An injectable photopolymerized hydrogel with antimicrobial and biocompatible properties for infected skin regeneration

Skin is a critical barrier against microbial invasion, but wounds from trauma, burns, or surgery compromise integrity and are prone to infection, with Staphylococcus aureus a common cause. Such infections delay closure, reduce immune function, and can lead to severe complications. Overreliance on antibiotics contributes to resistance, and many dressings lack biocompatibility or do not provide a moist healing environment. The study aims to develop an injectable, visible light–photopolymerized hydrogel based on ε-poly-l-lysine (ε-PL) and γ-poly(L-glutamic acid) (γ-PGA) modified with glycidyl methacrylate (GMA) to combine broad-spectrum antimicrobial activity with biocompatibility and moisture retention. The hypothesis is that ε-PL-GMA/γ-PGA-GMA hydrogels will be rapidly gelled in situ, exhibit antimicrobial activity against Gram-positive and Gram-negative bacteria, be biocompatible, and promote healing of infected skin wounds while potentially reducing antibiotic use.

Hydrogels are three-dimensional, water-rich networks widely used in drug delivery, implants, and tissue engineering due to their biocompatibility and ability to maintain a moist environment that supports tissue repair. Antimicrobial hydrogels can be created from inherently antimicrobial polymers (for example, chitosan) or by loading antimicrobials (e.g., silver ions or antibiotics) and employing photothermal strategies, but many systems face limitations such as inadequate biodegradation, suboptimal gelation behavior, bacterial resistance, and narrow therapeutic windows. ε-Poly-l-lysine (ε-PL) is a biocompatible, water-soluble antimicrobial polypeptide used as a safe food preservative; it disrupts microbial membranes and reduces resistance development. γ-Poly(L-glutamic acid) (γ-PGA), a microbially produced polypeptide, mimics extracellular matrix features and supports tissue repair in bone and skin. Combining ECM-like polymers with antimicrobial polypeptides is anticipated to yield clinically relevant, biodegradable, and effective wound dressings.

Materials: γ-PGA (Mn ~2000 kDa), ε-PL (Mn 2–5 kDa), GMA (97% purity with MEHQ), tetrabutylammonium bromide (TBAB), and lithium phenyl(2,4,6-trimethylbenzoyl) phosphinate (LAP) as the visible-light photoinitiator. Synthesis of precursors: γ-PGA-GMA was prepared by dissolving γ-PGA at 5% (wt/vol) in deionized water at 60 °C, adding GMA and TBAB, adjusting to pH 5.0, and stirring for 6 h. The mixture was dialyzed (MWCO 8–12 kDa) for 3 days and lyophilized. ε-PL-GMA was prepared similarly by dissolving ε-PL at 3% (wt/vol) at 60 °C, adding GMA and TBAB, adjusting to pH 5.0, stirring for 8 h, dialyzing (MWCO 1000 Da) for 4 days, and lyophilizing. Hydrogel preparation: γ-PGA-GMA (0.5 g) was dissolved in 5 mL PBS (0.01 M, pH 7.4). LAP was added at 0.05 wt% (relative to solution volume). Mixtures of γ-PGA-GMA and ε-PL-GMA at varying ratios (four formulations) were irradiated with visible light (λmax 405 nm, 60 mW/cm²) at room temperature to form hydrogels. The hydrogels were designed to be injectable and rapidly gel upon light exposure. Characterization: Chemical modification was confirmed by 1H-NMR (400 MHz, D2O) and FT-IR (400–4000 cm⁻¹). Microstructure was examined by SEM on gold-sputtered freeze-dried samples. Gelation time was determined by tube inversion during 405 nm irradiation. Swelling ratio (SR) was measured by immersing identically sized, freeze-dried hydrogels in PBS at 37 °C, blotting, and weighing (SR = (Wh − W0)/W0 × 100%). Rheological properties were tested on a HAAKE Rheostress 6000 rheometer with a 20 mm cone plate (4°) using disk samples (radius 10 mm, height 1 mm). In vitro antibacterial testing: Escherichia coli and Staphylococcus aureus were grown overnight in Mueller-Hinton Broth (MHB), diluted to 10^6 CFU/mL, and 100 µL of bacterial suspension was seeded on 200 µL hydrogel formed in 48-well plates. After 2 h at 37 °C, viable bacteria were resuspended with 1 mL PBS, plated on LB agar, and incubated 24 h at 37 °C. Kill% was calculated as ((control count − survivor count on hydrogels)/control count) × 100. Bacterial morphology after 2 h and 24 h exposure was assessed by SEM following fixation in 2.5% glutaraldehyde and graded ethanol dehydration. Biocompatibility assays: Extract-based cytotoxicity was evaluated via MTT on NIH 3T3 cells (3×10^3 cells/well, 96-well plates) exposed to hydrogel extracts for 24, 48, and 72 h, with OD measured at 490 nm. Direct contact/proliferation was assessed by seeding NIH 3T3 cells (2×10^4 cells/well) on UV-sterilized hydrogels in 24-well plates, followed by live/dead staining (Calcein-AM/PI) and fluorescence imaging at 24, 48, and 72 h; control cells were cultured without hydrogels. In vivo infection and wound-healing model: Full-thickness 10 mm circular dorsal wounds were created in anesthetized female Sprague–Dawley rats. Wounds were infected with 100 µL of S. aureus inoculum at 10^11 CFU/mL. After 12 h (pus formation), 200 µL hydrogel precursor mixture was injected onto the wound and photopolymerized in situ with visible light. Controls included untreated wounds and wounds treated with 200 µL mupirocin ointment. Wounds were covered with sterile gauze and sutured. Wound appearance was photographed on days 4, 8, 12, and 16, and closure was quantified as percent of original wound area. Histology and immunofluorescence: At days 4, 8, 12, and 16, tissues were harvested, fixed in 4% paraformaldehyde, paraffin-embedded, sectioned at 10 µm, and stained with H&E and Masson’s trichrome. Immunofluorescence staining quantified IL-6 and TGF-β expression using primary antibodies and fluorescent secondaries; nuclei were counterstained with DAPI.

The ε-PL-GMA/γ-PGA-GMA hydrogels formed rapidly upon visible-light irradiation, were injectable, and demonstrated biocompatibility. They exhibited broad-spectrum antibacterial activity against both Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria in vitro. In a rat model of S. aureus–infected full-thickness skin wounds, in situ–formed hydrogels inhibited infection and accelerated wound healing compared with controls, effectively shortening healing time. The materials also adhered to bacterial cells and supported wound regeneration. Specific quantitative antibacterial kill percentages, gelation times, swelling ratios, and wound-closure percentages were not provided in the available text.

The study addresses the clinical need for biocompatible, antimicrobial dressings that can be applied in an infected wound environment and reduce reliance on systemic or topical antibiotics. By combining ε-PL, a broad-spectrum antimicrobial polypeptide, with γ-PGA, an ECM-mimetic, biocompatible polymer, and enabling in situ gelation with visible light (avoiding potential UV-induced tissue damage), the hydrogel system provides rapid coverage, moisture retention, and localized antimicrobial action. The observed inhibition of S. aureus infection and enhanced wound closure in vivo align with the hypothesis that such a composite hydrogel can both control bacterial burden and support tissue repair. The approach has relevance for clinical wound care by potentially lowering antibiotic usage and offering a conformal, injectable dressing for irregular wounds while maintaining cytocompatibility with mammalian cells.

This work presents an injectable, visible light–photopolymerized hydrogel composed of ε-PL-GMA and γ-PGA-GMA that is rapidly formed in situ, biocompatible, and exhibits broad-spectrum antimicrobial activity. In a rat S. aureus–infected wound model, the hydrogel reduced infection and accelerated healing. These findings support the hydrogel as a promising candidate for antimicrobial wound dressings and infected skin regeneration. Future research could quantify long-term biodegradation and mechanical properties in vivo, evaluate efficacy across diverse pathogens and polymicrobial infections, optimize formulation ratios for performance, assess integration with drug delivery (e.g., growth factors), and compare outcomes against standard-of-care dressings in larger animal models.

The provided text does not include detailed quantitative results (e.g., exact gelation times, rheological parameters, swelling ratios, in vitro kill percentages, or wound-closure rates), nor long-term in vivo degradation or immune response data. Spectrum of efficacy is demonstrated only for E. coli and S. aureus, and broader antimicrobial testing is not reported here. Safety beyond NIH 3T3 cytocompatibility (e.g., hemocompatibility, systemic toxicity) and comparisons to multiple clinical standard dressings beyond mupirocin are not described in the available excerpt.