Antibiofilm Activity of Activated Carbon Film Wound Dressing

Abstract

Background: Biofilm is a structured community of bacterial cells enclosed in a self-produced matrix containing 80% extracellular polymeric substances (EPS). When biofilms are present on the wound surface, they impair optimal penetration of antimicrobials resulting in delayed healing. Chronic wounds are likely to be impacted since the wound may be moist and purulent which are conducive for biofilm growth. Objective: To develop and characterise hydrogel films containing AC at various concentrations and to study the antibiofilm activities of the produced films through in vitro approach on two common biofilm strains, Staphylococcus aureus, and Pseudomonas aeruginosa. Methods: An approach was suggested in this study by utilising the high adsorptive capacity of activated carbon (AC) loaded into PVP/CMC hydrogel film wound dressing to disrupt and inhibit the biofilms present on the wound surface. Results: The results obtained showed that the developed hydrogel films containing AC at different concentrations (0.1%, 0.5%, 1.0%, 1.5% and 2.0%) provided a balanced moist environment to the wound surface with adequate swelling properties. Characterisation of the AC films also showed satisfactory mechanical properties indicating the films were durable and flexible, thus suitable for wound application. Antibiofilm study was conducted via XTT assay by treating biofilms of two common bacterial strains, Staphylococcus aureus and Pseudomonas aeruginosa with the AC hydrogel films. Findings showed AC exerted antibiofilm activity by inhibiting more than 50% of biofilm growth on P. aeruginosa biofilm, with AC 0.5% achieving the highest biofilm inhibition (62.41±8.48%). However, there was lack of evidence to support AC hydrogel films in inhibiting biofilm produced by S. aureus because there was no significant difference (p<0.05) in biofilm inhibition when compared to AC 0%. Conclusion: Although AC hydrogel films showed promising results on P. aeruginosa biofilm inhibition, its application in wound dressing as antibiofilm agent may be limited especially on multispecies biofilm due to its lack of activity towards S. aureus. Further investigations are required to assess antibiofilm activity of AC hydrogel films on other bacterial strains and its mechanism of biofilm inhibition.

Introduction

Wound is formed when there is any disruption to the epithelial integrity of the skin resulting in the impairment of normal tissue function and structure. Wound can be categorised into acute or chronic wound depending on their ability to heal. Acute wounds are wounds that occur recently and heal in a short period of time within four weeks such as traumatic wounds and surgical wounds whereas chronic wounds are wounds that fail to heal under a normal expected healing period and require more than 12 weeks to heal [1]. Examples of chronic wounds

include diabetic ulcers, pressure ulcers and venous ulcers. Nevertheless, when a wound becomes infected, the natural healing process is impaired resulting in delayed wound healing commonly seen in chronic wounds [2, 3]. Bacterial biofilms pose a significant issue in chronic wound management. It was reported that up to 75% of microbial infections are due to the colonisation of bacteria in the form of biofilm [3]. Biofilm is a structured community of bacterial cells enclosed in a self-produced matrix and adherent on an inert or living surface. It is made up of 80% extracellular polymeric substances (EPS) consisting of polysaccharides, proteins, and nucleic acids. Bacteria in biofilms behaved differently than planktonic bacteria since mostly are sessile. Multi-species bacteria can also adhere to a surface and form biofilm. Due to the nature of biofilms, they are recalcitrant to antimicrobials, including antibiotics [4]. It is estimated that bacteria in a biofilm are 50-1000 times more resistant to conventional antibiotic treatment [5].  Wound infection persists because biofilms are resistant to killing by host defence mechanisms and antibacterial treatments. The wound is unable to resolve from the inflammatory stage resulting in poor wound healing, and the formation of chronic wounds [4]. The current management of biofilms consists of physical debridement and chemical approaches. Physical debridement involves the removal of the tissue layer containing biofilm, which causes pain on the wound surface. hence chemical approaches were favoured over physical debridement. Chemical approaches include the development of antibiofilm agents such as antimicrobials, ethylenediaminetetraacetic acid (EDTA) and xylitol.

A more specific approach is required to manage biofilms present in chronic wounds. In this research, hydrogel films were chosen as a wound dressing of choice. Hydrogel dressings are insoluble hydrophilic materials made up of natural or synthetic polymers that can provide a moist environment due to their high-water content promoting granulation of tissues and epithelium. They are also skin-friendly and easy to apply to wounds [6-8].

Novel approaches are intensively studied to find ways to disrupt the biofilm layer and improve the penetration of antimicrobials. Activated carbon (AC) was studied to investigate its antibiofilm activities in vitro. Activated carbon is also known as activated charcoal. Activated charcoal is characterised by a carbon matrix. It incorporates a large number of pores of various diameters and lengths. The available surface for adsorption ranges from 400 to 1200 m2 per gram of charcoal and can be up to 5000 m2 [9]. Clearly, AC possessed powerful adsorptive capability resulting in its uses in the biomedical field for the removal of toxins and malodour [10]. It is hypothesised that AC may also have potential antibiofilm activity aimed at combating biofilms present on the wound surface.

Thus, this research aimed to develop and characterise hydrogel films containing AC at various concentrations and to study the antibiofilm activities of the produced films through in vitro approach on two common biofilm strains, Staphylococcus aureus, and Pseudomonas aeruginosa. These objectives were made to find out the potential of AC to be loaded into hydrogel films since the majority of the wound dressing containing AC available in the market was either in fibre or cloth form indicated for malodour management of wounds. Nevertheless, there was also a lack of study in investigating the ability of AC in inhibiting biofilm growth. Therefore, this research was conducted to address these issues and produced a practical wound dressing with advantages to wound healing.

Materials And Methods

Materials

Formulation of hydrogel films consist of two polymers, namely low viscosity sodium carboxymethylcellulose from Merck (Darmstadt, Germany) and polyvinylpyrrolidone K-90 from Acros Organics (Massachusetts, USA). Agar powder bacto grade and Glycerol 99.5% were obtained from Sisco Research Laboratories Chemicals (Mumbai, India). A premium grade activated carbon was purchased from Noble Healthcare (Kuala Lumpur, Malaysia). Next, potassium chloride was obtained from Merck (Darmstadt, Germany). Gelatine from bovine skin was obtained from Sigma Aldrich (Missouri, USA).

For antibiofilm study, Gram-positive bacteria, Staphylococcus Aureus (ATCC 25923) and Gram-negative bacteria, Pseudomonas aeruginosa (ATCC 27853) were used. Mueller-Hinton agar (MHA) and Mueller-Hinton broth (MHB) were purchased from Merck (Darmstadt, Germany). Phosphate-buffer saline (PBS) tablet was obtained from Sigma Aldrich (Missouri, USA). The chemical reagent used consists of tetrazolium sodium (XTT) purchased from Bio Basic (Toronto, Canada), menadione crystalline and acetone from Sigma Aldrich (Missouri, USA).

Preformulation and Film Preparation

Preformulations of hydrogel consist of incorporating two polymers, polyvinylpyrrolidone (PVP) and sodium carboxymethylcellulose (Na-CMC) based on a method described by  by varying the ratios of the polymers 80:20 and 20:80. A total of six formulations were prepared with different ratio of the polymers and glycerol concentration (Table I). Then, the formulation that possessed the best physical attributes such as satisfactory appearance and flexibility was chosen to be incorporated with activated carbon (AC) at different concentrations (0%, 0.1%, 0.5%, 1.0%, 1.5% and 2.0% w/v).

The hydrogel films were prepared by heating 30 mL of distilled water to 80°C. PVP and Na-CMC were then poured slowly into the heating water and mixed with a magnetic stirrer for 20 minutes until all powders were completely dissolved and no lumps were present. Then, glycerol   was added slowly into the solution while stirring. Next, 2 g of agar powder was added to the solution and stirred for another 30 minutes. The solution was made to 100 mL by adding distilled water. A cloudy solution was formed and placed under moist heat treatment at a condition of 121°C and 103 kPa for 20 minutes to allow crosslinking of the polymers resulting in a transparent gel solution [11]. Then, the hydrogel solution (20 g) was carefully

poured into a petri dish (85 mm diameter) following the solution casting method and allowed to solidify at room temperature for 30 minutes. A total of four films was obtained from the preparation of 100 mL hydrogel solution. The hydrogels were set to dry in the oven at 50 ± 2°C for 24 hours resulting in the formation of hydrogel film. The dried films were peeled gently from the petri dish and conditioned in a desiccator (50 – 60% RH, 27 ± 2°C) prior to testing for temperature stabilisation as described by  Ng and Leow [12].

The most satisfactory formulation was chosen based on physical observation and incorporated with AC at different concentrations. AC was added into the chosen formulation after moist heat treatment by continuous stirring at 350 rpm for 10 minutes until a homogenous, black-coloured gel solution was formed. Characterisation and further studies were performed on AC-containing hydrogels at different concentrations.

Physical Assessment

Physical assessments of the films were done macroscopically which included assessing their appearance, colour, odour, and flexibility. The film thickness and diameter were also measured at four points at every 45-degree angle by using a digital vernier calliper, Absolute Digimatic Calipers from Mitutoyo (Kanagawa, Japan). The measurements were expressed as the mean ± standard deviation (SD).

The moisture content of each film was also assessed by measuring the weight of the films before drying and after drying at 50 ± 2°C in the oven for 24 hours. The weight before drying was taken when pouring the hydrogel solutions (20 g) into the petri dish whereas the weight after drying was taken after storing the films in the desiccator for 30 minutes for temperature stabilisation. The moisture content was determined as the percentage of the initial film weight lost during drying using the following equation:

Where W₀ is the initial weight of the film (g) and W₁ is the weight of the film after drying (g). All tests were done in triplicate.

Swelling Ratio Test

A method described by Matthews, Stevens [13] was adapted with some modifications. It consisted of using a simple non-animal model to demonstrate swelling properties of hydrogel films on suppurating wounds. The swelling medium consisted of 4% w/v gelatine that was prepared by adding 4 g of gelatine powder into 100 mL of distilled water that had been heated to 60°C. The mixture was slowly stirred for 10 minutes until homogenous. When a clear solution was formed, 20 g of the gelatine solution was poured into petri dish and allowed to cool at room temperature. Once cooled, the petri dishes were covered and allowed to be set overnight at room temperature.

Next, the films were cut into a circular shape with a diameter of 20.50 mm and weighed accurately. Then, the films were placed at the centre and on top of the gelatine medium. The weight of the films was measured at different time intervals (1 hour, 3 hours, 5 hours, and 24 hours). The swelling properties of the films were calculated as shown in Eq. (2). The experiment was done in triplicate for each formulation.

Moisture Absorption Study

A method described by Ng and Leow [12] was adapted with modifications. Films of known diameter (20.50 mm) were cut, weighed accurately, and placed in a desiccator containing silica beads for 24 hours (50 – 60% RH, 27 ± 2°C). Another desiccator containing saturated potassium chloride solution was prepared and left to condition for 24 hours to form a humid environment (84% RH, 27 ± 2°C). The films were then placed inside the desiccator and left for 24 hours. After 24 hours, the films were weighed, and the percentage of moisture absorption was computed as the difference in the initial and final weight of the films with respect to the initial weight. The equation used to compute the percentage was shown in the following equation.

Where W_f is the final weight of the film (g) and W₀ is the initial weight of the film (g).

Moisture Vapour Transmission Rate (MVTR) Study

Films of known diameter (20.50 mm) were cut and gently placed on an opened glass vial that contained 2 g of silica beads. A double-sided tape was used to ensure the films adhered firmly to the top of the glass vial. The exposed film diameter was measured (15.50 mm) and used in the calculation of the exposed surface area. An opened glass vial without any film and a glass vial sealed with aluminium foil acted as controls in this study. Then, the glass vials with films and controls were placed in a desiccator containing saturated potassium chloride solution (84% RH, 27 ± 2°C). The weight of each glass vial was measured including the silica beads and film every hour for the first 6 hours followed by every 24 hours for two days. The moisture transmitted through the films was determined gravimetrically by the increment of weight of the glass vials. The rate of moisture transmission was calculated from the difference in mass in a period of time divided by the exposed film area. The equation used to calculate the MVTR was as follows:

Where  is the weight of the glass vial at a given time (g),  is the initial weight of the glass vial (g),  is the time presented in hours, and  is the exposed surface area of film in cm². The test was run in triplicates.

Mechanical Properties

The mechanical properties of the films were evaluated by utilising the Universal Testing machine model 5567 from Instron (Warwick, UK). The computed value obtained consists of the maximum load at break withstand by the film and the extension of film at break. These parameters were used to assess the tensile strength (TS), percentage of elongation at break (E%) and Young’s modulus.

To prepare the film, firstly the films were trimmed according to the ASTM standard dumbbell shape template with a length of 30 mm and width of 5 mm. The films were stretched to break at a crosshead speed of 5 mm/min. The test was run in triplicates. The calculation of TS, E%, and Young’s modulus were demonstrated below:

Where F_max is the maximum force at break (N) and  is the cross-sectional area of the film (mm²).

Where L_f is the extension of the film at the point of break (mm) and L₀ is the initial length of the film prior to stretching (30 mm).

Where TS is the tensile strength computed in Eq. (5), L_f  and the initial length of the film prior to stretching (30 mm), L₀

Fourier-Transform Infrared Spectroscopy (FTIR)

The dried hydrogels were analysed by using a Perkin Elmer Spectrum 100 FT-IR spectrophotometer using a Perkin Elmer universal attenuated total reflection (ATR) sampling assembly. The samples that were tested include hydrogel films containing AC 0%, 0.1%, 0.5%, 1.0%, 1.5% and 2.0% as well as AC powder only.

Firstly, the hydrogel films were cut into a circular shape with a diameter of 10 mm and placed on top of the ATR crystal. Adequate force was applied to the ATR pressure clamp to ensure optimal contact of samples on the ATR crystal. As for the AC powder, the powder was gently loaded on top of the crystal and ATR pressure clamp was firmly placed on the powder. The FTIR spectra of samples were recorded with a uniform resolution of 2 cm^-1 and the selected range was between 4000 and 650 cm^-1.   

Antibiofilm Study (XTT assay)

The antibiofilm activities of the AC-containing films were quantified by performing XTT assay. Aseptic techniques were practised throughout the study and the biosafety cabinet class II (BioAir, Italy) were utilised in carrying out this experiment. The methods described by Peeters, Nelis [14] were adapted with some modifications.

Firstly, two bacterial strains (Staphylococcus Aureus and Pseudomonas Aeruginosa) were grown in MHB to form bacterial suspensions with turbidity equal to 0.5 McFarland standard. Adjustments were done accordingly to achieve the desired turbidity. The adjusted suspensions were used within 30 minutes. In the preparation of treatments, the hydrogel films containing AC were trimmed into a circular shape, with a diameter of 5 mm and placed carefully at the bottom of the wells of a 96-well-flat-bottom tissue culture plate (Biofil Chemicals and Pharmaceuticals Ltd., India). 100 µL of the bacterial suspension was added to each well by using a micropipette. Untreated wells were also prepared to contain MHB only and bacterial suspension only. The plates were incubated at 37°C for 24 hours under static conditions. After 24 hours, the remaining bacterial suspension and the hydrogel films in the wells were removed. The plates were then rinsed with PBS twice to remove planktonic bacteria and any residue from the films.

Next, XTT assay was conducted to study the antibiofilm activities of the films. The light-yellow colour of the XTT solution prepared in this experiment will be reduced to a dark orange water-soluble formazan salt facilitated by bacterial enzymes. In this antibiofilm study, the first step was to prepare fresh XTT solution prior to use by dissolving 4 mg of XTT powder in 10 mL of prewarmed (37°C) PBS followed by 100 µL of menadione stock solution. Menadione stock solution was prepared beforehand by dissolving 55 mg of menadione in 100 mL of acetone. Next, 100 µL of PBS and 100 µL of XTT-menadione solution were added to each well, then the plates were incubated in the dark for 5 hours at 37°C. After 5 hours, the plates were taken out of the incubator and the supernatant contained in the wells was withdrawn (100 µL) and transferred to a newly opened 96-well-flat-bottom tissue culture plate. The new tissue culture plate containing the supernatant was analysed by using Thermo Scientific™ Multiskan™ GO microplate reader and the absorbance was measured at 490 nm. The percentage of biofilm inhibition was calculated by using the following equation:

Biofilm inhibition (%)

Statistical Analysis

All the collected data are presented as the means ± standard deviation and analysed using Microsoft Excel 2019. Statistical tests such as t-test and one way-ANOVA were also computed using IBM SPSS^® for Windows version 22. The significant difference was set to be p < 0.05.  

Results and Discussion

Preformulation and Film Preparation

In the preformulation stage, six formulations (F1-F6) were prepared by varying the ratio of PVP/CMC and the amount of glycerol. The films produced were thin, transparent, odourless and had good adherence to the skin without leaving any sticky residue. A study by Roy, Saha [11] reported that PVP/CMC ratios of 20:80 and 80:20 appeared to have better swelling and viscoelastic properties compared to other ratios of PVP/CMC (0:100, 50:50, 100:0). F1 and F4 were the formulations adopted from Roy, Saha [11]  . Modifications were made to the amount of glycerol to investigate the effects of glycerol concentration on film flexibility. The amount of glycerol (%w/v) used were 1%, 3% and 5%. It was found that when the concentration of glycerol increased, the films produced seemed to be more flexible. This was due to the effect of glycerol as plasticizer that reduced the intra-molecular attraction between the polymeric chains, by favourably forming hydrogen bonds resulting in reduced polymer-polymer interactions and improve mobility of the polymer chains [15, 16]. Therefore, among the six formulations, F6 had the most satisfactory physical appearance, flexibility, and adherence to the skin. F6 was further incorporated with AC at various concentrations (0%, 0.1%, 0.5%, 1.0%, 1.5% and 2.0%).

The AC films prepared were opaque and exhibited an increasingly intense black colour when the concentration of AC increased (Figure I). Hydrogels are well known for their transparency because of the ease of observing the wound without detaching the dressing [17]. However, the addition of AC led to an opaque appearance that impaired the ability to observe the wound during application. It was also observed that the films became drier when the concentration of AC increased. The moisture content of AC films at different concentrations was significantly different (p < 0.05) from one another, with AC 2.0% exhibiting the lowest moisture content (89.65 ± 0.31). Nevertheless, the films remained flexible and pliable at all AC concentrations. They were easily trimmed into desired shapes and further characterisations of the films were conducted.

Physical Assessments

The weight, thickness, diameter, and moisture content of AC films were measured and summarised in Table II. Based on the measurements obtained, the weight and thickness of the AC films gradually increase when the concentration of AC increases. This was relatively due to the increased mass of AC added into the formulation. As for the thickness of the films, only AC 1.5% and AC 2.0% showed a significant increase (p < 0.05) when compared to AC 0%. On the other hand, shrinkage was observed as the diameter of AC 1.5% and AC 2.0% films were significantly smaller than AC 0% (p < 0.05). A study by Illsley, Akhmetova [10] also reported similar findings where there was shrinkage when incorporating AC in plasticised agarose film composites. This was due to a surface adsorption effect on AC resulting in the carbons interacting with and retaining water, plasticizer (glycerol) or the polymeric chain during drying [10]. The moisture content of AC films was significantly higher (p < 0.05) than AC 0% due to the high adsorptive capacity and high pore volume of AC [18]. Generally, hydrogels possess high moisture content (70-90%) due to their complex hydrophilic polymers that facilitate autolytic debridement by providing hydration to necrotic tissues [17, 19]. Hence, the AC films prepared exhibited high moisture content that had potential in wound healing applications targeted for rehydrating the wound bed and facilitating debridement for improved wound healing.

Swelling Ratio Test

The wound model used to investigate the swelling properties of the AC films was gelatine medium due to its comparable characteristics to a suppurating wound, as described by Matthews, Stevens [13]. According to their methods, the diameter of the films was measured at time intervals and the degree of swelling was computed. However, as for the AC films, measuring the diameter may not provide an accurate representation of their swelling properties, because they did not expand uniformly. Instead, the film swells by increasing both its thickness and diameter. Hence, the weight of the films was measured to increase the accuracy in studying the swelling properties of the films.

The percentage of swelling of AC film preparations was presented in Figure II. As an overview, all films swelled at least twice their weight. The film that had the highest swelling capacity after 24 hours was AC 0% (373.33 ± 15.28%). The films containing AC were unable to retain their swelling capacities as a gradual decrease in the percentage of swelling was seen after 3 hours. At 24 hours, AC 1.5% and AC 2.0% swelling reduced to 194.97 ± 26.53% and 191.67 ± 9.55% respectively in comparison to its swelling at the first hour, 256.67 ± 20.82% and 263.33 ± 5.77% respectively. AC 1.0% reduced its swelling capacity after 5 hours while AC 0.5% was after 24 hours. Only AC 0.1% showed a gradual increase in swelling after 24 hours. These findings suggested that there is a limit to the degree of swelling of films containing AC as a bigger loss in swelling capacity at certain time intervals was seen when AC concentrations increased. A reduction in swelling capacity in the function of time also suggested that the absorbed fluid may be released to the surrounding, which is a study by Minsart, Mignon [20] also reported similar findings, where PEG-based hydrogels containing AC 0.5% also possessed significantly lower swelling capacities (p < 0.05) when compared to hydrogel with no AC. It was deduced that the hydrophobicity of AC contributed to the reduction of swelling capacities [20].

Swelling capacity is an important factor in the prevention of bacterial growth on the wound bed due to fluid accumulation such as exudates [21]. Hence, the AC film wound dressings prepared may be beneficial for low and medium exudates as they are able to absorb exudates with limitations to promote wound debridement [22].

Moisture Absorption Study

Moisture absorption study was conducted to find out the ability of the film to absorb moisture from its surroundings in providing a moist environment to the wound. Figure III showed the percentage of moisture absorption of all films. It was found that films containing AC at different concentrations had a significantly higher percentage of moisture absorption (p < 0.05) than film with no AC (15.02 ± 0.63%). AC 0.5% had the highest percentage of moisture absorption (36.66 ± 6.05%). Films containing AC were able to absorb more moisture than the control film due to AC adsorptive properties. AC is highly porous and has a large surface area that enhances the adsorption of moisture from the surrounding (18). Additionally, there were no significant differences (p < 0.05) in moisture absorption among different concentrations of AC.

Besides, maintaining a moist environment is important in wound healing. Wound dressings that have higher moisture absorption had an improved healing rate compared to dressings that had lower moisture absorption [23]. Thus, the AC hydrogel films prepared have potential benefits for wounds that require adequate moisture to facilitate healing such as burn wounds, and necrotic wounds [22].

Moisture Vapour Transmission Rate (MVTR) Study

Maintaining optimal moisture on the wound surface is one of the determining factors to facilitate wound healing. MVTR study can be utilised to assess the ability of wound dressings in controlling moisture gain and moisture loss (24). Figure IV summarised the findings obtained for all film formulations. It was found that in the first three hours, all film formulations showed a rapid increase in moisture gain per area followed by a gradual increase after 6 hours. AC 0.1% had the highest MVTR of 0.111 ± 0.01 g/cm2/day, although there is no significant difference (p < 0.05) of MVTR in all film formulations. MVTR of normal skin is 0.020 g/cm2/day, whereas, for wounded skin, MVTR may rise within the range of 0.028 to 0.514 g/cm2/day due to the disruption of the skin barrier resulting in the release of fluids and electrolytes (25, 26). A study by Xu, Xia (24) also reported that wound dressings that had MVTR of ≤ 0.2028 ± 0.024 g/cm2/day were capable of maintaining optimal moisture for proliferation and regular function of epidermal cells and fibroblasts. They also noted that MVTR should not be too low as it will lead to the accumulation of fluids while MVTR that is too high may lead to dehydration of the wound surface. Hence, the prepared film formulations containing AC were considered suitable for wounded skin because MVTR obtained was within the specified range ( ≤ 0.2028 ± 0.024 g/cm2/day) for wounded skin and capable of advancing wound healing.

Mechanical Properties

Factors such as durability and flexibility are important for the application of film wound dressings. An ideal wound dressing should be able to withstand adequate pressure as well as possess suitable flexibility to ensure the film does not tear easily and is able to follow body movements providing optimal protection of the wound towards mechanical stress [27]. The evaluation of mechanical properties of AC-containing films .was done by assessing their tensile strength, percentage of elongation at break and Young’s modulus. The results obtained were summarised in Figure V. The tensile strength of AC 0% film (0.69 ± 0.02 MPa) was the highest among formulations containing AC. It was also significantly higher than other formulations (p < 0.05), although there was a significant difference in TS among concentrations of AC 0.1%, 0.5%, 1.0%, 1.5% and 2.0%. This showed that the addition of AC in the formulation had an impact on the maximum force required to cause breakage per unit area. It took lesser force to inflict tearing on the AC hydrogel films, with AC 1.5% requiring the least force per unit area (0.36 ± 0.08 MPa) to tear.

Nevertheless, the highest percentage of elongation at break was exhibited by AC 2.0% (69.00 ± 8.19 %). It was not significantly different (p < 0.05) from AC 0% (57.00 ± 7.55 %). Only AC 1.5% (38.33 ± 2.08 %) was significantly different (p < 0.05).

These findings suggested that AC may not have a significant impact on the ability of the films to be more elastic that may be due to the low concentration of AC present in the hydrogel films to contribute to a stronger intermolecular interaction. To investigate further on elasticity, Young’s modulus was computed. Generally, Young’s modulus of the skin is 0.5 – 2.0 MPa [28]. According to Figure V, it showed that Young’s modulus of the films containing AC at various concentrations was not significantly different (p < 0.05) from AC 0% (1.23 ± 0.13 MPa), other than AC 0.1% (0.87 ± 0.08 MPa) that was significantly lower (p < 0.05).

The evaluation of mechanical properties of hydrogel films is important to assess the performance of the films in the presence of external force. A hydrogel film that is easily torn can impair adequate wound protection. Hence, it is crucial that the hydrogel films should be able to withstand satisfactory pressure throughout the application on the wound surface to ensure optimal protection of the wound from contaminants [22, 29, 30]. All AC films prepared were almost as elastic as the human skin. Based on the obtained findings of mechanical properties of the

films, it could be summarised that the addition of AC in the formulation may not significantly enhance the mechanical properties of the films.

Fourier-Transform Infrared Spectroscopy (FTIR)

FTIR analysis was utilised in this study to investigate the presence of functional groups in the films, and to observe any possible chemical bonding occurring in the presence of AC. Figure VI showed the FTIR spectra of all film formulations containing different concentrations of AC. Control FTIR spectrum refers to film formulation that had AC 0%. As a recap, the film formulations were adapted from Roy, Saha [11] with some modifications. Hence, the findings from Roy, Saha [11] were used as a reference to compare and contrast the obtained FTIR spectra. Based on the FTIR spectra (Figure VI), it showed that spectra for control, AC 0.1%, AC 0.5%, AC 1.0%, AC 1.5%, and AC 2.0% were identical. A strong broad peak was observed between the range of 3500 – 3000 cm-1 indicating O- H stretching due to the presence of hydroxyl group. Then, a weak doublet peak (2830 – 2695 cm-1) were seen representing

aldehyde functional group. According to Roy, Saha [11] findings, the presence of a single broad peak between 1620 – 1650 cm-1 showed that there was good interaction among components of the hydrogel film (PVP, CMC, agar and glycerol). Thus, the spectra obtained from the films prepared were coherent with Roy, Saha [11]. As an overview, there is no noticeable difference between the control film and the films containing AC at various concentrations. There were no additional peaks seen in films containing AC. It could be deduced that AC may not have an impact on the physical and chemical interactions that occurred in the films. The nearly identical spectra may be due to the small amount of AC added to the films, which was less than 2% w/v.

Antibiofilm Study (XTT assay)

The XTT assay was conducted under aseptic conditions to maintain sterility. XTT assay is a reproducible and relatively easy method to quantify the presence of biofilm formation by measuring its activity through optical density (OD). A high OD reflects a positive indication of biofilm formation because the XTT solution was reduced from a light-yellow colour to dark orange water-soluble formazan salt by bacterial enzymes [14]. The antibiofilm study was conducted by using two bacterial strains, Staphylococcus aureus (Gram-positive) and Pseudomonas aeruginosa (Gram-negative). These bacterial strains were chosen in this study because they are known to be high biofilm producers resulting in increased cases of nosocomial infections, delayed wound healing in chronic wounds and resistance towards antimicrobials [3, 31]. Figure VII summarised the findings obtained for all films containing different concentrations of AC. It was found that the films containing AC exhibited antibiofilm activities towards both bacterial strains. The highest percentage of P. aeruginosa inhibition was achieved by film AC 0.5% (62.41 ± 8.48%) followed by AC 0.1% (60.33 ± 4.27 %), AC 1.5% (53.99 ± 5.08%), AC 1.0% (51.37 ± 1.61%) and AC 2.0% (44.69 ± 5.51%). The formulations containing AC demonstrated more than 50% of biofilm inhibition and were significantly different (p < 0.05) when compared to AC 0%, which showed an increase in biofilm growth indicated by the negative value of biofilm inhibition (-16.72 ± 3.87%). The result obtained also showed that the highest concentration of AC which is AC 2.0% had the lowest percentage of biofilm inhibition. The mechanism of the reduced biofilm inhibition with the increase of AC concentration was not studied.

On the contrary, antibiofilm activities of AC hydrogel films on S. aureus bacteria strain were less promising in comparison to P. aeruginosa. The highest percentage of biofilm inhibition was achieved by AC 1.5% (39.83 ± 5.84%) followed by AC 1.0% (34.49 ± 3.22%), AC 0.5% (26.50 ± 5.28%), AC 0.1% (26.50 ± 6.64%) and AC 2.0 (14.43 ± 1.65%). The findings showed that the highest concentration of AC, AC 2.0%

exhibited the lowest percentage of biofilm inhibition (14.43 ± 1.65%). Besides, the film that contained no AC (18.81 ± 11.07%) showed some antibiofilm activities which suggest that other components of the hydrogel films (PVP/CMC polymer, glycerol and agar) may be contributed to the antibiofilm activity. Therefore, there is a lack of evidence to indicate that the films containing AC have antibiofilm activity towards S. aureus biofilm since there was no significant difference (p < 0.05) of biofilm inhibition of AC hydrogel films when compared to AC 0%. The rationale for the lack of antibiofilm activity towards S. aureus biofilm was not further studied.

Based on the obtained results, it could be deduced that AC had antibiofilm properties towards P. aeruginosa bacterial strains. P. aeruginosa, a gram-negative bacteria, was found to be more sensitive towards the inhibitory effect of AC resulting in more than 50% of the biofilms present being disrupted. Gram- negative bacteria are commonly known to be more resistant towards antimicrobials because of the presence of the outer membrane that can undergo alterations by increasing hydrophobicity or mutations [32]. Theoretically, it can be deduced that due to the hydrophobicity properties of AC, it possesses the ability to adsorb the outer membrane of P. aeruginosa resulting in the disruption and inhibition of biofilm. However, this research did not study the mechanism of biofilm inhibition of AC and further investigations are required to assess the adsorptive ability of AC films to adsorb the outer membrane of P. aeruginosa.

Furthermore, a study by Ng and Leow [12] showed that gentamicin at a concentration of ≤ 0.06 mg/mL showed an inhibitory effect towards biofilm at ≤ 60% (P. aeruginosa). Gentamicin (≤ 0.06 mg/mL) also showed biofilm inhibition towards S. aureus, but at a lower percentage (<50%) because it had lower activity towards gram-positive bacteria. It can be said AC had potential equivalent antibiofilm activities with ≤ 0.06 mg/mL of gentamicin. Theoretically, antimicrobials that are effective towards gram-positive bacteria include vancomycin which works by inhibiting cell wall formation. It is done so through binding of the hydrophilic part of vancomycin to the alanine residue present on the cell wall linking enzyme [33]. It could be deduced that AC had no antibiofilm activity towards S. aureus due to its high hydrophobicity, and unable to bind and adsorb optimally to the surface of the cell wall of S. aureus. However, this hypothesis should be assessed with further investigations on the mechanism of AC in exhibiting its antibiofilm activity.

Next, 1.5% w/w of xylitol had antibiofilm activity of 36.9 ± 6.7% while AC 1.5% showed 53.99 ± 5.08% of biofilm inhibition towards P. aeruginosa. Xylitol had almost the same activity towards both S. aureus and P. aeruginosa. For bacterial strain S. aureus, xylitol 1.5% was able to exhibit 32.9 ± 8.4% of antibiofilm activities in comparison to AC 1.5% (39.83 ± 5.84%). Besides, 0.05% w/w of EDTA showed 38.4 ± 4.5% of antibiofilm activity towards P. aeruginosa in comparison to AC 0.1% (60.33 ± 4.27 %) [34].

In summary, AC hydrogel films had antibiofilm properties as discussed above and were found to be competitive with other antibiofilm agents available commercially such as antimicrobial (gentamicin), xylitol and EDTA, when in vitro results were compared. It can be theorised that the mechanism behind AC antibiofilm activities was due to its powerful adsorptive capacities with high porosity, and large surface area for the site of adsorption [18]. Previously, AC had shown effectiveness for wounds with malodour [10]. Thus, the results obtained from this study showed the promising potential of AC being an antibiofilm agent towards P. aeruginosa biofilm. However, the application of AC as an antibiofilm agent in wound dressing may be limited due to the lack of evidence to indicate antibiofilm activity towards S. aureus. Biofilms that are formed on the wound surface may consist of multispecies of bacteria, thus selective inhibition of biofilms by AC may reduce its effectiveness when applied onto the wound.

Conclusion

Hydrogel film wound dressings containing activated carbon were successfully developed with satisfactory physical appearance, swelling and mechanical properties. According to the characterisation results, the produced AC hydrogel films were found to be suitable for low to medium-suppurating wounds as they were shown to maintain adequate moisture balance to the wound bed while possessing good swelling

properties to absorb exudates. The prepared AC hydrogel films were also found to have good mechanical properties that were flexible and durable with moderate pressure.

Furthermore, AC hydrogel films were also tested on their antibiofilm activities. It was shown that AC had potential antibiofilm activities with more than 50% of inhibition towards P. aeruginosa biofilm whereas there was a lack of evidence that showed antibiofilm activity of AC towards S. aureus which limits AC application in biofilm-targeted therapies for wound healing. Therefore, this study concluded that the AC hydrogel film produced required further investigations to evaluate the mechanism of AC in biofilm inhibition and understand why AC showed no significant antibiofilm activity towards S. aureus prior to establishing AC as an antibiofilm agent.

Acknowledgement

We also would like to thank the Centre for Drug Delivery Research and Vaccines for research facilities support. This research is supported by UKM grant no. (GUP-2019-003).

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