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

<b>Background/Objectives:</b> 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 <i>Staphylococcus aureus</i> strains overexpressing efflux pump genes. <b>Methods:</b> 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 <i>S. aureus</i> 1199B and K2068 strains. <b>Results:</b> 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 <i>1199B</i> was notably superior to the NOR + CCCP (carbonyl cyanide <i>m</i>-chlorophenylhydrazone) combination. <b>Conclusions:</b> 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.