Gentamicin–rhDNase liquid crystalline nanoparticles for pathogen-dependent antibiofilm activity against Gram-positive and Gram-negative biofilms

Biofilm-associated infections are difficult to eradicate because the extracellular matrix and altered bacterial physiology limit antibiotic activity. Here, monoolein-based liquid crystalline nanoparticles (LCNPs) were evaluated for co-delivery of gentamicin (GEN) and recombinant human DNase I (rhDNase) against single-species Staphylococcus aureus ATCC 25923, methicillin-resistant Staphylococcus aureus (MRSA) USA300 and Pseudomonas aeruginosa PAO1 biofilms. The formulations had mean hydrodynamic diameters of approximately 150–170 nm, low polydispersity and negative surface charge. Representative cryo-TEM images showed multiple particles, several with periodic internal contrast; because small-angle X-ray scattering was not performed, a specific mesophase was not assigned. GEN and rhDNase encapsulation efficiencies were approximately 97–99%. In the pressure-driven in vitro release assay, LCNP association slowed the initial release of GEN relative to diffusion of free GEN across the membrane, although approximately 90% of GEN was released within 2 h; rhDNase release from the LCNP formulations remained slower than diffusion of free rhDNase throughout 4 h. GEN-LCNPs reduced planktonic minimum inhibitory concentrations by approximately two-fold and minimum biofilm inhibitory concentrations by approximately two- to four-fold, depending on the strain. At 10 μg/mL GEN-equivalent and 4 μg/mL rhDNase-equivalent, GEN+rhDNase-LCNPs produced the largest decreases in attached biomass and recoverable viable bacteria. After 24 h, viable counts were approximately 3 log10 colony-forming units (CFU)/mL for S. aureus , 4–5 log10 CFU/mL for MRSA USA300 and 5 log10 CFU/mL for PAO1. The findings support pathogen-dependent antibiofilm activity of the co-loaded formulation, while the absence of mesophase assignment, human-cell biocompatibility testing and a physiologically predictive release model limits translational interpretation.