*3.1. Preparation of BC Hydrogels and Characterisation*

The successful biosynthesis of bacterial cellulose is shown in Figure 3. The BC pellicles were harvested from the top of the growth media (A) and are opaque in colour (B), following purification by boiling in water and 2% (*w/v*) sodium hydroxide and again in water (C), then, the pellicles were removed and have become transparent after the purification process (D) as all remanence of growth media and *G. xylinus* cells were removed.

**Figure 3.** Biosynthesis of bacterial cellulose in Hestrin and Schramm medium, including pre- and post-purification. (**A**) static biosynthesis of BC which can be seen floating on top of the medium, (**B**) unpurified BC post harvest, (**C**) purification of BC in 2% (*w/v*) sodium hydroxide and, (**D**) purified BC.

After biosynthesis, both unpurified and purified BC were lyophilised for 96 h and analysed using a scanning electron microscope. Figure 4A shows an unpurified sample of BC, which has been false coloured to highlight the closed matrix as it is laden with growth media and biomass, the bacteria, *G. xylinus*, can be seen highlighted in blue. Figure 4B shows an SEM micrograph of purified BC, and the nanofibrous matrix is now visible (blue) with typical voids (purple) also present.

**Figure 4.** False coloured scanning electron micrographs of (**A**) unpurified (*G. xylinus* coloured blue), and (**B**) purified bacterial cellulose (cellulose nanofibres highlighted in light blue).

Once the bacterial cellulose was purified and lyophilised, SEM analysis was undertaken to confirm the pore size. Our results are concordant with previously published data [51], which show an average pore size of 117.9 nm to 3.4 μm (Figure 5A,B).

**Figure 5.** (**A**,**B**) Scanning electron micrographs of purified bacterial cellulose highlighting the size of the pores found within the matrix with an average range of 117.9 nm to 3.4 μm. *n* = 14, error bar 1 μm.

Following the characterisation of the purified BC via SEM, the lyophilised BC was aseptically cut using biopsy punches into 4 mm disks to be used in cytotoxicity assays and 8 mm disks used in disk diffusion assays. After this, the BC disks were placed into solutions of amphotericin b, thymoquinone, ocimene, or miramistin and were agitated overnight to ensure maximum loading of the antifungal agent. To confirm the successful loading, FTIR was performed on each of the samples (Figure 6A–F).

**Figure 6.** FTIR spectrographs of (**A**) purified BC, (**B**) BC: Amphotericin B, (**C**) BC: Miramistin, (**D**) BC: Ocimene, and (**E**) BC: Thymoquinone. (*n* = 3, 16 scans).

Each sample displayed characteristic O-H stretching between 2500 and 3300 cm−<sup>1</sup> due to hydroxyl groups found in the bacterial cellulose matrix (Figure 6A). It is worth noting that all samples analysed displayed similar characteristic wavelengths to pure cellulose of 3330, 2894, 1614, 1370, 1159, and 1056 cm<sup>−</sup>1, as seen in research previously conducted [52]; however, subtle additions to these wavelength peaks in the fingerprint region were used to confirm the presence of the antifungal agents in the respective samples.

The FTIR spectrum for BC: amphotericin B (Figure 6B) shows three principal vibrations of the amphotericin B molecule: the band with a maximum at 1730 cm−1, which can be assigned to the vibration of C=O in the –COOH group, the CH2 scissoring vibrations (the band centred at 1460 cm−1), and the C=C stretching vibration, which is represented by peaks between 1486 and 1631 cm−1. Additionally, the peaks in the region of 3300–3400 cm−<sup>1</sup> indicate stretching vibrations of the O–H group and the N–H stretching in concordance with previous research [53]. The appearance of peaks between 3400 and 3300 cm−<sup>1</sup> in the FTIR spectra, Figure 5B, can be also attributed as a band of OH groups of BC involved in forming hydrogen bonds with the antifungal agent, indicating an interaction between BC and amphotericin B.

The FTIR spectrum for BC: miramistin (Figure 6C) shows two principal vibrations of the miramistin molecule: the signals from the amide group (NH–CO) were observed in the range of 3400–3200 cm−<sup>1</sup> and C=O in the range 1650–1700 cm−1. In addition, peaks seen at 1650–1580 cm−<sup>1</sup> suggest bending in N-H groups and stretching vibrations of C=C in the phenolic ring [54]. Moreover, the broad band seen between 3400 and 3300 cm−<sup>1</sup> in Figure 6C, can also indicate on the formation of the hydrogen bonding between functional amide groups NHCO of miramistin and BC.

A strong peak observed at 3000–2900 cm−<sup>1</sup> in Figure 6D corresponds to C–H stretching in the alkyne of ocimene. Peaks at 1605 cm−<sup>1</sup> and 1640 cm−<sup>1</sup> suggest C=C bonding. However, the confirmation of successful loading of ocimene was achieved by identifying a sharp peak at 892 cm−<sup>1</sup> which corresponds to C=C bending in the vinylidene group, which is similar to other research [55].

The results of FTIR spectra for thymoquinone-loaded BC (Figure 6E) have assigned the existence of a variety of sharp, strong, and weak peaks as well as crucial functional groups that correspond to C=O, C-H, –CH2, –CH3, C=C, and C–O, suggesting the successful loading of thymoquinone within the BC sample. The intense band present at 2967 cm−<sup>1</sup> corresponds to the C-H stretching of aliphatic groups, while the band observed at a higher wavenumber ≈3040 cm−<sup>1</sup> was assigned to the stretching observed in the vinylic C–H in the C=C–H groups, which had previously been reported [56]. Additionally, the characteristic strong band of the carbonyl groups of a cyclohexadiene ring is observed at the wavenumber ≈1650 cm−1. As a result of FTIR analysis, we can confirm that the bacterial cellulose was successfully loaded with each antifungal drug, respectively.
