Establishing a chicken embryo model for studying infection control of a novel lipid nanoparticle.

The rise of antibiotic-resistant infections highlights the urgent need for alternative therapeutic strategies. Monoglyceride-based lipid nanoparticles (LNPs) offer both antimicrobial activity and the potential for controlled drug delivery. In this study, LNPs with diameters of 230-350 nm and ζ-potential of approximately -35 mV demonstrated high colloidal stability and strong antibacterial efficacy in vitro, achieving complete growth inhibition of Staphylococcus aureus (S. aureus) at 0.04 mg/mL and Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) at 0.9 mg/mL. To bridge the gap between in vitro and in vivo evaluation, the chicken chorioallantoic membrane (CAM) model was optimized as a physiologically relevant platform to study infection progression and treatment response. Incubation temperature and humidity, inoculum concentration, infection site, and imaging conditions were refined to ensure reproducibility and minimize embryo variability. LNP administration improved embryo survival: S. aureus from 35 % to 65 %, and S. Enteritidis from 40 % to 65 % at 48 h post-inoculation. Fluorescently labeled Escherichia coli JM105-GFP and Nile Red-tagged LNPs enabled real-time monitoring of infection and nanoparticle biodistribution using the IVIS Spectrum imaging system, with GFP intensity strongly correlating with bacterial density (R2 = 0.985). At concentrations above 109 CFU/mL, Escherichia coli JM105-GFP was readily visualized despite the eggshell's autofluorescence. This study establishes a reproducible CAM infection model for Gram-positive and Gram-negative bacteria and demonstrates the therapeutic potential of monoglyceride-based LNPs as localized antimicrobial agents.