% (v/v) penicillin/streptomycin. The cultured cells were maintained in a humidified atmosphere of 95:5 (v/v) air:CO₂ at 37 °C. The cells were seeded at a cell density of 1 × 10⁴ cells/well/200 μL in a standard 96-well plate, grown for 24 h to obtain a confluent monolayer, and used for further experiments.