Development, Characterization, and Antimicrobial Evaluation of Hybrid Nanoparticles (HNPs) Based on Phospholipids, Cholesterol, Colistin, and Chitosan Against Multidrug-Resistant Gram-Negative Bacteria

<b>Background:</b> Colistin, a lipopeptide antibiotic usually used as a last resort against multidrug-resistant bacterial strains, has also begun to address the challenge of antimicrobial resistance. <b>Objective</b>: this study evaluates whether hybrid nanoparticles (HNPs) composed of Phospholipon^® 90G, cholesterol, and colistin can enhance its effectiveness against resistant clinical isolates of <i>Klebsiella pneumoniae</i>, a clinically significant Gram-negative bacterium. <b>Methods:</b> HNPs were developed using the ethanol injection method and coated with chitosan through a layer-by-layer technique. HNP characterization included measurements of particle size, polydispersity index (PDI), and zeta potential, along with thermal (DSC) and spectrophotometric (FT-IR) analyses. Ultrafiltration and ATR-FTIR were employed to assess colistin's association and release efficiencies. The biological evaluation followed CLSI guidelines. <b>Results:</b> uncoated hybrid nanoparticles (U-HNP) and chitosan-coated hybrid nanoparticles (Ch-HNP) described monodisperse populations, with respective PDI values of ~0.124 and ~0.150, Z-averages of ~249 nm and ~250 nm, and zeta potential values of +17 mV and +20 mV. Colistin's association and release efficiencies were approximately 79% and 10%, respectively. Regarding antimicrobial activity, results showed that colistin as part of HNPs is poorly effective against this microorganism. However, in the most resistant strain, colistin activity increased slightly when the HNP was coated with chitosan. <b>Conclusions:</b> HNPs described high stability against disaggregation, limiting the colistin release and, therefore, affecting antimicrobial performance.