Introduction

The human skin being the largest organ of the body is a multifaceted and heterogeneous organ, with diverse attributes like thermal insulation and regulation, defense against foreign pathogens, touch based sensation, vitamin D production and prevention of water loss1. The sturdiness of the skin is also essential for the continuance of biological activities. However, any abuses like heat, chemical, mechanical and radiation can disturb the skin barrier; structural and functional loss, which may lead to a wound or injury2. Although skin have inherent regenerative properties, but sometimes it is halted or delayed due to multiple factors3. The complex and dynamic process of wound healing involves the regeneration and growth of cells and tissue, which is also affected by wound nature i.e. acute or chronic. After tissue damage, the body generally starts a chain of complicated and interrelating biological events such as hemostasis, inflammation, proliferation and maturation or re-modeling for healing of wounds2. Each phase is firmly governed with the regulation of different cells and substrates, i.e. fibroblasts, macrophages, platelets, and keratinocytes, endothelial epidermal growth factors4.

Protection of wounded skin from further damage or infection is essential, which is generally provided by a wound dressing. Traditionally, biomaterials like honey, ash, leaves, cobwebs, cotton swabs and gauzes were used as wound dressings. These dressings can excessively absorb secretions and stick to regenerated cells leading to a painful removal along with microbial contamination because of their non-sterile nature. With the improvement of medical treatment, a lot of modern wound dressings have been developed such as hydrogels, foams, films and hydrocolloids5 as moisture-retentive dressing materials. Among the various modern wound dressings, hydrogel membranes (cross-linked three dimensional structures) made of hydrophilic polymers have been drawing researchers attention because of their exceptional features, such as maintaining moist environment, oxygen permeability, biocompatibility, biodegradability and excellent swelling behavior. Hydrogel membranes are highly porous, permitting entrapment of multiple therapeutic agents and their release6.

Biopolymers are preferred to prepare hydrogel films as they are non-toxic and biodegradable but they give weak mechanical strength. For effective wound repair, biopolymers can be incorporated with a variety of therapeutic ingredients like natural or synthetic polymers, metal/metal oxide nanoparticles and essential oils. Incorporation of these materials results in the formation of composite hydrogel films with enhanced properties and functionalities.

Among various gum polysaccharides, gellan gum (GG) is an anionic, high molecular weight, linear tetra saccharide with repeating fractions of glucose, glucuronic acid, glucose and rhamnose. It is an end product of bacterial fermentation of Pseudomonas elodea and Sphingomonas paucimobilis7. Being biologically inert, GG with low mechanical strength is too weak to produce an effective wound dressing because its chemical structure lacks integrin-binding domains. These issues can be resolved by the introduction of another polymer, either physically or chemically bonded components8 leading to a composite system. So, it’s feasible to incorporate another integrin binding protein polymer, such as collagen to bestow GG based films with bio-adhesiveness9. Collagen, the main element of extracellular matrix, is widely used in biomedical engineering because collagen includes RGD (arginine glycine-aspartic) chains in its structure, which are responsible for adhesion and propagation of stem cells10.

Essential oils (EOs) are complex mixture of highly volatile hydrophobic phyto-therapeutic components, widely employed in pharmaceutical industry, beautifying products and food packaging industry11,12. They are reported to possess enhanced antioxidant, anticancer, antibacterial and anti-inflammatory properties13. EOs serves as an alternative therapeutic ingredient to conventional synthetic compounds and thus become an attractive approach in the biomedical area14. Now a days, the biomaterials encapsulated with EOs have been developed to cope with volatility, low stability and high sensitivity of EOs, followed by enhanced biological activities (i.e. antioxidant and antibacterial) in particular composite applications15. Lemongrass essential oil (LEO) exhibited antibacterial activity against E. coli and S. aureus, when encapsulated in nanocapsules of polylactic acid and in edible coating, LEO prolonged the shelf life of various fresh-cut fruits, i.e. apples and pineapples16. Geranium essential oil (GEO) is also reported to have antibacterial and antioxidant properties, widely used in food preservation, biomedical and cosmeceutical industries.

Herein we prepared bioinspired GG based composite hydrogel membranes (CHMs) infused with GEO and LEO. The combination of biopolymers, i.e. gellan gum and collagen will not only provide definite mechanical properties, but also offer an appropriate microenvironment for cell adhesion and proliferation. Unlike previously reported hydrogel systems, our study uniquely integrates essential oils into a gellan gum–collagen composite platform, thereby uniting mechanical robustness, biocompatibility, and therapeutic activity in a single membrane. Several CHMs encapsulated with GEO, LEO will be systematically characterized for the physicochemical properties and functional performance, thus providing new insights into the design of multifunctional wound dressings and expanding the scope of essential oil utilization in biomedical materials.

Materials and methods

Materials

Gellan gum (low acyl, molecular weight 500 kDa, CAS No: 71010-52-1) was bought from Alfa aesar (USA). Collagen type II (CAS No: 9007-34-5) was purchased from Daejung (Korea). Glycerol (CAS No: 56-81-5), Tween® 80 (CAS No: 9005-65-6), ethanol (CAS No: 64-17-5), calcium chloride (CAS No: 10043-52-4), and 2, 2-diphenyl-1-picrylhydrazyl (DPPH, CAS No: 1898-66-4) were bought from Sigma Aldrich (USA). Geranium and lemongrass oil was purchased from Go Natural Pakistan. All other chemicals used were of analytical grades.

Preparation of CHMs

CHMs were prepared by solution casting crosslinking method with slight modification as reported previously. Initially, aqueous solution of GG (% w/v, at 50 °C) and collagen (% w/v, at 25 °C) were prepared by dissolving both polymers separately in distilled water with continuous stirring (at 700 rpm) for 30 min until clear liquid solutions were obtained (Table 1). The GG solution was kept at room temperature (25 °C) and later on collagen solution was added slowly in it with continuous stirring. Meanwhile, different concentrations of LEO and GEO (previously mixed with 0.4% w/v, Tween® 80) were incorporated in above prepared solution under constant stirring for 15 min. Then glycerol (15% w/w based on polymers mass) was added in polymeric solution under constant stirring to impart plasticity to the membranes. Afterward, the whole mixture was casted in sterile disposable petri dishes followed by spray of calcium chloride solution (0.5%w/v) over the membrane surface (Fig. 1) to cross-link the polymeric contents via ionic interaction. Membranes were allowed to dry at room temperature (25 °C) and peeled off easily after drying. These dried membranes were stored in a desiccator for further analytical studies17.

Table 1 Various formulations of CHMs.
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Fig. 1
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Schematic representation of synthesis of CHMs.

Physical characterization

The thickness of composite hydrogel membranes was measured by a digital micrometer (Shang Chong Co. Ltd, China)18, while the folding endurance was checked manually by folding the hydrogel films repetitively at the same point. The number of times hydrogel films can be folded without any break or crack is termed as the folding strength of each film17. For weight uniformity, ten different samples (1.5 × 1.5 cm2) were taken from each film and weighed individually by using electronic weighing balance (OHAUS adventurer analytical balance, USA) and their results were reported as mean ± SD (Kaur et al., 2019).

Swelling index

The swelling index of hydrogel membranes (blank and essential oil-encapsulated) was determined by immersing the pre-weighed membranes of fixed dimensions (1.5 × 1.5 cm2) in phosphate buffer solution (pH 7.4) for 24 h at room temperature. Then swollen films were removed at specific time intervals, and weighed after gently wiped off excess water with filter paper. For each formulation, three samples were taken (n = 3) and the swelling index was calculated by using the following formula 1;

$$\:Swelling\:\left(\%\right)=\frac{(Wt-Wi)}{Wi}\times\:100$$
(1)

where, Wt refers to the final weight of swollen films at specific time intervals, and Wi states the initial weight of dried films19.

Solid-state characterization

FTIR analysis

FTIR study was performed to identify functional groups and possible chemical interactions between polymers and essential oils. Analysis was carried out using Bruker FTIR in the scanning range of 4000–400 cm− 1 at the resolution of 2 cm− 117.

XRD analysis

XRD analysis was performed to evaluate the state of crystallinity of blank and essential oil encapsulated composite membranes. The XRD spectra were obtained by using X-ray diffractometer (X’pert PRO, PANalytical, Netherlands) having Cu-Kα radiation source. All data recorded over a scanning range of 5–40° (2θ) operated at 30 mA current and 40 kV voltages17.

SEM analysis

The surface morphology of blank and essential oil encapsulated composite hydrogel films was visualized by scanning electron microscope (VEGA3 TESCAN) at 20 KV accelerating voltage17.

Biological evaluation

Antibacterial activity

The prepared composite membranes were assessed for their antibacterial activity against the gram-positive bacteria (S. aureus) and gram-negative bacteria (E. coli), as these bacteria colonize around the injury within 48 h, by using previously reported agar well diffusion method. In this method, nutrient agar medium was prepared and maintained its pH and sterilized for 20 minutes at 121 °C, and poured into sterile plastic plates. After solidification of media, surface was inoculated by streaking the prepared inoculum containing 106–107 CFU/mL of the test microorganisms. Under sterilized conditions, the composite membrane discs (10 mm) were placed in the wells drilled in solidified agar with sterilized borer and 50–80 µL of PBS (pH 7.4) was added over the membrane discs in the well. Then, the agar plates were incubated for 24 h at 37 °C, and the zone of inhibition was calculated for each membrane in mm (n = 3)20.

Antioxidant activity

Radical scavenging activity of blank and essential oil encapsulated composite hydrogel films was measured by using DPPH (2,2-diphenyl-1-picrylhydrazyl radical) with some modification in previously described method. Approximately 25 mg of film sample was vortexed with 10 mL of deionized water and methanol mixture (1: 1) for 3 min and mixture was left to stand for 3 h at room temperature, then again vigorously vortexed for 3 min. Afterward, one aliquot (2 mL) of the diluted film solution was mixed with 2mL of 0.2 mM DPPH in methanol. The mixture was vigorously shaken and kept in the dark at room temperature for 30 min. Absorbance was determined at 517 nm using a UV–visible spectrophotometer (CECIL CE7400S). The assay was carried out in triplicate and the antioxidant capacity of membranes was calculated by the following Eq. 2:

$$\:\%\:Scavenging\:activity=\frac{\left({Abs}_{blank}-{Abs}_{sample}\right)}{\left({Abs}_{blank}\right)}$$
(2)

where Absblank states the absorbance of DPPH, and Abssample is the absorbance of tested sample21.

In vivo wound healing activity

A wound healing study was conducted by a full-thickness excisional wound model in albino rats weighing 200 g in accordance with the protocols approved by the institutional Ethical Review Committee (ERC) of Government College University, Faisalabad, Pakistan (Ref No: GCUF/ERC/16). For this study, all purchased animals were acclimatized under standard laboratory conditions for one week at relative humidity (55 ± 5%), a 12 h light/dark cycle, and a temperature of 22 ± 2 °C. The animals were also given free access to water and a standard rodent diet.

Healthy rats were divided into seven groups each containing three rats (n = 3). Group (i) animals considered as positive control (disease group), group (ii & iii) rats treated with blank formulations (G2 & GC7) respectively, group (iv & v) animals were treated with only oil encapsulated composite hydrogel membrane (GG4 & GL1), group (vi & vii) rats were treated with oil encapsulated optimized polymeric membranes (GCG4 & GCL1) respectively. Rats were first intraperitoneally anaesthetized with Ketamine/Xylazine (100 mg/kg; 20 mg/kg), and then the desired area was shaved off and washed with 70% ethyl alcohol. Afterwards, a biopsy punch was used to create two full-thickness wounds of 5 mm diameter on the dorsal side of the rats. Composite hydrogel membrane patches (1.5 cm2) were placed over the wound site, secured with an elastic bandage, and replaced daily. On several days i.e. zero, 2nd, 4th, 6th, 8th, 10th , and 12th day, wound dimensions were measured by using digital callipers and per cent wound closure was calculated by the following formula;

$$\:\%\:Wound\:contraction=\:\frac{Ao-At}{Ao}\times\:100$$

where “Ao” refers to the initial wound dimensions and “At” refers to the wound dimensions at the time of biopsy17.

Detection of pro-inflammatory cytokines

Level of pro-inflammatory cytokines such as Tumor Necrosis Factor-α (TNF-α) and Interlukin-6 (IL-6) in rat serum were determined according to the manufacturer’s procedure by using enzyme-linked immunosorbent assay (ELISA) kits (Elabscience®, Wuhan, China). The absorbance of each film sample was taken at 450 nm with the ELISA plate reader in triplicate (n = 3) and optical density of each sample were verified by using standard curve.

Histopathological examination

For histopathology, rat skin samples were taken immediately on the 12th day after decapitation of anesthetized animals (Ketamine/Xylazine, 100 mg/kg; 20 mg/kg). They were fixed overnight in a 10% v/v buffered formalin solution before being embedded in paraffin tissue blocks. One histological section was obtained per skin explant. Representative sections were stained with hematoxylin and eosin (H & E) and Masson’s Trichrome. The photomicrographs of each section were taken with the Accu-scope 3000-LED microscope to evaluate the extent of wound healing in all groups22.

Histopathological and morphometric analysis

For histopathology, rat skin samples were taken immediately on the 12th day after euthanasia. They were fixed overnight in a 10% v/v buffered formalin solution before being embedded in paraffin tissue blocks. One histological section was obtained per skin explant. Representative sections were stained with hematoxylin and eosin (H & E) and Masson’s Trichrome. The photomicrographs of each section were taken with the Accu-scope 3000-LED microscope to evaluate the extent of wound healing in all groups. Morphological quantification was performed with ocular field number 18 with field area 2.54 mm2 per field of view at higher magnification. From 5 selected sections epithelial thickness (µm) was measured by using calibrated ocular micrometer and mean values were counted. Number of fibroblast, inflammatory cells and blood vessels were expressed as mean per field of view and counted from 10 randomly selected non-overlapping fields/section. Histopathological hallmarks were scored on 0–5 scale, 0 indicated healthy physiology and 05 represented severe histopathological changes.

Statistical analysis

All experiments were carried out in triplicate (n = 3) and data was represented as mean ± SD. For statistical comparison, one way ANOVA or Student t-test was used where p ˂ 0.05 states a statistical significant difference.

Results and discussion

Preformulation

During Preformulation several CHMs were prepared by employing the different concentrations of polymers and essential oils. All of them were analyzed for their physical attributes and integrity. Results were recorded and presented in supplementary data (Supplementary table S1). Preformulation and physical evaluation of CHMs revealed that films prepared from gellan gum alone have poor mechanical strength and brittle in nature. Therefore, collagen was added at different concentrations to enhance bio-adhesion (being a prominent part of extracellular matrix) and physicochemical stability of membranes. Before encapsulation of Eos in CHMs, we tested the antibacterial activity of pure EOs. We tested the pure essential oils (Supplementary figure S1). Moreover, we also tested the films with various concentration of oils for choosing suitable concentration oil for optimum antibacterial effect. The tested concentrations of EOs (i.e. 25%, 50% and 100% w/w of polymer) have more or less the same inhibitory activity against S. aureus and E. coli the most common inhabitant of wounds (Supplementary table S2 and figure S2). Thus, only 25% w/w of EOs appeared more effective against tested bacteria. It is proposed that cross-linked hydrogel matrix can limit the diffusion of EOs and reduce the antimicrobial activity. This observation is in agreement with previously reported studies17,23,24. Hence, we selected minimum concentration of EOs, i.e. 25% w/w for encapsulation in CHMs to avoid material loss.

Therefore, on the account of physical and antibacterial evaluation of CHMs, we selected six optimum formulations (i.e. G2, GC7, GG4. GL1, GCG4, and GCL1) for further testing to confer their wound healing potential. From here onwards, only these formulations will be discussed.

Physical evaluation of CHMs

The prepared films appeared thin, flexible, transparent, and free from wrinkles and bubbles. Moreover, after curing time, they were easily removed from cast petri dishes. Thickness is an important parameter that influences mechanical properties. These attributes demonstrate CHMs’ integrity during handling, transport, application, and the uniformity of contents. The thickness of the composite hydrogel membranes ranged from 0.038 ± 0.001 to 0.060 ± 0.001 mm, as shown in Table 2. Thickness increased with the addition of collagen and EOs. This effect could be due to the higher total solid content of the films and also indicates a uniform distribution of the film contents. The weight of all optimized composite hydrogel membranes ranged from 0.007 ± 0.0001 to 0.014 ± 0.001 g. Adding essential oils to CHMs resulted in a slight increase in membrane weight and further suggests content uniformity within the gel matrix (Mahmood et al., 2021). The folding endurance of all optimized composite hydrogel membranes ranged from 202 ± 5 to 218 ± 9 folds, indicating high mechanical strength and elasticity necessary for wound applications. These results suggest the employed method is suitable for developing uniform CHMs.

Table 2 Thickness, folding endurance and weight variation of CHMs (n = 10).
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Swelling behavior of optimized CHMs

Swell ability and the degree of swelling are main attributes of ideal wound dressings as they govern the release of therapeutic moiety from the gel matrix, ability to absorb high amount of wound exudates, bio-adhesion of membrane on wound and retain sufficient moisture in wound area to speed up healing process by stimulating proliferation and migration of fibroblasts and keratinocytes25.

Swelling degree of blank (G2 and GC7) and EOs encapsulated CHMs, i.e. GG4, GL1, GCG4 and GCL1 were evaluated gravimetrically phosphate buffer saline (PBS) of pH 7.4. The intact skin has a pH of 5.4 and it changes continuously in the healing process as in case of full thickness wounds, it varies between 7 and 926. The swelling indices of all optimized CHMs are depicted in Fig. 2, which demonstrates the outstanding swelling ability of films with a rapid rise in swelling initially and is a characteristic trait of hydrogel films. After immediate swelling phase, swelling index starts to decrease, which is probably due to erosion of polymeric matrix20.

Fig. 2
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Swelling indexes of CMHs in buffer solution having pH 7.4. Error bars indicate SD (n = 3).

Swelling of CHMs comprises of three distinct stages; first stage involves increasing adsorption of media on membrane surface, which has high affinity to absorb media owing to hydrophilic functional groups of GG and collagen. In second stage, repulsion of carboxylate ions produced by carboxylic acid groups of GG27, leading to increased swelling index of membranes. In the third stage, highly saturated membrane surfaces ingest more water media into the gel matrix that weakens the intermolecular hydrogen bonding and expansion of hydrogel matrix, which finally leads to degradation or erosion17,28.

Furthermore, the swelling degree of optimized formulations was found to be less as compared to blank hydrogel membranes, which might be due to encapsulation of hydrophobic essential oils29. Geranium (citronellol, citronellyl acetate, citronellyl formate, and geranio) and lemongrass (geranial, neral, isoneral, isogeranial, geraniol, geranyl acetate, citronellal, citronellol, germacrene-D, and elemol) oil lessen the propensity of CHMs to affix water by limiting the intermolecular interaction between polymers and swelling media thus leading to alteration of microstructure of the gel matrix30. Our results are in agreement with various reported studies where incorporation of lavender oil or tea tree oil17, flaxseed oil31 and zataria oil32 in hydrogel membranes reduces their swelling index. Thus, our results indicates that optimized CHMs have potential to be used as wound dressing, as they can effectively absorb and retain swelling media for a long duration with less polymeric erosion.

Solid-state characterization

FTIR analysis

FTIR spectroscopy was employed to confirm the chemical structure of the polymers and for identification of interactions between functional groups of EOs and polymeric chains. The FTIR spectra of G2, GC7, GG4, GL1, GCG4, and GCL1 are presented in Fig. 3.

The spectrum of G2 membrane showed a characteristic broad band at 3316 cm− 1, which is assigned to stretching of -OH group (3000–3500 cm− 1) of gellan gum and was in agreement with previous results33. This peak prevails in all optimized GG based composite membranes with slight variation, which indicates possible intermolecular interactions between polymeric chains and hydrophobic constituents of EOs. The absorption bands at 1604 cm− 1 and 1409 cm− 1 are attributed to carbonyl group and C—C aromatic stretching vibrations of GG. The peak at 1029 cm− 1 represented glycosidic bonds between galacturonic sugar units. While the peaks appeared at 2869 cm− 1 to 2936 cm− 1 were assigned to C-H stretching and bending of GG34.

Similar peaks appeared in spectra of GG4 and GL1 membranes, with minor differences in intensities and peak positions owing to interactions between EOs and GG polymeric functional groups. For instance, in GG4 and GL1, the broad bands of the –OH group have shifted to 3365 cm− 1 and 3375 cm− 1 respectively. The intense peaks observed at 2922 cm− 1 and 2866 cm− 1 could be attributed to the symmetric and asymmetric stretching of –CH2 and –CH3 groups respectively. The medium peaks at 1735 cm− 1 are probably due to the symmetric stretching of C = O bonds and deep intense peaks at 1608 cm− 1 and 1609 cm− 1 are possibly due to the vibration of C = C, confirming the presence of conjugated double bonds (C = C_CHO) in citral, which are common in acyclic monoterpenes of EOs35.

In spectrum of GC7 composite membrane, collagen showed peaks at 1636 cm− 1, 1544 cm− 1 and 1236 cm− 1, which are attributed to the presence of amide I, II and III bands36. A broad absorption band at 3267 cm− 1 was also observed due to hydroxyl group –OH group stretching37.

Similar peaks were observed in GCG4 and GCL1 with slight differences in peaks positions and intensities, which indicate that EOs have been successfully incorporated within a polymeric network. For example, sharp peaks at 1456 cm− 1 in GCG4 and GCL1 could be due to asymmetric stretching of C-H bonds, while these peaks were less intense in GC7 at 1406 cm− 135. The characteristic bends at 2926 cm− 1, 2925 cm− 1in GCG4 and GCL1 respectively denoted asymmetric stretching of –CH3 and symmetric, asymmetric stretching of –CH2 corresponding to an alkyl saturated aliphatic group attributed to the presence of citral, consistent with results reported previously38. The peaks in the range of 1750 –1650 cm− 1 were attributed to the presence of C = O bonds in spectra of GCG4 and GCL1. Furthermore, peaks appearing at 1083 cm− 1 and 1086 cm− 1 in GG4 and GL1 are possibly due to C-O bonds stretching present in EOs, whereas these particular peaks appeared less intense in GC7. These results are in agreement with previous studies39. Based on FTIR results, it is concluded that miscibility between the EOs and CHMs is due to the formation of intermolecular hydrogen bonds.

Fig. 3
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FTIR spectra of G2, GG4, GL1, GC7, GCG4, and GCL1.

XRD analysis

X-ray diffraction was employed to determine the physical state of optimized formulations i.e. state of crystallinity or amorphicity. The diffractograms of blank (G2 and GC7) and EO encapsulated CHMs (GG4, GL1, GCG4, GL1) are presented in Fig. 4. The XRD diffractogram of G2 showed one characteristic peak at 2θ of 28°, which indicates the partly crystalline nature of GG, consistent with the previous results17. The diffractograms of GG4 and GL1 also revealed the same peaks at 2θ of 28°. It indicates that encapsulation of oils has negligible effect on the crystallinity of the composite hydrogel membrane. The XRD pattern of GC7 membrane did not showed characteristic peak of GG, which is an indication of amorphous nature of blended polymers. Here, it is possible that collagen has partially masked the crystalline nature of GG. Similar patterns were observed with oil encapsulated CHMs i.e. GCG4 and GCL1, which are in agreement with the previous results where lemongrass oil was loaded on cellulose nanofibre-poly ethylene glycol composite38.

Fig. 4
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XRD diffractograms of G2, GG4, GL1, GC7, GCG4, and GCL1.

SEM analysis

SEM was employed to demonstrate the surface morphology of blank and CHMs encapsulated with essential oils as shown in Fig. 5. SEM micrographs of blank formulations i.e. G2 and GC7 composite membrane revealed smooth, homogenous and compact surfaces with no phase separation as reported previously17,40, which could be attributed to stable interactions between polymeric hydroxyl groups and amides of collagen41 that leads to uniform blend of polymers.

The SEM images of GG4, GL1, GCG4, and GCL1 showed heterogeneous surfaces owing to encapsulation of GEO and LEO40,42. For CHMs employed as wound dressings, surface integrity is of utmost importance to prevent the entry of invading microorganisms. Although, EOs encapsulated film’s surfaces are crack-less, bubble free with no oil droplets that confirmed the membrane integrity and complete dispersion of oils in the matrix. However, in comparison to the blank films, EOs loaded CHMs are relatively rough, irregular but nonporous in nature16,17,43. The roughness makes developed CHMs good candidates for the attachment of cells, like fibroblasts and keratinocytes that are helpful for curing of wound40.

Fig. 5
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SEM images of surface view of (a) G2, (b) GG4, (c) GL1, (d) GC7, (e) GCG4, and (f) GCL1.

Biological evaluation

Antibacterial assay

Currently, medical science is facing several issues for treatment of open wounds such as excess microbial growth, prevalence of infections at wound side and resistance of common pathogens of wounds such as S aureus and E coli against currently used synthetic molecules. EOs are considered as promising alternative to synthetic molecules owing to their potent antibacterial, antifungal and anti-inflammatory properties.

Initially, we tested the antibacterial effect of pure EOs on S aureus and E coli as shown in supplementary figure S1. Then oils were encapsulated in CHMs in various concentrations and 25%w/w oil concentration was chosen as optimum formulation owing to suitable inhibitory effect against tested bacterial strains as compared to the other concentrations. Here, blank CHMs did not showed antibacterial activity. While, the CHMs encapsulated with LEO and GEO showed significant inhibitory action as shown in Table 3; Fig. 6.

The antimicrobial activity of CHMs was attributed to the hydrophobic constituents of geranium and lemongrass oils. In GEO, the major constituents responsible for antimicrobial activity are citronellol, geraniol, linalool and citronellyl formate44,45,46. Whereas, LEO comprises a mixture of 28 constituents, among which geranial, neral (citral), and geranyl acetate form the major proportion of essential oil. They are mainly responsible for antibacterial activity as these compounds can alter the permeability of bacterial membrane by degradation of proteins and phospholipids47.

Moreover, it was observed that pure EOs (Supplementary figure S1) and CHMs loaded with EOs showed better antibacterial effect against S aureus (Table 3; Fig. 6). Generally, EOs have a higher inhibitory effect against gram-positive bacteria i.e. S aureus than gram-negative bacteria i.e. E coli, possibly due to differences in their cell wall structure. Gram-negative bacteria have an additional hydrophilic lipo-polysaccharide layer, which restricts the permeation of hydrophobic oil constituents. Similar trend of antimicrobial activity of hydrogel film loaded with EOs was also reported by Huma et al.17.

Table 3 Zone of Inhibition CHMs membrane (n = 3).
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Fig. 6
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Antibacterial activity of CHMs encapsulated with various concentrations of essential oils against E coli and S aureus.

Antioxidant assay

The antioxidant activity was measured on the basis of radical scavenging abilities by DPPH in GG based composite hydrogel membranes encapsulating LEO and GEO. DPPH is a stable free radical, which is reduced by e− transfer or H+ atom donation from the EOs encapsulated in polymeric chains of optimized CHMs38. The results are presented graphically in Fig. 7, where the antioxidant ability of EOs encapsulated composite films is compared with bare essential oils.

The essential oils of geranium and lemongrass have shown potential antioxidant capacities due to the presence of flavonoids and phenolic components48,49. Figure 7 revealed a minor difference in antioxidant capacities of bare EOs and encapsulated EOs, that means EOs sustain their scavenging ability even after encapsulation within a polymeric network of GG based composite membranes50. The differences in scavenging activities of composite films could be attributed to the release of active components, which is dependent on crosslinked structure of composite membranes38. GEO was found to have low antioxidant ability as compared to LEO, which is mainly because of the presence of citral in LEO, attributed to its tendency to donate H+ and thus, quenched the free radicals of DPPH51,52.

Fig. 7
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Antioxidant capacities of pure GO, LGO, GG4, GL1, GCG4, and GCL1 (n = 3). * indicates significant difference (p ˂ 0.05) compared to LGO. # indicates significant difference (p ˂ 0.05) compared to GO. @ indicates significant difference (p ˂ 0.05) compared to GG4.

In-vivo wound healing assay

The in-vivo wound healing was tested on albino rats (full thickened excisional wound model) using G2, GC7, GG4, GL1, GCG4, and GCL1. Untreated group which is wound sweep with normal saline was also included for the purpose of comparison. Percent wound contraction of optimized formulations was compared with untreated and blank films (Fig. 8a). LEO and GEO encapsulated GG membranes had a significant wound closure effect as compared to control and blank membranes, when observed on the 4th day. Similar trend for wound size reduction was observed at 6th and 8th day where up to 70% contraction was recorded for optimum formulations. All wounds progressively close over the duration of study, reaching complete wound closure by day 12 of the treatment (Fig. 8b).

Hence, the results from in-vivo study have demonstrated that the optimum formulations (GEO and LEO loaded GG based composite membranes) enhanced cell migration and promoted early wound closure. These effects could be attributed to the presence of collagen and GG based hydrogel membranes capable of promoting local cell-cell interaction could potentiate the release of paracrine wound healing factors and maintenance of such microenvironment that promote wound healing9. Moreover, antimicrobial, antioxidant, and anti-inflammatory properties of essential oils which are due to citral of LEO and citronellol, geraniol of GEO have accelerated the healing process. During the whole study, no reaction of skin irritation was observed, which supported the use of GG as a safe dressing material for cutaneous wounds.

Fig. 8
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(a) Macroscopic images of wound treated with different formulations. (b) Graphical illustration of wound closure (n = 3), * indicates significant difference (p ˂ 0.05) compared to untreated. α indicates significant difference (p ˂ 0.05) compared to G2. β indicates significant difference (p ˂ 0.05) compared to GC7. # indicates significant difference (p ˂ 0.05) compared to GL1.

Histopathological study

H & E being one of the most common staining methods is regarded as essential to illustrate pathological changes of treated and untreated wounds (12 days post wounding). H & E results (Fig. 9) clearly showed that groups treated with optimized formulations had better granulation tissue formation and tissue repair as compared to the control group and the groups treated with blank films. The control and blank groups showed the abundance of inflammatory cells such as neutrophils and macrophages near the wound edges along with dilated blood vessels.

Regeneration of epithelium occurs during the period of day 3 to day 10 of the healing process. All treated groups exhibited a well formed epidermis with irregular thickness, accompanied by an integral layer of keratin on day 1253. The groups treated with GG4, GL1, GCG4, and GCL1 revealed almost complete wound healing with well-developed epidermis, dermal microstructures, collagen deposition and neovascularization54. Herein, a normal skin like structure and relatively low intensity of macrophages was seen in groups treated with optimized formulations, indicating the anti-inflammatory activity of GEO and LEO55,56. Essential oils containing aldehyde monoterpenes (citral) and alcoholic terpenoids (citronellol and geraniol) contribute antioxidant, anti-inflammatory, and antibacterial properties that help in rapid wound contraction and healing. Delaying of wound healing in control and blank groups may be attributed to the persistent inflammation and lack of antibacterial agents22.

Fig. 9
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H&E stained histological images of wound healing on day 12 of post wounding in different groups (a) healthy, (b) untreated/control, (c) G2, (d) GC7, (e) GG4, (f) GL1, (g) GCG4, (h) GCL1. Here green: epidermis, red: inflammatory cells, black: macrophages, maroon: blood vessels, yellow: hair follicles, and orange bracket: connective tissues. While in MT stained histological images green arrows represent epidermis, yellow: hair follicles, black: collagen, and red: blood vessels.

As we know that collagen formation is essential for wound healing, the skin tissues were stained by Masson’s trichrome to reveal the collagen reforming and maturation at wound site (Fig. 9). MT staining differentiates different morphological parameters of wound healing i.e. hemoglobin, keratin, and muscles (red color), cytoplasm and adipose tissue (pink), and collagen fibers stained blue53.

The control group showed a very poor level of loosely packed thin collagen fibers, whereas the groups treated with optimized formulations had abundant, mature and compact collagen deposition along with hair follicles and sebaceous glands9. Optimized formulations treated groups exhibited relatively well organized collagen fiber alignment in contrast to the control and blank groups, more similar to the basket weave arrangement of collagen in normal healthy rat skin that attributes structural integrity and flexibility57. The groups treated with blank hydrogel films also exhibited thin collagen fibers with irregular alignment.

It is well known that the wounds rapidly re-epithelialize in moist environments, thus our tested GG based composite hydrogel membranes with remarkable water absorption capacity and optimal water permeability had effectively promoted wound healing by maintaining a moist bed at wound sites. Moreover, these composite membranes fit the wound very well with an easy removal58. The encapsulation of essential oils in GG based composite hydrogel membranes results in rapid epithelialization, collagen deposition, and neovascularization59,60,61.

Histopathological and morphometric analysis

Morphometric analysis of wound healing model revealed a significant difference among treatment groups when compared to healthy and untreated group (Table 4). Untreated group wound site showed impaired healing as evident by decreased epithelial thickness, impaired angiogenesis, infiltration of inflammatory cells, reduced count of fibroblast as manifested by high pathological score (4.2 ± 0.5) showing tissue damage and impaired recovery. However, compared to other treatment groups, which showed improved indices of wound repair, as manifested by restoration of epithelial thickness, decreased count of inflammatory cell showing re-epithelialization and proliferation of keratinocytes after treatment with GCLI and GCG4. Neovascularization with wound closure observed in treatment groups especially GCLI (11.0 ± 0.1) and GCG4 (10.21 ± 0.3). GCLI and GCG4 treatment groups showed higher vascular densities compared to healthy group as an evident of angiogenesis support for oxygen and nutrients to augment regeneration of tissue and repair. Reduced count of inflammatory cells at wounded site after treatment with GCLI and GCG4 showed tissue remodeling with suppression of inflammation. For deposition of extracellular matrix and formation of granulation tissues fibroblast proliferation is essential which is poor in untreated/control group. However, all treatment groups especially GCLI showed significant recovery in fibroblast count, which showed that treatment increased collagen synthesis with activated fibroblast recruitment improved tissue repair and healing.

Table 4 Quantitative measurement of histopathological score of wound model by morphometric analysis.
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Detection of pro-inflammatory cytokines

Pro-inflammatory cytokines TNF-α and IL-6 are pivotally attributed for the inflammation process. Therefore, the levels of TNF-α and IL-6 in the serum of rats (full-thickness excisional wound models); after 12 days of treatment with G2, GC7, GG4, GL1, GCG4, and GCL1 were detected by ELISA. The Fig. 10 showed that the TNF-α and IL-6 levels in the serum of treated groups were significantly reduced as compared to the diseased group. Whereas, the groups treated with optimized formulations produced a highly significant reduction in both TNF-α and IL-6 levels in contrast to the groups treated with blank films. That might be due to the presence of GEO and LEO encapsulated within GG based composite hydrogel membranes.

Fig. 10
Fig. 10The alternative text for this image may have been generated using AI.
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Expression of (a) TNF-α and (b) IL-6 levels (n = 3) in full thickened wounds. # indicates significant difference (p ˂ 0.05) compared to healthy, * indicates significant difference (p ˂ 0.05) compared to diseased group.

The exact mechanism of the anti-inflammatory action of EOs is unclear. However, it has been suggested that some plant constituents, particularly alcohol terpenoids (geraniol and citronellol) and aldehyde monoterpenes (citral) contribute to the partial inhibition of the release of inflammation mediators, thus helpful in the management of inflammatory responses62,63. Citral, the major constituent of LEO, has been reported to suppress the production of pro-inflammatory cytokines IL-6 and TNF-α55,64,65. Literature has reported that the geraniol/citronellol of GEO suppress the adherence response of neutrophils in-vitro and lowered their recruitment to the infected site56. Our findings are in agreement with those reported in the literature for EOs rich in monoterpene alcohols and aldehydes, showing a strong anti-inflammatory effect66.

Conclusion

The current research work designates the effective development of bio inspired GG based CHMs cross-linked with CaCl2 by solution casting and evaporation method. The investigation stayed focused on the fabrication and characterization of antimicrobial polymeric membranes as wound dressings. GG and collagen conglomerated the polymeric matrix and EOs of geranium and lemongrass as bioactive agents. The optimized formulations were found to have excellent physical integrity and absorbance capacity. The swelling results evidenced the ability of prepared CHMs to provide a moist environment by meaningfully reducing the transmission of moisture from the wound site. The optimized CHMs have shown significant inhibitory effects against S. aureus and E. coli along with excellent antioxidant properties. SEM, FT-IR and XRD have revealed successful oil encapsulation and physicochemical stability of optimized CHMs.

Results of in-vivo study in full thickness excisional wound model confirmed the healing potential of optimized GG based CHMs with rapid re-epithelialization and collagen formation. Moreover, significantly reduced levels of TNF-α and IL-6 in serum of rats treated with optimized formulations i.e. GG4, GL1, GCG4, and GCL1 further evidenced the anti-inflammatory healing ability. Hence, it is concluded that the fabricated CHMs have potential to serve as a novel wound dressing for treatment of full thickened wounds and other skin infections.

In future, the toxicity evaluation of GG based composite hydrogel membranes can be carried out either in-vitro or in-vivo to validate their wound healing potential further.