Hydrogel films of chitosan and eucalyptus oil enhance wound healing in rats

2.1. Materials

Chitosan (CH) (Sigma Aldrich, USA; high molecular weight), Cinnamaldehyde (CIN) (assay minimum:98%; density: 1.040-1.050 pure; LOBA Chemie, Mumbai, India), Eucalyptus oil (EO) (Minimum assay as cineole content: 60%; density: 0.897-0.924; HiMedia Laboratories Pvt Ltd, Mumbai, India), Cedarwood oil (CW) (0.96g/ml at 20°C; BDH Chemicals Ltd Poole, England), Blackseed oil (BS) (purchased from local market), silver nitrate (extra pure from ScharlauChemie S.A., Sentmenat, Spain), Glycerol (anhydrous, 99.0-101.0%; SIGMA-ALDRICH, Germany), Tween 80 (SIGMA-ALDRICH, Germany), Methanol (MeO (99.8%; SIGMA-ALDRICH, Germany), Phosphate buffer saline (pH 7.4) (PBS) (Aldon Corporation, Avon, NY), Ethanol (EtOH) (AnalaR NORMAPUR, France), Nutrient broth (pH 7.4 ± 0.2 at 25°C) (NB) (TM Media, Titan Biotech Ltd., Rajasthan, India) and Moringa oleifera leaf powder (ML) (Family: Moringaceae; Country of origin: India; USDA Organic; procured from Superfoods, by MRM Nutrition, USA) was used as received. Deionized water was used to prepare hydrogels. For the wound healing study, all chemicals and reagents, including Carbomer 5% were purchased from SIGMA-ALDRICH, Germany-Saudi Arabia agent.

2.2. Preparation of ML extract (MLE)

For preparing MLE, the ML powder was placed in MeOH (10% w/v) and macerated at room temperature for 24 h with constant agitation over a shaker. The methanolic extract was filtered and transferred to another flask, and a fresh batch of MeOH was added for maximum extraction. The collected macerate was filtered and concentrated in a rotary evaporator and stored for future use in a glass bottle in a refrigerator [27].

2.3. Biosynthesis of silver NP in methanolic MLE (NP/MLE)

NP were prepared according to a previously reported method [28,29]. The calculated weight of concentrated MLE (as in section 2.2) was dissolved in MeOH (10%w/v), and to 10 mL of this methanolic solution of MLE, 0.01M silver nitrate solution (10 mL) was added and placed in the dark for 24 h. The color change was noted, and the synthesis of NP was confirmed by transmission electron microscopy (TEM) analysis.

2.4. Preparation of hydrogel films

The CH solution was prepared in acetic acid (1%v/v) in deionized water at 2% concentration (w/v) while stirring on a magnetic stirrer at 60°C under agitation for 24 h and then cooled to room temperature. The solution was filtered with a Whatman filter paper to remove any undissolved matter or impurities. After filtration, glycerol and Tween 80 were added to the CH solution. This CH solution with glycerol (0.75 mL/g on weight of CH) and Tween 80 (0.2 g/g on weight of CH) was termed CHT. CHT contents (CH, glycerol and Tween 80) were stirred thoroughly on a magnetic stirrer for 2 h, after which the CHT solution was ready to be used for film formation. The composition of film solutions and their respective codes are given below:

Film A (CHT/CIN): is CHT with 1% w/w CIN (on weight of CH)

Film B (CHT/CIN/MLE): is CHT/CIN with 25% w/w MLE (on weight of CH)

Film C has CHT, CIN, MLE, and 3% v/v essential oil, coded as given below: CHT/CIN/MLE loaded with 3% v/v EO: C-EO

CHT/CIN/MLE loaded with 3% v/v CW: C-CW

CHT/CIN/MLE loaded with 3% v/v BS: C-BS

Film D has CHT, CIN, 3% v/v essential oil with NP/MLE, coded as given below:

CHT/CIN/EO and NP/MLE (as prepared) : D-EO

CHT/CIN/CW and NP/MLE (as prepared) : D-CW

CHT/CIN/BS and NP/MLE (as prepared) : D-BS

These were poured into glass petri dishes and dried at room temperature for 72 h to investigate their potential as hydrogel film formers. Care was taken to eliminate the entrapment of air bubbles during mixing.

2.5. Characterization

3.1. Synthesis

The films were prepared from natural products (CH, CIN, MLE, EO, CW, BS), which are cost-effective and environment-friendly, being natural in origin. Film A, CHT/CIN, was modified by the incorporation of MLE and EO/CW/BS, drop-wise under agitation, followed by blending. Glycerol was added as a plasticizer, and Tween 80 as an emulsifier. The films were further reinforced by silver NP that had been biosynthesized in methanolic MLE. The crosslinking of the films was accomplished using CIN. The film solutions in equal amounts were poured on glass petri dishes, left to dry at room temperature, and after 72 h, the films were peeled off the petri dishes (Figure 1).

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Figure 1.

Hydrogel films.

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3.2. Swelling studies

The maximum swelling percent attained by the films in distilled water was 35%-48%, PBS 40%-60%, and in SWF was found to be 38%-45%, respectively. The films were stable in water, PBS, and SWF after 24 h of swelling in these respective media. This revealed that the films were adequately crosslinked with the help of CIN.

3.3. FTIR

The presence of characteristic functional groups typical for the components of the hydrogel films was confirmed by FTIR (Appendix). All films showed the presence of a broad absorption band at 3200-3400 cm^-1 due to stretching vibrations of -OH bands. The absorption bands around 3300 cm^-1, corresponding to the -NH bond of the primary amine of CH, have overlapped with the broad -OH band. The absorption band around 1600 cm^-1 could be attributed to the bending vibrational absorption of -NH. The absorption bands in the region between 2900-2800 belong to the -CH stretching vibrations of aliphatic -CH bonds, while those around 1370 relate to -CH bending vibrations. The bands that appear at about 1700 cm^-1 support the presence of -C=O, carbonyl group, while those around 1000 cm^-1 correspond to -C-O stretching vibrations [39].

3.4. Morphology

TEM of NP/MLE (Figure 2): The shape and size of NP were determined by TEM analysis. The presence of biosynthesized silver NP of size <100 nm was evident in TEM. Amongst these biosynthesized NP, the small-sized NP were spherical, with distinct boundaries. They appeared as round, dark grey cherries (NP), well-dispersed and well-embedded throughout the light grey cake matter (the matrix). The large-sized NP appeared somewhat spherical, with their boundaries touching and, in some cases, overlapping with each other. These could be seen occurring as agglomerates that either appeared covered by a grey sheath or seemed embedded in the grey matrix. The grey sheath or matrix was comprised of biomolecules of MLE.

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Figure 2.

TEM of NP/MLE.

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SEM of the films (D-EO, D-CW, D-BS) (Figure 3): Silver NP were homogenously distributed throughout the hydrogel matrices of D-EO and D-BS. The SEM micrograph of D-EO revealed a dense and interconnected network; nano-sized, spherical silver NP (size < 100nm, denoted by arrows in Figure 3) were homogenously distributed throughout the hydrogel matrix. In the SEM micrograph of D-BS, a compact and dense network was evident, with narrow channels. However, unlike D-EO and D-BS, the SEM micrograph of D-CW revealed agglomeration.

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Figure 3.

(a) SEM of D-EO, (b) SEM of D-CW, (c) SEM of D-BS. Size < 100nm, denoted by arrows.

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3.5. Thermogravimetric analysis (TGA)

TGA was performed to assess the percentage weight loss or degradation of the films at high temperatures (Figure 4). Additionally, 20wt% loss was observed in the hydrogel films between 200°C to 250°C, and 50wt% degradation occurred between 300°C to 350°C. The onset of the last step of degradation was observed at about 450°C. The thermograms followed a multi-step decomposition pattern; however, the decomposition steps were not well-demarcated in some of the thermograms. TGA analysis revealed that the films could be safely used up to 200°C.

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Figure 4.

(a-h) TGA thermograms of A, B, C-EO, D-EO, C-CW, D-CW, C-BS, and D-BS films.

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3.6. Antibacterial study

The antibacterial behavior of the test samples was studied by the disc diffusion method for E. coli, S. aureus, and P. aeruginosa. The antibacterial behavior of each film was evaluated based on the ZOI measured in mm. The films loaded with EO, C-EO, showed 9 mm and 8 mm ZOI for E. coli and S. aureus, respectively, while those containing BS exhibited a ZOI of 10 mm for S. aureus. The films D-EO, D-CW, and D-BS, reinforced with NP, exhibited higher ZOI values, i.e., 11mm, 10mm, 8 mm for E. coli, 15mm, 10mm, 11 mm for S. aureus, and 10mm, 10mm, and 9 mm for P. aeruginosa, respectively (Table 1). C-BS, D-EO, D-CW, D-BS, with respect to Amoxicillin, showed comparable or slightly improved antibacterial behavior against S. aureus. D-EO, D-CW, and D-BS films exhibited antibacterial behavior against P. aeruginosa, which was comparable to that of Amikacin. However, against E. coli, films of C-EO, D-EO, D-CW, and D-BS could not perform better than Amikacin.

3.7. Microbial penetration study

The test tubes covered with films, cotton balls, and an open test tube were observed for any change in clarity of solution or turbidity. All the test tubes had clear solutions for two weeks. After 3 weeks, turbidity was evident in the open test tube while the solutions in the rest of the test tubes were unaffected, as observed visually and noted by recording their respective transmittance values (Figure 5). The test samples did not show turbidity for one month (100% clear); the transmittance of their respective media showed values very close to or the same as that of the negative control test tube. There were no visual changes in the clarity of solutions (100% clear) and the percentage of transmittance, which were 99-100%, for the test samples, compared with the negative control.

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Figure 5.

Microbial penetration study.

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3.8. Soil biodegradation behavior

The hydrogel films were buried under moist soil for several days, and their biodegradation potential was investigated by pulling out the buried films from time to time and examining the changes in color, texture, and weight. During soil biodegradation, the films were weighed periodically, and it was found that the films had initially gained weight up to 15 days. After fifteen days of burial, although no notable loss in weight had occurred, the first degradation sign, i.e., discoloration, became evident. Films turned softer and discoloration became more pronounced with time, along with the development of very small dark brown spots. After 30 days of soil burial, the films had lost their color completely, gained more softness, and presented difficulty in handling; after 40 days, the films turned too fragile to handle, and fragmentation became pronounced.

3.9. Wound healing evaluation

The hydrogel films incorporating EO, i.e., C-EO and D-EO, showed the best antibacterial behavior, compared to those loaded with CW and BS. Therefore, only the sets of films comprising A, B, C-EO, and D-EO were tested for their wound healing efficiency. The progress in wound healing was primarily recorded as wound contraction in mm² by using transparent paper, a marker, and a ruler. The wound of each rat on day zero was then considered 100%. Each subsequent day, wounds were measured in mm² and converted to a reduction percentage in comparison with the day zero wound dimension.

Wound area contraction was calculated in percentage and given as wound healing (WH) % by the given formula:

$\text{WH}\%=\text{A}-\text{B/A}\times \text{1}00$

A: Initial wound area; B: Wound area on specific day (day 3, 6, 9, 12, 15, 18, 21, 24, 28)

During the wound healing study, no unusual illness or health symptoms were observed, and no significant increase or decrease in weights was recorded in the test animals. The wound of group 6 (film C-EO) healed in 18 days Figure 6(a) and (b).

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Figure 6.

(a) Wound healing in the six rat groups (group 1 “with no wound” is excluded). Curve showing the wound width % (WH%, mm²± SD, X-axis) and duration in days (Y-axis). Each group contained five rats, (b) Hydrogel treated wounds.

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3.10. Biochemistry profile

There was no significant change in the weight of the rats, increase or decrease, recorded during the investigation. The results of film C-EO were comparable to the healthy group except for the GLU level. The GLU level in the C-EO film-treated group was much improved compared to other groups.

3.11. RT-PCR analysis

The RT-PCR was performed for samples from different groups on the 28^th day (Figure 7). There is overexpression of all genes, especially 5S rRNA, in the film C-EO.

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Figure 7.

(a). The expression of WNT4, MMP9, TGFB1, 5S rRNA and CTNNB1 in different rat groups on day 28. A: Bar chart data is represented as mean ±SD (n = 3, two independent experiments). The results are expressed as fold-change compared to the untreated group (1-fold change: -ve control). The raw delta-Ct values (the difference between CT values obtained for the gene of interest and the housekeeping gene) were converted into relative expression levels (fold-change) using the formula 2−∆∆Ct. Statistical differences, compared to untreated control cells, were assessed using a one-way ANOVA with the Tukey’s post-hoc multiple comparison test. p < 0.001 (***) was taken as significant. (b): Cluster of all genes.

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3.12. Discussion

More than 3%v/v CW could not be incorporated into the films. At 6%v/v, CW was found to be oozing out of the films, due to phase separation. Thus, for consistency and comparison, the percent inclusion of EO, CW, and BS was kept as 3%v/v in C-EO, C-CW, and C-BS. It is well reported that MLE is rich in alkaloids, flavonoids, and polyphenols. These compounds are responsible for the phytoreduction reaction resulting in the biosynthesis of silver NP without using any solvent, stabilizer, or capping agent at room temperature [10,14,15]. Due to mild reaction conditions, this biosynthesis reaction is considered low-cost and environment-friendly. The color change of the solution from green to brown indicated the formation of silver NP, which was further confirmed by TEM analysis [27,40]. The films obtained were homogenous, free from any crack, pore, or bubble, soft, and easily foldable. The thickness of the films was found to be 124-130µm. The swelling studies indicated that all the films were swollen in water, PBS, and SWF; however, with the incorporation of NP, the % of imbibed solution decreased.

FTIR spectra (Appendix) confirmed the presence of functional groups such as -OH, -C=O, -NH, -CH, -CO-, however, as the films had dried, it was not possible to identify every functional group, due to the functional groups undergoing crosslinking by chemical reactions [39]. TEM micrograph confirmed the biosynthesis of NP in MLE, and SEM of the films confirmed the inclusion of NP in each crosslinked film. These results further confirmed the development of nanocomposite films of D-EO, D-CW, and D-BS, respectively. SEM micrograph showed narrow channels that can be correlated to crosslinking between film components. These were well-pronounced in D-BS and D-EO. The films of D-EO, D-CW, and D-BS exhibited higher thermal stability due to the inclusion of NP, as evident by their higher thermal degradation temperatures.

A microbial penetration study was performed to assess the ability of the films to combat microbial penetration. The test samples could completely block the penetration of microbes and demonstrated their capability to safeguard the wounds from secondary bacterial infections. The reason might be the small pore size of the films and the presence of antibacterial components such as CIN, NP as well as the CH base matrix that contains functional groups, bonding with the bacterial surface and inhibiting their activity [30,41].

The films incorporating EO showed good antibacterial behavior compared to those with CW and BS. The results of the antibacterial study confirmed that films incorporating NP showed improved antibacterial behavior against S. aureus and E. coli, as well as P. aeruginosa, as evident from their higher ZOI values compared to those of C-EO (9 mm and 8 mm against E. coli and S. aureus, respectively) and C-BS (10 mm against S. aureus), respectively. Thus, these films could be applied as antibacterial coatings and films. The films loaded with EO showed the best antibacterial activity. Therefore, these films were subjected to wound healing studies.

During the soil biodegradation study, initially, the films had gained weight due to the absorption of moisture from the soil. Gradually, they got discolored due to the action of soil components. After 30 days of soil burial, the films had turned soft and had lost their original color completely. At this stage, they were not subjected to weighing, lest they might have broken while handling at the point where they were held. And this might have impaired the degradation study. After 40 days, films had turned too fragile, and some even fragmented into very small pieces [29]. Thus, the films were found to be biodegradable in soil by the combined action of soil microbes, moisture, and pH, after a considerable period.

Since the films incorporating EO showed superior antibacterial activity relative to those with CW and BS, only C-EO and D-EO were selected for wound healing study. WH percentage was calculated by wound area quantification (mm² reduction of wound over time) by measuring contraction in wound area in mm² over the number of days of treatment. The healing of wounds on skin is associated with inflammation, proliferation, and remodeling. The duration of wound healing is significantly governed by wound contraction. Film C-EO, which had CH, CIN, MLE combined with EO, exhibited wound contraction in 18 days, the quickest relative to all the other films. Thus, film C-EO enhanced wound contraction through improvement in the epithelization step due to the presence of EO, which accelerated wound healing [42,43]. Films A and B showed wound contraction in 21 days, which is the same as in the case of Fucidin, compared to the control, which took 28 days for wound healing. Crosslinking is crucial for ensuring the structural integrity and performance of the CH hydrogel. CIN is considered a biosafe crosslinking agent. During the crosslinking reaction, the aldehyde group of CIN reacts with an amino group of CH by Schiff base reaction [44]. Proper crosslinking of hydrogel films confers the necessary structural integrity and strength required for application and removal of films from wounds, ensures adequate swelling ability, and supports absorption of wound exudates without film impairment.

For investigation of the possible molecular mechanism for the response of hydrogel films to the wound healing process, gene expression was examined using real-time polymerase chain reaction. It was found that film C-EO caused a significant increase in 5S rRNA expression, amongst all the films [42].

From the results of blood chemistry, the response of the body to the treatment is reflected, and this helps to assess the effectiveness of the treatment. During the wound healing process, keeping an eye on lipid and kidney parameters is very pivotal. The group treated with film C-EO, consisting of EO and MLE, showed a biochemistry profile almost similar to the healthy animal. Amongst all the test groups, the C-EO-treated group showed the best response to Glucose (GLU) level (129.0). The film D-EO-treated group demonstrated slightly elevated glutamic-oxaloacetic transaminase (GOT), glutamic-pyruvic transaminase (GPT), and creatinine and very high GLU levels (135.94) relative to the healthy group, in response to inflammation. From the results obtained it can be interpreted that all the groups have shown better response to wound healing relative to positive control as revealed from the parameters given in Table 3.

The film C-EO-treated group exhibited the best wound healing efficiency amongst the test groups. D-EO, which had silver NP dispersed in the matrix, showed wound contraction in 24 days compared to film C-EO, which showed wound contraction relatively earlier (18 days). NP exhibit a profound influence on wound healing as they promote wound contraction, accelerate wound healing, stimulate proliferation, migration, differentiation, and maturation of keratinocytes [45,46], due to the high surface area: volume ratio of NP. However, it is possible that in the case of MLE biosynthesized silver NP, their surface area: volume ratio is lowered as they are clustered or agglomerated (as observed in TEM; Figure 2). Therefore, in the D-EO-treated group, the wound healing efficiency was suppressed. However, the antibacterial behavior of D-EO was still enhanced, despite agglomeration. Test animals treated with film B, containing MLE, and film A without MLE exhibited similar wound healing duration as those treated with Fucidin. EO is rich in terpene 1,8-cineole, besides other phytochemicals. Test animals treated with C-EO showed improved wound healing behavior amongst all the test animals due to antimicrobial, antioxidant, and proliferative properties of phytochemicals of EO [43,47].

The hydrogel components were carefully selected based on their properties for end-use application, i.e., wound dressing. CH was used in film preparation as the base matrix due to its minimum allergenicity, non-toxicity, biocompatibility, and biodegradable nature [48]. Essential oils, as used in this study, are naturally infused with antibacterial and anti-inflammatory properties, required for wound healing, that render them a good substitute for synthetic antibiotics [49]. MLE has alkaloids, flavonoids, and polyphenols that participate in the biosynthesis of NP, obviating the use of any other chemical. CIN was used as a natural crosslinking agent that conferred adequate integrity and strength to the hydrogel films.