A Topical Alginate-Based Wound Gel Fixated with Metal-Based Nanoparticles and Tranexamic Acid as a Hemostatic Wound Healing System

Wounds are usually accompanied by complications such as excessive bleeding and bacteria invasion. The design of wound dressings that rapidly stop excessive bleeding and inhibit bacterial invasion, is crucial to promoting accelerated wound healing. To meet the abovementioned requirements in wound dressings, topical gels were prepared from sodium alginate (SA) and carbopol. The wound dressings were loaded with an antifibrinolytic agent, tranexamic acid, and a variety of metal-based nanoparticles. The prepared topical gels displayed significant antibacterial efficacy against Gram-negative bacteria and moderate activity against Gram-positive bacterial strains. These gels exhibited good spreadability and viscosity, signifying suitability for skin application, and ease of application. Moreover, the prepared gels showed good cytocompatibility, promoted cell proliferation, and exhibited > 85% wound closure on day 3 compared to the untreated which showed a 38% wound reduction in vitro. Excellent blood clotting absorbance values were observed for the gels compared to the control, showing that they can promote blood clotting. Features presented by the prepared gels reveal that they are promising wound dressings for treating bleeding and infected wounds.


Introduction
Wounds are prone to the invasion of infectious pathogens if appropriate wound dressings are not used [1,2].Wound healing is the body's innate response to injury [3], and it involves four phases: hemostasis, the first phase after injury where the body induces clotting to stop bleeding [4], then the inflammatory phase followed by proliferation and finally remodeling where the wound fully closes and may form a scar [5,6].Managing bleeding after an injury is critical in achieving a progressive wound-healing process.Excessive bleeding is a leading cause of death in emergency and prehospital cases [7,8].
Excessive bleeding was reported to constitute over 10% of annual mortality translating to more than 5.8 million deaths worldwide in 2016 [9,10].Liang et al. reported hemorrhage to be responsible for over 40% of deaths during injury [11].Besides the threat to human life, uncontrolled bleeding also impedes wound healing and is a platform for complications, such as bacterial infection, acidosis, organ failure, coagulopathy, hypothermia, or anemia [12,13].Controlling excessive bleeding is crucial in pre-hospital treatment and is a life-saving protocol after an injury [11,14,15].Excessive loss of blood after an injury can be avoided if suitable and ideal hemostatic agents/dressings are used immediately [7].
Hemostatic dressings offer life-saving treatment for bleeding wounds in pre-hospital cases.The use of topical hemostatic dressings for hemorrhaging wounds is effective in preventing excessive loss of blood.A variety of hemostatic dressings such as Celox™, Quikclot combat gauze, Traumacel, Floseal, Tisseel, GelFoam®, Kaltostat™, WoundStat™, or Arista™ are commercially available [16][17][18][19][20][21][22].However, these dressings suffer from several limitations, such as high cost, host rejection, prolonged rates of functioning, exposure of the wounds to infection, can cause severe pain, and increase temperatures at the wound site, causing superficial burns, etc. [8,23,24].Therefore, developing effective hemostatic agents with fewer limitations and high hemostatic ability is crucial.
Liu et al. designed alginate pastes loaded with lipidated tissue factors to induce hemostasis within 75 s [25].The hemostatic capability of the dressings was attributed to the concentration of alginate used for the development of the wound dressings.Increasing the alginate concentration, increased the clotting time of the dressing.Huang et al. designed hemostatic microspheres from silk fibroin with alginate [26].A low concentration of alginate in the wound dressings reduced the blood clotting time.Tranexamic acid is a commercial antifibrinolytic material used to reduce blood loss [27], it is commonly used orally (tranexamic acid tablets IP: tranefit 500) or intravenously (tranexamic acid injection) [28].Huang et al. reported that the use of tranexamic acid in neurosurgical settings can cause neurotoxicity and hyperexcitability [29].Zhong et al. prepared CMCS-SA composite films impregnated with tranexamic acid to control bleeding.The films promoted more rapid blood clotting kinetics than the pure SA and CMCS films [30].A high SA content induced shorter blood clotting time with a rapid drug release, showing a synergistic effect of tranexamic acid and SA in controlling bleeding.The pure SA films also exhibited shorter clotting time than the pure CMCS films.Feng et al. reported hemocompatible alginatebased TEMPO-mediated oxidized cellulose nanocrystals with a good capability to promote minimal blood loss of over (0.539 ± 0.069 g) within 70 s in vivo [31].Jin et al. reported formulations of berberine-coated alginate microspheres that exhibited clotting time influenced by the concentration of berberine and a reduced blood loss of 26.3 mg than the microporous polysaccharide hemostatic powder with a blood loss of 54.5 mg [32].Zhao et al. formulated Gel-rGO/tannic acid cryogel with hemolysis ratios below 5% [33].Lv et al. reported cotton-like mat dressings of SA and poly (l-lactic acid) grafted with dopamine and impregnated with Fe 3+ that displayed excellent cytocompatibility, antibacterial activity, hemocompatibility, histocompatibility, and a short blood clotting time of 47 s [34].A recent report by Buyana et al. from our research group showed that the encapsulation of an antifibrinolytic agent, aminocaproic acid in combination with ZnO nanoparticles into SA-based gels synergistically promoted enhanced hemostatic effect in vitro [35].
It is important to select an ideal wound dressing for the management of hemostasis and infections in wounds.In this study, topical hemostatic gels were prepared from a combination of sodium alginate and carbopol.The gels were fixated with tranexamic acid, an antifibrinolytic agent together with a variety of metal-based nanoparticles (AgNPs, MgONPs, ZnONPs, F 3 O 4 NPs) and carbon-based biomaterials (graphene oxide (GO) and reduced graphene oxide (rGO) to explore the efficacy of metal-based nanoparticles and carbon-based biomaterial in the design of wound dressings for bleeding and infected wounds.The prepared topical gels were analyzed for their physicochemical properties by Fourier transform infrared spectroscopy (FTIR), pH profiling, viscosity, spreadability, in vitro cytotoxicity, wound healing assay, hemolysis assay, and antibacterial activity.

Ag Nanoparticles Preparation
The nanoparticles were prepared by heating silver nitrate solution (1 mM) with stirring at 90 °C.Trisodium citrate solution (10 mM) was added dropwise to the solution of silver nitrate with continuous stirring.A color change was observed from a colorless solution to a light yellow solution, confirming the formation of silver nanoparticles.The silver nanoparticles solution was allowed to cool to room temperature and then stored in the fridge at 4 °C for further use [36].

ZnO Nanoparticles Preparation
The zinc oxide nanoparticles were prepared according to the procedure reported by Jyoti et al. [37].A solution of zinc chloride was prepared by dissolving 2.75 g of zinc chloride in 50 mL of distilled water.The solution was stirred at 90 °C, and sodium hydroxide solution (5 M) was added dropwise with continuous stirring.A white milky colloidal solution was formed, and it was stirred for 2 h after the complete dropwise addition of sodium hydroxide.The solution was allowed to cool to room temperature and centrifuged.The precipitates formed were washed with distilled water and dried at 100 °C for 6 h to obtain zinc oxide nanoparticles.

MgO Nanoparticle Preparation
Magnesium oxide nanoparticles were prepared according to a procedure reported by Chandrappa et al. [38].Solutions of magnesium chloride (203.3 mg) in 50-mL ethanol and sodium hydroxide (80 mg) in 50-mL ethanol were prepared.Both solutions were stirred at 60 °C.The solution of sodium hydroxide was added dropwise to the solution of magnesium chloride under vigorous stirring at 60 °C.The resultant solution was centrifuged (2000 rpm, 10 min), and the precipitates obtained were dried at 60 °C followed by calcination at 300 °C for 1 h.

Fe 3 O 4 Nanoparticles Preparation
Iron oxide nanoparticles were prepared based on the method reported by Fatima et al. [39].FeSO 4 •7H 2 O (0.2780 g) was dissolved in 5 mL of ethylene glycol resulting in a homogenous solution.A dropwise addition of KOH (0.5 M, 8 mL) with continuous stirring was performed.The homogenous solution formed was stirred at 200 °C for 24 h resulting in the formation of black solids which were separated using the magnet separation method.The magnetically separated solids were 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 which shows the quantity of reagents used for the preparation of the gel formulations.Sodium alginate:carbopol (1:1) ratio (used to promote drug loading efficiency) was used to prepare the topical gels in 10 mL of distilled water with continuous stirring at 200-600 rpm for 2 h.A clear gel was formed and methylparaben was then added with further stirring.It was used as a preservative for the gels.TA, nanoparticles, or carbon-based biomaterials (GO and rGO) were added to the prepared gels with continued stirring for 20 min as indicated in Table 1.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 at 4 °C.A schematic diagram showing the preparation of the nanoparticles is shown in Fig. 1.

FTIR
It was performed using an FTIR spectrometer (Spectrum Two) PerkinElmer on all the topical gels within the range of 4000-500 cm −1 .

Spreadability
Spreadability is used to determine the rate at which the gels can be spread throughout the wound.Gel (0.1 g or an initial gel diameter of 1.0-1.3cm) was placed on a glass slide and a second glass slide was carefully placed on top of the slide containing the gel.A known mass of 100 g was placed on top of the two glass slides for 1 min, and the spreadability was measured [35].

Viscosity
The viscosity (cP) of the gel formulations was measured using a Brookfield viscometer (DV-1).Spindle 63 (LV3) was rotated at a speed of 50 and 100 rpm, at time intervals of 1 and 2 min, and at a temperature of 37 °C.

pH Evaluation and Stability Studies
The pH of the topical gel formulations was evaluated using pH strips and a digital pH meter.The pH of the gel formulations was adjusted using triethylamine.

Scanning Electron Microscope
The surface morphology and elemental composition of the prepared nanoparticles were evaluated using SEM.It was performed at an accelerating voltage of 15 kV on a JEOL (JSM-6390 LV) scanning electron microscope, Japan.

X-Ray Diffraction
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 = 5-80°, scanning at 1.5° min −1 with a time constant filter of 0.38 s per step and 6.0 mm slit width The X-ray diffraction data were handled using the Profex program (evaluation curve fitting).It was used to study the crystalline nature of synthesized nanoparticles [42].

Scanning Transmission Electron Microscopy
The TEM micrographs were obtained using a field emission scanning electron microscope (Zeiss MERLIN, Oberkochen, Germany).The sample solutions of the nanoparticles were placed on carbon-coated copper TEM grids which were loaded into a STEM sample holder.The beam conditions used were a 20 kV accelerating voltage and a 250 pA probe current.
The hydrodynamic results are provided as mean ± standard deviation (SD).

In Vitro Antibacterial Analysis
The antibacterial evaluation was performed using selected bacterial strains shown in Table 2.The MIC evaluation of the gels was performed according to the protocol reported by Fonkui et al. [41].The gels were dissolved in 5 mL of a mixture of DMSO and dH 2 O (4:1, v/v) to obtain stock solutions followed by 6 times dilution in 100 μL of Muller-Hinton nutrient broth in 96 well plates.Selected concentrations were obtained namely, 12.5, 25, 50, 100, 200, and 400 μg/ mL. 100 μL each of the aforementioned solutions was placed in duplicate followed by seeding with 100 μL of an overnight bacterial culture.The positive controls used were nalidixic acid, ampicillin, and streptomycin while the negative control was composed of 50% nutrient broth in DMSO.

In Vitro Whole Blood Clot Assay
The in vitro whole blood clot assay was performed according to the protocol reported by Catanzano et al. on selected gel formulations at a wavelength of 540 nm [42].Statistical significance was investigated using a Student's t-test by comparing the averages and standard deviations of test samples to the negative control.To 200 μL of the whole blood, 2 mg of the topical gel was immersed.CaCl 2 (20 μL) was added to activate blood coagulation on the topical gel formulations.Incubation was then performed for 10 min using a thermostatic incubator with gentle shaking at a temperature of 37 °C.6 mL of deionized water was added to hemolyze the red blood cells (RBCs) by dripping.The blood samples' relative absorbance was evaluated after dilution to 25 mL [43].

Cytotoxicity Evaluation
It was performed on the wound dressings to evaluate the biocompatibility of the developed topical gels employing the MTT assay.It was performed using HaCaT cells (immortalized human keratinocytes).A 96-well plate at a volume of 90 μL/well and a density of 5 × 10 4 cells/mL was used for cell seeding [44].The negative controls were the cells treated with 10 μL of 1 × PBS while the positive control was cells treated with 10% DMSO.The incubation of the 96-well plates was performed for 48 h followed by the addition of an MTT reagent (10 μL) per well.A 4-h incubation period was performed at 37 °C.The solubilized formazan crystals were prepared using a solubilization reagent.Absorbance values were measured at 570 nm using a multimode microplate reader (Victor X3, Perkin Elmer, MA, USA).The experiments were performed in triplicate and the calculation of the percentage cell viability was performed using Eq. ( 1): where OD S is the absorbance of the test compound and OD c is the absorbance of the control.OD u is the absorbance of the untreated compound.

Wound Healing Assay
The in vitro wound healing assay was evaluated based on a procedure adapted from Cheng et al., Felice et al., and Suarez-Arnedo et al. [45][46][47].The assay was performed on selected formulations to evaluate their wound-healing capability within 4 days.HaCaT cells were cultured in a humidified incubator at 37 °C and 5% CO 2 to 90% confluency in Dulbecco's modified eagle's medium (DMEM) containing 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin-streptomycin antibiotics.The trypan blue dye exclusion method was used to trypsinized the cells, and viable cells were quantified.The cell density employed was 2.5 × 10 5 cells/mL in DMEM and 2 mL of the cell suspension was incubated per well in 6-well plates until cell monolayer formation which occurred 48 h later.A micropipette tip (200 μL) was used to make uniform single scratch wounds per well.The cells were washed once using 2 mL of 1 × PBS to aspirate dislodged floating cells.DMEM containing 1% FBS (serum-starved medium) was added to the wells (1800 μL per well), and cells were treated with 200 μL of samples at various concentrations that showed the highest cell viability on the MTT assay.Untreated cells seeded in DMEM containing 10% FBS were used as positive control while those cultured in 1% FBS in DMEM were employed as the negative control.The images were captured in duplicates at 0, 24, 48, 72, and 96 h using the 4 × objective and phase- contrast feature of an inverted light microscope (Olympus CKX53, Olympus, Tokyo, Japan).Cell migration was quantified using ImageJ image processing software and wound closure was calculated using Eq. ( 2) [48]:

Statistical Analysis
The data obtained from the in vitro studies were evaluated via Student's unpaired t test on GraphPad Prism version 9

FTIR Analysis of the Gels
The FTIR spectra of the prepared topical gels are shown in Fig. 2a-f.The topical gels containing TA and nanoparticles were compared with the ones containing only nanoparticles.Similar characteristic peaks were observed for all the prepared topical gels, which can be attributed to a similar polymer composition used in their preparation.The spectra also showed that the nanoparticles or TA did not interact with the polymer network.The FTIR of all the prepared topical gels revealed absorption bands at 3310 cm  1).The FTIR spectra of the nanoparticles and rGO are shown as supplementary figures.The FTIR spectra of iron oxide nanoparticles revealed a characteristic peak of Fe-O stretch at 661 cm −1 .The peak at 661 cm −1 is attributed to Fe-O stretching of iron oxide and similar peaks have been reported between 620 and 660 cm −1 for maghemite nanoparticles [51,52].The FTIR spectra of magnesium oxide nanoparticles displayed a characteristic peak of Mg-O vibration at 873 cm −1 , revealing the formation of hexagonal nanoparticles [53].Zinc oxide nanoparticles also showed a characteristic peak of Zn-O stretching at 699 cm −1 (Supplementary Fig. 1) [54].

Spreadability
The prepared topical gels were analyzed for their spreadability, pH, homogeneity, stability, and appearance (Table 3).The pH values of the topical gels were in the range of 6.7-7.2.The pH of human skin is reported 4.5-5.5.However, some studies have reported that gel formulations which are compatible with the skin exhibit pH in the range of 5.3-7.6 [55][56][57][58].The good spreadability of the gels signifies that they can be easily administered on the skin.The prepared topical gels exhibited spreadability in the range of 5.2-9.3cm, a feature useful for uniform application and are also an ideal quality in topical formulations.It is also an important feature that reveals the adherence of the topical gel to the skin.The loading of the nanoparticles, TA, or carbon-based biomaterials did not have an obvious effect on the spreadability of the gel formulations.The healing efficiency of topical gels is also influenced by their spreadability which aids in their uniform application [41].The good spreadability range of the gels reveals that they can be easily applied to the wounds without flowing off the wound bed, thereby indicating a good contact time that can provide moisture to the wound environment [59].The prepared formulations were clear and homogenous with no phase separation.After storage of the topical gels for 10 months, all the gels maintained their physical state, showing their good stability.The good spreadability of the gels reveals their capability to improve therapeutic efficacy [60].Topical gels with good spreadability have been reported by some researchers in the range of 5.0-9.0 cm [61,62].However, it is important to indicate that there is still a need for more research to fully understand the appropriate range of spreadability suitable for topical gels employed for wound dressings.

Viscosity Evaluation of the Wound Dressings
The viscosity of topical gels was within the range of 1116-5940 cP at 50 rpm and 828-4590 cP at 100 rpm (Table 4).The viscosity of the blank gel was high when compared to the nanoparticles loaded formulations which revealed the loaded nanoparticles may have attributed to reduced viscosity.The permeation enhancer/gelling agent (carbopol) effect was not significant in the gel formulations.The viscosity revealed consistency for most of the formulations except for X3.More studies are needed to fully understand the high viscosity of formulation X3.An increase in the shear rate from 30 to 120 s, decreased the viscosity of the gels, indicating a non-Newtonian flow (shear thinning).A non-Newtonian flow is acceptable because when applied at high shear conditions there is a low flow resistance [63,64].The reduced viscosity also suggests pseudoplastic behavior which confirms their good spreadability and capability to be retained on the wound site after application without flowing off the site of application [65].The viscosity of the gel formulations also showed good stability of these formulations.3a) [66].SEM of ZnO NPs showed a combination of spherically and cube-shaped morphology with a solid dense structure (Fig. 3b).Similar SEM morphology was reported by Mohammadi et al. for ZnO NPs [67].The SEM image of MgO NPs (Fig. 3c) showed spherically and flake-shaped morphology with large particle clusters.Similar morphology was reported by Sushma et al. [68].GO appeared blockshaped morphology with a smooth surface (Fig. 3d).rGO exhibited coarse and irregular morphology (Fig. 3e).

TEM and Hydrodynamic Analysis of the Nanoparticles
The nanoparticles were further characterized by FTIR, TEM, and UV-vis which confirmed the successful preparation of the nanoparticles.The UV-vis, FTIR, and XRD are presented as supplementary figures.Table 5 shows the hydrodynamic properties of the nanoparticles and the particle sizes of the nanoparticle obtained.The zeta potential of the nanoparticles was low for Fe 3 O 4 and MgO nanoparticles, suggesting low stability.It is important to indicate that factors such as the pH and dispersion media viscosity affect the surface charge of the nanoparticles.Zeta potential provides information on the electrostatic repulsive forces.However, it does not provide information on the attractive van der Waals forces.There have been reports of stable colloids displaying low zeta potential and vice versa [72].
MgO nanoparticles prepared by fungus-mediated biological synthesis exhibited a zeta potential of -6.66 mV and were  [74].The polydispersity index of the nanoparticles was in the range of 0.141-0.877,revealing they are moderately polydispersed [75].The TEM images revealed the morphology of the nanoparticles (Fig. 4).The TEM image of AgNPs revealed spherical-shaped nanoparticles with an average particle size of 19.77 nm.Tak et al. reported a higher uptake of spherically shaped AgNPs through the skin than rod-shaped AgNPs.
The uptake of AgNPs into the skin was via different layers of skin and dependent on the shape of the nanoparticles [76].
The TEM image of MgO nanoparticles revealed a combination of rectangular and rod-shaped morphology.A similar finding was reported by Fouda et al. [77].The TEM image of ZnO revealed a combination of hexagonal and rectangularshaped morphology [78].The TEM image of Fe 3 O 4 showed a combination of spherical and cubic shapes [79], confirming the successful synthesis of the nanoparticles.

In Vitro Antibacterial Assay
Antibacterial activity was reported as shown in Table 6.
The prepared topical gels showed a selective antibacterial efficacy against gram-negative strains of bacteria.X1, X2, X3, X8, X10, X11, and X13 displayed similar antibacterial activity against P. vulgaris and P. aeruginosa, and this antimicrobial effect can be attributed to the presence of SA.However, the antibacterial activity against P. vulgaris was more significant for X2 than X1, suggesting that the loading of TA induced a synergistic antibacterial activity of the gel.This can be supported by the antibacterial activity exhibited by pure TA against the same two bacterial strains, P. vulgaris and P. aeruginosa.This finding further revealed that TA induces antibacterial activity.Moreover, X9 loaded with only iron oxide nanoparticles did not display any obvious antimicrobial activity against all the tested bacterial strains.Dressings loaded with magnetite iron oxide NPs have been reported to display limited or moderate antimicrobial effects [80,81].However, the pure iron oxide NPs presented in  E. coli [84].These findings suggest that MgO NPs have a wide variety of antibacterial activity; however, it is dependent on their particle size, concentration etc. X5 displayed a significant antibacterial effect against all strains of gram-negative bacteria except for E. cloacae, and the high antibacterial activity can be attributed to the presence of ZnO NPs.Several reports have proven ZnO NPs to be effective against gram-positive and gram-negative strains of bacteria [85,86].The antibacterial activity of ZnO NPs is influenced by the electronic state, surface energy, roughness, surface charge, shape, surface area, and particle size [87].Farooq et al. report the mode of action of ZnO-NPs on bacteria is via oxidation of bacterial protein, radical stress, and destruction of DNA synthesis, thus inhibiting bacterial growth [88].X6 was only effective against K. oxytoca.X7

In Vitro Whole Blood Clot Assay
Haemostatic material should be able to form clots and thus stop bleeding within a limited amount of time to hinder excessive bleeding.Huang et al. stated that lower absorbance values yield a larger volume of blood clots [26].Absorbance values of all the prepared topical gels were lower compared to that of the control (Fig. 5) with (p value = 0.0021 "X2", 0.0014 "X3", 0.0043 "X4", 0.0017 "X9", 0.0121 "X10", 0.0054 "X12", and 0.0002 "X14").Xuan et al. reported that the addition of rGO to cryogel enhanced the blooding-clotting capability [94].Zhao et al. reported formulations containing rGO with high hemostatic effect [33].X14 containing rGO exhibited the lowest absorbance values (p value < 0.05).The statistical difference was not significant when compared to X3 (p value = 0.4585).This shows that loading reduced graphene oxide in the sodium alginate-based gels promoted platelet aggregation.X3 containing both iron oxide and TA exhibited slightly lower absorbance values than formulations containing only TA (X2) with a p value of 0.5334 and X9 loaded with iron oxide nanoparticles p value was 0.5140.The statistical difference between these formulations was not significant.Similarly, X4 loaded with TA and MgO-NPs exhibited significantly lower absorbance values than X10 loaded with only MgO-NPs p value (0.5504).This shows that loading a combination of TA and MgO-NPs resulted in a synergistic effect that is not statistically different from that of a single drug scaffold.Buyana et al. reported the blood clotting effect of combining aminocaproic acid with metal-based nanoparticles in topical gel [35].X2 and X9 exhibited a similar blood clotting effect with a p value of 0.9778, suggesting that TA and the iron oxide nanoparticles possibly promoted hemostasis through a similar pathway.Sasmal and Datta in their findings reported that a combined effect of the polymer and TA played a significant role in promoting rapid coagulation time [95].X12 loaded with AgNPs also promoted blood clotting, suggesting that the loading of AgNPs induced the hemostatic potential of the gel.Nanoparticles reported in this study induced hemostasis, and loading then in combination with TA resulted in a synergistic effect.The findings reported in this study suggest that the combination of an antifibrinolytic agent with metal nanoparticles in alginate-based wound dressings plays a significant role in the management of bleeding wounds.Moreover, loading rGO to alginate-based dressings is a good approach to developing scaffolds that inhibit rapid blood loss from bleeding wounds.

In Vitro Cytotoxicity
A hemostatic material will be in direct contact with the open wound, and hence, its safety profile is critical.The in vitro cytotoxicity assay on selected topical gel formulations was evaluated using HaCaT cells tested at different concentrations, 9.8, 4.9, 2.45, 1.225, and 0.6125 μM (Fig. 6).The cytotoxic effect of the prepared gels was concentration dependent with the highest concentration (200 μM), displaying the lowest cell viability for all the gel formulations.X1 (blank) was more cytotoxic against the HaCaT cell regardless of a change in its concentration compared to all other gels with 65% maximum cell growth at 50 μM.X9 was also toxic to the HaCaT cells, displaying 76% viability only at 100 μM.All other gels were cytocompatible and promoted cell growth and migration (Fig. 5).A synergistic effect attributed to the combination of biopolymers and nanoparticles has been reported to promote high cell viability.Fixating TA-loaded metal nanoparticles within the gels significantly improved their cell viability, migration, and cell growth.These results revealed topical gels' capability to enhance angiogenesis and rapid wound closure.In particular, X12 and X4 exhibited higher cell viability compared to other SA-based formulations due to the synergistic effect between alginate and the nanoparticles (AgNPs and MgONPs) and were thus selected for in vitro wound scratch assay.A similar finding was reported by Ontong et al. for AgNPs loaded in carbopol 940 gel with a high % cell viability in the range of 89-100% on human keratinocytes [64].

In Vitro Wound Healing Assay
Cell growth and migration are positive features of tissue regeneration and cellular interaction, useful for the progressive and complete wound healing process.In vitro wound healing of X4 and X12 was compared to the untreated cells for 96 h.The wound healing of scratched cells was evaluated through a microscope using HaCaT cells (Fig. 7a and b) for the treated and untreated groups.Topical gel-treated groups stimulated fibroblast proliferation higher than the untreated group after 96 h post scratching as shown in Fig. 7c.The untreated group displayed a slower rate of wound reduction (38%) in 96 h while X12 promoted a higher rate of HaCaT cell migration and proliferation with a 62% wound closure.X4 exhibited moderate wound closure (48%) after 96 h.This shows that the prepared topical gels promote cell migration.The migration of the HaCaT cells was high.Maximum wound closure/cell migration characteristic was observed after 72 h post-treatment in all treatment groups.However, higher cell migration was observed for the formulation, X4 showing ± 88% wound reduction.X12 displayed ± 81% reduction and untreated cells showed ± 42%.Findings presented by the in vitro wound healing correlate with that of in vitro cell viability, as X12 maintained the highest wound closure recorded on the final day post-treatment compared to X4 and untreated.
The decreased rate of wound reduction on day 4 could be linked to the biodegradation characteristics of the topical gels and could be also linked to the decreased viability of the HaCaT cells.Moreover, in line with viscosity results, X4 revealed a shear thinning behavior while X12 exhibited a robust behavior; hence, X4 displayed a rapid cell degradation compared to X12.However, a statistical difference was observed between untreated cells and X12 on day 4 (p Fig. 5 A graph of the whole blood clot analysis of (X2, X3, X4, X9, X10, X12, and X14) (error bars " ± std) ***p value ≤ 0.0002, **p value ≤ 0.002, *p value ≤ 0.02 value: 0.0065), showing decreased cytotoxicity compared to the untreated groups.This data demonstrates that the prepared SA-based gel formulations can stimulate cell migration crucial in wound healing.However, additional complementary tests in vivo are still needed.Chen

Conclusion
The prepared topical gels displayed good wound-healing features.The FTIR spectra of the gels confirmed the successful loading of the nanoparticles and also revealed that neither TA nor the nanoparticles affected the polymer composition of the gels.The high viscosity displayed by the gels suggests that they can be easily applied to the skin without flowing off during and after application.The decrease in viscosity of the gels with increased time showed that the gels have good shear-thinning behavior.Encapsulation of TA and nanoparticles significantly increased cell viability and antibacterial activity of the prepared topical gels, as pure topical X1 exhibited a cytotoxic effect and the least microbial efficacy.Formulations containing both tranexamic acid and nanoparticles displayed a synergistic effect against cell viability, antimicrobial efficiency, and hemostasis.Formulations X3, X4, X5, X6, X7, and X8 displayed improved bacterial inhibition, good blood clotting potential, and excellent cell migration features compared to formulations without tranexamic acid.There was a slight difference between the hemostatic ability of formulations with or without tranexamic acid.The topical gels displayed promising antibacterial activity.X5 displayed antibacterial activity against a range of gram-negative strains, while X6 was only effective against Klebsiella oxytoca.X1, X2, X3, X8, X10, X11, and X13 exhibited antibacterial activity against Proteus vulgaris and Pseudomonas aeruginosa.All other topical gels exhibited antibacterial activity against both strains of bacteria.X9 did not induce antimicrobial inhibition with moderate cell viability of 76%, showing that it is not ideal for application on bacteria-infected wounds.However, this gel formulation exhibited good hemostatic ability.Encapsulation of nanoparticles in the gels improved their antibacterial activity; however, in some cases, it decreased their activity which can be linked to the concentration/content of the nanoparticle in the gel.All the prepared gel formulations exhibited lower absorbance values compared to the control (p value < 0.05).X14 exhibited the lowest absorbance values than other formulations.However, it was not statistically significant than X3 (p value > 0.05).Gel formulations, X4 and X12 displayed higher cell proliferation features compared to other gel formulations and were selected for in vitro wound healing assay.These formulations reduced the wound size with increased HaCaT cell migration in the affected region.This shows that the prepared topical gels are promising scaffolds for the management of bleeding and microbial-infected wounds.However, in vivo studies are still needed, to further confirm the wound-healing properties of these formulations.

Fig. 1
Fig. 1 Schematic diagram showing the preparation of the formulations wound area day 0 − wound closure day 96 wound closure day 0 × 100

Figure 3a -
Figure 3a-e depicts the SEM images of the prepared nanoparticles.The SEM image of Fe 3 O 4 nanoparticles revealed spherical morphology and similar morphology was reported by Gul et al. for Fe 3 O 4 nanoparticles (Fig.3a)[66].SEM of ZnO NPs showed a combination of spherically and

Fig. 2 a
Fig. 2 a FTIR spectra of X1, X3 and X9; b FTIR spectra of topical gels X2, X5, and X11; c FTIR spectra of topical gels X4 and X10; d FTIR spectra of topical gels X8 and X14; e FTIR spectra of topical gels X7 and X13; f FTIR spectra of topical gels X6 and X12

Fig. 4
Fig. 4 TEM images of the nanoparticles: a Ag nanoparticles, b zinc oxide nanoparticles, c Fe3O4 nanoparticles, and d MgO nanoparticles

Fig. 6 Fig. 7 a
Fig.6 The % cell viability of the prepared wound dressing formulations (error bars ± std)

Table 2
Different bacterial strains used for the in vitro antibacterial studies Inc., San Diego, CA, USA).The data obtained are expressed as mean ± standard deviation, in triplicate (n = 3) and a p value ≤ 0.05 is considered significant.

Table 4
The zeta potential of ZnO nanoparticles was + 27.3 ± 73.4 mV.Kavitha et al. reported ZnO nanoparticles with a zeta potential of 17.6 mV

Table 6
displayed antibacterial activity against E. faecalis, B. subtilis, P. vulgaris, and P. aeruginosa, suggesting that encapsulation of iron oxide NPs into the gels revealed decreased antimicrobial activity.X4 displayed antibacterial activity against B. subtilis, S. aureus, E. cloacae, E. faecalis, P. vulgaris, P. mirabilis, and K. oxytoca strains of bacteria.Liu et al. reported electrospun nanofibers with antibacterial activity dependent on the concentration of MgO NPs against S. aureus, S. epidermidis, and E. coli [82].Similar results were reported by Sukumaran et al. for chitosan/gelatin films incorporated with MgO NPs that exhibited antibacterial activity against S. aureus and E. coli [83].Yaroslavovytch et al. reported MgO NPs with a particle size of less than 10 nm promoted cell death of B. subtilis and S. aureus while those with an average particle size of 50 nm displayed partial effect against B. subtilis and et al. investigated the wound healing potential of PVA/alginate hydrogel formulations using scratch assay and reported them to enhance mobility and migration of HaCaT cells, showing more than 90% wound reduction after 12 h with a p value (< 0.05) compared to the control [96].Kong et al. reported similar PVA/ alginate hydrogels loaded with different concentrations of 5-hydroxymethylfurfural [97].The wound healing scratch assay of these formulations revealed that higher concentrations of 5-hydroxymethylfurfural induced a faster wound reduction, showing ± 80%, and ± 38% at the lowest concentration for 48 h against human skin fibroblast cells.Zheng et al. reported injectable thermos-sensitive hydrogels that stimulated migration of HUVECs cells and showed 100% wound reduction in 12 h of treatment, imaged using fluorescent [98].