Development of a synthetic antimicrobial peptide targeting MDR wound pathogens and biofilms: effective therapeutics...

Multidrug-resistant (MDR) pathogens are a major contributor to chronic and non-healing wound infections, where biofilms, high ionic strength and protease-rich exudates severely limit the performance of conventional antimicrobials. By 2050, antimicrobial resistance has been projected to cause 10 million deaths annually, raising an urgent need for therapeutics that can remain active in harsh wound microenvironments. Although antimicrobial peptides (AMPs) offer broad-spectrum activity, their clinical applicability is limited due to poor stability, low bioavailability and host toxicity, thus restricting their translational value in wound care. To overcome these limitations, two synthetic peptides, one with N-/C-terminal capping (b-DP1) and the other, an enantiomeric form, i.e., D-form (D-DP1), were tested for improved antimicrobial efficacy, protease resistance and suitability for wound-like physiological conditions. Both analogues showed markedly enhanced antibacterial potency, with minimum inhibitory concentrations (MICs) in the range of 1-32 µM (b-DP1) and 1-8 µM (D-DP1), against both Gram-positive and Gram-negative bacteria, including MDR clinical isolates and a representative fungal pathogen, extending the antimicrobial spectrum to include fungal pathogens frequently encountered in polymicrobial chronic wounds. The novel peptides retained their activity under protease-rich, high-salt and serum-rich conditions that mimic chronic wound exudates, with D-DP1 showing complete resistance to trypsin degradation. Time-kill assays demonstrated rapid bactericidal action within 30 minutes and no bacterial regrowth was observed for up to 48 hours. Serial passage studies confirmed that neither analogue induced bacterial resistance even after 10 consecutive cycles of exposure. Mechanistic analyses supported by field emission-scanning electron microscope (FE-SEM) imaging and confirmed by impedance spectroscopy revealed extensive bacterial membrane disruption. Both peptides inhibited biofilm formation at concentrations of 8 µM and above, resulting in visible disruption of mature biofilms. D-DP1 was particularly effective in reducing biomass by more than 90%. Importantly, the peptides displayed excellent biocompatibility, with hemolytic and cytotoxic thresholds exceeding 64 µM, representing an eight-fold safety margin above their effective antimicrobial doses. In addition, in vitro wound healing models mimicking mechanical, punch-type and thermal injuries demonstrated that D-DP1 supports cellular migration and recovery even under inflammatory conditions, highlighting its compatibility with tissue regeneration processes. In scratch wound assay, D-DP1 treatment resulted in around 76% wound closure within 24 hours, compared to 40% in untreated controls. In patch wound assays, it promoted migration of approximately 1500 cells into the wound region, compared to 1200 cells in controls. Thermal burn assays demonstrated enhanced cellular recovery to 285 ± 47%, compared to 312 ± 48% in control cells. Notably, D-DP1 maintained these regenerative effects even under inflammatory conditions (LPS co-treatment), with no significant difference compared to D-DP1 treatment alone (p > 0.05), while LPS alone severely impaired healing across all models. Together, the results of this study establish these synthetic peptides as powerful molecules for producing protease-resistant, broad-spectrum and biocompatible antimicrobials, making them well-suited for integration into next-generation therapeutic and regenerative scaffolds for MDR-infected wounds.