Immobilization of Bacillus-derived protease onto carboxymethyl chitosan aerogel: A bioactive platform for bacterial inactivation, skin cancer therapy, and accelerated wound healing.

The development of multifunctional wound care materials capable of addressing bacterial infections, promoting tissue regeneration, and providing localized anticancer therapy is a growing biomedical priority. In this current study, we designed a novel therapeutic scaffold by immobilizing Bacillus-derived protease (PR)onto carboxymethyl chitosan aerogel (CMCsAE), forming aerogel matrix (PR-CMCsAE), and evaluated its antibacterial, antibiofilm, anticancer, and wound healing properties. Two loading concentrations (0.15 and 0.3 g) of PR enzyme were tested. PR0.3-CMCsAE showing superior immobilization yield (90 ± 3.7%) and retained enzymatic activity (72.5 ± 1.91%). Compared with the free enzyme, the immobilized protease demonstrated enhanced thermal and pH stability, reusability (65% activity after 10 cycles), and long-term storage performance (79.7% activity retained after 4 weeks). PR0.3-CMCsAE exhibited broad-spectrum bactericidal effects against some skin-causing bacterial infections, achieving complete bacterial inactivation at 400 μg/mL within 60-90 min. It also significantly inhibited biofilm formation over a 7-day period. Anticancer evaluation on A431 skin carcinoma cells revealed a dose-dependent reduction in viability (down to 21.6%), with apoptosis as the primary mode of cell death, supported by flow cytometry and AO/EB fluorescence imaging. Additionally, PR0.3-CMCsAE promoted the wound healing (in vitro), increasing scratch closure to 54.6% compared to 41.5% in controls. Toxicity testing confirmed the formulation's biocompatibility (EC50 > 100). Overall, PR-CMCsAE represents a therapeutic platform that combines enzymatic debridement, antimicrobial defense, anticancer activity, and regenerative support. This bioengineered aerogel holds strong potential for use in advanced wound dressings and localized skin cancer treatments.