Citotoxicidade de micropartículas de poliméricas mucoadesivas para liberação controlada de nistatina

Abstract

Polymeric microparticles (MPs) for controlled release of nystatin (N) were tested for cytototoxicity at their MIC50 and MIC80 against C. albicans in vitro, by the Alamar Blue® and MTT methods. The MPs obtained with the polymers Eudragit L-100® (E), Gantrez MS-955® (G) or the combination of both (EG), plus N 10% or 20%.Human keratinocytes (NOK) were proliferated in vitro and seeded (2 X 104 cells / mL), in DEMEM culture medium with 10% fetal bovine serum in 96-well plates and incubated for 24 h for semiconfluency. Then, the culture medium was replaced with new medium (200 µl) containing 2 µl of stock solutions, in DMSO, of the studied materials, to reach the MICs, conforming the experimental groups: GN1050, GN1080; GN2050, GN2080, EN1050, EN1080, EN2050, EN2080, EGN1050, EGN1080, EGN2050, EGN2080 (all MPs with N) and G0 and E0 (white MPs, without drug). The solvent was also evaluated at 1% (DMSO group). As a control group (C), cells were cultured that received only the culture medium (100% growth). Along with the treatments, 20 µl of the Alamar Blue® reagent were added to the wells containing the cells, which were incubated for 24 h. Fluorescence readings were taken at 6, 12 and 24 h (emission of 544 nm and 590 nm of transmission). In the MTT test, the reagent was inserted at the end of the incubation period with the treatments (24 h). For this, the cells were washed with PBS and received 200 µl of MTT at 0.5 mg / mL, sealed and returned to the oven for 4 h. The solution was discarded and the formazan crystals solubilized in 200 µl of methanol, for 5 min. The supernatant was read at 570 nm. The tests were carried out in triplicate and on three occasions. The fluorescence or absorbance measurements were calculated as a percentage of inhibition of metabolic activity compared to the control and classified qualitatively. The data were analyzed using the Shapiro-Wilk test, which showed a non normal distribution. The factors considered were material (independent, ten levels), MIC (dependent, two levels: 50/80) and incubation time (only Alamar Blue®, dependent, three levels). The complementary tests used were Kruskall-wallys and Friedman. Significant difference was considered with p˂0.05 values. The qualitative analysis of cytotoxicity of the different MPs showed that in the fluorescence readings, no MP showed moderate or highly cytotoxic behavior. In the quantitative analysis, there was no significant difference between the groups evaluated in the periods of time tested, showing a similar behavior for the MPs. In the absorbance readings, in the qualitative analysis, the MPs also showed a mild or non-cytotoxic behavior. In the quantitative analysis, the MPs showed a significant difference in relation to the control group, but not between them. MP GN1080 was found to be non-cytotoxic in all tests. No MP showed moderate or high cytotoxicity.