Desenvolvimento, caracterização e avaliação in vitro da atividade antibacteriana de formulações lipossomais

Abstract

The incorrect use of antimicrobials, one of the main causes of the emergence of resistance, may be associated with factors such as subtherapeutic doses and ineffectiveness of the selected drug, which is why the use of modified drug delivery systems in pharmaceutical preparations with great potential for modifying properties pharmacological effects of compounds for therapeutic purposes, such as liposomes, are candidates for a new therapy condition. This thesis aimed at the development, characterization and in vitro evaluation of the antibacterial activity of different liposomal formulations. In the preparation of the liposomes, the lipids dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidyl serine (DPPS) and cholesterol (COL), were used in the proportion 5: 3: 1 (DPPC: COL: DPPS). Analyzes of the vesicle diameter, Polydispersion Index and Zeta Potential were performed. Atomic Force Microscopy (MFA) and Scanning Electron Microscopy - SEM were used to determine the shape and morphology of the liposome surface. Microbiological tests were performed against standard and multiresistant bacteria (Staphylococcus aureus and Escherichia coli), and the Minimum Inhibitory Concentration-MIC was determined by the broth microdilution method. For the test of the modulating effect of the liposome without the incorporation with norfloxacin reference antibiotics and gentamicin, the fraction was used in a subinhibitory concentration (MIC / 8) and the drugs were microdiluted. In the assay on the efflux pump, the strains of S. aureus used were: SA-1199B, which expresses the NorA gene encoding the NorA efflux protein, and the wild strain SA-1199. The dynamic light scattering analysis for liposomes without the incorporation of the drug showed a diameter of 185.46 nm (unilamellar), polydispersity index of 0.48, Zeta potential of -40.9 mV. Microscopic analyzes of the liposomes without the incorporation of the drug showed a typical structure of affine systems, spherical and slightly flattened. The results of the evaluation of antibacterial activity for the determination of MIC of liposomes without the incorporation of the drug for all multidrug-resistant species and standards were ≥ 1024 μg / mL. It was possible to observe that the association of antibiotics to the liposome without incorporation of drugs (control), in the 24h and 48h times, potentiated the effect in decreasing the concentration of the microorganisms analyzed. The liposomes associated with antibiotics tested in the wild type strain SA 1199 and in the carrier strain of the pump 1199B, showed a better representation of growth inhibition compared to the wild type strain SA 1199. In the evaluation of PVOJ 56 (derived from quinoxaline) encapsulated in liposome, we obtained antagonistic results for strains of E. coli and S. aureus, where the drug (PJOV 56) has an isolated bacterial growth inhibition effect, but when encapsulated and compared with the control liposome, an antagonistic effect is observed. In the results for antibiotics when encapsulated, there were no antibacterial results. In the modulation of the liposome without incorporation with gentamicin, thus showing a much more efficient effect when compared to gentamicin alone with the bacteria. And, with the time of 48 hours, an antagonistic effect was seen in the modulation of norfloxacin with the liposome. Given the potential for inhibition of the efflux pump seen in the results, we highlight the potential development of new formulations that can play a pharmacological role without having their drugs removed from the target cell by the efflux pumps present in many resistant microorganisms. Therefore, according to the positive results for the synergistic effect of liposomes without the incorporation of the drug with antibiotics, the work follows the prospective approach to develop new formulations based on nanoparticles with antimicrobial properties.