Formulation of pH-responsive lipid-polymer hybrid nanoparticles for co-delivery and enhanced antibacterial activity of 18β-glycyrrhetinic acid and vancomycin against MRSA.
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Background: Due to the rise in antimicrobial resistance and the challenges accompanied by conventional antibiotic dosage forms, there is a need for developing drug delivery systems that enhance, protect and potentiate the current antibiotics in the market. Furthermore, natural derivatives from plants have proven to be potent antimicrobial agents. Therefore, their combination with antibiotics could be effective in overcoming antimicrobial resistance. Aim: The aim of this study was to co-deliver vancomycin and 18β-glycyrrhetinic acid via pH-responsive lipid-polymer hybrid nanoparticles (VCM-GAPAH-LPHNPs) formulated from polyallylamine and oleic acid (OA) and to explore its potential for enhanced activity and targeted delivery. Methods: Molecular dynamics and stability studies were used to determine the stability of the oil and water phases independently as well as VCM-GAPAH-LPHNPs as a complex. VCM-GAPAH-LPHNPs were prepared using the micro-emulsion technique. The size, polydispersity index and zeta potential of VCM-GAPAH-LPHNPs were determined using the dynamic light scattering technique. Transmission electron microscopy analysis was conducted to determine the morphology of VCM-GAPAH-LPHNPs. The entrapment efficiency and drug loading were determined using the ultrafiltration method. Differential scanning calorimetry was used to determine the thermal profiles of VCM-GAPAH-LPHNPs and its components. In vitro drug release studies were performed using the dialysis bag technique. Drug release kinetics were analysed using the DDSolver program. Cytotoxicity of VCM-GAPAH-LPHNPs were determined using the MTT assay. Haemolysis of VCM-GAPAH-LPHNPs were performed at different concentrations using sheep blood. In vitro antibacterial activity of VCM-GAPAH-LPHNPs were determined against SA and methicillin-resistant Staphylococcus aureus (MRSA) at pH 6 and 7.4. Time killing assay was performed using the plate colony count method. MRSA biofilm study was performed using the crystal violet assay. Results: Molecular dynamics indicated VCM-GAPAH-LPHNPs to be stable. VCM-GAPAH-LPHNPs were successfully prepared using the micro-emulsion technique. VCM-GAPAH-LPHNPs size, polydispersity index, zeta potential and encapsulation efficiency were found to be 198.4 ± 0.302 nm, 0.255 ± 0.003, - 3.8 ± 0.335 mV and 69.46 ± 2.52 % respectively. Thermal profiles of lyophilized VCM-GAPAH-LPHNPs showed transformation from crystallization to amorphous form. In vitro drug release studies revealed that VCM-GAPAH-LPHNPs released 60% of VCM after 24 h whereas bare VCM released 90% of VCM after 24 h hence VCM-GAPAH-LPHNPs showed sustained drug release compared to bare VCM. At pH 6 VCM-GAPAH-LPHNPs released 82% of VCM after 24 h whereas at pH 7.4 VCM-GAPAH-LPHNPs released 60% of VCM after 24 h indicating VCM-GAPAH-LPHNPs had a faster drug release at pH 6 compared to pH 7.4. The Weibull model was considered the best fit model for VCM-GAPAH-LPHNPs. The MTT assay revealed 75% > cell viability which indicated VCM-GAPAH-LPHNPs to be non-cytotoxic. At 0.5 mg/ml VCM-GAPAH-LPHNPs showed < 1% haemolysis. Stability studies at 4 °C and room temperature indicated VCM-GAPAH-LPHNPs to be stable. In vitro antibacterial activity against MRSA treated with VCM-GAPAH-LPHNPs demonstrated a 16-fold lower minimum inhibitory concentration than bare VCM at acidic conditions. The time-killing assay study at 12 h revealed that VCM-GAPAH-LPHNPs eliminated 100% of MRSA cells whereas bare VCM eliminated 55% of MRSA cells. The crystal violet assay analysis revealed VCM-GAPAH-LPHNPs ability to eliminate MRSA biofilms. Conclusion: VCM-GAPAH-LPHNPs could effectively treat MRSA infections at a faster rate as compared to bare VCM. Therefore, this novel pH-responsive LPHNPs may serve as a promising nanocarrier for enhancing antibiotic delivery and antibacterial activity.