ENGINEERING A COLLAGEN-BASED WOUND-MIMETIC PLATFORM FOR IN VITRO P. AERUGINOSA BIOFILM GROWTH AND DNASE I–CIPROFLOXACIN POST TREATMENT

Abstract

Biofilm-associated wound infections caused by Pseudomonas aeruginosa are difficult to eradicate due to the protective extracellular polymeric substance (EPS), which restricts antibiotic penetration and promotes antimicrobial resistance. The present study aimed to develop a collagen-based wound-mimetic biosimilar surface that closely simulates the in vivo wound microenvironment for in vitro biofilm formation and to evaluate the efficacy of DNase I-assisted antibiotic therapy against pre-formed biofilms. The biosimilar surface was developed using rat-tail collagen Type I and simulated wound fluid (SWF) and optimized by Response Surface Methodology (RSM) using Design-Expert 13.0 software. The optimized formulation consisted of 90.09 µL SWF and 30.23 µL collagen Type I, producing the highest absorbance (0.5684, p < 0.05). P. aeruginosa formed significantly greater biofilm biomass on the collagen-based biosimilar surface than on conventional polystyrene plates, demonstrating its suitability as a wound-mimetic model. The synergistic activity of DNase I and ciprofloxacin was optimized using Combenefit software, and the selected combination reduced the minimum biofilm inhibitory concentration (MBIC) of ciprofloxacin from 256 to 128 µg/mL. Furthermore, the combination therapy achieved 92.21% biofilm inhibition within 10 min and markedly reduced biofilm thickness, as confirmed by bright-field microscopy, scanning electron microscopy, and confocal laser scanning microscopy. These findings demonstrate that the developed collagen-based biosimilar surface provides a reliable in vitro wound biofilm model and that DNase I mediated matrix disruption significantly enhances ciprofloxacin efficacy against pre-formed P. aeruginosa biofilms, offering a promising therapeutic strategy for chronic wound infections.