**4. Conclusions and Future Prospects**

The development of novel antifungal treatment options is an ever-pressing issue as the rise in the incidence of wound infections among both civil and military traumatic injuries, along with burns, trauma, and ulcers, are becoming more frequent. These injuries and subsequent infections by fungal pathogens are linked with increased mortality, risk of limb loss, prolonged stays in hospital, failure of the treatment regimen, and further systemic infection. As shown in this study, thymoquinone, ocimene, or miramistin-loaded bacterial cellulose wound dressings are promising antifungal hydrogels that could address the aforementioned issues. As shown through the MFC and cytotoxicity assays, these materials could be potentially used in the groups mentioned above owing to the high level of antifungal activity and low cytotoxic effects when compared to conventional antifungal drugs that could exasperate pre-existing conditions, namely amphotericin B [70].

The results of this study indicate that the implementation of thymoquinone, ocimene, or miramistin hydrogel wound dressings may depreciate the need to use other systemic antifungal compounds in the treatment of superficial and deep tissue wounds, which is also in line with currently antimicrobial stewardship agendas and has been developed to specifically target and reduce the incidence of antimicrobial resistance [71]. Prolonged states of trauma are usually associated with inappropriate therapy, which is well documented in being a causative factor toward the development of antimicrobial resistance [72,73]. The issue of antifungal resistance and the need to develop newer and more efficacious products

was highlighted by The World Health Organisation in 2020 during the first meeting of the WHO antifungal expert group tasked with identifying priority fungal pathogens and subsequent treatment options [6].

Through this study, the preliminary antifungal properties of thymoquinone, ocimene and miramistin have been conducted against four commonly encountered fungi, which are causative agents in wound infections. The agents have been shown through internationally recognised disk diffusion and broth dilution assay protocols to possess a similar antifungal profile compared to amphotericin B. However, the marked difference is found within our agents' cytotoxicity profiles, which are significantly lower (*p* < 0.01) than that of amphotericin B.

Future perspectives of this study will focus on the loading capabilities of bacterial cellulose and combinations of potential antifungal agents to increase the bioactivity to a broader spectrum of organisms. Additionally, as the water-holding capability of the material is so high, this introduces issues of adherence of the wound dressing to the skin. Further investigations are required to develop a method by which the biomaterial can be modified to become adherent, possibly taking advantage of the ionic pores (Figure 5) within the cellulose matrices. Additionally, further investigations into the time release of each compound from BC will be conducted with appropriate time kill studies to determine how long these materials remain bioactive.

Following SEM characterisation of purified BC, it is clear that the pore sizes found within the matrices are generally uniform in size, around 117.9 nm to 3.4 μm, which is in concordance with existing research [38]. It is also postulated that there are two forms of pore that can be seen. The main pores, which are easily observed, fall within the mentioned dimensions; however, there are also indications that there are more superficial pores that are much larger in the region of 200 μm in diameter. This development in identifying various sizes and conformations of pores would allow future developments to be pursued in the area of incorporating additional compounds with a much larger structure, such as bioactivated zeolites loaded with antimicrobial metal ions. Table 3 highlights the efficacy of bacterial cellulose to absorb a large amount of free compound within a solution, showing up to 78.95 ± 17.5% successful loading and retainment throughout the entire material via physical absorption. This is agreeable to published data for the swelling ratio of bacterial cellulose [36].

The data collected during this study have highlighted the efficacy and high in vitro tolerability of our agents in use as an antifungal and would benefit greatly from further investigation. As we have highlighted in this study, the importance of utilising naturally occurring products is paramount in fighting the burdening issue of antifungal resistance [74,75]. It is clear that pharmaceutical agents such as amphotericin B are usually accompanied by severe side effects that make their use intolerable to individuals while having low efficacy to their target organism [76,77]. To the best of our knowledge, we are the first to successfully load thymoquinone into bacterial cellulose hydrogels, thus producing a novel wound dressing. We have also determined that thymoquinone and amphotericin B have similar antifungal potencies; however, the thymoquinone's overall cytotoxicity is significantly lower (*p* < 0.01).

**Author Contributions:** S.S. writing original draft; I.R. and S.S. were the main persons involved in the planning of experiments and interpretation of the data; G.A. was involved in FTIR interpretation, I.R., S.S., H.G., A.G., W.H., M.K. and G.A. editing and assisting with the preparation of the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** Partial financial support from the European Regional Development Fund Project EnTRESS No. 01R16P00718.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are openly available.

**Acknowledgments:** The authors would like to thank Biorender.com (2021) for their paid subscription in creating a graphical abstract and Figure 2. We would like to thank Keith R. Jones form the University of Wolverhampton, UK for his help with scanning electron microscopy images. The authors would also like to thank Enas Al-Ani (University of Wolverhampton, Wolverhampton, UK) for her very kind assistance and work with producing confocal images of Hep-2 cells in this study.

**Conflicts of Interest:** The authors declare no conflict of interest.
