In Vitro Cytotoxicity Studies

Cytotoxicity of cisplatin and cisplatin loaded silk reservoirs on human neuroblastoma KELLY cells (Sigma-Aldrich, St Louis, MO) were evaluated using AlamarBlue® assay (Invitrogen, Carlsbad, CA). RPMI 1640 with GlutaMAX (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum, 100 IU/ml penicillin and 100 μg/ml streptomycin was used to maintain the cells that were incubated at 37°C, in a 5% CO₂ atmosphere. Once the cells reach 80% confluence, they were passaged using Trypsin-EDTA 0.25% (Gibco, GrandIsland, NY). For evaluation of cisplatin cytotoxicity, the cells were exposed to varying concentrations of cisplatin in a range of 1ng/mL to 50μg/mL in culture medium described above (n=4) and LC50 (lethal concentration to kill 50% of the cells) was calculated. Toxicity of cisplatin reservoirs were tested by exposing the cells to in vitro release samples of each formulation in triplicate. For AlamarBlue® assay, 10,000 KELLY cells (P7) per well were seeded in 96-well plates and incubated overnight to attach and adhere the plates. Culture medium was removed and either cisplatin solutions in medium (100 μL, 1ng/mL to 50μg/mL) or medium plus the cisplatin release samples (1:1, 100 μL medium + 100 μL release sample) were added to the cells. The plates were incubated 48 hours in presence of cisplatin samples before adding 10% AlamarBlue® solution and incubating an additional 4 hours at 37°C as suggested by the manufacturer’s protocols. A plate reader (SpectraMax m2e, Molecular Devices) was used to read fluorescence with excitation wavelength of 570 nm and emission wavelength of 600 nm settings. One Way ANOVA was performed using IBM SPSS Statistics 22 Software (New York, USA) for statistical analysis (p<0.05).