*2.6. Cytotoxicity of Ib*−*M1 Peptides and Ib*−*M1/Alg*−*Chi Bioconjugate*

The indirect cytotoxicity of the membranes against Vero cell cultures was analyzed using the MTT (3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) reduction method. VERO cells at a concentration of 3 <sup>×</sup> <sup>10</sup><sup>4</sup> cells/mL were cultured in 96-well, flat-bottom plates with DMEM medium supplemented with 10% inactivated fetal bovine serum and incubated at 37 ◦C in a 5% CO<sup>2</sup> atmosphere until reaching a confluence greater than 90%. Cells were then exposed to serial 1:2 dilutions of peptide Ib−M1 at concentrations in the range of 200 µM to 0.78 µM, with concentrations of the MIC and 1/2 MIC in the bioconjugates and the Alg−Chi NPs at 0.4 mg/mL for 24 h. Subsequently, 20 µL of MTT in PBS pH 7.4 was added to each well at a concentration of 5 mg/mL and incubated for 4 more hours under the same conditions described above, after which time the culture medium was removed from the wells, and 100 µL/well of DMSO was added to solubilize the formazan crystals, which were measured by the absorbances obtained in the spectrophotometric readings at 570 nm to calculate the percentage of cytotoxicity of the compounds. Cells in culture medium receiving no treatment were employed as a negative control.
