**1. Introduction**

There are an overwhelming number of wound dressings available in the market. The high demand for wound dressing is due to the increasing number of wound cases recorded. It is reported that the treatment costs for chronic wounds are substantial and are estimated to account for approximately 1–3% of the total healthcare expenditure in developed countries [1,2]. For example, Wales was estimated to have a chronic wound prevalence of 6% in the year 2012–2013, corresponding to 5.5% of National Health Service expenditure [1], and in the United Kingdom as a whole, the cost associated with wound management was estimated to be GBP 4.5 and GBP 5.1 billion in 2012 [3]. In the United

**Citation:** Muktar, M.Z.; Bakar, M.A.A.; Amin, K.A.M.; Che Rose, L.; Wan Ismail, W.I.; Razali, M.H.; Abd Razak, S.I.; in het Panhuis, M. Gellan Gum Hydrogels Filled Edible Oil Microemulsion for Biomedical Materials: Phase Diagram, Mechanical Behavior, and In Vivo Studies. *Polymers* **2021**, *13*, 3281. https://doi.org/10.3390/ polym13193281

Academic Editor: Domenico Acierno

Received: 24 August 2021 Accepted: 15 September 2021 Published: 26 September 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/).

States, it has been reported that over 6.5 million patients with wounds cost the health care system USD 25 billion annually [4]. The point of using wound dressing is to cover the wound from infection and provide appropriate conditions to enhance the healing process of the wounds. However, there is a concern about the usage of hazardous chemicals in the development of wound dressing such as various inorganic nanoparticles. Inappropriate chemicals in wound dressing may lead to other diseases such as skin cancer [5]. The usage of biopolymer and natural products for a better and safer wound dressing is needed. Hence, gellan gum produced by the Gram-negative bacterium of Pseudomonas elodea is chosen due to its biocompatibility and unique properties [6,7].

Gellan gum (GG) is nontoxic, biocompatible, and biodegradable, and the resulting hydrogel is transparent and stable [8]. GG hydrogels are three-dimensional crosslinked polymer networks, a result of transformation from a disordered single coiled structure at high temperature (80 ◦C) to a double helix and bonded by internal hydrogen bonding between D-glucoronate and D-glucose C residues upon cooling between 30 to 50 ◦C [9]. In a swollen state these are soft and elastic, resembling the living tissue and exhibiting excellent biocompatibility. The unique properties of hydrogels lead to the wide use of these biomaterials in different fields, including pharmaceutical and biomedical [10,11]. Gellan gum has been proven by the United States Food and Drug Administration (US FDA) and the European Union (EU) to be safely utilized in the food industry. Gellan gum has been studied as matrices to repair and regenerate a wide variety of tissues and organs [12]. The material has also been used as scaffold materials for the application of tissue engineering [6], in the development of wound dressing materials [13], as a vehicle for drug delivery [14], and in eye drops [15]. Gellan gum also has shown good compatibility against various live cells such as mouse fibroblast (L929 cell line) [13], mouse fibroblast cell (3T3) [16], human fetal osteoblast (HFOBs 1.19) [17], human skin fibroblast (CRL2522) [6], and human nasal cartilage [18].

Virgin coconut oil (VCO) is essentially colorless, free from rancidity, and endowed with natural antioxidant and vitamin E that prevents the peroxidation reaction. VCO mainly consists of medium-chain triglycerides (MCT) and differs from animal fats that consist of long-chain saturated fatty acids, which is the one main risk factor for cardiac compilation. Based on studies, VCO has been reported to have the potential in promoting the healing process [19]. The oil has been applied in treating wounds in young rats and healed faster by decreasing time for complete epithelization, and results in a significant increase of collagen production, which indicates higher collagen crosslinking. VCO also showed a significant effect in reducing inflammation in acute and chronic inflammation on ethyl phenylpropionate-induced ear edema in rats [20]. A few studies have been reporting the use of biopolymers with essential oil to produce a dressing material [21]. Gellan gum hydrogel films with lavender/tea tree oil showed 98% wound contraction in rats after ten days of treatment and histological images displayed completely healed epidermis [21]. Another study used poly(lactic acid) (PLA) polymer and babassu oil and reported that this material provides a good option for use as wound dressings—films showed a recommended value of the water vapor transmission rate (WVTR), maintained a humid environment above the wound, had good cytotoxicity on normal human keratinocytes (HaCaT), stimulated the keratinocytes migration, and inhibited Pseudomonas aeruginosa growth [22].

Based on the demands to produce a more efficient and safer wound dressing, this study optimizes the ratios of VCO, water, and surfactant by developing a ternary phase diagram and producing a VCO microemulsion. The optimum concentration of VCO microemulsion is selected for incorporation into GG solution and characterized for their chemical interaction, mechanical performance, physical properties, and thermal behavior. Furthermore, the qualitative in vitro antibacterial activities were examined against two Gram-negative (*Escherichia coli* and *Klebsiella pneumoniae*) and two Gram-positive (*Staphylococcus aureus* and *Bacillus subtilis*) bacteria. The in vivo studies were carried out to study the healing properties of the samples on Sprague–Dawley rats, observing the ultrasound images of wound skin and the histological evaluation after the 14th day of post-wound.

#### **2. Materials and Chemicals**

#### *2.1. Materials*

Low-acyl gellan gum (GG, batch no: 5C1574A) was obtained from CP Kelco, Atlanta, GA, USA. Glycerin (product number—G2289), anhydrous calcium chloride, CaCl<sup>2</sup> (product number—C5670), Tween 80 (product number—P1754), and Triton X-100 (product number—T9284) were obtained from Sigma Aldrich, St. Louis, MO, USA. Virgin coconut oil (product number—VCO0216) was obtained from Phyto Biznet Sdn Bhd, Skudai, Johor, Malaysia. All materials were used as received without further purification.

#### *2.2. Construction of Phase Diagrams*

Phase diagrams were constructed by mixing two of the components (VCO and water) and titrated using surfactant (Tween 80—Method I or TritonX-100—Method II) as a third component. The surfactant, i.e., Tween 80, was added into the VCO containing distilled water at different ratios (Table 1) and was vortexed (Vortex 3, Eppendorf, Germany) for 3 min. The mixtures were then centrifuged (Minispin Eppendorf, Germany) at 400 rpm for 10 min and later placed in a water bath at room temperature (26 ± 2 ◦C) for 24 h to record the stability of the mixtures produced. The same procedure was carried out with TritonX-100 (Method II) surfactant at the specific ratios (Table 1).

**Table 1.** Ratios of VCO, water, and surfactants (Tween 80—Method I and TritonX-100—Method II) to develop a ternary phase diagram and produce a stable VCO microemulsion.


### *2.3. Preparation of GVCO Hydrogel*

The gellan gum (GG) solutions were prepared by dissolving 2% (*w*/*v*) gellan gum in deionized water (18 MΩ) with 50% (*w*/*w*) glycerin under continuous stirring at 500 rpm for 2 h at 80 ◦C. VCO microemulsions were prepared by mixing the VCO: Water with Tween 80 surfactant at a specific percentage, as shown in Table 1. For example, to produce the VCO microemulsion of VCO60, 21.38% of VCO was mixed with 14.25% of water with an addition of 64.37% of Tween 80 (Table 1). The same process was carried out for VCO70 and VCO80. To produce GVCO60 hydrogel, 5% (*v*/*v*) of the VCO60 microemulsions were added into the GG solution and stirred for 20 min at 80 ◦C. The methods were repeated for VCO70 and VCO80 microemulsions. The mixtures were then poured into a Petri dish and allowed to cool at room temperature for 24 h before use for characterization. The GG solution with VCO60 was then known as GVCO60 hydrogels, and the same naming was applied for VCO70 and VCO80 hydrogels.

#### *2.4. Characterisation of the Sample*

2.4.1. Ultraviolet–Visible Spectroscopy

Ultraviolet–visible (UV–Vis) absorption and transmission spectra of solutions and hydrogels were performed using a spectrophotometer (Varian, Cary 50 UV–Vis NIR) with data interval = 5 nm, scan speed = 24,000 nm/min, and wavelength range 500–800 nm. The UV–Vis transmittance was conducted by attaching the hydrogel to the cuvette surface.

#### 2.4.2. ATR–FTIR Spectroscopy

ATR–FTIR spectra were collected using a Perkin Elmer Spectrum 100 FTIR spectrophotometer with a PIKE Miracle ATR accessory (single-bounce beam path, 45◦ incident angle, 16 scans, 4 cm−<sup>1</sup> resolution). An advanced ATR correction was applied to all spectra in the region from 4000 to 600 cm−<sup>1</sup> .

#### 2.4.3. Mechanical Properties

Mechanical properties of hydrogel films were performed using an Instron Universal Testing Machine (model 3366) with a load capacity and cross-speed according to ASTM standard method D882 (ASTM, 2001). Each sample was cut to 2.0 <sup>×</sup> 2.0 cm<sup>2</sup> for stress–strain measurements. The thicknesses of hydrogel films were measured using a handheld micrometer (Mitutoyo Corporation, Mitutoyo, Japan). Stress-at-break (σ), strain-at-break (γ), and Young's modulus (E) were recorded and a minimum of three independent measurements were obtained per sample.

#### 2.4.4. Swelling Properties

The swelling properties were determined according to the ASTM Standard Test Methods for One-Dimensional Swell (D4546-08). The swelling was measured by weighing dried films (Wdry) before immersion into 50 mL phosphate buffer solutions of pH 7.2 at 37 ± 0.5 ◦C in a water bath. The hydrogels were removed after 24 h, gently wiped with a tissue to expel the surface water, and weighed (Wwet). Swelling degree (%) was determined according to Equation (1):

$$\text{Swelling degree (\%)} = (\text{W}\_{\text{wet}} - \text{W}\_{\text{dry}}) / \text{W}\_{\text{dry}} \times 100\tag{1}$$

where Wdry and Wwet are the initial weight and final weight, respectively. A minimum of three independent measurements was obtained per sample.

#### 2.4.5. Water Vapor Transmission Rate (WVTR)

The water vapor transmission rate (WVTR) was measured following a modified ASTM International standard method ASTM E96-95. Each hydrogel was fixed on the circular opening of a permeation bottle with an effective transfer area (A) of 1.33 cm<sup>2</sup> . The permeation bottle was placed in the temperature–humidity chamber at 37 ◦C and 50 ± 5% relative humidity. The equilibrium moisture penetration was determined by weighing the bottles at 0 and 24 h. The WVTR was calculated according to Equation (2):

$$\text{WVTR} = \text{(m/\Delta T)} / \text{A} \tag{2}$$

where m/∆T is the amount of water gain per unit time of transfer and A is the area exposed to water transfer (m<sup>2</sup> ).

#### 2.4.6. Thermogravimetric Analysis

Thermogravimetric analyses were performed on a Pyris 6, Perkin-Elmer-TGA6. Hydrogel samples were analyzed in platinum pans at a heating rate of 10 ◦C/min to 275 ◦C in an atmosphere of N<sup>2</sup> atmosphere at a flow rate of 50 mL/min. The sample used was approximately 10 mg.

#### 2.4.7. Scanning Electron Microscopy (SEM)

The cross-section morphology of the hydrogels was acquired using a JOEL JSM 6360 LA electron microscope. Scanning electron microscopy (SEM) images of cross-sections were obtained by freeze-dried technique. Samples were freeze-dried in liquid nitrogen (−160 ◦C) and fractured at −150 ◦C at the middle of hydrogels. It was then coated with Auto Fine Coats (JFC-1600) before microscopic observation.
