Development and Optimization of Grape Skin Extract-Loaded Gelatin-Alginate Hydrogels: Assessment of Antioxidant and Antimicrobial Properties

Abstract

Background: In this study, we aimed to develop and optimize unique eco-friendly gelatin-alginate hydrogels enriched with sustainable grape skin extract for advanced wound healing applications. Methods: Following confirmation of the extract's antioxidant activity, hydrogels were synthesized by varying gelatin content and CaCl₂ concentration to achieve desirable crosslinking density, mechanical properties, and extract release behavior. Physicochemical characterization of hydrogels included equilibrium swelling analysis, mechanical testing, FTIR analysis, and in vitro release of extract from hydrogel. Moreover, the biocompatibility of hydrogels enriched with extract was assessed via MTT assay, while antimicrobial activity was tested against Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 10145, and Candida albicans ATCC 10231. The antioxidant capacity of the hydrogels was evaluated using DPPH, ABTS, and FRAP assays. Results: Our results showed that higher gelatin and CaCl₂ concentrations produced denser crosslinked networks, leading to reduced swelling and increased stiffness. Additionally, the extract exhibited a biphasic release profile from hydrogels, featuring an initial rapid release followed by sustained release over 24 h. Conclusions: The hydrogels maintained high biocompatibility and demonstrated selective antimicrobial activity, particularly against Escherichia coli, and satisfactory antioxidant activity. Obtained multifunctional sustainable hydrogels enriched with grape skin extract represent promising agents for managing skin conditions associated with oxidative stress and bacterial infections.

Keywords: antimicrobial activity; antioxidant; biopolymers; grape skins; hydrogels.

Figure 1

Dependence of the equilibrium swelling degree of hydrogels on gelatin content and CaCl₂ solution concentration.

Figure 2

Cross-section micrographs of freeze-dried hydrogels: Alg/Gel-1 (A), Alg/Gel-2 (B), and Alg/Gel-3 (C).

Figure 3

Stress–strain curves of alginate–gelatin hydrogels crosslinked in 0.5 M (A) and 1.0 M CaCl₂ solutions (B). The influence of gelatin content under different crosslinking conditions on compression modulus (C) and compression strength (D) of alginate–gelatin hydrogels.

Figure 4

FTIR spectra of alginate–gelatin hydrogels with various % of gelatin and CaCl₂.

Figure 5

Mass of released extracts (%) as a function of time for E extract.

Figure 6

Direct contact cytocompatibility assessment of grape extract-enriched hydrogels on MRC-5 cells: (A) Bright-field images, 20× objective, of MRC-5 cells after 24 h exposure to hydrogels base (1, 2, 3), grape skin extract-enriched formulations with lower and higher concentrations (EL and EH respectively), positive control Tween 20, and untreated (negative) control (DMEM). (B) MTT assay results showing MRC-5 viability (% of untreated control) after direct contact with hydrogels. Data are presented as M ± SD. Normality and homogeneity of variances were confirmed using the Shapiro–Wilk and Levene’s tests, respectively, prior to ANOVA. Significance: p < 0.05 (*), p < 0.01 (**), p < 0.001 (***).

Figure 7

Cytocompatibility of grape-based hydrogel extracts on MRC-5 cells: (A) Bright-field and live/dead fluorescence microscopy images, 20× objective, of MRC-5 fibroblasts after 24 h exposure to extracts released from hydrogels base (1, 2, 3), grape skin extract-enriched formulations with lower and higher concentration (EL and EH respectively) at 25%, 50%, and 100% concentrations. Teen 20 was used as a positive control at 0.025%, 0.05%, and 0.1% concentrations. DMEM was used as the untreated (negative) control. (B) MTT assay showing cell viability expressed as a percentage relative to the untreated (negative) control. (C) Red/green fluorescence ratio after EO/AB staining as an indicator of cell membrane integrity. Data are presented as M ± SD. Normality and homogeneity of variances were confirmed using the Shapiro–Wilk and Levene’s tests, respectively, prior to ANOVA. Statistical significance is indicated as p < 0.001 (***).

Figure 8

Antimicrobial effects of grape-based hydrogels against S. aureus, E. coli, and P. aeruginosa in direct contact assay: (A) Fluorescence images, 40× objective, of S. aureus ATCC 25923, E. coli ATCC 25922, and P. aeruginosa ATCC 10145 after 24 h direct contact with hydrogel base (1, 2, 3), extract-enriched formulations with lower and higher concentration of grape skin extract (EL and EH, respectively), stained with AO/EB. Live bacteria appear green; dead/damaged bacteria appear red. MHB was used as an untreated (negative) control. (B) Bacterial growth inhibition (% of untreated control) after direct hydrogel contact was determined by measuring OD₆₀₀. ERT = erythromycin (S. aureus control), GENT = gentamicin (E. coli, P. aeruginosa controls). (C) Red/green fluorescence ratio after AO/EB staining indicating bacterial membrane integrity loss. Data are presented as M ± SD. Normality and homogeneity of variances were confirmed using the Shapiro–Wilk and Levene’s tests, respectively, prior to ANOVA. Significance: p < 0.05 (*), p < 0.01 (**), p < 0.001 (***).