Introduction:: When a wound is formed on the skin, various pathogenic bacteria proliferate in the wound. Wound-infecting bacteria are typically Pseudomonas aeruginosa and Staphylococcus epidermidis, both forming biofilms and becoming resistant to antibiotics. Antibiotic-resistant bacteria, especially in hospitals, is a major problem worldwide. To solve this problem, graphene oxide (GO) and copper nanoparticles (Cu) were used as antibacterial materials. However, since excessive antibacterial substances are fatal to normal tissues, GO/Cu was encapsulated with a gelatin complex to lower the cytotoxicity. Among the catechol-based substances, gallic acid, which has anti-inflammatory and antibacterial properties, was used in this study to impart stability to the gelatin complex. This study shows the potential of the antibacterial gelatin-gallic acid microcomplex in prohibiting antibiotic-resistant bacteria and their biofilms and controlling the release of antimicrobial substances to improve biocompatibility.
Materials and Methods:: Gelatin (GE) and gallic acid (GA) were combined by a crosslinking method using EDC/NHS as a crosslinker. And the synthesized GO/Cu nanocomposites were placed in a GE-GA conjugate solution to fabricate a microcomplex using a water-in-oil method. The antibacterial and anti-biofilm effect of the GO/Cu@GE-GA microcomplexes was confirmed against gram-positive bacteria (Staphylococcus epidermidis) and gram-negative bacteria (Pseudomonas aeruginosa) using a microdilution broth method. And the cytotoxicity evaluation for human skin cells (HDF) at the same concentration was confirmed.
Results, Conclusions, and Discussions:: GO/Cu@GE-GA microcomplex inhibited the growth of S. epidermidis by more than 97% at a concentration of 0.5 mg/mL or more. And they inhibited the growth of P. aeruginosa by 56% at 0.5 mg/mL and by 98% at 1 mg/mL. Also, when 4 wt % GO/Cu@GE−GA microcomplex was treated at a concentration of 1 mg/mL, 86.1 and 100% of biofilms of S. epidermidis and P. aeruginosa were removed, respectively. However, 95% of HDF cells survive up to 0.25 mg/mL, and more than 80% of cells survive up to 1 mg/mL.
GO/Cu nanocomposites are released as the microcomplexes are dissolved, and the released GO/ Cu can be on the cell surface. Nevertheless, the cytotoxicity of the microcomplexes for up to 24 h is much lower than when bare GO/Cu was used. Therefore, the cytotoxicity of GO/Cu nanocomposites is lowered by loading GO/Cu in the gelatin microcomplex. A comparison of the antibacterial ability evaluation results of the microcomplexes with GO/Cu content of 1 and 4 wt % shows that the antibacterial ability depends on the amount of GO/Cu loaded in the microcomplex. Additionally, loading GO/Cu onto GE-GA microcomplexes did not significantly change its antibacterial ability compared to using GO/Cu alone. Therefore, the GO/Cu@GE-GA microcomplex not only maintains antibacterial ability but also enhances biocompatibility by reducing cytotoxicity when compared to using only GO/Cu.
This study confirms that the prepared GO/Cu@GE-GA microcomplex exhibits an antimicrobial effect on both gram-positive bacteria (S. epidermidis) and gram-negative bacteria (P. aeruginosa). At the same time, it maintains biocompatibility with human cells (HDF). Thus, these antibacterial materials minimize tissue damage while effectively eradicating bacteria and their biofilm. The synthesized material has the potential for various wound infection treatment applications. However, further research is necessary to confirm the cytotoxicity to other human cell lines, the antibacterial and anti-biofilm effects on other bacteria related to wound infection, and the mechanisms of antibacterial and anti-biofilm activity of the microcomplexes.
