Antioxidant-biocompatible and stable catalase-based gelatin-alginate hydrogel scaffold with thermal wound healing capability: immobilization and delivery approach

Abstract

Hydrogel-based matrix prepared using biopolymers is a new frontier of emerging platforms for enzyme immobilization for biomedical applications. Catalase (CAT) delivery can be effective in inhibiting reactive oxygen species (ROS)-mediated prolongation of the wound healing process. In this study, to improve CAT stability for effective application, gelatin(Gel)-alginate (Alg) biocompatible hydrogel (Gel-Alg), as immobilization support, was prepared using calcium chloride as an ionic cross-linker. High entrapment efficiency of 92% was obtained with 2% Gel and 1.5% Alg. Hydrogel immobilized CAT (CAT-Gel-Alg) showed a wide range of pH from 4 to 9 and temperature stability between 20 to 60 °C, compared to free CAT. CAT-Gel-Alg kinetic parameters revealed an increased K _m (24.15 mM) and a decreased V _max (1.39 µmol H₂O₂/mg protein min) × 10⁴. CAT-Gel-Alg retained 52% of its original activity after 20 consecutive catalytic runs and displayed improved thermal stability with a higher t _1/2 value (half-life of 100.43 vs. 46 min). In addition, 85% of the initial activity was maintained after 8 weeks' storage at 4 °C. At 24 h after thermal injury, a statistically significant difference in lesion sizes between the treated group and the control group was reported. Finally, our findings suggest that the superior CAT-Gel-Alg stability and reusability are resonant features for efficient biomedical applications, and ROS scavenging by CAT in the post-burn phase offers protection for local treatment of burned tissues with encouraging wound healing kinetics.

Keywords: Burn wound healing; Catalase immobilization; Gelatin alginate biopolymers; Half-life; Hydrogel; Stabilization; Thermal injury.

Fig. 1

Swelling behavior of cross-linked hydrogel matrix (Gel–Alg) (2% (w/v) gelatin and 1.5% (w/v) sodium alginate) at 1, 2, 3, 4, 6, 8, and 12 h in 0.1 M potassium phosphate buffer, pH 7.0 at 25 °C. Each point represents the average of three experiments ± SD

Fig. 2

Effect of temperature on catalytic activity of free CAT and immobilized CAT (CAT–Gel–Alg) after 15 min incubation at various temperatures from 20 to 60 °C (pH 7.0: phosphate buffer, 50 mM). CAT activity assay was carried out under standard assay conditions. Each point represents the average of three experiments ± SD

Fig. 3

Determination of pH stability on free CAT and immobilized CAT (CAT–Gel–Alg) enzyme at various pH values pH 4.0–pH 9.0 (used buffers: 50 mM sodium acetate buffer for pH values 4.0 and 5.0 and 50 mM potassium phosphate buffer for pH values from 6.0 to 9.0) at 25. Each point represents the average of three experiments ± SD

Fig. 4

Reusability of free CAT and immobilized CAT (CAT–Gel–Alg) enzyme in consecutive hydrolytic cycles. Residual activity of the immobilized CAT (in %) after 20 cycles of repeated use (initial activity was taken as 100%). CAT activity assay was carried out under standard assay conditions. Each point represents the average of three experiments ± SD

Fig. 5

Storage stability of free CAT and immobilized CAT (CAT–Gel–Alg) enzyme for 60 days (50 mM potassium phosphate buffer, pH 7.0, 4 °C). Initial activity at day zero was taken as 100% and CAT activity assay was carried out under standard assay conditions. Each point represents the average of three experiments ± SD