Abstract
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.
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1 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 pre-hospital 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 alginate-based 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 Fe3+ 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, F3O4NPs) 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.
2 Materials
2.1 Solvents and Reagents
Distilled water was used to prepare the topical gels. The materials used (sodium alginate, carbopol 940, methylparaben, propylene glycol, triethylamine, graphene oxide (GO) (carbon content 42.0–52.0%), reduced graphene oxide (rGO) (carbon content > 75%), and tranexamic acid (TA) (grade: pharmaceutical secondary standards), were all purchased from Sigma Aldrich, South Africa. They were all used without further purification.
2.2 Experimental
2.2.1 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].
2.2.2 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.
2.2.3 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.
2.2.4 Fe3O4 Nanoparticles Preparation
Iron oxide nanoparticles were prepared based on the method reported by Fatima et al. [39]. FeSO4•7H2O (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.
2.2.5 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.
Schematic diagram showing the preparation of the formulations
2.3 Characterization
2.3.1 FTIR
It was performed using an FTIR spectrometer (Spectrum Two) PerkinElmer on all the topical gels within the range of 4000–500 cm−1.
2.3.2 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 1.0–1.3 cm) was placed on a glass slide and a second glass slide was carefully placed on top of the slide containing the gel. A known mass of 100 g was placed on top of the two glass slides for 1 min, and the spreadability was measured [35].
2.3.3 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.
2.3.4 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.
2.3.5 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.
2.3.6 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].
2.3.7 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.
2.3.8 Hydrodynamic Properties of the Metal Oxide Nanoparticles
The hydrodynamic properties of the nanoparticles were analyzed using a Malvern Zetasizer Nano ZS (Malvern, UK). [Refractive index(es): 1.74 (MgO); 2.23 (Fe3O4); 2.00 (ZnO); 0.150 and absorption value of 0.588 (Ag). An absorption value of 0.001 was used unless stated otherwise. The hydrodynamic results are provided as mean ± standard deviation (SD).
2.3.9 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 dH2O (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.
2.3.10 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. CaCl2 (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].
2.3.11 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 \(\times\) 104 cells/mL was used for cell seeding [44]. The negative controls were the cells treated with 10 μL of 1 \(\times\) 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 ODS is the absorbance of the test compound and ODc is the absorbance of the control. ODu is the absorbance of the untreated compound.
2.3.12 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% CO2 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 \(\times\) 105 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 \(\times\) 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 \(\times\) 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]:
2.3.13 Statistical Analysis
The data obtained from the in vitro studies were evaluated via Student’s unpaired t test on GraphPad Prism version 9 (GraphPad Software, 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.
3 Results and Discussion
3.1 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 for the O–H stretch, 1637 cm−1 for the C = O stretch, and 1042 cm−1 for the C-O stretch. Similar peaks were reported for alginate-based topical gels which further confirmed the successful preparation of the gel formulations [49, 50]. Pure SA powder revealed C = O stretch at 1604 cm−1, O–H carboxylic group peaks at 3302 cm−1, C-O stretch at 1026 cm−1, and C-H stretch at 2936 cm−1. The O–H stretch in the prepared topical gel confirmed the successful interaction of the polymers, SA, and carbopol. The C-O stretch further confirmed an interaction between SA and carbopol. The FTIR spectrum of TA revealed absorption signals at 2936 cm−1, 2830 cm−1, 2959 cm−1, 2205 cm−1, 1651 cm−1, 1549 cm−1, 1379 cm−1, and 1041 cm−1, which is attributed to the O–H stretch carboxylic group, C-H stretch, C = O stretch, and C-O stretch, respectively (Supplementary Fig. 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].
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
3.2 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 5.2–9.3 cm, 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.
3.3 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.
3.4 SEM Analysis of the Prepared Nanoparticles
Figure 3a–e depicts the SEM images of the prepared nanoparticles. The SEM image of Fe3O4 nanoparticles revealed spherical morphology and similar morphology was reported by Gul et al. for Fe3O4 nanoparticles (Fig. 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 block-shaped morphology with a smooth surface (Fig. 3d). rGO exhibited coarse and irregular morphology (Fig. 3e).
SEM images of the prepared nanoparticles: (a) Fe O NPs (b) ZnO NPs (c) MgO NPs (d) GO (e) rGO
3.5 XRD Analysis of the Prepared Nanoparticles
The XRD graphs of the prepared nanoparticles are presented as supplementary Figures. The XRD graphs of the Fe3O4 nanoparticles 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 (Supp Fig. 2a). Similar findings of XRD graphs were reported for hematite and magnetite nanoparticles [69]. The prepared ZnO nanoparticles revealed crystal 2Ɵ peaks at 11.8°, 30°, 32°, 34.9°, 36.5°, 48°, and 57° (Supp Fig. 2b). Similar ZnO nanoparticles 2Ɵ peaks were also reported at 33.2° and 36.3° by Shalumon et al. [70]. The MgO nanoparticles XRD graph revealed diffraction of 2Ɵ peaks at 11°, 18°, 25.5°, 27.9°, 33.5°, 36.5°, 43°, 45.5°, 53.1°, 56.3°, and a sharp peak at 31.9° (Supp Fig. 2c). Sukumaran et al. reported that they peaks were attributed to (111) (200), (220), (311), and (222) planes [71].
3.6 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 Fe3O4 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 stable for over 90 days [73]. 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 [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 rectangular-shaped morphology [78]. The TEM image of Fe3O4 showed a combination of spherical and cubic shapes [79], confirming the successful synthesis of the nanoparticles.
TEM images of the nanoparticles: a Ag nanoparticles, b zinc oxide nanoparticles, c Fe3O4 nanoparticles, and d MgO nanoparticles
3.7 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 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 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 exhibited an antibacterial effect against gram-positive (E. faecalis, S. epidermidis, and S. aureus) and gram-negative strains of bacteria (E. cloacae, P. vulgaris, K. oxytoca, P. aeruginosa, and P. mirabilis). Similar results were reported by Li et al. where hydrogels loaded with rose Bengal and GO exhibited antibacterial activity against gram-positive and gram-negative bacterial strains [89]. Prema et al. reported GO/ZnO (Cu)O nanocomposites with a significant antibacterial activity which was attributed to the GO’s large surface area [90].
Yang et al. reported GO nanosheet’s antibacterial activity is due to reactive oxygen species and destructive of bacterial phospholipids leading to damaged cell membrane integrity and bacterial death [91]. X12 containing only AgNPs induced antibacterial activity against E. faecalis, S. aureus, E. cloacae, P. vulgaris, K. oxytoca, P. aeruginosa, and P. mirabilis. These results are similar to those reported by Masood et al. where AgNPs encapsulated in CS/PEG hydrogels, inhibited bacterial cell growth of P. aeruginosa, B. subtilis, E. coli, and S. aureus than the plain AgNPs [92]. Choudhary et al. formulated AgNPs and calcium alginate nanoparticle-impregnated CS hydrogels with good antibacterial activity against E. coli, S. aureus, B. subtilis, and P. aeruginosa [41]. AgNPs bind to the bacterial cell wall and disturb enzymes essential for their metabolism. X14 was more effective against B. subtilis, E. faecalis, S. aureus, E. cloacae, K. oxytoca, P. aeruginosa, and P. mirabilis. Ali et al. reported carboxymethyl cellulose hydrogels incorporated with rGO inhibited the bacteria growth of S. aureus and P. aeruginosa [93].
3.8 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.
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
3.9 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].
The % cell viability of the prepared wound dressing formulations (error bars ± std)
3.10 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.
a Scratch assay images showing wound healing potential of untreated, X4, and X12 for 0–48 h; b Scratch assay images showing wound healing potential of untreated, X4, and X12 for 0–48 h; and c A graph showing the percentage of wound closure in vitro for untreated, X4, and X12 for 96 h post-treatment
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 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 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].
4 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.
Data Availability
All data and materials used for the reported work are all reported in the manuscript.
References
Pawar, H. V., Tetteh, J., Debrah, P., & Boateng, J. S. (2019). Comparison of in vitro antibacterial activity of streptomycin-diclofenac loaded composite biomaterial dressings with commercial silver based antimicrobial wound dressings. International Journal of Biological Macromolecules, 121, 191–199
Gupta, A., Kowalczuk, M., Heaselgrave, W., & Britland, S. T. (2019). The production and application of hydrogels for wound management: A review. European Polymer Journal, 111, 134–151
Tvl, H. B., Vidyavathi, M., Kavitha, K., Tp, S., & Rv, S. (2010). Preparation and evaluation of ciprofloxacin loaded chitosan-gelatin composite films for wound healing activity. Int J Drug Deliv, 2, 173–182
Stricker-Krongrad, A. H., Alikhassy, Z., Matsangos, N., Sebastian, R., Marti, G., Lay, F., & Harmon, J. W. (2018). Efficacy of chitosan-based dressing for control of bleeding in excisional wounds. Eplasty, 18, 122–130
Negut, I., Grumezescu, V., & Grumezescu, A. M. (2018). Treatment strategies for infected wounds. Molecules, 2392, 1–23
Rivera, A. E., & Spencer, J. M. (2007). Clinical aspects of full-thickness wound healing. Clinics in Dermatology, 25, 39–48.
Bennett, B. L. (2017). Bleeding control using hemostatic dressings: Lessons learned, wilderness environ. Med., 28, S39-49
Barba, B. J. D., Tranquilan-aranilla, C., & Abad, L. V. (2016). Hemostatic potential of natural/synthetic polymer based hydrogels crosslinked by gamma radiation. Radiation Physics and Chemistry, 118, 111–113
Wang, Y., Fu, Y., Li, J., Mu, Y., Zhang, X., Zhang, K., Liang, M., Feng, C., & Chen, X. (2018). Multifunctional chitosan/dopamine/diatom-biosilica composite beads for rapid blood coagulation. Carbohydrate Polymers, 200, 6–14
Tavakoli, S., Kharaziha, M., Nemati, S., & Kalateh, A. (2021). Nanocomposite hydrogel based on carrageenan-coated starch/cellulose nanofibers as a hemorrhage control material. Carbohydrate Polymers, 251, 117013
Liang, Y., Xu, C., Li, G., Liu, T., Liang, J. F., & Wang, X. (2018). Biointerfaces graphene-kaolin composite sponge for rapid and riskless hemostasis. Colloids and Surfaces B, 169, 168–175
Wang, Y., Yin, M., Li, Z., Liu, Y., Ren, X., & Huang, T. S. (2018). Preparation of antimicrobial and hemostatic cotton with modified mesoporous particles for biomedical applications. Colloids and Surfaces B, 165, 199–206
Chan, L. W., Kim, C. H., Wang, X., Pun, S. H., White, N. J., & Kim, T. H. (2016). PolySTAT-modified chitosan gauzes for improved hemostasis in external hemorrhage. Acta Biomaterialia, 31, 178–185
Aydemir, U., Kocer, Z., Sahin, İ, Aru, B., & Yan, G. (2018). Oxidized regenerated cellulose cross-linked gelatin microparticles for rapid and biocompatible hemostasis: A versatile cross-linking agent. Carbohydrate Polymers, 200, 624–632
Liu, J., Li, Y., Hu, Y., Cheng, G., Ye, E., Shen, C., & Xu, F. (2018). Hemostatic porous sponges of cross-linked hyaluronic acid/cationized dextran by one self-foaming process. Materials Science and Engineering C, 83, 160–168
Barba, B. J., Aranilla, C. T., Relleve, L. S., Cruz, V. R. C., Vista, J. R., & Abad, L. V. (2018). Hemostatic granules and dressing prepared from formulations of carboxymethyl cellulose, kappa-carrageenan and polyethylene oxide crosslinked by gamma radiation. Radiation Physics and Chemistry, 144, 180–188
Chen, J., Ai, J., Chen, S., Xu, Z., Lin, J., Liu, H., & Chen, Q. (2019). Synergistic enhancement of hemostatic performance of mesoporous silica by hydrocaffeic acid and chitosan. International Journal of Biological Macromolecules, 139, 1203–1211
Neuffer, M. C., McDivitt, J., Rose, D., King, K., Cloonan, C. C., & Vayer, J. S. (2004). Hemostatic dressings for the first responder: A review. Military Medicine, 169, 716–720
Sezer, U. A., Kocer, Z., Aru, B., Demirel, G. Y., Gulmez, M., Aktekin, A., Ozkara, S., & Sezer, S. (2016). Combination of gelatin and tranexamic acid offers improved haemostasis and safe use on internal hemorrhage control. RSC Advances, 6, 95189–95198
Spotnitz, W. D., & Burks, S. (2012). Hemostats, sealants, and adhesives III: A new update as well as cost and regulatory considerations for components of the surgical toolbox. Transfusion, 52, 2243–2255
Wang, Y., Liu, G., Wu, L., Qu, H., Song, D., Huang, H., Wu, C., & Xu, M. (2020). Rational design of porous starch/hyaluronic acid composites for hemostasis. International Journal of Biological Macromolecules, 158, 1319–1329
Wang, Y., Zhao, Y., Qiao, L., Zou, F., Xie, Y., Zheng, Y., Chao, Y., Yang, Y., He, W., & Yang, S. (2021). Cellulose fibers-reinforced self-expanding porous composite with multiple hemostatic efficacy and shape adaptability for uncontrollable massive hemorrhage treatment. Bioact. Mater., 6, 2089–2104
Liu, X., & Jia, G. (2018). Modern wound dressing using polymers/biopolymers. Journal Material Science Engineering, 7, 7–10
Liu, X., Niu, Y., Chen, K. C., & Chen, S. (2017). Rapid hemostatic and mild polyurethane-urea foam wound dressing for promoting wound healing. Materials Science and Engineering C, 71, 289–297
Liu, C., Hi, Z., Sun, H., Mujuni, C. J., Zhao, L., Wang, X., & Huang, F. (2020). Preparation and characterization of tissue-factor-loaded alginate: Toward a bioactive hemostatic material. Carbohydrate Polymers, 249, 116860
Huang, X., Fu, Q., Deng, Y., Wang, F., Xia, B., Chen, Z., & Chen, G. (2021). Surface roughness of silk fibroin/alginate microspheres for rapid hemostasis in vitro and in vivo. Carbohydrate Polymers, 253, 117256
Mahmoodzadeh, A., Moghaddas, J., Jarolmasjed, S., Ebrahimi Kalan, A., Edalati, M., & Salehi, R. (2021). Biodegradable cellulose-based superabsorbent as potent hemostatic agent. Chemical Engineering Journal, 418, 129252
Wong, Y., Low, J. A., & Chio, M. T. W. (2016). Role of topical tranexamic acid in hemostasis of locally advanced basal cell carcinoma. JAAD Case Reports, 2, 162–163
Huang, L., Liu, G. L., Kaye, A. D., & Liu, H. (2020). Advances in topical hemostatic agent therapies: A comprehensive update. Advances in Therapy, 37, 4132–4148
Zhong, Q. K., Wu, Z. Y., Qin, Y. Q., Hu, Z., Li, S. D., Yang, Z. M., & Li, P. (2019). Preparation and properties of carboxymethyl chitosan/alginate/tranexamic acid composite films. Membranes, 9, 1–8
Cheng, F., Liu, C., Wei, X., Yan, T., Li, H., He, J., & Huang, Y. (2017). Preparation and characterization of 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO)-oxidized cellulose nanocrystal/alginate biodegradable composite dressing for hemostasis applications. ACS., 5, 2–11
Jin, J., Xu, M., Liu, Y., Ji, Z., Dai, K., Zhang, L., Wang, L., Ye, F., Chen, G., & Lv, Z. (2020). Alginate-based composite microspheres coated by berberine simultaneously improve hemostatic and antibacterial efficacy. Colloids Surfaces. B., 194, 111168
Zhao, X., Zhang, Z., Luo, J., Wu, Z., Yang, Z., Zhou, S., Tu, Y., Huang, Y., Han, Y., & Guo, B. (2022). Biomimetic, highly elastic conductive and hemostatic gelatin/rGO-based nanocomposite cryogel to improve 3D myogenic differentiation and guide in vivo skeletal muscle regeneration. Applied Materials Today, 26, 101365
Lv, C., Li, L., Jiao, Z., Yan, H., Wang, Z., Wu, Z., Guo, M., Wang, Y., & Zhang, P. (2021). Improved hemostatic effects by Fe3+ modified biomimetic PLLA cotton-like mat via sodium alginate grafted with dopamine. Bioact. Mater., 6, 2346–2359
Buyana, B., Aderibigbe, B. A., Ndinteh, D. T., Fonkui, Y. T., & Kumar, P. (2020). Alginate-pluronic topical gels loaded with thymol, norfloxacin and ZnO nanoparticles as potential wound dressings. J. Drug. Deliv. Sci. Technol., 60, 101960
Chugh, H., Sood, D., Chandra, I., Tomar, V., Dhawan, G., & Chandra, R. (2018). Role of gold and silver nanoparticles in cancer nano-medicine. Artif. Cells Nanomedicine Biotechnol., 46, 1210–1220
Jyoti, M., Vijay, D., & Radha, S. (2013). To study the role of temperature and sodium hydroxide concentration in the synthesis of zinc oxide nanoparticles. International Journal of Scientific and Research Publications, 3, 2250–3153
Chandrappa, K. G., Praveen, B. M., & Shylesha, B. S. (2015). Generation of nanostructured MgO particles by solution phase method. Res. J. Chem. Sci., 5, 13–18
Fatima, H., Lee, D. W., Yun, H. J., & Kim, K. S. (2018). Shape-controlled synthesis of magnetic Fe3O4 nanoparticles with different iron precursors and capping agents. RSC Advances, 8, 22917–22923
Rahman, S. S. U., Qureshi, M. T., Sultana, K., Rehman, W., Khan, M. Y., Asif, M. H., Farooq, M., & Sultana, N. (2017). Single step growth of iron oxide nanoparticles and their use as glucose biosensor. Results Phys., 7, 4451–4456
Choudhary, M., Chhabra, P., Tyagi, A., & Singh, H. (2021). Scar free healing of full thickness diabetic wounds: A unique combination of silver nanoparticles as antimicrobial agent, calcium alginate nanoparticles as hemostatic agent, fresh blood as nutrient/growth factor supplier and chitosan as base matrix. International Journal of Biological Macromolecules, 17, 41–52
Fonkui, T. Y., Ikhile, M. I., Muganza, M., Fotsing, M. C. D., Arderne, C., Siwe-Noundou, X., Krause, R. W. M., Ndinteh, D. T., & Njobeh, P. B. (2018). Synthesis, characterization and biological applications of novel Schiff bases of 2-(trifluoromethoxy)aniline. Journal of Chinese Pharmaceutical Sciences, 2, 307–323
Catanzano, O., D’Esposito, V., Formisano, P., Boateng, J. S., & Quaglia, F. (2018). Composite alginate-hyaluronan sponges for the delivery of tranexamic acid in postextractive alveolar wounds. Journal of Pharmaceutical Sciences, 107, 654–661
Shukr, M. H., & Metwally, G. F. (2013). Evaluation of topical gel bases formulated with various essential oils for antibacterial activity against methicillin-resistant Staphylococcus aureus. Tropical Journal of Pharmaceutical Research, 12, 877–884
Felice, F., Zambito, Y., Belardinelli, E., Fabiano, A., Santoni, T., & Stefano, R. D. (2015). Effect of different chitosan derivatives on in vitro scratch wound assay: A comparative study. International Journal of Biological Macromolecules, 76, 236–241
Suarez-arnedo, A., Figueroa, F. T., Clavijo, C., Arbela, P., Cruz, J. C., & Munoz-Camargo, C. (2020). An image J plugin for the high throughput image analysis of in vitro scratch wound healing assays. PLoS One, 15, e0232565
Cheng, Y., Hu, Z., Zhao, Y., Zou, Z., Lu, S., Zhang, B., & Li, S. (2019). Sponges of carboxymethyl chitosan grafted with collagen peptides for wound healing. International Journal of Molecular Sciences, 20, 3890
Devi, D. R., Lakshna, S. S., Parvathi, S. V., & Hari, B. N. V. (2018). Investigation of wound healing effect of topical gel of Albizia amara leaves extract. South African J. Bot., 119, 400–409
Md, S., Abdullah, S., Alhakamy, N. A., Shaik, R. A., Eldakhakhny, B. M., Omar, U. M., Eid, B. G., Ansari, A. R., Alamoudi, A. J., Rizg, W. Y., & Riadi, Y. (2022). Development and evaluation of ginkgo biloba/sodium alginate nanocomplex gel as a long-acting formulation for wound healing. Gels, 19, 1–19
Siddique W, Zaman M, Waheed S, Sarfraz RM, Bashir S, Minhas MU, Farooq U, Saeed A (2022) Development and optimization of ganciclovir-loaded carbopol topical gel by response surface methodology for enhanced skin permeation. Polymer Bulletin 1–28
Niraimathee, V. A., Subha, V., Ravindran, R. E., & Renganathan, S. (2016). Green synthesis of iron oxide nanoparticles from Mimosa pudica root extract. Int J Environ Sustain Dev, 15, 227–240
Demirezen, D. A., Yıldız, Y. Ş, Yılmaz, Ş, & Yılmaz, D. D. (2019). Green synthesis and characterization of iron oxide nanoparticles using Ficus carica (common fig) dried fruit extract. Journal of Bioscience and Bioengineering, 127, 241–245
Essien, E. R., Atasie, V. N., Okeafor, A. O., & Nwude, D. O. (2020). Biogenic synthesis of magnesium oxide nanoparticles using Manihot esculenta (Crantz) leaf extract. Int Nano Lett, 10, 43–48
Maheo, A. R., Vithiya, B. S., & Prasad, T. A. (2022). Biosynthesis of chitosan and Eupatorium adenophorum mediated zinc oxide nanoparticles and their biological and photocatalytic activities. MaterToday Proc, 65, 298–312
Omar, M. M., Hasan, O. A., & El Sisi, A. M. (2019). Preparation and optimization of lidocaine transferosomal gel containing permeation enhancers: A promising approach for enhancement of skin permeation. International Journal of Nanomedicine, 14, 1551–1562
Thakur, N., Jain, P., & Jain, V. (2018). Formulation development and evaluation of transferosomal GEL. J. Drug Deliv. Ther, 8, 168–177
El-Gizawy, S. A., Nouh, A., Saber, S., & Kira, A. Y. (2020). Deferoxamine-loaded transfersomes accelerates healing of pressure ulcers in streptozotocin-induced diabetic rats. J Drug Deliv Sci Technol, 58, 101732
Opatha, S. A., Titapiwatanakun, V., Boonpisutiinant, K., & Preparation, C. R. (2022). Characterization and permeation study of topical gel loaded with transfersomes containing asiatic acid. Molecules, 27, 1–16
Barkat, R. A., Pottoo, F. H., Singh, S. P., & Ahmad, F. J. (2018). Therapeutic intervention of aloe gel containing nano-sized and micron-sized silver sulfadiazine gel on second-degree burn: A comparative study. Int J Lower Extremity Wounds., 17, 176–183
Ahmed, M. M., Anwer, M., Fatima, F., Alali, A. S., Kalam, M. A., Zafar, A., Alshehri, S., & Ghoneim, M. M. (2028). Development of apremilast nanoemulsion-loaded chitosan gels: In vitro evaluations and anti-inflammatory and wound healing studies on a rat model. Gels, 8, 253
Chinatangkul, N., Phosrithong, N., Nuchtavorn, N., Rattanapit, T., Mongkol, C., & Chutipanyaboot, P. (2015). Formulation and physicochemical evaluation of topical preparations containing extract of Clerodendrum disparifolium. Advances in Materials Research, 1060, 95–98
Fatima, F., Aleemuddin, M., Ahmed, M. M., Anwer, M., Aldawsari, M. F., Soliman, G. A., Mahdi, W. A., Jafar, M., Hamad, A. M., & Alshehri, S. (2023). Design and evaluation of solid lipid nanoparticles loaded topical gels: Repurpose of fluoxetine in diabetic wound healing. Gels., 9, 1–20
Saffarian, M., Mohammadi, M., & Mohammadi, M. (2015). Non-Newtonian shear-thinning fluid passing through a duct with an obstacle, using a power law model. J. Mech. Eng., 61, 594–600
Ontong, J. C., Singh, S., Nwabor, O. F., Chusri, S., & Voravuthikunchai, S. P. (2020). Potential of antimicrobial topical gel with synthesized biogenic silver nanoparticle using Rhodomyrtus tomentosa leaf extract and silk sericin. Biotechnology Letters, 42, 2653–2664
Carvalho, F. C., Barbi, M. S., Sarmento, V. H. V., Chiavacci, L. A., Netto, F. M., & Gremia˜o MPD,. (2010). Surfactant systems for nasal zidovudine delivery: Structural, rheological and mucoadhesive properties. Journal of Pharmacy and Pharmacology, 62, 430–439
Gul, W., Alrobei, H., Riaz, S., Shah, A., & Khan, A. (2020). Effect of iron oxide nanoparticles on the physical properties of medium density fiberboard. Polymers, 12, 1–18
Mohammadi, F. M., & Ghasemi, N. (2018). Influence of temperature and concentration on biosynthesis and characterization of zinc oxide nanoparticles using cherry extract. J Nanostructure Chem, 8, 93–102
Sushma, N. J., Prathyusha, D., Swathi, G., Madhavi, T., Prasad Raju, B. D., Mallikarjuna, K., & Kim, H. S. (2016). Facile approach to synthesize magnesium oxide nanoparticles by using Clitoria ternatea—characterization and in vitro antioxidant studies. Applied Nanoscience, 6, 437–444
Qureshi, A. A., Javed, S., Javed, H. M., Jamshaid, M., Ali, U., & Akram, M. A. (2022). Systematic investigation of structural, morphological, thermal, optoelectronic, and magnetic properties of high-purity hematite/magnetite nanoparticles for optoelectronics. Nanomaterials, 12, 1635.
Shalumon, K. T., Anulekha, K. H., Nair, S. V., Nair, S. V., Chennazhi, K. P., & Jayakumar, R. (2011). Sodium alginate/poly (vinyl alcohol)/ nano ZnO composite nanofibers for antibacterial wound dressings. International Journal of Biological Macromolecules, 49, 247–254
Shalumon, K. T., Anulekha, K. H., Nair, S. V., Nair, S. V., Chennazhi, K. P., & Jayakumar, R. (2011). Sodium alginate/poly (vinyl alcohol)/nano ZnO composite nanofibers for antibacterial wound dressings. International Journal of Biological Macromolecules, 49, 247–54.
Bhattacharjee, S. (2016). DLS and zeta potential–what they are and what they are not? Journal of Controlled Release, 235, 337–351
Raliya, R., Tarafdar, J. C., Singh, S. K., Gautam, R., Choudhary, K., Maurino, V. G., & Saharan, V. (2014). MgO nanoparticles biosynthesis and its effect on chlorophyll contents in the leaves of clusterbean (Cyamopsis tetragonoloba L.). Adv. Sci. Eng. Med., 6, 538–545
Kavitha, S., Dhamodaran, M., Prasad, R., & Ganesan, M. (2017). Synthesis and characterisation of zinc oxide nanoparticles using terpenoid fractions of Andrographis paniculata leaves. Int. Nano Lett., 7, 141–147
Takechi-Haraya, Y., Ohgita, T., Demizu, Y., Saito, H., Izutsu, K. I., & Sakai-Kato, K. (2022). Current status and challenges of analytical methods for evaluation of size and surface modification of nanoparticle-based drug formulations. An Official Journal of the American Association of Pharmaceutical Scientists, 23, 1–20
Tak, Y. K., Pal, S., Naoghare, P. K., Rangasamy, S., & Song, J. M. (2015). Shape-dependent skin penetration of silver nanoparticles: Does it really matter? Science and Reports, 5, 1–11
Fouda, A., Hassan, S. E. D., Saied, E., & Hamza, M. F. (2021). Photocatalytic degradation of real textile and tannery effluent using biosynthesized magnesium oxide nanoparticles (MgO-NPs), heavy metal adsorption, phytotoxicity, and antimicrobial activity. Journal of Environmental Chemical Engineering, 9, 1–15
Nagarajan, S., & Arumugam Kuppusamy, K. (2013). Extracellular synthesis of zinc oxide nanoparticle using seaweeds of gulf of Mannar. India. J. Nanobiotechnol., 11, 1–11
Ba-Abbad, M. M., Benamour, A., Ewis, D., Mohammad, A. W., & Mahmoudi, E. (2022). Synthesis of Fe3O4 nanoparticles with different shapes through a co-precipitation method and their application. JOM Journal of the Minerals Metals and Materials Society, 74, 3531–3539
Sathiyaseelan, A., Saravanakumar, K., Mariadoss, A. V., & Wang, M. H. (2021). Antimicrobial and wound healing properties of FeO fabricated chitosan/PVA nanocomposite sponge. Antibiotics., 10, 1–20
Kanagasubbulakshmi, S., & Kadirvelu, K. (2017). Green synthesis of iron oxide nanoparticles using Lagenaria siceraria and evaluation of its antimicrobial activity. Def Life Sci J, 2, 422–427
Liu, M., Wang, X., Li, H., Xia, C., Liu, Z., Liu, J., Yin, A., Lou, X., Wang, H., Mo, X., & Wu, J. (2021). Magnesium oxide-incorporated electrospun membranes inhibit bacterial infections and promote the healing process of infected wounds. J. Mater. Chem. B., 9, 3727–3744
Attayil Sukumaran, S., Kalimuthu, B., Selvamurugan, N., & Mani, P. (2022). Wound dressings based on chitosan/gelatin/MgO composite films. International Journal of Polymeric Materials and Polymeric Biomaterials., 71, 1252–1261.
Yaroslavovytch, P. O. (2017). Antimicrobial effect of wound healing nano-containing polymer materials. The Moldovan Medical Journal., 60, 35–38
Norouzi, M. A., Montazer, M., Harifi, T., & Karimi, P. (2021). Flower buds like PVA/ZnO composite nanofibers assembly: Antibacterial, in vivo wound healing, cytotoxicity and histological studies. Polymer Testing, 93, 106914
Soubhagya, A. S., Moorthi, A., & Prabaharan, M. (2020). Preparation and characterization of chitosan/pectin/ZnO porous films for wound healing. International Journal of Biological Macromolecules, 157, 135–145
Zhang, M., Qiao, X., Han, W., Jiang, T., Liu, F., & Zhao, X. (2021). Alginate-chitosan oligosaccharide-ZnO composite hydrogel for accelerating wound healing. Carbohydrate Polymers, 266, 118100
Farooq, A., Patoary, M. K., Zhang, M., Mussana, H., Li, M., Naeem, M. A., Mushtaq, M., Farooq, A., & Liu, L. (2020). Cellulose from sources to nanocellulose and an overview of synthesis and properties of nanocellulose/zinc oxide nanocomposite materials. International Journal of Biological Macromolecules, 154, 1050–1073
Li, Y., Wang, J., Yang, Y., Shi, J., Zhang, H., Yao, X., Chen, W., & Zhang, X. (2021). A rose Bengal/graphene oxide/PVA hybrid hydrogel with enhanced mechanical properties and light-triggered antibacterial activity for wound treatment. Materials Science and Engineering C, 118, 111447
Prema, D., Binu, N. M., Venkatasubbu, P. J., & GD,. (2021). Photo induced mechanistic activity of GO/Zn(Cu)O nanocomposite against infectious pathogens: Potential application in wound healing. Photodiagnosis and Photodynamic Therapy, 34, 102291
Yang, Y., Dong, Z., Li, M., Liu, L., Luo, H., Wang, P., Yang, X., Zhou, K., & Lei, S. (2020). Graphene oxide/copper nanoderivatives-modified chitosan/hyaluronic acid dressings for facilitating wound healing in infected full-thickness skin defects. International Journal of Nanomedicine, 15, 8231–8247
Masood, N., Ahmed, R., Tariq, M., Ahmed, Z., Masoud, M. S., Ali, I., Asghar, R., Andleeb, A., & Hasan, A. (2019). Silver nanoparticle impregnated chitosan-PEG hydrogel enhances wound healing in diabetes induced rabbits. International Journal of Pharmaceutics, 559, 23–36
Ali, N. H., Amin, M. C. I. M., & Ng, S. F. (2019). Sodium carboxymethyl cellulose hydrogels containing reduced graphene oxide (rGO) as a functional antibiofilm wound dressing. Journal of Biomaterials Science, Polymer Edition, 30, 629–645
Xuan, H., Du, Q., Li, R., Shen, X., Zhou, J., Li, B., Jin, Y., & Yuan, H. (2023). Shape-memory-reduced graphene/chitosan cryogels for non-compressible wounds. International Journal of Molecular Sciences, 24, 1389
Sasmal, P., & Datta, P. (2019). Tranexamic acid-loaded chitosan electrospun nanofibers as drug delivery system for haemorrhage control applications. J Drug Deliv Sci Technol, 52, 559–567
Chen, G., He, L., Zhang, P., Zhang, J., Mei, X., Wang, D., Zhang, Y., Ren, X., & Chen, Z. (2020). Encapsulation of green tea polyphenol nanospheres in PVA/alginate hydrogel for promoting wound healing of diabetic rats by regulating PI3K/AKT pathway. Materials Science and Engineering C, 110, 110686
Kong, F., Fan, C., Yang, Y., Hoon, B., & Wei, K. (2019). 5-hydroxymethylfurfural-embedded poly (vinyl alcohol)/sodium alginate hybrid hydrogels accelerate wound healing Int. J. Biol Macromol., 138, 933–949
Zheng, Z., Bian, S., Li, Z., Zhang, Z., Liu, Y., Zhai, X., Pan, H., & Zhao, X. (2020). Catechol modified quaternized chitosan enhanced wet adhesive and antibacterial properties of injectable thermo-sensitive hydrogel for wound healing. Carbohydrate Polymers, 249, 116826
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Open access funding provided by University of Fort Hare. The financial support of the Govan Mbeki Research and Development Centre, University of Fort Hare, South Africa Medical Research Council, and National Research Foundation, South Africa, towards this research is hereby acknowledged.
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Conceptualization- B.A.A., S.A.A., P.U., D.T.N., P.K., and Y.E.; Methodology and investigation- X.N., S.A.A., P.U., D.T.N., P.K., and Y.E.; formal analysis- X.N., S.A.A., P.U., D.T.N., P.K.; writing—original draft preparation and writing— X.N.; review and editing- X.N., S.A.A., P.U., D.T.N., P.K., Y.E., and B.A.A.; supervision-B.A.A, funding and acquisition-B.A.A, All authors have read and agreed to the published version of the manuscript.
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Nqoro, X., Adeyemi, S.A., Ubanako, P. et al. A Topical Alginate-Based Wound Gel Fixated with Metal-Based Nanoparticles and Tranexamic Acid as a Hemostatic Wound Healing System. BioNanoSci. 13, 2400–2417 (2023). https://doi.org/10.1007/s12668-023-01208-9
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DOI: https://doi.org/10.1007/s12668-023-01208-9