Development of a Novel Nanoparticle-Antibiotic Conjugate System for Overcoming Multidrug Resistance in Staphylococcus aureus Biofilm Infections: In Vitro and Ex Vivo Studies

Staphylococcus aureus biofilm infections represent one of the most intractable challenges in clinical microbiology, combining resistance to conventional antibiotics with physical impermeability of the biofilm matrix. Nanoparticle-antibiotic conjugate systems offer a rational strategy to overcome these barriers by enhancing antibiotic penetration, enabling controlled release, and providing synergistic biocidal activity. This study developed and evaluated a novel chitosan-vancomycin nanoparticle (CS-VAN-NP) conjugate system designed to penetrate S. aureus biofilms from MRSA clinical isolates obtained at the University of Benin Teaching Hospital, Edo State, South South Nigeria. CS-VAN-NPs were synthesized by ionic gelation and characterized by dynamic light scattering, zeta potential measurement, encapsulation efficiency, and scanning electron microscopy. In vitro anti-biofilm efficacy was assessed on 24-hour and 72-hour preformed MRSA biofilms using crystal violet quantification and colony-forming unit recovery. Ex vivo efficacy was evaluated using a porcine skin infected biofilm model. Reactive oxygen species generation and membrane integrity assays were performed to elucidate mechanisms. CS-VAN-NPs (mean diameter: 182 nm, zeta: +28 mV, encapsulation efficiency: 84%) demonstrated 8-fold lower minimum biofilm eradication concentration (MBEC) than free vancomycin against 72-hour biofilms. Ex vivo experiments showed 4.2 log reduction in viable biofilm cells compared to 1.7 log for free vancomycin. ROS generation was 3.4-fold higher for CS-VAN-NPs. Cytotoxicity in human fibroblast cell lines was within acceptable limits at therapeutic concentrations. This work constitutes an original nanotechnological contribution to MRSA biofilm infection management relevant to the Nigerian clinical context. Keywords: chitosan nanoparticles, vancomycin, MRSA biofilm, nanoparticle-antibiotic conjugate, anti-biofilm therapy.