Development and Evaluation of Graphene Oxide-Enhanced Chitosan Sponges as a Potential Antimicrobial Wound Dressing for Infected Wound Management

Abstract

Chronic infected wounds remain a major medical challenge, particularly in the context of increasing antibiotic resistance. The objective of this study was to develop and evaluate chitosan-based (CS) sponges enhanced with graphene oxide (GO) as potential antimicrobial wound dressings. The composite sponges were fabricated using microcrystalline CS (MKCh) and 5% (w/w) GO, followed by freeze-drying and γ-sterilization (25 kGy). Physico-mechanical characterization showed that GO incorporation did not significantly alter tensile strength, while absorption and sorption capacities were improved, especially after sterilization. Structural and spectroscopic analyses confirmed increased porosity and molecular interaction between CS and GO. Cytocompatibility was verified in vitro using L-929 fibroblasts, with no cytotoxic effects observed in indirect contact. Antimicrobial activity tests demonstrated that GO-modified dressings exhibited enhanced activity against E. coli and S. aureus, though results were strain-dependent and not uniformly superior to CS alone. Notably, antifungal efficacy against C. albicans was reduced with GO addition. Overall, the developed GO-enriched CS sponges present favorable biocompatibility, mechanical resilience, and selective antimicrobial activity, supporting their potential application in chronic wound management. Further optimization of GO concentration and formulation is warranted to maximize antimicrobial efficacy across a broader spectrum of pathogens.

Keywords: antimicrobial activity; biocompatibility; chitosan; dressings; graphene oxide; infected chronic wound.

Figure 1

The representative photographic images of the (A) sponges made of MKCh and (B) sponge made of MKCh with the addition of GO. SEM image of the outer surface of the sponge made of MKCh with and without the addition of GO, where (C) MKCh sponge surface before sterilization, (D) MKCh + GO sponge surface before sterilization, (E) MKCh sponge surface after sterilization (25 kGy), (F) MKCh + GO sponge surface after sterilization (25 kGy). Magnification 1500×, presented scale bar indicates 100 μm.

Figure 2

FTIR spectra of GO, CS, and CS–GO composite. The characteristic absorption bands are assigned as follows: for GO—broad O–H stretching (~3360 cm⁻¹), C=O stretching (~1720 cm⁻¹), H-O–H bending from adsorbed water (~1620 cm⁻¹), C–OH stretching (~1380 cm⁻¹), C–O (epoxy or phenolic) stretching (~1220 cm⁻¹), and C–O–C stretching (~1070 and ~980 cm⁻¹); for CS—O–H and N–H stretching (~3350–3280 cm⁻¹), C–H stretching (~2922 and ~2868 cm⁻¹), Amide I (C=O, ~1647 cm⁻¹), N–H bending (~1583 cm⁻¹), CH₂ and CH₃ deformations (~1417 and ~1375 cm⁻¹), C–N stretching (Amide III, ~1325 cm⁻¹), C–O–C asymmetric stretching (~1153 cm⁻¹), and C–O stretching (~1080 and ~1028 cm⁻¹).

Figure 3

Raman spectra of GO, pure CS-based dressing (without the addition of GO), and CS-GO dressing, respectively. The characteristic D (~1350 cm⁻¹) and G (~1580–1600 cm⁻¹) bands of GO are clearly visible in both GO and CS-GO samples, confirming the successful incorporation of GO into the dressing matrix. The CS sample does not exhibit these bands. On the right, representative Raman microscope images of the analyzed regions for CS and CS-GO are shown, indicating the precise locations from which the spectra were acquired.

Figure 4

(A) Morphology of L-929 cells after 24 h in contact with extracts from sponges made of CS and CS-GO in increasing dilutions (1:1, 1:2, 1:4, 1:8, respectively), as well as morphology of the cells in contact with PE as a negative control, SLS in increasing dilutions (1:1 and 1:2, respectively) as a positive control. Cells cultured in fresh, complete medium served as the reference in this study. Scale bar indicates 200 μm. (B) MTT cell viability (mean and standard deviation) expressed as % in comparison to cells cultured in fresh, complete medium (i.e., reference).

Figure 5

(A) The viability (mean and standard deviation) of bacteria and fungi in relation to the negative control (referred to as NC, which was sterile blotting paper) for dressings made of CS and CS enriched with GO (marked as CS-GO). The dressing samples were applied to freshly inoculated tested microorganism species—wound contamination. (B) The viability (mean and standard deviation) of bacteria and fungi in relation to the negative control. The dressing samples were applied to inoculated test microorganism species, incubated for 24 h, and then assessed for wound colonization.