Three-dimensional microfluidic nanoprecipitation of zein-loaded polyethylene glycol poly(lactic-co-glycolic acid)...

Antimicrobial resistance calls for innovative drug delivery strategies that enhance natural antibacterial agents while minimizing off-target toxicity. Most cationic antimicrobial nanocarriers rely on electrostatic membrane binding as a prerequisite for bactericidal activity, which may compromise biocompatibility through nonspecific interactions with host cells. Here, we propose a nanoparticle-bacterium contact-independent sustained-release antibacterial approach using zein-loaded PEG-PLGA nanoparticles fabricated via three-dimensional (3D) microfluidic nanoprecipitation. The optimized nanoparticles (112.3 ± 7.1 nm, -35.4 ± 4.1 mV, 8.4 ± 1.0% drug loading) exhibited excellent colloidal stability, attenuated burst release, prolonged sustained release, preserved zein's intrinsic antioxidant activity, and supported favorable hemocompatibility and low cytotoxicity. Antibacterial assays revealed significantly enhanced activity against Staphylococcus aureus (S. aureus) (minimal inhibitory concentration, MIC 1.0 mg/mL, minimum bactericidal concentration, MBC 2.0 mg/mL) and Escherichia coli (E. coli) (MIC 1.5 mg/mL, MBC 3.0 mg/mL) versus free zein. Three independent lines of evidence supported the proposed nanoparticle-bacterium contact-independent sustained-release antibacterial mechanism. Scanning electron microscopy showed dose-dependent bacterial membrane rupture, while zeta potential measurements revealed no change in bacterial surface charge, indicating no detectable electrostatic adsorption. Propidium iodide uptake assays further demonstrated membrane permeabilization of approximately 50% at MIC and over 85% at MBC. Blank nanoparticles caused no membrane damage, suggesting that sustained drug liberation from the non-adherent carrier is a major contributor. This work provides evidence that potent antibacterial activity can occur without detectable electrostatic membrane adsorption under the tested conditions, offering a differentiated design principle for next-generation antibacterial nanomedicines.