Introduction

Wound healing is a complex process comprising various pathways and the involvement of different cell types (i.e. keratinocytes, immune, myoblast, endothelial, fibroblast, etc.) that promote the wound healing process. The process of wound healing can be classified based on the type of wound, i.e. (acute or chronic wound). Acute wounds heal within a limited time of 1–12 weeks, whereas chronic wounds exceed the predicted period [1]. Several limitations antagonize the wound healing process, such as alcohol, bacterial infection, old age, using inappropriate wound dressings and health conditions (i.e. haemophilia, diabetes, obesity, cancer, etc.). These factors not only delay the wound healing process but also result in a wound becoming chronic [2]. Therefore, it is of great importance to take good care of wounds using appropriate wound dressings irrespective of their size to avoid further complications. Over the years, different wound dressings have been developed to accelerate the wound healing process. An ideal wound dressing should be non-toxic, protect against bacterial invasion and the external environment, be biocompatible, retain a moist environment, be biodegradable, stimulate cell proliferation and migration and induce rapid wound healing [3,4,5]. Biopolymers exhibit interesting properties that make them suitable for developing wound dressings with the aforementioned features of an ideal wound dressing, and among them is sodium alginate (SA), a brown seaweed-derived polysaccharide [6].

Alginate is composed of α- L-guluronic acid (G- blocks) and β-D-mannuronic acid (M- blocks) linked by 1 → 4 glycosidic bonds [7, 8]. Alginate-based dressings have attracted great attention due to their unique features, including good gelling properties, controlled release properties, high haemostatic effects, hydrophilicity and stabilization of dispersions and the capability to induce regeneration of tissue [1, 3]. Moreover, they protect wounds from bacterial invasion and promote an accelerated wound healing process [2]. Additionally, they induce a pro-inflammatory signal and activate macrophages useful in resolving inflammation [9]. Batista et al. reported hybrid alginate/chitosan aerogels with antibacterial activity against S. aureus and K. pneumoniae that induced a 75% wound scratch reduction in 48 h post-injury [10]. Raguvaran et al. formulated sodium alginate/gum acacia hydrogels encapsulated with ZnO to inhibit the bacterial growth of P. aerigunosa and B. cereus [4]. Alginate biomaterials are good carriers of antibiotics, metal nanoparticles, bioactive agents, etc. [11].

The use of metal-based nanoparticles due to their outstanding features such as optical, catalytic, magnetic, bio-separation of DNA and proteins and antibacterial properties has enabled their use in various biomedical applications. Iron oxide nanoparticles (FenOn) occur in different forms as hematite (α-Fe2O3), maghemite (g-Fe2O3) and magnetite (Fe3O4) [12]. The most commonly used iron oxides in wound management are maghemite (g-Fe2O3) and magnetite (Fe3O4); however, there is not enough information on their wound healing mechanism. Shabanova et al. reported thrombin@ Fe3O4NPs for the management of internal bleeding; these NPs displayed non-toxic effects against HeLa and HELF cells, and they rapidly promoted local haemostasis effect [13]. Bioactive agents such as tranexamic acid, a synthetic compound derived from amino acid lysine, are responsible for the inhibition of fibrinolysis by hindering plasminogen activation, and thus, reducing plasmin formation and preventing fibrin network destruction that leads to stable blood clot formation [14,15,16,17]. Bhattacharya et al. reported tranexamic acid-loaded gellan gum-based polymeric beads with a prolonged cumulative release of over 90% of tranexamic acid at pH 7.4 [18].

The loading of essential oils (EO) in biopolymer-based dressing has also drawn a lot of attention in wound management. Mahmood et al. reported gellan gum-based hydrogel films loaded with lavender or tea tree EO with ofloxacin with significant antibacterial activity against E. coli and S. aureus. The films also promoted a 98% wound reduction within 10 days [19]. lavandula aspic L. (lavender EO)-based ointment prepared by Djemaa et al. revealed a 98% wound reduction after 14 days post-injury [20]. Khezri et al. prepared polymer-based nanostructured lipids carriers (NLC) loaded with rosemary oil. The lipids promoted complete wound healing within 12 days [21]. The prepared NLCs induced collagen production, re-epithelialization, fibroblast infiltration, increased vascularization and displayed antibacterial activity against S. epidermidis, E. coli, L. monocytogenes, P. aeruginosa and S. aureus. Liakos et al. reported SA-based films loaded with a variety of EO (lavender, eucalyptus, elicriso italic, lemongrass, cinnamon, peppermint, tea tree, chamomile blue and lemon). The films exhibited promising wound healing and antimicrobial effects against C. albicans and E. coli [22].

It is important to select an ideal wound dressing for the management of bleeding and infected wounds. Based on the good reports of the wound healing effects of essential oils, we developed SA/EO-based topical gels composed of sodium alginate, carbopol and different essential oils (i.e. rosemary, lavender and eucalyptus oil). These topical formulations were also loaded with iron oxide (Fe3O4) nanoparticles and tranexamic acid. The formulations were characterized using FTIR, pH, viscosity and spreadability, followed by in vitro cytotoxicity, blood clotting assay, antibacterial evaluation and wound healing studies.

Materials

Solvents and reagents

Distilled water was used to prepare the topical gels. The materials (sodium alginate, carbopol 940, methylparaben, propylene glycol, triethylamine, potassium hydroxide, ethylene glycol and tranexamic acid (TA)) were of ≥ 95% purity and purchased from Merck Chemicals, South Africa. The essential oils (EO), eucalyptus oil (Euc), lavender oil (Lav) and rosemary (Rose) with a 98–99.9% purity were purchased from Clicks Pharmacy, South Africa. The materials were all used without further purification.

Experimental

Fe3O4 nanoparticles preparation

Iron oxide nanoparticles were prepared based on the method reported by Fatima et al. [23]. FeSO4·7H2O (0.2780 g) was dissolved in 5 mL ethylene glycol to form a homogenous solution. KOH (0.5 M) was dissolved in 8 mL, and then, this solution was added dropwise to the solution of FeSO4·7H2O (0.2780 g) in ethylene glycol with constant stirring. The resultant solution was then stirred at 200 °C for 24 h, and black solids was formed. The black solids were separated using a magnet separation method, washed with ethanol/water and dried at 40 °C for 6 h.

Preparation of topical gels

The topical gel formulations were prepared according to Table 1. A ratio of 1:1 sodium alginate to carbopol was used to prepare the topical gels in 10 mL distilled water, with continuous stirring at 200–600 rpm until a clear gel was formed. The essential oil was added with continuous stirring at room temperature, resulting in the formation of a gel emulsion. Carbopol was used as a gelling agent and a surfactant/emulsifier to disperse the essential oil. Methylparaben was also added and used as a preservative. TA and iron oxide were added to the prepared gel emulsions with continued stirring for 20 min. Triethylamine was used to adjust the pH of the prepared gels, and propylene glycol was used as a penetration enhancer. The prepared gel formulations were stored in the refrigerator for further analysis.

Table 1 The composition of the topical gel formulations
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Characterization

Attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR)

Attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) was performed on the gel formulations using Spectrum Two PerkinElmer. Their spectra were recorded within the range of 4000–500 cm−1 using Origin software.

Spreadability

Spreadability is used to determine the rate at which the gels spread on the wound surface. 0.1 g of the gel with an initial diameter in the range of 1–1.3 cm was placed on a glass slide, and a second glass slide was carefully placed on top of the slide containing the gel. A 100 g mass was placed on top of the two glass slides for 10 min, and the spreadability was determined in cm [24,25,26,27,28].

Viscosity

Brookfield viscometer (DV-1) was used to determine the viscosity (cP) of the prepared gel formulations. Spindle 63 (LV3) was rotated at a speed of 50 and 100 rpm. The reading was recorded at 1- and 2-min time intervals and at a temperature of 37 °C.

pH evaluation

The pH of the gel formulations was evaluated using a digital pH metre.

Scanning electron microscope (SEM)

The SEM was used to evaluate the surface morphology and elemental composition of the prepared nanoparticles. It was performed on a JEOL (JSM- 6390 LV) scanning electron microscope, Japan, at an accelerating voltage of 15 kV [29].

X-ray diffraction (XRD)

XRD thermographs were reported on a Bruker D8 Discover equipped with a proportional counter using Cu-Kα radiation. Data were collected within the range of 2ϴ = 5–60°, scanning at 1.5° min−1 with a time constant filter of 0.38 s per step and 6.0 mm slit width. Samples were mounted on a slide with a silicon wafer. The X-ray diffraction data were handled using the Profex program (evaluation curve fitting). Baseline correction was achieved by the elimination of a spline feature adapted to the curved base on each diffraction pattern. It was used to study the crystalline nature of magnetite nanoparticles.

In Vitro antibacterial analysis

The antibacterial evaluation was performed to determine the antibacterial activity of the wound dressings against selected strains of bacteria. Three controls were used, ampicillin (AMP), streptomycin (STM) and nalidixic acid (NLD) (Table 2). The minimum inhibitory concentration (MIC) of the formulations was evaluated according to the procedure by Fonkui et al. [30]. The stock solutions were prepared by dissolving 4 mg of each gel formulation in 5 mL of a mixture of DMSO and dH2O (4:1, v/v). These solutions were then serially diluted (6 times) in 100 µL of nutrient broth in 96 well plates to the desired concentrations (400, 200, 100, 50, 25 and 12.5 µg/mL). 100 µL of each of these solutions was then placed in duplicate and seeded with 100 µL of an overnight bacterial culture. Streptomycin, ampicillin and nalidixic acid were used as positive controls. The negative control contained 50% nutrient broth in DMSO.

Table 2 Selected bacterial strains used and abbreviations
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In vitro whole blood clot assay

The in vitro haemostasis analysis was performed according to the procedure described by Catanzano et al. on the gel formulations, SA/Rose, SA/Lav, SA/Lav/Fe3O4, SAT/Euc/Fe3O4, SAT/Rose/Fe3O4 and SAT/Lav/Fe3O4 at a wavelength of 540 nm [31]. A two-tailed t-test using GraphPad was used to evaluate the significant difference between the formulations and the control. The wound dressing, 2 mg was immersed in 200 µL of the whole blood and placed in 15-mL tubes. Blood coagulation on the formulations was activated by adding 20 µL of CaCl2. The wound dressings were then further incubated in a thermostatic incubator for 10 min at 37 °C with gentle shaking. Deionized water (6 mL) was used to haemolyse the red blood cells (RBCs) trapped in the clot. A spectrophotometer was used to measure the relative absorbance (A) of the blood samples diluted to 25 mL at a wavelength of 540 nm [31].

Cytotoxicity evaluation

The in vitro cytotoxicity of the wound dressings was performed to evaluate their biocompatibility using a (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)(MTT) assay. The gels SA-Blank, SA/Lav/Fe3O4, SAT/Euc/Fe3O4, SAT/Rose/Fe3O4 and SAT/Lav/Fe3O4 were screened against HaCaT cells (immortalized human keratinocytes) which were seeded at a density of 5 × 104 cells/ml in 96-well plates at a volume of 90 µL/well. The experiments were performed in triplicate, and the cytotoxicity results of these gels were analysed by calculating the percentage cell viability using Eq. 1 [32]:

$${\text{\% Cell viability}} = \frac{{\left( {{\text{ODs}} - {\text{ODc}}} \right)}}{{\left( {{\text{ODu}} - {\text{ODc}}} \right)}} \times 100$$
(1)

where ODS is the absorbance of the test compound, ODc is the absorbance of the control, and ODu is the absorbance of the untreated compound.

Wound healing assay

In vitro wound healing assay was performed based on a procedure adapted from Felice et al., Suarez-Arnedo et al., Cheng et al. and Ranzato et al. [33,34,35,36]. It was only performed on SAT/Lav/Fe3O4 formulation to evaluate its wound healing ability within 3 days. The cell migration was quantified using ImageJ image processing software. The wound closure was calculated using the following Eq. 2 [37]:

$${\text{\% Wound closure}} = \frac{{{\text{wound area day }}0 - {\text{wound closure day }}72}}{{{\text{wound closure day }}0}}{ } \times 100$$
(2)

Statistical analysis

Cytotoxicity and wound healing experiments were performed in triplicates. An unpaired Student’s t-test was performed using GraphPad to study the statistical significance of the treated versus untreated samples, and p ≤ 0.05 was considered statistically significant.

Results and discussion

FTIR

The ATR-FTIR spectra of the prepared gels are depicted in Fig. 1a–c. The pure lavender oil exhibited an absorption peak at 3468 cm−1, resulting from O–H stretch due to the presence of terpene-4-ol and linalool [38]. The intense peak at 2946 cm−1 is attributed to the aliphatic C–H stretch. The strong peak at 1740 cm−1 is attributed to the C=O carbonyl group, indicating the presence of the major components of lavender oil, such as lavandula acetate, linalool, camphor and linalyl acetate [39, 40]. The C–O stretch bend visible at 1253 cm−1 further confirms the presence of an ester component of the lavender oil [38, 39]. The peaks at 917, 1018 and 1253 cm−1 correspond to the C–H deformations [39, 40]. SA/Lav/Fe3O4 and SAT/Lav/Fe3O4 displayed broad characteristic peaks at 3460 cm−1 corresponding to the O–H stretching [24]. The vibration bend at 2923 cm−1 is attributed to C–H stretch present in both polymer and lavender oil components. The twin peaks at 1641 and 1732 cm−1 are due to the C=O stretching carbonyl group [38]. There was no major difference in the absorption peaks found in the formulations, SA/Lav, SA/Lav/Fe3O4 and SAT/Lav/Fe3O4. The characteristic peaks of lavender oil were retained in all the formulations, SA/Lav, SA/Lav/Fe3O4 and SAT/Lav/Fe3O4. The FTIR spectra of eucalyptus oil showed vibration bend at 2948 cm−1 attributed to asymmetrical and symmetrical bending of C–H stretching [41]. The vibration bends at 968, 1079–1203 and 1354 cm−1, corresponding to the symmetrical bending of the CH2 plane, C–O–C stretching and C–O–H deformation, indicating the presence of 1,8-cineol and citronella, active components of eucalyptus oil.

Fig. 1
figure 1

FTIR spectra a SA/Euc, b SA/Rose and c SA/Lav-based formulations

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In the FTIR spectra of the formulations containing lavender oil, SA/Euc, SA/Euc/Fe3O4 and SAT/Euc/Fe3O4, there was no major difference. The presence of eucalyptus oil components was retained in all the formulations with major characteristic peaks visible at 968, 1079–1203, 1354 and 2948 cm−1 relating to CH2 plane, asymmetric C–O–C stretching, C–O–H deformation and C–H symmetric bending, respectively. Additionally, a broad vibrational bend was observed at 3457 cm−1 due to O–H bending in alginate [42]. The FTIR spectrum of rosemary oil revealed vibration bending for C–H methylene group and O–H at 2932 cm−1 and 3486 cm−1, respectively [43, 44]. The characteristic peak at 1723 cm−1 is attributed to the C=O carbonyl group [44]. The vibration bends at 833, 984, 1085, 1236 and 1330 cm−1 indicate the monoterpenes, such as 1,8-cineole present in rosemary oil [45, 46]. The FTIR spectra of SA/Rose/, SA/Rose/Fe3O4 and SAT/Rose/Fe3O4 revealed similar absorption bends and a broad O–H peak at 3443 cm−1. These formulations showed the presence of rosemary oil which was stable in the polymeric matrix and did not interact with the polymer matrix. The major vibration bends for monoterpenes were retained [47]. The FTIR spectra of the gel formulations revealed the successful loading of the essential oils.

Spreadability

The successful preparation of the gel formulations was confirmed by FTIR analysis. The gels were then further evaluated for their spreadability, pH, stability and viscosity. The pH of the gels was in the range of 6.8–7.2 which is compatible with skin application. The prepared topical gels exhibited spreadability within the range of 5.4–10.1 cm (Table 3). SA/Rose exhibited lower spreadability values compared to the formulations loaded with either iron oxide alone or a combination of iron oxide and tranexamic acid. The SA/Lav formulation revealed a decreased spreadability behaviour. SA/Euc topical gels showed no significant spreadability trend. The contact time of the topical that aids the healing efficiency of topical gels is influenced by their spreadability [26]. The viscosity of the prepared formulations was recorded at 50 rpm (revolution per minute) in 30 and 120 s. The viscosity of all topical gel formulations was significantly lower compared to the pure gel (SA-Blank). SA-Blank, SA/Rose/Fe3O4 and SAT/Rose/Fe3O4 revealed a shear-thinning behaviour as time increased from 30 to 120 s when compared to other formulations. After 12 months of storage in a refrigerator, all the prepared formulation maintained their physical and chemical state, showing their stability under long-term storage.

Table 3 Spreadability, pH and stability results of the prepared topical gels
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SEM, UV–vis and XRD

The SEM images of the prepared Fe3O4 nanoparticles (Fig. 2a, b) revealed aggregated and a cluster of spherical-shaped morphology. Pirsa et al. reported similar morphology, and the particle aggregation is influenced by an increase in the particle’s surface energy and decreased particle size [48]. Similar findings were also reported by Kulkarni et al., Hariani et al. and Awwad et al. [49,50,51]. The UV–vis of Fe3O4 nanoparticles revealed absorbance at 299 nm, confirming the successful preparation of the nanoparticles (Supp Fig. 1). The XRD graph displayed prominent crystalline characteristic 2Ɵ peaks at 21.7°, 30.01°, 31.01°, 36°, 43.6° and 52.4° that were assigned to the crystal planes of (111), (220), (311), (222), (400) and (422) inverse cubic spinel magnetite, respectively, for Fe3O4 nanoparticles (Supp Fig. 2). A similar finding was reported by Qureshi et al. for hematite and magnetite nanoparticles [52]. The peaks confirmed the crystalline nature of the prepared Fe3O4 nanoparticles [48]. The SEM images, UV spectra and XRD characteristic peaks confirmed the successful synthesis of Fe3O4 nanoparticles.

Fig. 2
figure 2

SEM images of iron oxide nanoparticles at different magnifications

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In vitro antibacterial assay

The antibacterial activity (MIC values) of the topical gel formulations is shown in Table 4. The antibacterial activity of the formulations was compared to the controls, AMP, STM and NLD. SA-Blank revealed antibacterial activity mostly against gram-negative strains of bacteria (P. vulgaris, P. aeruginosa, K. pneumonia and S. aureus) which is attributed to alginate. Euc-EO exhibited antibacterial activity against B. subtilis, E. faecalis, S. aureus, E. cloacae, K. oxytoca, P. aeruginosa and P. mirabilis. SAT/Euc/ Fe3O4 showed microbial inhibition against P. mirabilis, a species under the same genus as P. vulgaris. The microbial inhibition effect displayed by these gel formulations against S. aureus, E. coli and P. aeruginosa; the commonly resistant strains of bacteria that cause sepsis and other diseases are also found in infected and chronic wounds [53,54,55]; P. vulgaris is reported to be responsible for biofilm and antibiotic resistance [56]. These formulations were effective against gram-negative strains which are considered the most resistant and harmful strains of bacteria [57].

Table 4 Antimicrobial activity of the prepared SA gels against different strains of bacteria
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Lav-EO exhibited a microbial inhibition effect similar to Euc-EO. SA/Lav against S. aureus, P. vulgaris, P. aeruginosa, E. coli and K. pneumonia. The loading of iron oxide nanoparticles in this gel formulation led to a broad spectrum of microbial activity against (B. subtilis, E. faecalis, S. epidermidis, S. aureus, E. cloacae, P. vulgaris, K. oxytoca, P. aeruginosa and P. mirabilis) visible in SA/Lav/Fe3O4. Similar results were reported by Mahmood et al. for hydrogel films containing Lav-EO which were more effective against S. aureus than E. coli [19]. SAT/Lav/Fe3O4 exhibited antimicrobial activity similar to SA/Lav/Fe3O4 except that it did not inhibit B. subtilis. As a spore-forming bacteria, B. subtilis is known to possess a much thicker proteinous cell wall which hinders the permeability of nanoparticles [58].

The antibacterial activity of Rose-EO was significant against B. subtilis, E. faecalis, S. epidermidis, S. aureus, E. cloacae, K. oxytoca, P. aeruginosa and P. mirabilis strains of bacteria. Gel formulations, SA/Rose and SA/Rose/Fe3O4 inhibited the same bacteria strains and displayed similar MIC values. The identical antibacterial activity displayed by SA/Rose and SA/Rose/Fe3O4 suggests that the encapsulation of iron oxide nanoparticles played no significant role in their antibacterial activity. SAT/Rose/Fe3O4 displayed significant antibacterial activity against S. aureus, P. vulgaris, P. aeruginosa and K. pneumonia, suggesting that the loading of two or more bioactive agents into the gels composed of SA/Rose decreased their antibacterial activity. Khezri et al. reported polymer-based nanostructured lipid carriers loaded with rosemary oil to inhibit the growth of P. aeruginosa and S. aureus [21]. The antimicrobial activity of the formulations reveals that they are promising therapeutics for treating infected wounds.

In vitro whole blood clot assay

Apart from wound dressings exhibiting promising antibacterial activity, haemostatic activity is a crucial feature needed in wound dressings used in treating bleeding wounds. Haemostatic material should be able to form clots and thus stop bleeding within a limited amount of time to hinder excessive bleeding. Absorbance values of all the prepared topical gels were lower compared to the control with (p-values = 0.0051, 0.0255, 0.0177, 0.0188, 0.0196 and 0.0371 for SA/Rose, SA/Lav, SA/Lav/Fe3O4, SAT/Euc/Fe3O4, SAT/Rose/Fe3O4 and SAT/Lav/Fe3O4, respectively. These results suggest that the prepared gels can form prevent excessive bleeding, as stated by Huang et al. that lower absorbance values are equivalent to larger blood clot size [59]. SA/Rose exhibited the lowest absorbance values compared to all other formulations with (p-value > 0.05) except for SA/Lav (p-value = 0.0156). Among the formulations, there was no significant difference in terms of the p-value. Iron oxide nanoparticles and tranexamic acid played no significant role when loaded on the prepared SA/EO gels, and similar blood clotting behaviour was observed across all the gels used in this study. However, rose oil formulations revealed promising blood clotting potential compared to lavender oil formulations (p-value < 0.05). SA/EO gels displayed good potential in faster clot formation and this compliments their antibacterial efficiency results, suggesting that they are potential wound dressings for treating bleeding and infected wounds. The biocompatible nature of wound dressing is also important when designing a wound dressing (Figs. 3 and 4).

Fig. 3
figure 3

Whole blood clot analysis (Error bars ± std * “p-value < 0.05”)

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Fig. 4
figure 4

In vitro % cell viability of the control and selected SA/EO-based topical gels (Error bars ± std)

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In vitro cytotoxicity

A wound dressing must have low toxicity because it will be in direct contact with the affected area, to avoid further complications. The in vitro cytotoxicity assay was investigated using HaCaT cells, and the formulation was tested at different concentrations. In relation to the antibacterial activity and blood clotting results, the essential oil-loaded gels revealed promising wound healing activity and showed no signs of toxicity even at a higher concentration of 200 µM. The loading of the essential oils and nanoparticles significantly improved the viability of these scaffolds (p-value < 0.05) at both 100 and 12.5 µM compared to the SA-Blank gel formulation. However, there was no significant difference between the blank and SAT/Rose/Fe3O4 at a concentration of 50 µM (p-value 0.0984). The prepared SA/EO-based gels displayed the highest cell viability at 100 µM and showed no significant difference compared to the control except for SAT/Rose/Fe3O4 (p-value = 0.0037) and SAT/Lav/Fe3O4 (p-value = 0.0024). According to the International Standard ISO10993-12:2009, a compound whose treatment results in a cell viability percentage of less than 70% is considered cytotoxic [60]. The SA/EO-based topical gels reported in this study revealed a non-cytotoxic effect against HaCaT cells, revealing their safe use as topical wound dressings. SAT/Lav/Fe3O4 exhibited a 75% cell viability across all the concentrations tested and was selected for in vitro wound healing.

In vitro wound healing assay

SAT/Lav/Fe3O4 exhibited outstanding antibacterial activity, high cell viability and rapid blood clotting potential, and its wound healing potential was investigated in vitro and compared to the untreated cells from 0 to 72 h (Table 5). Cell proliferation and migration are essential phases of the wound healing process [61]. Cell migration was evaluated using an inverted light microscope, and the micrographs are reported in Fig. 5a, b for treated and untreated groups. The untreated group displayed a slower rate of wound reduction (24%) in 72 h post-injury when compared to the SAT/Lav/Fe3O4 gel formulation that induced a higher rate of HaCaT cell migration and proliferation with 99% wound closure. These findings suggest that SAT/Lav/Fe3O4 promoted high cell migration to the affected area. The rapid wound closure presented by this formulation is influenced by the synergistic effect resulting from the combination of lavender oil, tranexamic acid and Fe3O4 nanoparticles. The spreadability and viscosity of this formulation also influenced its wound healing potential [26]. Elmowafy et al. stated that the viscosity of the topical formulations prolongs their contact time at the site of application and the rate of drug delivery [62]. Djemaa et al. developed a Lav-EO-based ointment that induced a 98% wound contraction in 14 days post-injury, revealing the efficacy of lavender oil in wound healing [20]. Lavender oil enhances wound healing by inhibiting lipid peroxidation [20]. It also accelerates wound closure by a rapid reduction in granulation tissue induced by platelet-derived growth factors, promotes granulation tissue formation in the early phase of wound healing and induces collagen replacement from type III to type I, an essential process for tissue remodelling and accelerated wound healing. It also induced the expression of TGF-β, a key molecule that is essential to induce angiogenesis, the proliferation of fibroblasts and matrix production by fibroblasts [63]. TGF-β is also prominent in the rapid formation of granulation tissue and increased production of collagen by fibroblasts, secretion of MMP-13, a collagenase-3 which is essential for the degradation of type III collagen for a replacement of type I collagen [63, 64]. Lavender oil induces the differentiation of fibroblasts to myofibroblasts in the early phase of wound healing, a process that is stimulated by TGF-β and useful for wound contraction [63, 65].

Table 5 The wound healing effect of SAT/Lav/Fe3O4 compared to that of untreated (mean ± STD, n = 3)
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Fig. 5
figure 5

a Micrographs showing migration of cells for untreated and SAT/Lav/Fe3O4 from 0 to 72 h, b a graph showing the percentage of wound closure in vitro over 72 h post-treatment

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Similarly, Kozics et al. reported the high antibacterial activity of lavender oil. At a higher concentration of 0.0469% (w/v), lavender oil did not induce a toxic effect on HaCaT cells in vitro [66]. Prashar et al. reported the non-toxic effect of lavender oil which was dose-dependent. A concentration of 0.125% (v/v) showed 80–100% cell viability and a concentration of 0.25% (v/v) revealed a toxic effect on human skin cells in vitro [67]. Moreover, Miastkowska et al. revealed that the cytotoxic effect of lavender oil on HaCaT cells in vitro is influenced by its concentration. A concentration of 0.025% (v/v) of lavender oil induced a 100% cell viability and 0.390% (v/v) of lavender oil displayed less than 25% HaCaT cell viability [68]. Kazemi et al. reported nanoemulsions composed of lavender EO and licorice (rhizome and root of Glycyrrhiza glabra) extract that accelerated wound contraction by 98% on the 14th day with an increased collagen type I and III expressions, including TGF-β1 [65].

Tranexamic acid is an anti-fibrinolytic drug that is useful in reducing blood loss. However, it also increases collagen synthesis and tensile strength within the granulation tissue, thereby retaining the fibrin matrix. It accelerates normal wound healing by stabilizing the fibrin structures [69]. Fe3O4 nanoparticles, on the other hand, induce wound healing by penetrating bacterial biofilm. The combination of Fe3O4 nanoparticles with silver nanoparticles revealed a significant penetration of bacterial biofilms [70]. Fe3O4 nanoparticles also enhanced the migration of normal adult human dermal fibroblast cells and wound closure [71]. Based on the aforementioned efficacy of Fe3O4 nanoparticles, lavender oil and tranexamic acid, combining them in topical gels revealed a synergistic effect. However, more studies are needed to fully understand the mechanisms of action of the formulation in wound healing.

Conclusion

The FTIR spectra of the prepared formulations revealed that the loaded tranexamic acid and iron oxide did not interact with the polymer matrix. The good spreadability and shear-thinning nature of the gels suggest that they will not flow off easily from the site of application. The gels exhibited pH values between 6.7 and 7.2 compatible with skin application. The SEM and XRD results confirmed the successful formation of iron oxide nanoparticles. The MIC values exhibited by SA/EO-based gels showed a broad antibacterial spectrum, even against the common resistant strains of bacteria, S. aureus and E. coli. SAT/Lav/Fe3O4 gel displayed the highest antibacterial efficacy across all the tested strains of bacteria when compared to other gel formulations. The blood clotting potential displayed by these formulations also revealed their capability to promote blood clotting rapidly. The gel formulations loaded with rosemary and eucalyptus oil exhibited lower absorbance values compared to the formulations loaded with lavender oil. Although SAT/Lav/Fe3O4 exhibited higher absorbance values when compared to other formulations, it is still an ideal wound dressing for controlling bleeding and wound healing. The prepared formulations were not toxic to HaCaT cells when tested in vitro and displayed cell viability above 75% at 100 and 12.5 µM. Interestingly, SAT/Lav/Fe3O4 displayed high cell migration, revealing its potential to accelerate wound healing. The prepared topical gels are promising dressings for the management of bleeding and microbial-infected wounds. However, further studies entailing biological and in vivo studies of these formulations are still needed.