Introduction

Bacterial cellulose (BC) is an ideal polymer for many applications in biomedical fields due to its non-toxic effect, high water retention capacity, biocompatibility and easy shaping properties [1,2,3,4]. Moreover, its characteristic properties are important in antimicrobial agents and anti-cancer treatments. However, unmodified BC has been restricted for therapeutic applications [4]. Therefore, BC needs to be modified to increase its biological properties as a biomedical material. Many studies have modified BC. For example, Fernandes et al. (2013) modified antimicrobial BC membranes by adding amino alkyl groups [5]. In other studies, BC nanoribbons were partially acetylated by catalysation with citric acid or hydrolysed using different acid concentrations (H2SO4 and HCl) and reaction times for nanocrystal formation [6, 7]. Some researchers have modified BC by changing its surface charge using carboxyl groups [8]. Hyaluronic acid–BC composites, chitosan–BC composites, cactus/BC hydrogels and metallic nanoparticles combined with BC have also been obtained by numerous researchers to improve the biological properties of BC [9,10,11]. Moreover, researchers have reported that BC composites with pullulan, chitosan, cactus and acrylic acid hydrogels increase the healing rate of burn wounds and may be used as wound dressing composites [11,12,13,14,15]. In addition, BC for skin wound treatment have antimicrobial efficacy. In this regard, antimicrobial peptides with quorum quenching enzymes, silver nanoparticles, zinc oxide nanoparticles, and carbon quantum dots–titanium dioxide (CQD–TiO2) nanoparticles have been used to impart antibacterial and antimicrobial properties to BC [16,17,18,19]. Biofilm, produced by pathogenic bacteria, is a complex community of microbes adhering to natural or synthetic structures and firmly embedded in the extracellular matrix [20]. Pathogenic bacteria in biofilms exchange genetic material with each other, and the genetic modifications in pathogens cause them to develop resistance to antibiotics. Currently, antibiotic resistance has led to an increase in infectious diseases and epidemics. Additionally, biofilms play an essential role in the infection processes of pathogens. For example, the presence of biofilms in chronic wounds is one of the factors that impair the healing process [21, 22]. Therefore, there is a need for materials that can penetrate and disrupt bacterial biofilms in the health sector. Modified BC can also be used to prevent biofilm formation, which is a structure that increases the pathogenicity of bacteria and causes many treatments to fail in the clinic [23]. In this regard, BC nanoparticles inhibit bacterial biofilm production, which is also being produced due to the anti-biofilm effects of nanoparticles, such as Ag, Zn and boron [24]. These BC nanoparticles could serve as important biomedical materials, especially in wound dressing applications or to minimise biofilm growth. Some researchers have determined the antibacterial activities of metal oxide nanoparticles, such as zinc oxide, copper oxide [25, 26] and boron [27, 28]. While these nanoparticles and boron show specific toxic properties on bacteria, they have little effect on human cells (25–28).

As previously mentioned, metal oxide (ZnO and CuO) nanoparticles and borax may be alternatives for limiting bacterial biofilm formation. According to the literature, antimicrobial agents, such as boron, silver, copper and zinc, have been incorporated to endow BC with antibacterial efficacy [19, 28,29,30,31,32,33]. Still, the anti-biofilm activities of synthesised composites have yet to be reported. CuO and ZnO nanoparticles are nanoparticles that have many applications such as sensors, biomedical and antibacterial. In addition, the mentioned nanoparticles were preferred due to easy to obtain by green synthesis method, cheap and harmless to nature [25, 32]. Thus, we aimed to develop BC loaded with nanoparticles (BC-NPs) with anti-biofilm efficacy. BC formed from Gluconobacter xylinus S4 isolate was loaded with zinc oxide nanoparticles (ZnONPs), borax and a combination (ZnOBoraxNPs and ZnCuO2NPs). The BC-NPs were analysed to determine their physicochemical properties by scanning electron microscopy (SEM). Moreover, we study their anti-biofilm, cytotoxic, and wound-healing properties.

Materials and Methods

Preparation of BC Loaded with Nanoparticles (BC-NPs)

Copper oxide nanoparticles (CuONPs) and zinc oxide nanoparticles (ZnONPs) used in the experiment were synthesised by the green synthesis method [34]. Borax pentahydrate (analytical grade) was purchased from Merck. Composites prepared with BC were synthesised ex situ [1]. After that, four BCs ( 4 × 4 cm plates) were placed in 200 mL of water that contain either ZnONPs (0.1 g), borax (0.1 g), ZnOBoraxNPs (0.05 g ZnONPs and 0.05 g borax) or ZnCuO2NPs (0.05 g ZnONPs and 0.05 g CuONPs). Then, they were agitated at 200 rpm at 50 °C for 48 h. The synthesised four different BC-NPs were dried and stored for analysis [35].

Analysis of the BC Loaded with Nanoparticles (BC-NPs) by SEM

The morphologies and elemental compositions of the BC-NPs were investigated by SEM (using a scanning electron microscope, Zeiss Supra 40 VP, Oberkochen, Germany) and energy dispersive X-ray spectroscopy (EDX), respectively. The samples were sputter-coated with gold–palladium particles (Quorum Q150R ES, Quorum Technologies Ltd, Lewes, UK), and the accelerator voltage was adjusted to 30 kV.

Determination of Biofilm Inhibition and Biofilm Degradation Activity of BC-NPs

Crystal Violet (CV) Assay

The activities of biofilm inhibition and degradation of the BC–NPs were determined via the CV assay method [36, 37]. In this study, we used Listeria innocua ATCC 33090, Staphylococcus aureus ATCC 29213, Pseudomonas aeruginosa PAO1, Escherichia coli ATCC 10536, Streptococcus parasanguinis ATCC 15909, Bacillus cereus RSKK 863, Enterococcus hirae ATCC 10541 and Candida albicans ATCC 64548 as human pathogens. Sabouraud Dextrose Broth (SDB, for Candida albicans), Todd Hewitt Broth (for Streptococcus strains) and Tryptic Soya Broth (for other bacteria) were used as growth media. Each cell culture (10% microbial inoculum/medium, 0.5 MacFarland) was added to wells containing the growth medium with BC–NPs (as sheet) and incubated at 37˚C for 48 h to determine the biofilm inhibition activity. Then, the samples were removed from the wells and the wells were washed with PBS (0.01 M, pH 7.4) to remove non-adherent cells. Each well in the plate was stained with 0.1% CV for 15 min. Then, 33% glacial acetic acid for gram-positive bacteria and Candida albicans and 95% ethanol for gram-negative bacteria were added to the wells. The optical density of the wells was read at 570 nm in a microtitre plate spectrophotometer (Epoch, Biotech). All tests were performed in duplicate. The formula below was used to determine the activity percentage of the BC–NPs.

Anti-biofilm activity (%) = [(Control OD – Sample OD) / Control OD] × 100.

To determine the biofilm degradation efficacy of the BC–NPs, microbial biofilms were grown at 48 h, and then, non-adherent cells were removed by washing with PBS. The BC–NPs were added to each well, and the plates were incubated at 24 h. Finally, the biofilm degradation was determined using the above method [38].

XTT Assay

The viability of each microbial strains in the biofilm was evaluated via the XTT assay [39,40,41,42]. After incubation for 48 h, the plates were washed with phosphate-buffered saline (PBS, 0.01 M, pH 7.2). Then, 100 µL of PBS and 50 µL of XTT reaction solution were loaded (Cell Proliferation Kit (XTT-based), Biological Industries, Israel). Then, the plates were incubated in a dark room at 37 °C for 5 h. Absorbance was measured at 450–630 nm. To determine the biofilm degradation effect of the BC–NPs, we added them to the wells containing pre-formed biofilms and incubated them for 12 and 24 h. At the end of the incubation period, all BC–NPs were removed, and each well was gently washed three times with PBS. A standard XTT reduction assay was performed to quantify the viability of the biofilm via bacterial metabolic activity. The decrease in metabolic activity compared to the control biofilm formed (100%) in the wells without BC–NPs is defined as the biofilm inhibition and degradation effect. The experiments involved two replicates. The formula below was used to determine the activity percentage of the BC–NPs.

Anti-biofilm activity (%) = 100- [(Sample OD / Control OD) × 100]

Cell Culture and Treatment

Mouse fibroblast cell lines (L929) are typically used as model cells in biocompatibility studies of biomaterials [43]. The L929 cell line was purchased from the American Type Culture Collection (ATCC). It was grown in Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma) and enriched with 10% heat-inactivated foetal bovine serum (FBS, Capricorn) and 1% penicillin/streptomycin solution (100X, Capricorn), as described previously [44]. The cells were cultured in a 100-mm cell culture dish (Nest) and incubated at 37 °C and 5% CO2 in a humidified CO2 incubator. The viability of the L929 cells was examined using MTT (3-(4, 5-dimethylthiazol2-yl)-2, 5-diphenyl tetrazolium bromide) reagent (Merck), as previously described [45]. For this, cells were harvested with 0.25% Trypsin-EDTA (Capricorn) at 90% confluence and seeded at a density of 2 × 103 cells/well in a 96-well plate (Costar, Corning) in a complete medium. After 24 h, the medium was replaced with different concentrations (25, 50 and 100%) of the medium containing BC–NPs. For the extraction, The BC–NPs sheets were sterilised by autoclaving at 121 °C for 15 min and placed on a six-well plate. The extraction conditions were based on ISO 10993-12 as described previously with slight modifications [1]. Briefly, 2 × 3 cm cut BC–NPs sheets (6 cm2/mL) was extracted at 37 °C for 24 h in culture media in a shaker. After 24 h, the medium was removed and filtered. An equal amount of fresh medium was added to untreated cells (negative control). After incubation period of the cells with BC-NPs (24 h) culture medium was removed, and 10 µL of MTT (5 mg/mL) was added to each well and incubated under culture conditions. Cell viability was calculated as decribed previsouly [45].

In Vitro Cell Migration Assay

The migration efficacy of BC–NPs was evaluated using the wound healing assay described previously [46]. In this study, the L929 mouse fibroblast cell line was selected. Fibroblast cells have been proposed to test wound healing capacity in in-vitro studies [47]. Briefly, 3 × 104 cells were plated into 6-well plates (Jet Biofil) and incubated under culture conditions. After 24 h, the cells were scraped with a 200-µL pipette tip [48] and washed with phosphate-buffered saline (PBS, Bioshop) to discard detached cells. The medium containing the non-toxic concentrations of BC–NPs was replaced for 24 h. The wound area in the treated cells was compared with the vehicle group. The wound size was captured (0, 24, 48 and 72 h) using an inverted microscope (Oxion Inverso, Euromex) at 10× magnification, and the wound closure rate (%) was analysed using ImageJ software 1.53e, as described previously [49].

Statistical Analysis

GraphPad Prism 9 (San Diego, CA, USA) was used for statistical analysis. The experiments were performed in triplicate, and the data are expressed as means ± SD. Two-way ANOVA was used to analyse the statistical differences between the groups. Ns = not significant, * = p < 0.05, ** = p < 0.005, *** = p < 0005, **** = p < 0.0001.

Results and Discussion

Bacterial Cellulose Loaded with Nanoparticles

As shown in Fig. 1, the morphology of BC loaded with nanoparticles was determined via SEM analysis. As shown in the electron microscope images, the nanoparticles on BC generally had an oval geometry. The nanoparticles likely penetrated deeply into the BC fibre networks. In other words, we speculated that nanoparticles bind strongly to BC fibrils owing to the hydroxyl groups in the chemical structure of native BC. This positively affects many biological properties of BC. Moreover, the quantitative analysis of BC modified with nanoparticles is shown in Fig. 1. Eventually, strong peaks in the EDX spectrum showed the formation of BC–NPs.

Fig. 1
figure 1

Scanning electron microscopy (SEM) images at 50.00 kx magnification and energy dispersive X-ray spectrum of BC and the nanohybrids. a, b, c and d show ZnO, ZnOBorax, ZnCuO2 nanoparticles (NPs) and Borax, respectively

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Determination of Biofilm Inhibition and Biofilm Degradation

Biofilms in chronic wounds delay their healing [50]. This is because the biofilm-forming feature of pathogens increases their infectivity and makes it difficult to combat them. Moreover, the bacteria in the biofilm structure escape the host defence more easily. For example, P. aeruginosa biofilm delayed wound healing compared to a group without the biofilm because of its prolonged inflammatory phase of healing [51]. Because antibiotics cannot penetrate the biofilm structure, the pathogens in a biofilm are almost always protected from the effects of drugs. Therefore, bacterial biofilms are a significant problem for human health. In our previous paper, the pure BC membrane did not exhibit any anti-biofilm activity, showing that non-modified BC did not have anti-biofilm activity against tested strains in the study, except S. aureus with a biofilm inhibition percentage of 22% [1]. However, BC–NP research is a recently developing field due to nanoparticles’ strong antimicrobial and anti-biofilm activities. In this study, we evaluated BC–NPs and their biofilm inhibition effect, biofilm degradation, and metabolic activity of microorganisms in the biofilm structure. Various human pathogenic microorganisms were used, and the activities of biofilm inhibition and degradation of BC–NPs were screened using a CV assay. According to the CV staining results, the effects on the biofilm growth and the development of a preformed biofilm were different (Table 1). It has been reported that different antimicrobial activities against different bacteria could lead to different extents of biofilm inhibition by nanoparticles [52]. In this study, the S. aureus ATCC 29213 biofilm was reduced by 65.53% by BC–ZnONPs, 71.74% by BC–BoraxNPs, 66.60% by BC–ZnOBoraxNPs and 28.27% by BC–ZnOCuO2NPs. Of the four BC–NPs, BC–BoraxNPs showed the highest effect on S. aureus ATCC 29213, followed by BC–ZnOBorax and BC–ZNONPs. The lowest biofilm inhibition effect was observed with BC–ZnOCuO2NPs (Table 1). The biofilm inhibition against P. aeruginosa PAO1 could not be observed for all BC–NPs, whereas BC–ZnONPs and BC–Borax had moderate inhibitory potential with 38.44 and 39.97% on E. coli ATCC 10536, respectively. The BC–NPs showed a more potent inhibitory capacity against S. aureus than E. coli and P. aeruginosa because the cell wall structure of gram-negative cells is more sophisticated than the cell structure of gram-positive cells that have a more substantial defence capability [53, 54]. BC–ZnONPs, BC–Borax and BC–ZnCuO2NPs inhibited the biofilm formation of L. innocua ATCC 33090 at 44.79, 54.86 and 65.14%, respectively. BC–Borax showed the highest biofilm inhibition activity at 57.12% in C. albicans ATCC 64548 as a eukaryotic microorganism. The biofilm inhibition effect of BC–NPs was lower on other tested bacteria, as shown in Table 1. BC loaded with ZnOCuO2 nanoparticles did not show any effect on both degradation and inhibition of P. aeruginosa PA01, S. parasanguinis ATCC 15909, B. cereus RSKK 863, E. hirae ATCC 10541 and C. albicans ATCC 64548 biofilms. In comparing the 24-h biofilm degradation, BC–Borax was the most effective at reducing P. aeruginosa PAO1, L. innocua ATCC 33090 and S. parasanguinis ATCC 15909 biofilms by 58.98, 62.40 and 54.26%, respectively, BC–ZnOBoraxNPs degraded 64.02% (for S. aureus ATCC 29213) and 52.93% (for L. innocua ATCC 33090) of the bacterial biofilm. According to the CV assay, the biofilm degradation of BC–NPs was likely related to the biofilm degrading ability of ZnO, borax, and CuO. However, other BC–NPs had no significant degradation effect on the pre-biofilm. Borax and nanoparticles, such as ZnO and CuO, have been reported to have inhibitory effects on bacterial biofilm formation [28, 32, 55].

Table 1 Biofilm inhibition percentages and degradation results from the CV assay (%)
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To evaluate the biofilm inhibition and eradication activities of the BC–NPs by using the XTT reduction assay, we selected L. innocua ATCC 33090, S. aureus ATCC 29213, P. aeruginosa PAO1, E. coli ATCC 10536, S. parasanguinis ATCC 15909 and C. albicans ATCC 64548. The anti-biofilm efficacy of BC–NPs was further confirmed by measuring the metabolic activity of the cells in the biofilm. The results obtained from the CV assay were not correlated with the XTT reduction assay. In other words, the biofilm inhibition of BC–ZnCuO2NPs was determined by the XTT reduction assay despite no biofilm inhibition shown in the CV assay. Moreover, the biofilm inhibition activity of BC–ZnCuO2NPs had the highest effects against C. albicans ATCC 64548, S. aureus ATCC 29213, L. innocua ATCC 33090 and E. coli ATCC 10536. Moreover, there was no significant inhibition effect for ZnONPs, Borax, and ZnOBoraxNPs against the tested microorganisms. The metabolic activities of these cells continued in the presence of nanoparticles. During bacterial biofilm formation, the most resistant bacteria to nanoparticles were P. aeruginosa PAO1 and S. parasanguinis ATCC 15909. In other words, these bacteria continued their metabolic activities in the presence of BC–NPs. There was no inhibition in bacterial biofilm formation (Fig. 2). However, we still chose to test the degradation effect of BC–NPs due to the effects of these bacteria on public health. However, there was no inhibition against these two bacteria. Interestingly, BC–NPs were observed to have degradation abilities against the biofilm formed by these bacteria (Fig. 3).

Fig. 2
figure 2

Biofilm inhibition percentages of BC–NPs (a) and metabolic activities of the cells in the biofilm (b) compared to the control. The results shown are the means ± SD. Ns = not significant, * = p < 0.05, ** = p < 0.005, *** = p < 0005, **** = p < 0.0001

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According to the results, the biofilm degradation efficacy of BC–NPs upon treatment for 24 h was higher than with treatment for 12 h (Fig. 3). Moreover, the biofilm degradation percentage increased, indicating the degradation effectivity of some BC–NPs against pathogens. In particular, while all BC–NPs showed high degradation activity against S. parasanguinis ATCC 15909 at 24 h (BC–ZnONPs: 85.49 ± 3.24%, BC–Borax: 69.09 ± 0.00%, BC–ZnOBoraxNPs: 59.70 ± 0.00% and BC–ZnCuO2NPs: 69.04 ± 8.58%) (Fig. 4d), only BC–ZnOBoraxNPs (52.10 ± 1.84%) showed moderate degradation sensitivity at 12 h (Fig. 3b). For P. aeruginosa PAO1, BC–ZnONPs (77.21 ± 3.91%) and BC–Borax (76.40 ± 1.05%) showed a similar degradation percentage at 12 h. Still, this effect of BC–ZnONPs and BC–Borax decreased at 24 h. BC–ZnCuO2NPs interestingly degraded 87.740.61 ± 0.00% of the P. aeruginosa PAO1 biofilm in 24 h (Fig. 3c). Similarly, BC–ZnCuO2NPs induced degradation of the biofilm against L. innocua ATCC 33090 by up to 87.72 ± 4.03% in 24 h (Fig. 3c). Moreover, a decrease in the metabolic activity of cells of the biofilm was observed with the BC–NPs. Not all BC–NPs showed degradation activity against C. albicans ATCC 64548 with high levels of metabolic activity (Fig. 3c and d).

Fig. 3
figure 3

Biofilm degradation and metabolic activity of cells with BC–NPs in the biofilm structure compared to the control. The results shown are means ± SD. Ns = not significant, * = p < 0.05, ** = p < 0.005, *** = p < 0.0005, **** = p < 0.0001

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Our modified BC–NPs showed degradation of the bacterial biofilm. Similarly, BC loaded with chitosan nanoparticles showed biofilm eradication activities (36.09–90.43%) against pre-existing bacterial biofilms [21]. According to Zhang and co-authors, a BC + tannic acid + Mg composite showed a strong antibacterial effect against S. aureus, E. coli and P. aeruginosa bacteria and reduced biofilm formation of S. aureus (80%), and P. aeruginosa (87%) [56]. Moreover, a BC + chitosan nanocomposite showed antimicrobial activity against S. aureus, P. aeruginosa and C. albicans and prevented biofilm formation on the nanocomposite surfaces [57]. Furthermore, our results indicated that the anti-biofilm activity of BC–NPs was related to the variety of nanoparticles and tested microorganisms. Previous studies indicated that nanoparticles, such as copper, zinc, and borax, had been used to inhibit biofilm formation [28, 58, 59]. In our study, the reduction in biofilm formation after treatment with BC–NPs may be related to the anti-biofilm effect of possibly damaging the cell membrane after the interaction of the nanoparticles with the bacteria or damaging bacterial DNA, as Brahma and co-authors mentioned [60]. The mechanism of biofilm degradation is unclear. However, the rapid destruction of the biofilm may also be related to the interruption of bacterial cell signalling systems and degradation of the biofilm matrix [61]. Moreover, the biofilm inhibition and degradation results may be due to the differences in microorganism strains and the effect of nanoparticles in the BC modification. Finally, as expected, decreases in metabolic activity were noted where the anti-biofilm effect was high.

MTT Assay

The in vitro compatibility of BC–NPs was determined on mouse fibroblast L929 cells by MTT assay. Three different concentrations of BC–NPs (25, 50 and 100%) were applied for cytocompatibility investigations for 24 h. Results from the MTT assay of the BC–NPs showed no significant effects on the cell viability at a concentration of 25% (Fig. 4). We did not observe any cytotoxic effect due to BC itself. We observed a cytotoxic effect as the concentration increased due to the nanoparticles remaining in the nutrient substitute. We also observed a cytotoxic effect as the concentration increased due to the nanoparticles remaining in the culture medium. Therefore, the highest non-cytotoxic doses of nanoparticles were selected for further studies.

Fig. 4
figure 4

Cytocompatibility of BC extracts on L929 mouse fibroblast cells for 24 h. The results are shown as means ± SD. Ns = not significant, ** = p < 0.005, *** = p < 0005, **** = p < 0.0001

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Wound Healing Assay

A wound healing assay was performed to assess the effects of BC loaded with ZnONPs, ZnOBoraxNPs, Borax and ZnCuO2NPs (highest non-cytotoxic concentrations of 25, 50, 50 and 25%, respectively) on the migration of L929 cells (Fig. 5). Following 24 h of treatment, the medium containing BC–NPs was removed from individual wells, the cells were washed with PBS twice, and the medium was refreshed. Note that the control group contained only culture medium.

Fig. 5
figure 5

Microscope images from the in vitro wound healing assay of BC extracts. The photos were taken at 0, 24, 48, and 72 h

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BC and its different composites are considered suitable biomaterials for tissue engineering and regenerative medicine, especially for their significant contribution to wound healing [62,63,64]. Czaja et al. (2006) suggested that the unique nanofibrillar structure of BC–based wound dressings contributes to their biocompatibility by providing an ideal environment for wound healing [65]. BC accelerates skin healing compared to traditional wound dressings, such as ointments and wet gauze [66, 67]. Research has shown that BC–based coverings lower rates of wound infection and accelerate re-epithelization [65, 68, 69].

In our study, the migration of L929 cells was significantly induced by BC–Borax, and cell migration reached 99% (Fig. 6). The wound area in the BC–ZnONPs (5.2%) was better than in the control group (6.4%) at 72 h. However, the wound area of cells treated with ZnOBoraxNPs and ZnCuO2NPs was found to be 18.5 and 22.3%, respectively, after 72 h. In other words, cell migration was restricted approximately three- and four-fold compared to the control cells in the same order. Similar to our findings, BC nanocomposites containing different nanoparticles showed wound-healing properties in prior studies [63, 70,71,72]. It is well established that zinc oxide nanoparticles have many properties, including wound healing by changing cell migration, re-epithelialization, and angiogenesis [73]. Similarly, BC has wound-healing activity due to its unique properties, including a uniform nanofiber network, high crystallinity, high water absorption, and retention capacity [74]. Moreover, borax loading could increase wound healing activity [75]. In this regard, BC–ZnO and BC–Borax showed better wound-healing properties. Additionally, our results showed for the first time that BC–Borax has potential wound-healing activity for L929 fibroblast cells.

Fig. 6
figure 6

Percentage of wound area for treated cells compared to the control at 0, 24, 48, and 72 h. The data shown are the means ± SD. Control group wound areas were taken to be 100% and calculated using ImageJ software

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Conclusion

In the present study, BC was successfully modified with ZnONPs, borax, ZnOBoraxNPs and ZnCuO2NPs, and they were confirmed by SEM analysis. The BC- ZnCuO2NPs showed good antibiofilm activity against C. albicans ATCC 64548, E. coli ATCC 10536, S. aureus ATCC 29213 and L. innocua. Moreover, the biofilm degradation efficacy of BC–ZnONPs, BC–Borax, and BC–ZnCuO2NPs towards preformed biofilms of P. aeruginosa and L. innocua were significant. Also, BC–Borax and BC–ZnONPs have showed wound healing activity. All of these results suggest that BC-NPs can be considered for use in wound-healing dressings and other biomedical applications by restricting and inhibiting biofilm formation towards different bacteria in wounds.