Antibacterial polysaccharide-based hydrogel dressing containing plant essential oil for burn wound healing

Huanhuan Wang; Yang Liu; Kun Cai; Bin Zhang; Shijie Tang; Wancong Zhang; Wenhua Liu

doi:10.1093/burnst/tkab041

. 2021 Dec 22;9:tkab041. doi: 10.1093/burnst/tkab041

Antibacterial polysaccharide-based hydrogel dressing containing plant essential oil for burn wound healing

Huanhuan Wang ¹, Yang Liu ^2,^✉, Kun Cai ³, Bin Zhang ⁴, Shijie Tang ⁵, Wancong Zhang ⁶, Wenhua Liu ⁷

PMCID: PMC8693078 PMID: 34988231

Abstract

Background

Polysaccharide-based hydrogels have been developed for many years to treat burn wounds. Essential oils extracted from aromatic plants generally exhibit superior biological activity, especially antibacterial properties. Studies have shown that antibacterial hydrogels mixed with essential oils have great potential for burn wound healing. This study aimed to develop an antibacterial polysaccharide-based hydrogel with essential oil for burn skin repair.

Methods

Eucalyptus essential oil (EEO), ginger essential oil (GEO) and cumin essential oil (CEO) were employed for the preparation of effective antibacterial hydrogels physically crosslinked by carboxymethyl chitosan (CMC) and carbomer 940 (CBM). Composite hydrogels were prepared and characterized using antimicrobial activity studies, Fourier-transform infrared spectroscopy, X-ray diffraction, scanning electron microscopy, gas chromatography-mass spectrometery, rheological analysis, viscosity, swelling, water loss rate and water vapor transmission rate studies. In addition, the biocompatibility of hydrogels was evaluated in vivo by cytotoxicity and cell migration assays and the burn healing ability of hydrogels was tested in vivo using burn-induced wounds in mice.

Results

The different essential oils exhibited different mixing abilities with the hydrogel matrix (CMC and CBM), which caused varying levels of reduction in essential oil hydrogel viscosity, swelling and water vapor transmission. Among the developed hydrogels, the CBM/CMC/EEO hydrogel exhibited optimal antibacterial activities of 46.26 ± 2.22% and 63.05 ± 0.99% against Staphylococcus aureus and Escherichia coli, respectively, along with cell viability (>92.37%) and migration activity. Furthermore, the CBM/CMC/EEO hydrogel accelerated wound healing in mouse burn models by promoting the recovery of dermis and epidermis as observed using a hematoxylin–eosin and Masson’s trichrome staining assay. The findings from an enzyme-linked immunosorbent assay demonstrated that the CBM/CMC/EEO hydrogel could repair wounds through interleukin-6 and tumor necrosis factor-α downregulation and transforming growth factor-β, vascular endothelial growth factor (VEGF) and epidermal growth factor upregulation.

Conclusions

This study successfully prepared a porous CBM/CMC/EEO hydrogel with high antibacterial activity, favorable swelling, optimal rheological properties, superior water retention and water vapor transmission performance and a significant effect on skin repair in vitro and in vivo. The results indicate that the CBM/CMC/EEO hydrogel has the potential for use as a promising burn dressing material for skin burn repair.

Keywords: Carboxymethyl chitosan, Eucalyptus essential oil, Hydrogel, Antibacterial activity, Burn, Wound healing

Highlights.

CBM/CMC hydrogels were prepared with essential oils by physical crosslinking, and CBM/CMC/EEO hydrogel characterization was extensively determined.
CBM/CMC/EEO hydrogel displayed antimicrobial and cell migration activity.
CBM/CMC/EEO hydrogel was proved to have burn wound healing activities.

Background

Polysaccharide-based hydrogels have been developed for many years to treat burn wounds [1–5]. Burn and scald injuries of skin are ailments with high morbidity and mortality, affecting ~11 million people worldwide each year [6]. Burn and scald injuries damage the barrier function of the skin, thus leading to the loss of water, electrolytes and proteins from wounds. During the healing process, improper nursing care, like traditional bandages, cotton pads and ointments, generally delay wound healing as microorganisms invade the wounds to cause infection. Further, the neonatal granulation tissue easily adheres to the dressing, leading to secondary injury during dressing removal [7,8]. Compared to traditional materials, application of polysaccharide-based hydrogel dressings on wounds presents advantages, such as superior exudate absorbance, transparent material allowing the monitoring of healing, temporary and prompt wound coverage, wearing comfort, immediate relief from pain and minimization of possible damage due to the 3D and hydrophilic polymeric network of hydrogels [9].

To date, many macromolecular polysaccharides, such as starch, alginate, cellulose, chitoson, hyaluronic acid, carboxymethyl chitosan (CMC, a water soluble derivative of chitosan), etc., have been widely used to develop polysaccharide-based hydrogels. The use of polysaccharides as the primary hydrogel matrix to improve the hydrophilic performance of polymeric hydrogels has emerged as a significant research field [10–14]. In addition, the majority of polysaccharide-based hydrogels are usually comprised of additional cross-linked substances, owing to their poor mechanical properties and insufficient biological activity. For instance, in order to improve the biological activity and physical properties of polysaccharide hydrogels, a carbomer940 (CBM)/CMC /bletilla striata polysaccharide cross-linked hydrogel was reported in our previous study [15]. However, the hydrogel demonstrated poor antibacterial activity, which limited its application in the biomedical field.

A number of essential oils are frequently incorporated into polysaccharide-based hydrogels as active ingredients [16–18]. Essential oils extracted from aromatic plants contain numerous low molecular weight molecules derived from isoprene, including terpenoids and related terpenes, which generally exhibit superior biological activity. Essential oils have been reported to be active against gram-positive and gram-negative bacteria, along with having active skin tissue repair and antioxidation functions [18,19]. However, the direct application of essential oils is limited because they are unstable and fragile volatile compounds. Essential oils are easily degraded under various physico-chemical conditions, such as light, heat, oxidation, etc. [20]. In order to maintain their biological activity over a long period of time, along with reducing their volatility, increasing their effective utilization rate and providing controlled release [21], essential oils are generally encapsulated in hydrogel matrices, leading to their sustained release. Boccalon et al. reported that hydrogels prepared by incorporating polyvinyl alcohol (PVA), sodium alginate and borax displayed superior antimicrobial properties after loading with essential oil [16]. Moradi et al. also reported optimal antibacterial activity of chitosan/PVA/thyme oil hydrogels against Escherichia. coli (E. coli) and Staphylococcus aureus (S. aureus) [17]. Therefore, the addition of essential oils to polysaccharide-based hydrogel matrices, rather than using them directly on wounds, can be instrumental in enhancing their physical stability and decreasing their volatility, thus leading to superior biological activity of the polysaccharide hydrogels.

In summary, polysaccharide hydrogels with improved antibacterial activity have been developed in this study. Natural hydrogel dressings for the treatment of burn wounds were subsequently prepared by physical crosslinking of CBM, CMC and plant essential oils (eucalyptus essential oil (EEO), ginger essential oil (GEO) and cumin essential oil (CEO)). The dressings were prepared by employing the optimal proportion of raw materials to achieve superior antibacterial performance. The morphology, rheological behavior, swelling, water loss and water vapor transmission properties of the hydrogels were systematically investigated. In addition, the burn wound healing of the composite hydrogel dressing was evaluated through in vivo and in vitro animal experiments.

Methods

Materials

EEO, GEO and CEO were purchased from ZRZR Biotechnology Co. Ltd, Guangdong, China. CMC (molecular weight concentrated at 195.7 kDa and 2.0 kDa; the degree of substitution was 73.73% (Figures S1–S5, see online supplementary material); and CBM (molecular weight concentrated at 1894.7 kDa and 15.8 kDa; CAS:54182–57-9) were supplied by Yuanye biotechnology Co. Ltd, Shanghai, China. Triethanolamine was procured from Aladdin Reagent Co. Ltd, Shanghai, China. Luria–Bertani (LB) broth and the L-929 cell migration and cytotoxicity detection kit were supplied by Solarbio (Beijing, China). 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) was purchased from Sigma (Shanghai, China). Chloral hydrate was obtained from Shanghai Macklin Biochemical Co. Ltd, China. The reagents used in the study were of analytical grade.

Preparation of hydrogels

Preparation was carried out by following the previously reported method [15]. In a 200 mL beaker, 0.5% (w/v) CBM was prepared by dissolving 0.5 g of CBM in deionized water, and the resultant solution was kept at ambient temperature overnight. Subsequently, CMC (10% (w/v), 5 mL) was added, and the CBM/CMC hydrogel was stirred for 5 min after adjusting the pH to 7 using triethanolamine. Afterwards, EEO, GEO or CEO (5 mL) was added slowly to the beaker with 50 μL of Tween 20 (CAS:9005-64-5), followed by uniform mixing at 25°C for 5 min. Finally, CBM/CMC/EEO, CBM/CMC/GEO and CBM/CMC/CEO hydrogels were obtained by adding up to 100 g of deionized water to the gel system and stirring the mixture at 25°C for 10 min. A schematic diagram of hydrogel preparation is shown in Figure 1.

Figure 1. — Schematic diagram of antibacterial polysaccharide-based hydrogels preparation. *CBM* carbomer940, *CEO* cumin essential oil, EO essential oil

Antimicrobial activity

The antimicrobial activity of the hydrogels was determined against E. coli and S. aureus bacterial strains by employing turbidimetric analysis [22]. The bacterial strains were incubated in LB broth, with shaking at 150 rpm (37°C) until an absorbance at 600 nm (OD₆₀₀) of 0.5 was achieved. After incubation, the hydrogels or positive control (PC, 50 μL) were mixed with 150 μL of mixed bacterial suspension with a concentration of 10⁵–10⁶ CFU/mL, followed by reaction for 6 h. A 96-well microplate reader was used to measure the OD₆₀₀. Gentamicin sulfate was used as the PC, whereas sterile water was used as the negative control (NC). The antibacterial rate (AR) of the samples for E. coli or S. aureus was calculated as follows:

(1)

where OD denotes the absorbance of the solution at 600 nm, NC denotes the negative control solution and sample denotes the test sample solution. The measurements were repeated thrice, and an average value was reported.

Hydrogel characterization

Fourier-transform infrared spectroscopy

Fourier-transform infrared (FT-IR) spectroscopy of the freeze-dried CBM/CMC, CBM/CMC/EEO, CBM/CMC/GEO and CBM/CMC/CEO hydrogels was carried out by mixing with KBr for pressing the pellets. Attenuated total reflectance (ATR) mode was used on a Thermo (USA) Nicolet iS50 FT-IR Spectrometer. The spectra were collected over the wavenumber range 4000–650 cm⁻¹ with a resolution of 4 cm⁻¹ and 128 co-added scans.

X-Ray diffraction

The X-ray diffraction (XRD) curves of the freeze-dried CBM/CMC, CBM/CMC/EEO, CBM/CMC/GEO and CBM/CMC/CEO hydrogels were recorded using a Bruker D8 ADVANCE (Germany) X-ray powder diffractometer with Cu Kα radiation (λ =0.154 nm), in the diffraction angle range 10–60° at 40 kV and 40 mA.

Scanning electron microscopy

The surface morphology of the CBM/CMC, CBM/CMC/EEO, CBM/CMC/GEO and CBM/CMC/CEO hydrogels was analyzed using a scanning electron microscope (JSM-840, Japan) at 10.0 kV accelerating voltage. To avoid structural collapse, the hydrogels were kept at −80°C overnight before freeze-drying for 48 h. The freeze-dried hydrogels were sputtered with a thin layer of gold under vacuum for 20 min to ensure conductivity of the hydrogels.

Gas chromatography-mass spectrometry

Gas chromatography-mass spectrometry (GC–MS) analyses of three kinds of essential oils were carried out on an Agilent 7890-5977A GC–MS system (USA) equipped with a HP-5MS capillary column (30 m length × 0.25 mm diameter × 0.25 μm film thickness). The essential oils were diluted with ethyl ether and 0.2 μL was injected in split mode with a split ratio of 1:10. The oven temperature was initially set at 50°C for 1 min, then programmed to 80°C at a rate of 5°C/min and hold for 2 min, then programmed to 180°C at a rate of 10°C/min and hold for 5 min and finally programmed to 220°C at 10°C/min and hold for 2 min. Helium was used as carrier gas at a flow rate of 1.0 mL/min.

For MS determinations, the ionization voltage of the mass spectrometer was 70 eV, with an ion source temperature of 230°C, scan mass range of 40–500 amu and solvent delay time of 2 min. The essential oil compounds were identified by comparison with the NIST 14 spectra library and reported as relative percentages of the total peak area.

Hydrogel properties

Rheological analysis

As viscoelastic materials, hydrogels exhibit unique rheological properties. Oscillatory rheological analysis of the CBM/CMC, CBM/CMC/EEO, CBM/CMC/GEO and CBM/CMC/CEO hydrogels was carried out using an AR2000ex rheometer (TA, UK) in parallel-plate mode (50 mm diameter) at 25°C. The frequency sweep measurement (0.01% strain) was used for gaining information about the storage (G') and loss (G") moduli.

Viscosity and swelling rate

The viscosity of the CBM/CMC, CBM/CMC/EEO, CBM/CMC/GEO and CBM/CMC/CEO hydrogels was measured using an NDJ-5S rotary viscometer after placing the hydrogels (100 g) in a beaker.

The original mass (m₀) was calculated by placing the freeze-dried (for 2 d) hydrogels in deionized water at 25°C and storing for 5 min. Subsequently, the hydrogels were weighed after swelling and equilibriating (m_a). The swelling ratio was estimated from the following equation:

(2)

Water loss rate and water vapor transmission rate

The original mass (m₀) was calculated after placing the hydrogels (1 g) in 2 mL centrifuge tubes. The tubes were subsequently dried in an oven for 18 h at 37°C. The water loss rate was eventually calculated by weighing the tubes once every hour (m_a) during drying as well as at the end (m_b), using the formula:

(3)

Water vapor transmission rate (WVTR) of the CBM/CMC, CBM/CMC/EEO, CBM/CMC/GEO and CBM/CMC/CEO hydrogels was calculated using a gravimetric method, according to the ASTM E96–00 standard. For this, 10 mL of deionized water was added to a glass vial with a diameter of 18 mm, and the mouth of the vial was covered with a layer of gauze. Hydrogel dressing (0.5 g) was used to uniformly cover the gauze, and the mouth of the vial was subsequently sealed. The initial mass (W₀) was accurately weighed. The vial was placed in a silica gel dryer at 37°C and subsequently removed after 12, 24, 36 and 48 h for weighing (W_t). WVTR (g/m²/day) was calculated as follows:

(4)

where, A is the cross-sectional area of the vial (m²) and T is the duration for which the hydrogels are placed in the dryer (day). An average of three measurements was taken for each sample.

In vitro cell culture

L929 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% fetal bovine serum (FBS) and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin) at 37°C overnight in an incubator (Thermo Forma 3111) in an atmosphere containing 5% CO₂. The adherent cells were digested with 0.25% trypsin and sub-cultured. The L929 cells were inoculated in 96-well plates at a cell density of 1 × 10⁵ per well and were cultured for 9–12 h until the cells adhered to the wall.

Indirect cytotoxicity of hydrogels

The indirect cytotoxicity of the CBM/CMC, CBM/CMC/EEO, CBM/CMC/GEO and CBM/CMC/CEO hydrogels was evaluated using the MTT assay [23,24]. Briefly, the freeze-dried hydrogels were sterilized by exposure to the UV light for 2 h. Subsequently, hydrogel extracting liquid solutions were prepared by soaking the hydrogels at different concentrations (1, 10, 100, 1000, 10 000 μg/mL) in DMEM containing serum at 37°C for 24 h. After sterilization with a 0.22 μm filter, the culture medium in the 96-well plates was removed by using fresh DMEM culture medium as well as the extracting liquids corresponding to the CBM/CMC, CBM/CMC/EEO, CBM/CMC/GEO and CBM/CMC/CEO hydrogels for 24 h. Subsequently, 20 μL of 5 mg/mL MTT solution was added to the wells. The solution was further cultured at 37°C for 4 h. Afterwards, the solution was removed from each well and 150 μL of dimethylsulfoxide was added. The absorbance at a wavelength of 490 nm was subsequently measured with a microplate reader (DNM-9602), along with recording the number of each plate. The cell viability (%) of the hydrogels was calculated as:

(5)

Migration assay of L929 cells

The migration of the fibroblast L929 cells was examined by employing the method described by Balekar et al. [25]. Briefly, the L929 cells (8 × 10⁵ cells/mL) in DMEM containing FBS (10%) were seeded in each well of a 6-well plate, followed by incubation at 37°C with 5% CO₂. After a confluent monolayer of the L929 cells was formed, a sterile pipette tip was used to generate a horizontal scratch in each well. Any cellular debris was removed by washing with PBS and adding 1 mL of fresh medium. The cells were treated with PBS and hydrogel extracting liquids at a concentration of 1 μg/mL (PBS served as a negative control). The hydrogel extracting liquid solution concentration based on the performed cytotoxicity assay.

Images of each well were taken using a microphotograph (XDS-500C, Shanghai) at 0, 24 and 48 h. To determine the migration of the L929 cells, the images were analyzed using ImageJ software. For this, after calibration, the uncovered area was delimited with a specific tool to calculate its value. The covered area (%) of the samples was calculated as:

(6)

In vivo studies

Six-week-old male KM mice (n = 84, weight = 28.5 ± 2 g) were purchased from Hunan SJA Laboratory Animal Co. Ltd (Hunan, China). Animal experiments were performed following the National Institute of Health Guide for the Care and Use of Laboratory Animals, China. The procedures for care and use of animals were approved by Experimental Animal and Animal Experiments Center of Shantou University. All reasonable efforts were made to minimize the suffering of the mice.

For the analysis, 84 mice were randomly divided into three groups (28 mice in each group) and acclimatized to the laboratory conditions for ~7 days before wounding. Afterwards, an intraperitoneal injection of 5% chloral hydrate (10/mL) was administered to anesthetize the mice. Before surgery, the dorsal surface hair was removed with 8% (w/v) sodium sulfide, followed by sterilization with 75% alcohol. Subsequently, second-degree burn wounds with a diameter of 1.5 cm were created on the back of each mouse by using a scalding apparatus (YLS-5Q, Yiyan, China) at 100°C with 0.1 kg compression for 10 s. Afterwards, the wounded groups were cured with MEBO Shirun Shaoshang Gao (PC), CBM/CMC/EEO (GEL) and normal saline representing the negative control (NC). Images of the scalds were taken daily, and wound healing rate was calculated using ImageJ software.

For histological analyses, eight mice each were harvested at days 3, 7, 14 and 21, and the wound sites and surrounding skin were excised. After removing the fur in 8% (w/v) sodium sulfide and cleaning with cold saline, the samples were dehydrated in graded ethanol and subsequently embedded in paraffin. The central wound sections with a thickness of 5 μm were fixed on poly-L-lysine-coated glass slides and stained with hematoxylin and eosin (H&E) as well as Masson’s trichrome (Beyotime Institute of Biotechnology, China). Images of the processes of re-epithelialization, granulation tissue formation and collagen deposition of the tissue were acquired with a Nikon ECLIPSE 80i microscope (Nikon, Japan).

For enzyme-linked immunosorbent assay (ELISA) analyses, the wound skin was mixed with normal saline at a ratio of 1:9 and was placed in a tissue homogenizer (OES-Y50, Tiangen, Beijing) for full grinding to achieve a 10% tissue grinding solution. The supernatant of the tissue grinding solution was collected after centrifugation at 3000 rev/min for 20 min, and the concentrations of interleukin- 6 (IL-6), tumor necrosis factor-α (TNF-α), TGF-β1, vascular endothelial growth factor (VEGF) and EGF were determined using an ELISA kit (Solarbio, Beijing, China), following the manufacturer’s protocols. The concentrations were recorded as pg/mL. All calibrations and analyses were repeated in triplicate.

Statistical analysis

Data were analyzed with GraphPad Prism 7 (GraphPad Software, Inc., La Jolla, CA, USA). Statistical comparisons of hydrogel properties, cell culture data and mouse model were performed with Student’s t-test. Results are presented as mean ± SD. Statistical comparisons between hydrogel groups were performed by analysis of variance/Kruskal–Wallis non-parametric tests. IBM® SPSS® Statistical software (version 22.0) was used for multivariate regression analysis. P values ≤0.05 were considered to be significant.

Results

Antibacterial activity

The CBM/CMC hydrogel can effectively improve its antibacterial activity with the addition of plant essential oils (especially against gram-negative bacteria). E. coli and S. aureus were selected to assess the antibacterial properties of the developed hydrogels. As shown in Table 1, the antibacterial efficiency of the CBM/CMC hydrogel was 16.49 ± 1.58% for S. aureus and −28.41 ± 0.99% for E. coli; the inhibition rates of EEO, GEO and CEO towards S. Aureus were 49.06 ± 1.52%, 20.67 ± 2.36% and 16.08 ± 0.99%, respectively, whereas the rates towards E. coli were 2.37 ± 0.11%, 2.23 ± 0.08% and 2.96 ± 0.29%, respectively. Therefore, owing to their reliable antibacterial activity, EEO, GEO and CEO were confirmed to be effective antibacterial agents. Although the antibacterial activity significantly increased on incorporating EEO, GEO or CEO into the CBM/CMC hydrogel, the CBM/CMC/EEO hydrogel was observed to possess the highest antibacterial activity towards S. aureus (46.26 ± 2.22%) and E. coli (63.05 ± 0.99%).

Table 1.

Antibacterial activity of CBM/CMC, PC, EEO, GEO, CEO, CBM/CMC/PC, CBM/CMC/EEO, CBM/CMC/GEO and CBM/CMC/EEO

Sample code	*Escherichia coli*	*Staphylococcus aureus*
CBM/CMC	−28.41 ± 0.99%^a	16.49 ± 1.58%^a
PC (gentamicin)	41.04 ± 0.54%^b	71.28 ± 0.21%^b
EEO	2.37 ± 0.11%^c	49.06 ± 1.52%^c
GEO	2.23 ± 0.08%^c	20.67 ± 2.36%^d
CEO	2.96 ± 0.29%^c	16.08 ± 0.99%^a
CBM/CMC/PC	0.71 ± 0.14%^d	16.70 ± 0.24%^a
CBM/CMC/EEO	46.26 ± 2.22%^e	63.05 ± 0.99%^e
CBM/CMC/GEO	18.21 ± 1.20%^f	38.41 ± 0.21%^f
CBM/CMC/CEO	22.90 ± 2.15%^g	53.67 ± 0.42%^g

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CBM carbomer 940, CMC carboxymethyl chitosan, EEO eucalyptus essential oil, GEO ginger essential oil, CEO cumin essential oil, PC positive control. Values are given as mean ± SD (n = 3). The concentration of PC used was 50 μg/mL, the concentrations of essential oils used was 50 mg/mL. ^a–gDifferent superscript letters in the same column indicate significantly different values (p < 0.05)