**1. Introduction**

Wounds from various sources that have been in a prolonged state of irritation and rubor, with a high degree of exudate, are decidedly prone to becoming infected by various opportunistic and commensal organisms. The typical approach in treating these wounds is to reduce infection and increase more favourable conditions at the wound site for

**Citation:** Swingler, S.; Gupta, A.; Gibson, H.; Heaselgrave, W.; Kowalczuk, M.; Adamus, G.; Radecka, I. The Mould War: Developing an Armamentarium against Fungal Pathogens Utilising Thymoquinone, Ocimene, and Miramistin within Bacterial Cellulose Matrices. *Materials* **2021**, *14*, 2654. https://doi.org/10.3390/ma14102654

Academic Editor: John T. Kiwi

Received: 1 April 2021 Accepted: 15 May 2021 Published: 18 May 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

healing, thus encouraging a more suitable environment for successful re-epithelisation and angiogenesis [1,2].

Immunodeficiency arising from burn injuries poses a genuine and present threat to the successful healing of patients. The central difficulty in burn patients is the burn wound itself. Although the body as a whole reacts to a burn dependent on severity, thickness, and coverage, those with thermal injuries that are extensive typify a whole-body reaction. As a result of this reaction, the inflammation arising from the injury mandates that the wound is protected from infection and that there is a timely closure of the exposed areas. Burn wound infections and mortality rates are drastically decreased with the application of topical treatments such as silver sulfadiazine and mafenide acetate by nearly 50% [3,4].

Antifungal resistance is an ever-growing issue within the healthcare sector, seeing over 1.5 million people die annually from fungal infections [5,6]. Immunocompromised patients make up the bulk of these deaths, and with classical antifungal drugs such as the azoles becoming less effective in the treatment of infections as a result of decreased efficacy, this figure is most likely going to increase.

A dramatic increase in fungal infections in recent years is associated with several factors such as prolonged dosage of broad-spectrum antibiotics, immunosuppressive therapy, extended stays in hospitals, burns, and recently undergone surgery as well as malignancy [7,8].

*Candida* species are polymorphic yeasts that can exist in various morphological forms that facilitate survival under extreme microenvironments by forming biofilms or invading and destructing target tissues, primarily in the pseudo-hyphae and true hyphae morphological state [9]. Unlike other pathogenic organisms, morphogenic plasticity helps *Candida* species evade host immune responses and confers differential responses towards antifungal agents [10]. Being eukaryotic organisms, fungal specific drug targets are very few and thus, limited numbers of antifungal agents such as azoles, polyenes, allylamines, and echinocandins, et cetera are available on the market [11].

Thymoquinone (Figure 1A) and ocimene (Figure 1B) are the major constituents of *Nigella sativa* and *Azadirachta indica*, respectively. It has been shown that both of these compounds possess various pharmacological qualities, which include antibacterial, antiparasitic, anti-inflammatory, anti-viral, and antifungal properties [12–14]. They have also been shown to provide a level of protection against nephrotoxicity [15]. Miramistin (benzyl dimethyl [3-(myristoilamino) propyl] ammonium chloride monohydrate) (Figure 1C) is a topical antiseptic that was developed in the Soviet Union during the Cold War within the framework of the 'Space Biotechnology Program' [16].

Amphotericin B (Figure 1D) is the principal antifungal polyene agent with a broadspectrum activity used to treat burn patients. The mode of the action revolves around the drug destabilising ergosterol within the cell wall of the fungi, thus causing rapid ion leakage culminating in cellular death [17]. The administration of amphotericin B ranges from 0.6 to 1 mg/kg/day and poses significant intolerance in burn patients who exhibit side effects such as hypoxia, hypertension, fevers, and more significantly, nephrotoxicity although the introduction of liposomal formulations has eased this side effect [18]. This side effect limits the overall effectiveness of intravenously administering amphotericin B as tubular wasting is significantly reduced. Therefore, a method for delivering this drug to patients' burn site is greatly needed [19]. The topical administration of amphotericin B to burn patients' wounds would allow for rapid, localised control and prevention of fungal pathogens becoming disseminated, consequently minimising the risk of fungal infections becoming deep-rooted, which drastically increases the risk of death.

Although the overall mechanism of the antifungal actions of both thymoquinone and ocimene is not fully understood, it is well documented that both thymoquinone and ocimene are sources of reactive oxygen species (ROS), leading to oxidative stress, which could play a significant role in the fungicidal properties [20,21]. However, the antifungal activity of miramistin is poorly documented (Figure 2).

**Figure 1.** Chemical structures of thymoquinone (**A**), ocimene (**B**), miramistin (**C**), and amphotericin B (**D**).

The two most prevalent fungal genera responsible for burn wound infections are *Candida* and *Aspergillus* species. In the present study, *C. albicans, C. auris, A. fumigatus*, and *A. niger* are being investigated providing representative pathogenic organisms of both non-dermatophyte fungi and yeasts. *Candida* species are responsible for approximately 80% of nosocomial yeast infections, of which 4–11% of burn patients are diagnosed with invasive candidemia, which results in a 30–58% mortality rate [22]. However, *Aspergillus* species account for 0.3–7% of fungal wound infections in burn patients, resulting in invasive aspergillosis. Nevertheless, the highly angio-invasive nature of aspergillosis results in a 50–78% mortality rate [23–25].

Established dressings such as gauze and tulle form a barrier between the wound site and the external environment that keeps the wound site dry. However, they are unable to impart any anti-microbial activity directly or influence the wound-healing process [26]. Conversely, moist wound dressings such as bacterial cellulose dressings act as a barrier to infection and also maintain moisture levels around the wound. One advantage of these dressings is that they are easily removed from the wound site, thus avoiding further trauma during dressing changes. These dressings also respond to variations in moisture levels, thereby facilitating re-epithelialisation of the wound site [27]. In addition to absorbing and retaining excess wound exudate, hydrocolloid dressings also provide a cooling, soothing effect, which also reduces rubor and the sensation of pain [28].

**Figure 2.** Schematic diagram showing the target sites of currently used antifungal drugs (pink), pipeline antifungals (green), and the test drugs used in this study (yellow).

Bacterial cellulose (BC) can be used as part of a biocompatible system; it is nonpyrogenic and hydrophilic, which make it innately suitable for wound treatment applications [29]. In addition, BC also contours superbly to the undulating surface of the skin, providing a uniform covering even in areas that are usually difficult to dress, such as the groin and neck. BC also provides protection to healing wounds due to the thickness of the dressing, which reduces further injury from further trauma. This protection further aids in the promotion of angiogenesis and rapid wound healing [30,31].

Bacterial cellulose is biosynthesised through the conjugation of linear homopolysaccharides and β-D-glucose units which are linked by 1,4-β-glycosidic linkages [1]. Once the exopolysaccharides have formed, they then randomly become organised into chains consisting of 10 to 15 individual chains of cellulose, resulting in cellulose nanofibers. These single chains of nanofibers become further entangled to form microfibrils, which are up to 100 times smaller than commercially available vegetal analogues [32–34].

Then, the synthesised cellulose microfibrils form ribbon bundles, which are 3–4 nm thick and 70–80 nm wide, which are what form the cellulose pellicle. This is achieved through inter and extra-chemical bonding, primarily hydrogen bonding between hydroxyl groups within between the cellulose fibres [35].

Due to the high levels of hydrogen bonding between the cellulose fibres, pores are formed within the cellulose, which possess an overall negative ionic charge resulting from hydroxyl groups and permit additional compounds to be embedded. This intrinsic physical property of cellulose allows for an increased level of influence over the bioactivity of the material as additional compounds, providing there is some level of positive ionic charge owing to the negative ions charge in the bacterial cellulose arising from the hydroxyl groups, such as antifungal agents, which can be easily incorporated [36].

As bacterial cellulose is a biopolymer, there are naturally varying degrees of pore size and distribution. These pores can be further influenced by the method of fermentation itself, whether it be static or agitated. Previous research conducted by Revin et al. (2019) showed that under agitated conditions, the bacterial cellulose forms spheres with a pore

size of 165–330 μm, while under static conditions, the cellulose formed as a pellicle with an average pore size of 4 nm to 1000 μm in diameter [37,38].

The overall distribution and size of the pores within the bacterial cellulose pellicle greatly impacts both the physical and mechanical properties of the biomaterial, in that larger pores introduce greater voids resulting in a less dense material. Subsequently, these larger pores allow for larger molecules or substances to be loaded more easily as a result of increased porosity. However, the drawback with larger pores is that the overall mechanical strength of the material is negatively impacted [39]. In the lyophilised form, bacterial cellulose has an elongation at break of approximately 5% and a typical tensile strength of 340 mPa, a Young's modulus of 12 GPa, and a maximum strain order of 4%. However, wet bacterial cellulose has a maximum strain order of 20%, tensile strength of 400 mPa, and Young's modulus of 130–145 GPa, indicating that wet bacterial cellulose has a greater level of elasticity [40–42].

Due to bacterial cellulose only comprising approximately 1% of the overall pellicle weight, with water (approximately 99%) comprising the remainder, it was also deduced that this material also has a significantly higher swelling ratio compared to commercially available cellulose [42]. It was further shown that the biomaterial is capable of regulating moisture content, which is ideal for applications in heavily exuding wounds [43]. This water-holding capacity of bacterial cellulose has been found to be 50 to 100 times its dry weight and is directly related to the overall surface area and distribution of pores within the material.

Another pertinent difference between commercially available cellulose derived from plants is that in bacterial cellulose, the crystallinity ratio is much higher (85–90%) in comparison to its commercial counterpart (60–65%). Moreover, in bacterial cellulose, there are no additional by-products that need to be removed, such as lignin and hemicellulose, allowing for a more streamlined and efficient production process [39].

All of these properties of bacterial cellulose have led to the successful commercialisation of bacterial cellulose hydrogels (e.g., Dermafill® and Biofill®) for the treatment of burns, chronic ulcers, skin lesions, and periodontal disease [1]. Even though bacterial cellulose is not inherently antimicrobial itself, the unique 3D fibrillar network is highly porous and amenable to high loading with a controlled release of a range of antimicrobial agents, which can be delivered directly to the wound site [35,36].

The aim of this study was to evaluate the antifungal potential of biofunctionalised BC loaded with either thymoquinone, ocimene, and miramistin in comparison to the action of amphotericin B loaded in BC to determine whether alternative anti-fungal options can be used in the prevention and treatment of fungal wound infections.

#### **2. Materials and Methods**

### *2.1. Media and Reagents*

Thymoquinone, ocimene, and amphotericin B, foetal bovine serum, glutamine, and antibiotics (penicillin G, 60 mg/L; streptomycin, 100 mg/L; standard amphotericin B, 50 μL/L) were purchased from Sigma-Aldrich (Irvine, UK). Miramistin was purchased from Carbosynth Ltd. (Compton, UK). Dimethyl sulfoxide (DMSO) reagent grade, sodium hydroxide, disodium phosphate, and citric acid were purchased from Sigma-Aldrich (Irvine, UK). Dextrose, bacteriological peptone, yeast extract, agar number 2, Sabouraud dextrose agar (SDA), malt extract agar (MEA), Hestrin and Schramm agar (HSA), Hestrin and Schramm media (HS), and Ringer's solution were purchased from Lab M (Bury, UK). RPMI-1640 (Roswell Park Memorial Institute) was purchased from Fisher Scientific (Cramlington, UK). All media and reagents were prepared according to the manufacturer's instructions.

#### *2.2. Microorganisms*

*Gluconacetobacter xylinus* ATCC® 23770, Candida albicans ATCC® 10,231 and Aspergillus niger ATCC® 16,888 were obtained from the LGC Standards Ltd. (Middlesex,

UK). Aspergillus fumigatus NCPF 2140 and Candida auris NCPF 8971 were obtained from Public Health England, Porton Down, UK. All organisms were obtained in lyophilised form and maintained at −20 ◦C before use. C. albicans, C. auris, A. fumigatus, and A. niger were revived on sterile SDA made to manufacturers' specification, sterilised by autoclaving before use and incubated at 30 ◦C for 24 h to obtain maximum growth. G. xylinus was revived on sterile HSA at 37 ◦C for five days. Overnight cultures of G. xylinus were grown under agitation in HS broth medium at 37 ◦C for 24 h from stock plates to ensure the bacterial cells are well dispersed within the media, rather than conglomerating into a single bacterial pellicle.
