Nanostructured Lipid Carriers Containing Norfloxacin and 2-Aminothiophene Derivative Reduces Fluoroquinolone Resistance in Multidrug-Resistant Staphylococcus aureus Strains by Efflux Pump Inhibition

Background/Objectives: Multidrug resistance (MDR) remains a critical global public health concern, compromising the efficacy of currently available antibiotics. As the development of new antibiotics offers limited long-term solutions, alternative approaches such as efflux pump inhibition have gained attention. This study reports the development of nanostructured lipid carriers (NLCs) co-loaded with Norfloxacin (NOR) and the efflux pump inhibitor 2-amino-thiophen-6CN-Ethyl, to modulate NOR activity against resistant Staphylococcus aureus strains overexpressing efflux pump genes. Methods: NLCs were produced via the hot emulsion method followed by sonication. The formulations were characterized for encapsulation efficiency (EE%), particle size, polydispersity index (PDI), zeta potential, X-ray diffraction (XRD), infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), scanning electron microscopy (SEM), in vitro release kinetics, and stability. Antibacterial activity was evaluated against S. aureus 1199B and K2068 strains. Results: The NLC formulation containing norfloxacin and 6CN-Ethyl (NLC10NOR + 106CN) demonstrated high EE% for both compounds (99.50% for 6CN-Ethyl and 90.91% for NOR) and physicochemical stability over 60 days (particle size < 255 nm, PDI < 0.3, zeta potential < −20 mV). Structural analyses confirmed amorphization and effective encapsulation of the active constituents. Antibacterial assays showed that NLC10NOR + 106CN significantly increased NOR activity compared to the free drug and physical mixture; the effect in 1199B was notably superior to the NOR + CCCP (carbonyl cyanide m-chlorophenylhydrazone) combination. Conclusions: These findings highlight the potential of NLC-based co-delivery systems as innovative strategies to overcome bacterial resistance, particularly through efflux pump inhibition enhancing antibiotic efficacy.