Multifunctional Evaluation of Graphene Oxide–Sulfonamide Nanoconjugates: Antimicrobial, Antibiofilm, Cytocompatibility and Xenobiotic Metabolism Gene Expression Insight

The clinical utility of sulfonamide antibiotics is increasingly challenged by antimicrobial resistance and pharmacokinetic limitations. In this study, we synthesized five graphene oxide-sulfonamide nanoconjugates (GO-S1 to GO-S5) via covalent functionalization, comprehensively characterized them by IR, Raman, SEM, EDS, etc., and evaluated their antimicrobial, antibiofilm, cytotoxic, apoptotic, hemolytic and gene expression-modulating effects. While the free sulfonamides (S1-S5) exhibited superior antimicrobial activity in planktonic cultures (MICs as low as 19 μg/mL), their GO-functionalized counterparts demonstrated enhanced antibiofilm efficacy, particularly against <i>Pseudomonas aeruginosa</i> (MBIC: 78-312 μg/mL). Cytotoxicity studies using CellTiter assays and Incucyte live-cell imaging revealed low toxicity for all compounds below 250 μg/mL. Morphological and gene expression analyses indicated mild pro-apoptotic effects, predominantly via caspase-9 and caspase-7 activation, with minimal caspase-3 involvement. Hemolysis assays confirmed the improved blood compatibility of GO-Sx conjugates compared to GO alone. Furthermore, qRT-PCR analysis showed that GO-Sx modulated the expression of key xenobiotic metabolism genes (CYPs and NATs), highlighting potential pharmacokinetic implications. Among all tested formulations, GOS3, GOS4 and GOS5 emerged as the most promising candidates, balancing low cytotoxicity, high hemocompatibility and strong antibiofilm activity. These findings support the use of graphene oxide nanocarriers to enhance the therapeutic potential of sulfonamides, particularly in the context of biofilm-associated infections.