Cytotoxicity assay
Cytotoxicity test is adapted from ISO STANDARD procedure 10993-5. Balb 3T3 clone A31 cell line (mouse fibroblast) was purchased from ATCC (CCL-163). The culture medium used was DMEM High Glucose with stable glutamine with pyruvate sodium (L0103-500) supplemented with 10% v/v of Foetal Bovine Serum (S1810-500) from Dutscher and 1% v/v penicillin-streptomycin (15140-122) from Gibco. Preculture was performed in 24 wells-plate in order to obtain 80% confluency after 24 hours. Two ways for cytotoxicity were studied for our samples. First, PDA-PAR30 NPs were added in solution at different dilutions onto cell layer (direct assay) for 24 hours. We realized a PDA-PAR30 dilution of 1:10 in cell culture medium (ratio 1 to 9 Tris/Cell culture medium) to keep a normal cells viability, so the minimal dilution tested is 1:10. Then to increase the dilution of PDA-PAR30 NPs, we first diluted in Tris Buffer and then keep the same ratio of 1 to 9 Tris/Cell culture medium. Secondly, we tested gelatin hydrogels with PDA-PAR30 NPs at different dilutions, they were incubated in cell medium for 24 hours in order to test release from hydrogels, in parallel of cells preculture. Gel-PDA-PAR30 were prepared as previously explained. These vehicles extract was added onto cells layer (extract assay) for 24 hours. All volumes were fixed at 1 mL. After exposure of samples onto cell layer (extract and direct assays) we performed MTT test. We also took bright field pictures at x10 before and after samples exposition to observe cells morphology. We compared all results to a growth control condition (cells cultured in cell culture medium without contact with hydrogel or NPs) defined as 100% viability. We also tested Tris buffer (in solution or in the hydrogel) as a negative control to check its non-cytotoxicity. We used sodium azide 3M (in solution or loaded in the hydrogel) as positive control. All samples were performed in triplicates.