Carreadores lipídicos nanoestruturados contendo norfloxaxino e derivado 2-amino-tiofênico visando a modulação da resistência antibiótica em staphylococcus aureus

Abstract

Bacterial resistance is one of the greatest global public health challenges, highlighting the need for new antibacterial agents. This study aimed to develop nanostructured lipid carriers containing an efflux pump inhibitor, the thiophenic derivative 6CN-Ethyl, and norfloxacin, in order to modulate the sensitivity to norfloxacin of resistant Staphylococcus aureus strains that overexpress efflux pumps. 6CN-Ethyl was reobtained by the gewald reaction. The lipid carriers were obtained by the hot emulsion sonication method and characterized in terms of particle diameter (TMP), polydispersity index (PDI), zeta potential (PZ), encapsulation efficiency (EE%), X-ray diffraction (XRD) and infrared spectroscopy (FTIR), as well as a long-term stability study. A method for the simultaneous quantification of 6CN-Ethyl and norfloxacin by High Performance Liquid Chromatography (HPLC) was validated in accordance with the ICH guidelines (Q2) for the drugs. The potentiating activity of the antibiotic was assessed by the Minimum Inhibitory Concentration (MIC). The optimized CLN formulation (carrier without drug) after freeze-drying had a TMP of 196,3 ±1.87, PDI =0.084 ±0.06 and PZ = -17.52 ±1.70, after 90 days it maintained a TMP = 232, 3 ±3.21, PDI = 0.271 ±0.09 and PZ at -25.11 ±0.47, while the carriers containing norfloxacin and 6CN-Ethyl (CLN10NOR+106CN) after lyophilization showed TMP = 244, 4 ±7.44, PDI = 0.319 ±0.00 and PZ = -34.09 ±0.84, on day 90 it showed TMP = 349,2 ±125.15, but with PDI = 0.381 ±0.11 and PZ = -23.88 ±1.45, meeting expectations in terms of maintaining parameters and without the presence of precipitates in the formulation compared to the formulation in suspension. The EE% showed a result of 99.50% for 6CN-Ethyl and 90.91% for norfloxacin. The XRD showed a degree of amorphization in the lipid carriers, corroborating that the active ingredients were encapsulated, and in the FTIR analysis the CLN10NOR+106CN did not show peaks related to the active ingredients, indicating the encapsulation of the drugs. A validated analytical method was developed for the simultaneous quantification of 6CN-Ethyl and norfloxacin, meeting the ICH criteria. The evaluation of the antimicrobial activity of the nanostructured lipid carrier showed that CLN10NOR+106CN promoted a significant reduction (P < 0.0001) in the MIC (MIC = 62.5 μg/mL) compared to the group containing the physical mixture 6CN-Ethyl+norfloxacin (MIC = 125 μg/mL), being efficient in inhibiting the NorA efflux pump, and modulating the sensitivity of the S. aureus 1199B strain to norfloxacin. aureus 1199B strain to norfloxacin. An equivalent result was observed against the S. aureus K2068 strain, where the MIC of CLN10NOR+106CN = 78.41 μg/mL), versus the MIC of the physical mixture = 125 μg/mL. However, this result was even more promising, since the non-encapsulated physical mixture was unable to inhibit the MepA efflux pump and modulate sensitivity to norfloxacin. Therefore, the incorporation of norfloxacin and 6CN-Ethyl into a nanostructured lipid carrier proved to modulate the activity of the active ingredients against resistant S aureus strains, making it a promising approach for modulating the action of antimicrobials and an option for combating the problem of bacterial resistance.