Estudo das alterações da membrana celular de microrganismos por compostos de inclusão de clorexidina: beta-ciclodextrina em diferentes proporções molares usando Microscopia de Força Atômica e Microscopia Eletrônica de Varredura

Abstract

The formation of inclusion compounds of chlorhexidine for the controlled release of antimicrobials has shown decreasing in therapeutic concentrations effective. However, a lack of studies has been published about the mechanism which was inclusion compounds increase cellular permeability through the microorganisms membrane. Thus the objective of this work was to prepare and characterize inclusion compounds and study the mechanism of action of antimicrobial compounds for inclusion of chlorhexidine:beta-cyclodextrin (Cx: - cd) against the bacterial and fungal cells for Inclusion compounds have been in various prepared proportions molars by the method of freeze-dryed. The release of the drug was performed from samples for 10 days, bovine teeth were included in paraffin blocks, in the portion corresponding to the root dentin were opened windows of 4x 8mm was 75 mg gels based on hydroxy-metil-propyl cellulose at concentration 128 µg/mL. The gel the compounds tested in 128 were left in contact with the surface for 5 minutes after which the teeth were washed in distilled water and left in plastic pots containing 1 mL buffer solution which was withdrawn and replaced daily. The samples were subjected to UV-spectrophotometry of a visible at wavelength of 275 nm. The concentration of chlorhexidine released daily from the samples was obtained and the results submitted to the ANOVA statistical test. The Minimum Inhibitory Concentration (MIC) of Cx: - cd was determined to Aggregatibacter actynomicetemcomitans (A.a.) and Candida albicans (C.a.). The analysis of the mechanisms of action related to the membrane was made by scanning electron microscopy (SEM), atomic force microscopy (MFA) and transmission electron microscopy (MET) of microorganisms grown and the Technical SQM consisting (Sterol Quantification Method) in the extraction of ergosterol from fungal cell wall in contact with the 1µg / mL for compound. The MIC was 2 µg/ mL for Ca in all groups. For A.a and SEM could assess the cellular morphology and drugs. In the group of Cx was observed structures amorphous suggestive of bacterial cells surrounded by smaller particles similar to those compounds. The disorganization cell was significantly higher with the increase in the proportion of cyclodextrin compound, and that the molars 1:3 and 1:4 were observed large aggregates mixed of Cx bacterial remains in contrast with the group Cx where the microorganism presented a typical structure of coccus -bacilli characteristic or forming colonies. For the test of SQM groups 1:3 and 1:4 in the ergosterol was significantly decreased, with a range of more than 100% when compared to other substances tested. The MFA showed defects on the outcome of the solubilization of membrane lipids in the region of the areas of the drugs tested. Increasing the solubilization of the membranes areas is directly proportional to concentration of beta-cyclodextrin being more evident in the ratio 1:2. In conclusion the cyclodextrin increases the bondings with the cell membrane of the compounds for inclusion cd in nanoagregates possibly influenced: by the synergistic combination of factors such as electrostatic interactions, surface tension, thickness of the cell wall.