Nature-Inspired Bacterial Cellulose/Methylglyoxal (BC/MGO) Nanocomposite for Broad-Spectrum Antimicrobial Wound Dressing

M. Yang Institute for Materials Discovery Department of Chemistry University College London Roberts Building 1.08 Laboratory, London WC1E 7JE, UK Prof. J. Ward The Advanced Center for Biochemical Engineering Department of Biochemical Engineering University College London Room 6.09 Bernard Katz Building, London WC1E 6BT, UK Prof. K.-L. Choy Institute for Materials Discovery Faculty of Maths and Physical Sciences University College London Room 1.07 Roberts Building, London WC1E 7JE, UK E-mail: k.choy@ucl.ac.uk The ORCID identification number(s) for the author(s) of this article can be found under https://doi.org/10.1002/mabi.202000070.


Introduction
Bacterial cellulose (BC), also called microbial celluloses or bacterial nanocellulose, is a polysaccharide, produced by aerobic bacteria in both synthetic and nonsynthetic medium through oxidative fermentation. [1] When compared to plant cellulose, BC has a unique structure solely consisting of glucose monomer, great properties are exhibited such as the unique nanostructure, [2] high water-holding capacity, [3] high degree of polymerization, [4] high mechanical strength, [5] high crystallinity, [6] as well as biocompatibility and moisture ability for its ultrafine network structure of nonaggregated nanofibrils. [7][8][9] Previous studies have revealed that BC as well as its derivatives are promising materials for application in various fields of biomedical, electronic, and food industries. [10] Wound dressings can accelerate the healing process and reduce the debris influence by providing the functions of both permeability and protection of regenerated tissues [11,12] as well as maintaining suitable hydration. [13] Recent antimicrobial wound dressings are mainly made by sponges, hydrogels, hydrocolloids, and electrospinning membranes; however, those can have some drawbacks as sponges and hydrogel are often in poor mechanical strength which are not appropriate to treat wounds in late-stage, [14][15][16] while hydrocolloids can exhibit toxicity as well as electrospinning membranes due to the solvents used in fabrication process. [17][18][19] Therefore, a natural product such as BC is an excellent candidate for a dressing substrate. However, since BC has no antibacterial properties, the most important task is to introduce an antimicrobial property of BC. For wound infections, prevention of secondary infection and maintaining a suitable local environment has been proven to be the major issue in assisting the healing process. [14] Various types of solutions have been employed to deal with the problem, including BC/chitosan, [20] BC/silver nanoparticles (AgNP), [21,22] BC/copper nanoparticles, [23] BC/povidone-iodine, BC/poly hexamethylene biguanide (PHMB), [24] and BC/tetracycline hydrochloride (TCH). [25] However, some of the organic antimicrobial BC nanocomposites were just against Gram-positive bacteria, and those incorporated with Ag/AgNP are a threat because they can cause a danger to human health, such as Argyria or argyrosis. [26] Bacterial cellulose (BC) is a natural material produced by Acetobacter xylinum, widely used in wound dressings due to the high water-holding capacity and great mechanical strength. In this paper, a novel antimicrobial dressing made from BC/methylglyoxal (MGO) composite with a dip-coating method inspired by naturally antimicrobial Manuka honey is proposed, which to our best knowledge, has not yet to be reported. Characterizations by scanning electron microscope and atomic force microscopy show the intercon- Therefore, there is a need to find a natural material and greener method in antimicrobial BC composite fabrication.
Honey, especially Manuka honey, has been studied as a therapeutic management method of chronic wounds in both in vitro and in vivo research works. [27][28][29] Different from plain honey, Manuka honey contains a unique component, methylglyoxal (MGO), which can act as a antimicrobial agents in wound healing process. [30][31][32][33] The antimicrobial property of MGO in the form of a solution, [34] hydrogel, [35] polymer and polymer fibers, [36] nonwoven fabric [33] have been investigated. It has been found that MGO with the concentration of 0.0054 mg cm −2 was sufficient to reach 100% colony forming pathogenic bacteria reduction. Rabie et al. [37] found that MGO can damage the structural integrity and function of bacteria DNA and proteins by disrupting glutathione homeostasis, thus altering the permeability which leads to cellular lysis. Therefore, inspired by the naturally antimicrobial Manuka honey, it is of great interest to study the degree to which MGO coated BC has antimicrobial property against broad-spectrum bacteria as compared with other antimicrobial BC composites. In this report, we prepared BC/MGO composites by dipcoating MGO solutions onto BC membrane to produce BC/ MGO (   for the illustration of the fabrication process) and tested against a broad-spectrum of bacteria including Gram-positive Micrococcus luteus (M. luteus), Staphylococcus aureus (S. aureus) and Gram-negative Pseudomonas aeruginosa (P. aeruginosa), Escherichia coli (E. coli).

Structural Characterizations
Photos of pristine BC membrane and BC/MGO composite (Figure 2c-f) indicated that both untreated plain BC and BC/ MGO membranes are soft and translucent, while BC/MGO with a higher MGO content showed more yellowish. Scanning electron microscope (SEM) images of MGO/BC confirmed the presence of nanometer fibrils (≈60-200 nm in diameter) and 3D interconnected structure (Figure 2b), which is in good agreement with the structural characteristics of plain BC ( Figure 2a). As compared with plain BC sample, MGO/BC sample indicated that there was a change in the surface morphology where MGO acted as a coating filling the voids and covering the fibril network of the original BC. Fourier transform infrared (FTIR) analysis showed the presence of C─O─C at 980 cm −1 , C─H bending at 1088 cm −1 , [38] C─O stretching, and OH stretching at peaks of 1058.75 and 3351.75 cm −1 , indicating the chemical structure of the plain BC. [39] In addition, the second line in FTIR analysis graph showed the existence of a peak of C═O stretching group at 1750 cm −1 , [40] which confirmed the incorporation of MGO into the BC.
To further investigate and compare the surface morphology between BC and BC/MGO with different MGO coating concentrations, atomic force microscopy (AFM) characterizations were also conducted. As shown in Figure 3a,b, the mean diameter of the BC fibers was ≈50 nm, which is in accordance with other publications. [41,42] Meanwhile, BC/MGO nanocomposites in Figure 3c-h showed similar fibrils diameter as the plain BC; however, the surface roughness of the nanocomposites increased with increasing MGO content. In this case, Figure 3g,h revealed the most obvious difference in the surface morphology of BC/MGO nanocomposites from the plain BC, where the roughness was the highest, ≈114 nm. Furthermore, some knots were observed around the fiber-net, indicating that the incorporation of MGO coating on BC could increase the surface roughness by interpenetrating the nanonetwork structure, caused by the excellent physical absorption property of the interconnected nanonetwork of the plain BC.

Thermogravimetric Analysis (TGA)
In order to understand the thermal decomposition behavior of BC and BC/MGO composites, TGA was performed. Figure 4 shows the weight loss curves (TG) of BC, BC/MGO (4%), BC/ MGO (0.4%), and BC/MGO (0.04%), indicating that there was dehydration below 100 °C, depolymerization and decomposition of glucosyl unites from 320 to 400 °C. [43][44][45][46] When BC coated with MGO was analyzed using TGA, the nanocomposite was less stable than plain BC, with degradation at 320-370 °C, while plain BC degraded at 380-400 °C. This may be attributed to the weaker hydrogen bonding and decreased crystallinity when an increasing MGO content was introduced.

Mechanical Test
As mechanical strength is a key property of materials used in wound dressing applications, the tensile strength was tested in  untreated BC as well as BC/MGO with various MGO concentrations in the wet state. The tensile properties of the samples were illustrated in Figure 5 and summarized in Table 1. BC based samples exhibited high tensile strength due to the interconnected nanonetwork structure with strong hydrogen bonding. [47] Compared with other BC/MGO samples, untreated BC sample seems to be softer, more ductile, and flexible. Whereas, BC/MGO samples seemed to be harder and more brittle, with the increase of MGO concentration. BC/MGO 4% with the highest coating concentration of MGO exhibited highest tensile strength but lowest extension, which might be attributed to the presence of strongest carbonyl group due to increasing in the crystallinity of materials. [48] Overall all BC/MGO samples exhibited good mechanical properties, which make them promising flexible wound dressing materials.

Disk Diffusion Antimicrobial Test
In this work, the antimicrobial property of MGO coated BC nanocomposites against M. luteus, P. aeruginosa, S. aureus, and E. coli were investigated by the disk diffusion test method (Figure 6a-d). For each plate, a positive control group of two antibiotics dipped BC and plain BC control group were tested as comparison. The efficiency of antimicrobial activity was measured by the diameter of clear zones of inhibition (DIZ) around the round-shaped cut samples after 24 h of incubation in 37 °C. After 24 h incubation, DIZ of BC/MGO (4%) with highest MGO concentration were investigated as 14.3 ± 1.3, 12.3 ± 0.3, 17.1 ± 0.6, and 15.5 ± 0.5 mm in Figure 6e, while the positive control groups were 12.7 ± 1.4, 10.0 ± 0.5, 5.2 ± 0.4, and 8.5 ± 1 mm, and control groups of plain BC were none for all the samples, against M. luteus, P. aeruginosa, S. aureus, and E. coli, respectively. In addition, it was also obvious from Figure 6e that BC/MGO nanocomposites were slightly more effective on Gram-positive than biofilm contained Gram-negative bacteria strains while it was still lethal to multiantibiotic-resistant S. aureus. The results showed that for all these bacteria strains, with the increase of MGO concentrations, the antimicrobial activity was more pronounced. This indicated that MGO coated BC, with at least 0.04% concentration, sufficient to reduce the activity of most pathogenic bacteria which are the most likely   to exist in the mid to late stage of chronic wounds. Table 2 summarized recent publications on antimicrobial property of wound dressing materials including BC based nanocomposite.
The different values of DIZ against E. coli and S. aureus can be used to make comparisons. Although it cannot be comparable with antiseptic or antibiotic based BC composites, BC/MGO is still among the best in BC related antimicrobial materials. In addition, it is also of great significance to compare BC/MGO nanocomposite with other antimicrobial wound dressing materials. It is indicated that our proposed BC/MGO composite is among the best in natural antibacterial agents based and commercial silver based wound dressing materials against E. coli and S. aureus, though it is less effective than some nanoparticle embedded composites. Therefore, it can be confirmed that MGO/BC has great potential application in bacteria-infectious chronic wound dressing applications.

Conclusion
In this paper, we proposed an ecofriendly, nature-inspired, and cost-effective nanocomposite for chronic wounds dressing, made by antimicrobial BC/MGO nanocomposite via a simple dip-coating method. SEM and AFM characterizations indicated the difference in surface morphology of the interconnected nanostructure between BC and MGO, and FTIR analysis confirmed the introduction of MGO onto BC. Thermal analysis test revealed the great thermal stability of both BC and BC/ MGO; however, there was a slight reduction in the thermal stability of BC/MGO composite possibly due to the reduction of hydrogen bonds and increase of crystallinity. Despite this slight reduction in thermal stability, this should not affect the practical application of BC/MGO composite as a promising wound dressing material which is normally used at human body temperature (37 °C). Disk diffusion test showed BC/MGO based composites exhibited high antibacterial effect against most common pathogens in chronic wounds, even against antibiotic-resistant S. aureus, compared with other antimicrobial wound dressing composites in other publications. Tensile tests confirmed the excellent mechanical strength of BC/MGO composite. All these render BC/MGO composite a potential material for chronic wound dressings or skin substitute in chronic wound bed.
Characterizations: A ZEISS Ultra 55 field emission gun SEM was used to characterize the surface morphology of BC and BC/MGO based nanocomposites. Prior to investigation, each sample was subjected to a freeze-drying process in liquid nitrogen (−195 °C) and subsequently gold coated with a thickness of average 15 nm using a gold sputter. Surface morphology of samples was also characterized by AFM in a tapping mode. The composition of BC and BC/MGO composite was characterized by FTIR spectroscopy (Perkin Elmer, USA) at wavelength from 400 to 4000 cm −1 . TGA test was carried out using a Perkin-Elmer 2000 instrument, under continuous nitrogen flow of 70 mL min −1 , and A 10 mg sample was used and the weight loss was recorded from 25 to 600 °C with temperature ramp at 10 °C min −1 . Mechanical test was performed by Intron tensile tester, according to active standard test method D638 equipped with 1 kN static load cell. During the test, the tensile strength and elongation at break can be calculated based on measured thickness and width of each sample. A test speed of 10 mm min −1 was used. Each sample was tested at least five times.
Antimicrobial Activity: The antimicrobial activity of BC/MGO composites was investigated by disk diffusion test against M. luteus (NCIMB1327), P. aeruginosa (DSM8626), S. aureus (DSM111729), and E. coli (DH5α). Prior to the test, BC and BC/MGO samples were punched into 6 mm round-shaped discs. The tests were performed according to the standard Kirby-Bauer Disk Diffusion Susceptibility Test Protocol (American Society of Microbiology, 2009). Lawns of test bacteria (about 1 × 10 5 CFU per plate) were prepared from Luria-Bertani Broth cultivation overnight. The composites, positive control samples dipped with Kanamycin (50 mg mL −1 ) and Ampicillin (50 mg mL −1 ) and plain BC control groups were put onto the bacteria-spread agar plates and marked. After 24 h incubation at 37 °C, the plates were taken out and the diameter of inhibitions zones was measured with a ruler. The tests were done in triplicate.