Formulation of nanostructured lipid carriers using eugenol and D-α-tocopherol succinate for the inhibition of bacterial efflux pumps to enhance delivery of ciprofloxacin.

Abstract

The continuously growing antibacterial resistance crisis is decreasing the availability of the antibiotics to treat bacterial infections. Bacteria develop additional mechanisms to survive lethal concentrations of antibiotics such as efflux pumps and biofilms. Novel drug delivery systems are urgently required to enhance antibiotic efficacy and overcome resistance. Furthermore, natural derivatives from medicinal plants have shown great potential as bacterial adjuvants to inhibit these mechanisms. Therefore, employing these compounds in the formulation of nanocarriers could restore antibiotic efficacy and overcome antibiotic resistance. Aim: The aim of this study was to explore the potential of ciprofloxacin-loaded nanostructured lipid carriers (CIP-NLCs) designed using D-α-tocopherol succinate (TS) and eugenol for enhancing antimicrobial activity and overcoming resistance mechanisms against methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa (P. aeruginosa). Methods: CIP-NLCs were prepared using hot homogenization/ultrasonication method. The particle size, polydispersity index (PDI) and zeta potential (ZP) of CIP-NLCs were determined using the dynamic light scattering technique. Transmission electron microscopy analysis was conducted to confirm particle size and visualize the morphology of CIP-NLCs. The entrapment efficiency (EE %) of CIP-NLCs was determined using the ultrafiltration method and was quantified using High-Performance Liquid Chromatography (HPLC). In vitro drug release of CIP-NLCs were conducted using the dialysis bag technique and CIP released was quantified using HPLC. Drug release kinetics were analysed using the DDSolver program. Haemocompatibility of CIP-NLCs was performed using sheep blood. The in vitro antibacterial activity of CIP-NLCs were determined using micro broth assay against Staphylococcus aureus (SA), Escherichia coli (E. coli), MRSA, and P. aeruginosa. Bacterial killing kinetics were performed against MRSA and P. aeruginosa using the plate colony counting method. MRSA and P. aeruginosa biofilm inhibition of CIP-NLCs was evaluated using microtiter method. DPPH scavenging was used to study the antioxidant activity of CIP-NLCs. In vivo antibacterial activity of MRSA was studied using the systemic MRSA infection on BALB/c mice. Results: CIP-NLCs had a particle size, PDI, ZP, and EE % of 147.4 ± 0.59 nm, 0.219 ± 0.009, -9.64 ± 2.22 mV, and 82.8 ± 0.39 %, respectively. The in vitro biosafety evaluation revealed CIP-NLCs as non- haemolytic. The in vitro drug release study showed a biphasic release of CIP from the CIP-LNCs for 48 hours at physiological pH (7.4). The in vitro antibacterial activity of CIP-NLCs (SA:0.195 μg/mL and MRSA:12.5 μg/mL) showed 2-fold lower minimum inhibitory concentration (MIC) values over bare ciprofloxacin (SA: 0.39 μg/mL and MRSA: 25 μg/mL) against MRSA, SA, whereas the MIC values of CIP-NLCs (E. coli: 0.048 μg/mL and P. aeruginosa: 0.097 μg/mL) were 4-fold lower CIP ( E. coli: 0.195 μg/mL and P. aeruginosa: 0.39 μg/mL) against E. coli, and P. aeruginosa. The bacterial-killing kinetic test showed 100% elimination of MRSA and P. aeruginosa within eight and one hour(s) of treatment with CIP-NLCs, respectively. Conversely, 100% elimination of MRSA and P. aeruginosa was shown within 24 and 12 hours of treatment with bare ciprofloxacin, respectively. CIP-NLCs eliminated 3-fold MRSA biofilm compared to bare ciprofloxacin, whereas 1.25-fold P. aeruginosa biofilms were eliminated. The efflux pump inhibition potential of CIP-NLCs was confirmed using cartwheel assay, which showed high fluorescence intensity on bacteria treated with CIP-NLCs. The DPPH scavenging assay of CIP-NLCs proved antioxidant activity equivalent to Vitamin C (ascorbic acid), which is reported to be a potent antioxidant. The in vivo systematic infection in BALB/c mice reduced MRSA infection in kidney, liver, and blood by 12.27-fold, 4.47-fold, and 1613-fold, respectively. Conclusion: CIP-NLCs designed using TS and eugenol could enhance the antimicrobial activity against MRSA and P. aeruginosa infections and inhibit efflux pumps associated with these bacteria. Therefore, the CIP-NLCs may serve as a promising tool for enhanced delivery of ciprofloxacin and treatment of bacterial infections.