The antimicrobial peptide EM86 loaded to gamma-irradiated sodium alginate/polyvinyl alcohol electrospun nanofibrous dressing treated multidrug-resistant Pseudomonas aeruginosa wound infections in BALB/c mice

Background Treating infected wounds is challenging, and conventional dressings delay healing. Resistance to the last-resort antibiotics is on the rise. Antimicrobial peptides (AMPs) offer advantages over traditional antibiotics against multidrug-resistant (MDR) bacterial strains. We aimed to use a computer-aided approach to design an anti-Gram-negative AMP with reduced mammalian cell toxicity and to functionalize an electrospun nanofibrous dressing with the AMP for in vitro and vivo evaluation. Methods Colistin and polymyxin sequences were modified to generate analogs using a template-based approach. The membrane activity was predicted using the collection of antimicrobial peptide database. Three shortlisted sequences were synthesized, and their minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) against MDR Gram-negative bacterial isolates were determined. The time-kill kinetics of the potent candidate against Pseudomonas aeruginosa SM016 were analyzed, and toxicity to human skin fibroblasts (HSF) was determined by the MTT assay. A sodium alginate (SA)/polyvinyl alcohol (PVA) nanofibrous wound dressing was synthesized by electrospinning. The produced scaffolds were gamma-irradiated and examined using scanning electron microscopy (SEM). The AMP was loaded onto the scaffold by physical adsorption, followed by in vitro and in vivo testing against Pseudomonas aeruginosa SM016 in open infected wounds in BALB/c mice. Results EM86 exhibited bactericidal activity against all tested isolates. EM86 killed Pseudomonas aeruginosa SM016 within 60 min, with morphological changes indicative of cell death confirmed by SEM after 30 min of treatment. EM86 was safe to HSF(IC50 > 300 μg/mL). The polymeric mixture of 1:12.3 SA/PVA, crosslinked with 1.25% glutaraldehyde, electrospun at 30 kV, 0.045 mm/min, and 12.5 cm, produced nanofibrous dressings with a mean diameter of 238 ± 78 nm and a swelling capacity of 510%. EM86-loading was confirmed by Fourier-transform infrared spectroscopy. Fibers loaded with 35 ± 18 μg EM86 significantly reduced P. aeruginosa SM016 counts (P < 0.001) in open-wound infected BALB/c mice, compared to the untreated mice, after 4 days of a once-daily treatment. Conclusion Using computer-aided approaches, we designed EM86 with a bactericidal activity against MDR Gram-negative microorganisms and reduced toxicity to HSF compared to colistin. EM86-functionalized SA/PVA nanofibrous dressings offer a better treatment alternative for colistin-resistant infections in open wounds.