Design of polymeric nanoantibiotics: N-acetylcysteine-functionalized PLA-PEG nanoparticles for linezolid and pentamidine delivery

Polymeric nanoparticles bearing multifunctional features, including surface functionalization, hold great promise to address current limitations in antimicrobial drug delivery. Among bioactive ligands, N-acetylcysteine (NAC) and its derivatives have been investigated for their ability to interfere with biofilm formation and to disrupt mature biofilms. Here we propose novel N-acetylcysteine-decorated Nanoantibiotics based on poly(lactic acid)-poly(ethylene glycol) nanoparticles (PLA-PEG-NAC NPs) incorporating Linezolid (LNZ) and Pentamidine (Pent). NAC was selected to target methicillin-resistant Staphylococcus aureus (MRSA) biofilm and Pent for broadening the antimicrobial spectrum of LNZ. The PLA-PEG-NAC copolymer was synthesized through a multi-step pathway involving carbonyldiimidazole-mediated amidation, Steglich esterification, and copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC). Successful NAC grafting was confirmed by thiol-ene Michael addition using a crotonyl-substituted BODIPY dye. Nanoparticle’s formulation by dialysis achieved encapsulation efficiencies of 12% for LNZ and 20% for Pent, with sustained drug release profiles (78% and 25% cumulative release within 24 h, respectively). Despite the moderate encapsulation efficiency, the observed biological effects closely reflected the drug release profile and the achieved drug loading proved suitable for the aims of the present work. Cytotoxicity assays in Vero cells demonstrated no notable toxicity of PLA-PEG-NAC NPs, either in their drug-loaded or unloaded forms. Encapsulated LNZ preserved its antimicrobial activity, displaying Minimal Inhibitory and Bactericidal Concentrations (MIC and MBC) of 2 μg/mL and 16 μg/mL, respectively, against S. aureus, MRSA, S. epidermidis and Enterococcus faecium. Notably, sub-MIC concentrations of PLA-PEG-NAC@LNZ NPs reduced MRSA biofilm formation more effectively than free LNZ as demonstrated by biomass inhibition (62% vs 39%) and fluorescence microscopy. At 8 × MIC, PLA-PEG-NAC@LNZ NPs demonstrated activity against preformed MRSA biofilm either in the early (49% biofilm reduction) and late stages (30–41% biofilm reduction, depending on exposure time). Furthermore, the combined use of PLA-PEG-NAC@LNZ NPs and PLA-PEG-NAC@Pent NPs produced an additive antibacterial effect against Escherichia coli supporting the potential of Pent to sensitize Gram-negative bacteria to Gram-positive-targeting antibiotics such as LNZ.