Optimized TPL6 Peptide Gel Exhibits Broad‐Spectrum Antimicrobial Activity and Effectively Treats Drug‐Resistant Wound Infections in Diabetic Mice

Antibiotics are the primary treatments for diabetic foot infections (DFIs) but are often ineffective against severe, polymicrobial, or drug-resistant microbial strains. Antimicrobial peptides (AMPs) offer broad-spectrum activity, yet most fail in clinical translation due to known limitations. Here, TPL6 is developed, a TP4-derived AMP with glycine-rich segments deleted, yielding a variant with potent, robust antimicrobial activity and reduced cytotoxicity. TPL6 retains its α-helical structure and activity under high glucose and salt, and after heat stress. It also does not induce microbial resistance after prolonged exposure. Formulated with adjuvants into a hydrogel, TPL6 gel restores activity in serum-rich environments and outperforms Bacineocin ointment in simulated wound fluid. The gel further prevents formation and eradicates biofilms of multidrug-resistant (MDR) pathogens. In ex vivo porcine skin and diabetic mouse wound models infected with MDR Staphylococcus aureus or Candida albicans, TPL6 gel shows superior therapeutic efficacy versus Bacineocin ointment and Canesten 1% cream. Thus, this study shows that structural modification and adjuvant-based formulation of TPL6 can overcome key translational barriers that have hindered clinical application of AMPs, supporting its potential as an antibiotic-free treatment for drug-resistant wound infections.