Development and in vitro evaluation of borax cross-linked tamarind seed polysaccharide hydrogel films as an antioxidant and self-healing material for wound healing

Abstract

Conventional hydrogel wound dressings often suffer from mechanical weakness and an inability to self-repair, reducing lifespan and therapeutic benefits. Moreover, it cannot provide an ideal environment for wound healing due to excessive oxidative stress. To address this, we prepared a self-healing hydrogel film composed of tamarind seed polysaccharide (TSP), which possesses antioxidant properties. Fourier transform infrared spectroscopy (FTIR) confirmed the cross-linking between borax and TSP, while scanning electron microscopy (SEM) revealed a highly rigid cross-linked network structure. The thickness, transparency, water vapor transmission rate, tensile stress, and DPPH scavenging activity of formulations F1–F7 range from 0.247 to 0.476 mm, 8.07%–11.05% at 600 nm, 535 g m⁻² day⁻¹ to 1175 g m⁻2 day⁻¹, 2.023–10.75 MPa in the wet state and 11.431–38.15 MPa in the dry state, and 26.12%–53.39% in 6 h, respectively. The optimized formulation F6 exhibited desired properties, including thickness (0.472 ± 0.021 mm), water vapor transmission rate (1175.74 ± 10.675 g m⁻² day⁻¹), tensile stress (10.75 ± 0.620 MPa wet, 38.15 ± 0.795 MPa dry), % swelling ratio (506.04 ± 2.093%), and DPPH scavenging activity (43.64%). From visual inspection, it was observed that the cuts between the segments started to join instantly and were completely attached within 20 min. The optimized formulation showed a healing efficiency of 43.949 ± 0.566%, based on tensile stress measurements of self-healed films, recorded at 6 h. The developed hydrogel film exhibited an in vitro hemolytic rate of 0.33 ± 0.16%, which falls within the safe range, demonstrating good hemocompatibility.

Keywords

borax cross-linked tamarind seed polysaccharides hydrogel self-healing wound management

Introduction

Wounds pose a substantial challenge to global healthcare systems.¹ They compromise the skin’s protective barrier, leading to bleeding, inflammation, and pain. The affected area often becomes susceptible to infection and dehydration, further complicating the healing process.² Effective wound management is essential to prevent these outcomes, and maintaining a moist environment has been shown to accelerate wound healing.³ Hydrogels, due to their high water absorption capacity, can absorb exudates while maintaining a moist environment that promotes tissue remodeling.⁴ Consequently, hydrogel-based formulations have emerged as a vital treatment option for wounds. Among them, hydrogel-based films are gaining popularity in wound care, driven by increasing demand.⁵ These films protect wounds from external environment and are commonly used as dressings. Desired characteristics of hydrogel films include high moisture retention, good mechanical strength, an optimal water vapor transmission rate, biocompatibility, and antimicrobial properties.⁶

Hydrogel films can be formulated from synthetic or natural polymers. Synthetic polymer-based hydrogel films exhibit superior mechanical properties but often lack biocompatibility.⁷ In contrast, natural polysaccharide-based hydrogels are preferred due to their non-toxic, biodegradable, and inherently bioactive properties.^8–10 However, polysaccharide hydrogels have a significant limitation: their fragile network structure is susceptible to disruption, which compromises adhesion to wounds and may lead to the reopening of closed wounds.¹¹ The inherent fragility of polysaccharide-based hydrogel is primarily dependent on weaker interactions such as ionic bonding and hydrogen bonding between the polysaccharide chains, as compared to strong covalent bond formation in synthetic polymers.¹² The weaker mechanical strength of polysaccharide chains was attributed to the availability of bulky and complex branched structures. Due to this, an increase in steric hindrance between the bulky groups of polysaccharide limits the chain entanglement and flexibility of the polymeric network.¹³ This led to a decrease in the ability to entangle and dissipate energy during mechanical stress. Furthermore, excessive swelling led to dilution of polysaccharide matrices, which increases their susceptibility to mechanical failure. These reasons also indicate the inability of polysaccharide-based hydrogels to self-repair after mechanical damage.

The inability of traditional hydrogel films to self-repair stems from the irreversible nature of their cross-linking. Specifically, the covalent bonds connecting the monomers in conventional hydrogels are permanent and cannot be reformed once disrupted. This limitation reduces their lifespan and compromises their therapeutic efficacy. However, hydrogel networks featuring reversible dynamic covalent bonds have been shown to overcome this limitation by incorporating self-healing properties.¹⁴ Borate ester bonds have been utilized to prepare self-healing hydrogels for wound healing applications.¹⁵ Borax, a potent cross-linking agent, accelerates hydrogel formation in materials possessing active hydroxyl groups.¹⁶

Tamarind seed polysaccharides are highly branched, non-ionic, hemicellulose polysaccharides that are soluble in water.¹⁷ Due to their high branching, TSP exhibits a “mucin-like” structure, providing excellent adhesion to mucous membranes.^18,19 TSP exhibits a favorable biological profile, being biocompatible, biodegradable, non-toxic, and non-carcinogenic, with additional benefits such as hemostatic properties.²⁰ The recent discovery of its antioxidant and anti-inflammatory properties further enhances its appeal as a candidate for wound dressing applications.²¹ Additionally, TSP’s flexible film-forming properties, good tensile strength, high thermal stability, and drug-holding capacity further enhance its potential for wound dressing films.²² Moreover, the ample hydroxyl groups in TSP provide a unique opportunity for cross-linking with borax, forming dynamic covalent borate ester bonds that enable self-healing properties.

Borax cross-linked hydrogels have previously been explored with various natural polysaccharides for wound healing applications. Liu et al. developed a borax cross-linked hydrogel by complexing polyvinyl alcohol with carboxymethyl chitosan for wound healing.²³ A borax cross-linked self-healing hydrogel was also developed using xyloglucan and okra polysaccharide.²⁴ Another self-healing hydrogel was prepared using oxidized dextran, polyvinyl alcohol, and functionalized collagen peptide for wound management.²⁵ Zhang et al. prepared a borax cross-linked self-healing hydrogel using Bletilla striata polysaccharide and tannic acid for wound healing.²⁶

However, most of these borax cross-linked hydrogels are composites, combining polysaccharides with other polymers. In contrast, our study specifically focuses on the development of a borax cross-linked self-healing hydrogel film solely based on TSP, without the inclusion of any other polymers. To the best of our knowledge, this is the first report on the development of a borax cross-linked self-healing hydrogel based solely on TSP for wound healing applications.

The objective of this work is to develop a borax cross-linked tamarind seed polysaccharide based self-healing hydrogel film for wound healing applications. The film’s self-healing capability is expected to arise from reversible dynamic borate ester linkages. The inherent antioxidant and antimicrobial properties of TSP, combined with borax’s antimicrobial activity, may produce synergistic effects that enhance the film’s wound healing efficacy. This research aims to develop and evaluate the suitability of borax cross-linked TSP hydrogel films through physicochemical and in vitro biological assessments.

Materials and methods

Materials

TSP was kindly provided by Hindustan Gum & Chemical Limited, Bhiwani, India. Borax, glycerol, and ethanol were purchased commercially from Loba Chemie Pvt. Ltd., India. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) was purchased from SRL, India. All other reagents used were of analytical grade. Double distilled water was used throughout the study.

Preparation of hydrogel film

Hydrogel films were prepared by dissolving varying concentrations of TSP (2%–3% w/v) in water, under continuous stirring at 60 ± 2 °C until a homogeneous solution was obtained. The baseline non-cross-linked hydrogel film (F1) was prepared using 2% w/v TSP and 25% w/w glycerol (Gly; based on the dry weight of TSP) in 40 ml of water. Subsequently, cross-linked hydrogel films (F2–F4) were prepared using a fixed amount of borax (10% of the TSP weight), 25% Gly (relative to TSP), and varying concentrations of TSP (2%–3% w/v). Further optimization was carried out by varying the borax concentration from 5% to 20% w/w (based on TSP), while keeping the TSP concentration fixed at 3% w/v and Gly at 25% (Table 1). The resulting hydrogel solutions (40 ml) were cast into Petri dishes (ø = 7.7 cm) and dried at 60°C for 30 h, followed by air drying to constant weight. The reaction mechanism of hydrogel formation is shown in Figure 1.

Table 1.

Composition of hydrogel films.

Formulation code	Amount of TSP (% w/v)	Amount of borax (wt% of TSP)	Amount of glycerol (wt% of TSP)
F1	2	-	25
F2	2	10	25
F3	2.5	10	25
F4	3	10	25
F5	3	5	25
F6	3	15	25
F7	3	20	25

Fourier transform infrared (FTIR) spectroscopy

The FTIR spectra of TSP and borax cross-linked TSP-based hydrogel film were analyzed using an FTIR spectrophotometer (Alpha-E, Bruker, USA) at 4000–400 cm⁻¹.^27,28 The powdered samples were subjected to mixing with potassium bromide. The prepared mixture was then converted into pellets through a hydraulic press for suitable FTIR measurements.

Scanning electron microscopy (SEM)

The morphological structure of the hydrogel film was evaluated using SEM (SU3800, Hitachi, Japan).²⁹ The prepared hydrogel films were initially air-dried and placed in a desiccator containing silica gel to obtain a moisture-free hydrogel film. Afterward, a suitable amount of dried hydrogel films was put on conductive adhesive tapes, and samples were coated with gold under vacuum. SEM images were obtained under an acceleration voltage of 30 kV to characterize the microstructure of film samples.

Thickness of film

A digital vernier caliper (Mitutoyo, Japan) was used to determine the thickness of the film with a measurement precision of 0.001 mm at 10 separate locations on the film. The average thickness of the film was determined.

Transparency of films

The film samples (10 mm×30 mm) were cut and placed into a UV-visible spectrophotometer (UV-2450, Shimadzu, Japan) and analyzed in the range of 200–800 nm.³⁰ The transparency of films was determined using the following equation.³¹

T r a n s p a r e n c y = % T = 10^{(2 - A)}

(1)

%T is the transmittance of light, and A is the absorbance of the light.

Water vapor transmission rate (WVTR)

The WVTR of the developed films was evaluated by mounting each type of film sample (diameter = 1.5 cm) with Teflon and subsequently determining the mass change rate in a water-filled vial.³² The tube was put into a desiccator kept at 37°C with a 75% RH. A mixture of 10.23 g of water and 20 g of NaCl was placed at the base of the desiccator to maintain the relative humidity.³³ Test cells were taken out after 24 h, and the weight loss was determined. The WVTR of the film dressing was determined using the following equation.³⁴

W V T R (g . m^{- 2} . d a y^{- 1}) = \frac{Δ m}{Δ t} / A

(2)

Δm/Δt = Loss of water for 24 h (g/day), A = effective transfer area (m²).

Mechanical strength

The mechanical strength of the hydrogel films was assessed in both wet and dry conditions. For the determination of the mechanical strength of hydrogel films in wet states, films were soaked in simulated wound fluid (SWF), pH 8, for 30 s, and then extra SWF was eliminated using filter paper. Subsequently, the specimens were promptly subjected to tensile strength measurement in dry and wet conditions. The tensile stress (N/mm² or Megapascals) of the film is the stress at which the film specimen breaks. It was calculated by dividing the applied force (N) by the cross-sectional area (mm²; equation (3)). At the same time, the % elongation at break or tensile strain was determined by dividing the stretched length (mm) by the original length (mm) in both dry and wet states (equation (4)).³⁵

T e n s i l e s t r e s s (\frac{N}{m m^{2}} o r M P a) = \frac{M a x i m u m f o r c e a t b r e a k (N)}{I n i t i a l c r o s s - s e c t i o n a l a r e a o f a f i l m (m m^{2})}

(3)

% E l o n g a t i o n a t b r e a k = \frac{O r i g i n a l l e n g t h (m m)}{S t r e t c h e d l e n g t h (m m)} \times 100

(4)

Self-healing property

The self-healing ability of the borax cross-linked hydrogel film was determined by a gel-block fusion test. The self-healing ability of cross-linked films was determined visually. To help in the visualization of self-healing properties, hydrogel films were dyed in different colors, such as methyl orange and methylene blue indicators. Then, both the colored films were cut into 10 mm × 10 mm. After that, deionized SWF was sprayed over the slices of cross-linked films and kept in that condition for a short while, followed by the visual observation of the self-healing effect. Subsequently, the healing time at 30 s, 1 min, 5 min, 10 min, and 20 min and healing efficiency of the films were determined.

To determine the healing efficiency, the hydrogel film samples were cut into multiple sections and then the fracture surfaces were brought into contact and allowed to heal. A tensile test was carried out to determine the healing efficiency of the self-healed film immediately after healing, as well as after 1, 6, and 12 h of self-healing. The healing efficiency of the film can be determined using the following (equation (5)).³⁶

H e a l i n g e f f i c i e n c y (%) = \frac{M e c h a n i c a l v a l u e_{(h e a l e d)}}{M e c h a n i c a l v a l u e_{(i n i t i a l)}} \times 100

(5)

Mechanical value_(healed) is the tensile stress after self-healing and Mechanical Value_(initial) is the original tensile stress of the hydrogel film.

% Swelling ratio of the film

The gravimetric tea bag method was utilized to determine the % swelling ratio of hydrogel films.³⁷ Tea bags containing the non cross-linked (NCL) and cross-linked films with an area of 2 cm² immersed in a beaker containing 50 ml of SWF having a pH of 8.0 ± 0.2 at 37°C. The film specimens were weighed (W₀) before immersion in the SWF. After fixed time intervals, the tea bags with film specimens were removed from the fluid, excess fluid was blotted with tissue paper, and the weight of the swollen film (Wₛ) was immediately determined. The % swelling ratio was determined using the equation (6).

% S w e l l i n g r a t i o = \frac{w_{s} - w_{0}}{w_{0}} \times 100

(6)

w₀ = dry weight of film and w_s = weight of the swollen film.

In vitro degradation

NCL and cross-linked film specimens were cut in the area of 30 × 10 mm² and immersed into the 10 ml of degradation medium at 37°C. The sample was removed from the medium at predetermined time intervals. The samples were then put in an oven at 37°C until a constant mass was reached to measure the weight loss. The degradation solution was exchanged every 2 days. The tests were performed for 14 days.³⁸ The in vitro degradation of the film samples was determined using the formula of percentage weight loss (equation (7)).

W e i g h t l o s s (%) = \frac{w_{i} - w_{f}}{w_{i}} \times 100

(7)

W_i = initial weight of film at time 0 and W_f = final weight of film at time t.

Rheological characterization

Rheological characterization of the hydrogel samples before drying were carried out by using a rheometer (MCR 102 Rheoplus, Anton Paar, Austria) having parallel plate geometry. The viscosity (η) measurements of prepared hydrogel samples were determined at a shear rate range from 0.01 to 100 s⁻¹.³⁹ Additionally, the self-healing property of cross-linked hydrogel was determined through rheological recovery tests. The storage modulus (G’) and the loss modulus (G”) of original cross-linked and self-healed hydrogel samples (before drying) were determined by an oscillatory strain sweep test at a % strain (γ) of 0.1%–1000% at a constant oscillation frequency (ω) of 10 rad s⁻¹.²³ Furthermore, quantitative analysis of self-healing properties of prepared hydrogel samples before drying was analyzed through continuous alternative strain sweep measurements.²⁵ The hydrogel samples were subjected to alternating low and high oscillatory strains of 1% and 200%, respectively, for 20 s at a constant angular frequency of 10 rad s⁻¹. The cycle was repeated three times for 160 s, and the changes in G’ and G’’ were calculated as a function of time.

Antioxidant activity

The evaluation of the antioxidant properties of TSP and hydrogel films was conducted using the DPPH assay, which measures the ability to neutralize free radicals.⁴⁰ To assess the antioxidant capacity of TSP, it was tested at varying concentrations (2–10 mg/ml). About 0.1 mm solution of DPPH in ethanol (3 ml) was added to the above solutions. After mixing, the solutions were left to stand for 40 min in a dark environment at ambient temperature. The absorbance of both the DPPH solution and TSP-DPPH mixture was recorded at 517 nm using a UV-visible spectrophotometer (UV-2450, Shimadzu, Japan). Ascorbic acid served as the reference antioxidant (positive control), while a DPPH solution without TSP functioned as the negative control. The EC50 value, which is the concentration at which TSP and ascorbic acid exhibit a 50% scavenging rate was determined.

To assess the antioxidant efficacy of hydrogel films, samples of NCL and cross-linked films, each weighing 50 mg, were immersed in 0.1 mm solution of DPPH in ethanol and left to incubate for varying durations in a darkened environment at ambient temperature. A DPPH solution devoid of any hydrogel film served as the negative control. The absorbance was recorded at 517 nm at several time points (0.5, 1, 2, and 6 h) using a UV-visible spectrophotometer (UV-2450, Shimadzu, Japan). The transition of the solution’s color from a deep purple to yellow was observed as an indicator of antioxidant activity over these intervals.⁴¹ The % scavenging activity was determined using the equation given below.

S c a v e n g i n g a c t i v i t y (%) = \frac{A_{0} - A_{1}}{A_{0}} \times 100

(8)

A₀ = absorbance of the 0.1 mm DPPH-ethanolic solution (negative control) and A₁ = absorbance of the test sample and ascorbic acid (positive control).

Blood compatibility study

The blood compatibility through hemolysis test of the hydrogel film was performed to assess its efficacy in preventing lysis of red blood cells (RBCs).⁴² For the hemolysis test, blood of a goat was collected from a slaughterhouse in tubes containing anticoagulant (sodium citrate buffer). The collected blood was diluted with normal saline in the ratio of 1:1. Fifty milligram of the prepared cross-linked film samples were immersed in 10 ml of normal saline and incubated for 1 h at 37°C. After an incubation, 0.2 ml of prepared diluted blood (1:1) was added to the respective samples. For the preparation of negative and positive control, 0.2 ml of diluted blood was added to 10 ml of normal saline and 0.1% Na₂CO₃ solutions, respectively. The prepared groups were incubated at 37°C for an additional 1 h and centrifuged at 2000 rpm for 10 min. After centrifugation, the rate of hemoglobin release due to hemolysis was measured by absorbance of the obtained supernatants at 540 nm using a UV-visible spectrophotometer (UV-2450, Shimadzu, Japan). The percentage hemolysis rate was calculated by the equation given below.

H e m o l y s i s r a t e (%) = \frac{A s - A n}{A p - A n} \times 100

(9)

A_s = Absorbance of hydrogel sample; A_n = Absorbance of negative control (normal saline); and A_p = Absorbance of positive control (0.1% Na₂CO₃).

Statistical analysis

All results are expressed as mean ± standard deviation. p < 0.05 was used to make a statistically significant difference.

Results and discussion

Various hydrogel films were prepared using TSP (NCL) and borax cross-linked TSP. The cross-linked films were prepared by varying concentrations of both the polysaccharide and borax along with the addition of glycerol (25% w/w) as a plasticizer. TSP in solution forms a denser network structure due to its self-aggregation properties and strong intermolecular hydrogen bonding. Moreover, increasing the polymer concentration results in a highly viscous solution. A TSP concentration of 3% w/v among three concentrations (2, 2.5, and 3% w/v) cross-linked by borax (10 wt% of polymeric weight) showed an excellent hydrogel-forming ability due to the availability of abundant free hydroxyl groups. The availability of free 1,2-cis dihydroxyl groups forming a borate ester linkage resulted in the cross-linked network structure (Figure 2).⁴³ On increasing the TSP concentration to 3.5% w/v, a highly thick and viscous solution formed, which is not feasible for further development of the hydrogel film. Therefore, the TSP, having a concentration of 3% w/v, is used for further development of the hydrogel film.⁴⁴ Then, varying the borax concentrations from 5% to 20% of TSP weight, and developed a hydrogel film (Table 1).

An FTIR study was carried out to confirm the cross-linking between borax and TSP. The FTIR spectra of pure TSP and the hydrogel film without glycerol are shown in Figure 3. The TSP exhibited a broad absorption band conforming to O-H stretching at 3275 cm⁻¹ (Figure 3(a)). The signals of C-H stretching and C=O stretching in the glucose structure of TSP were represented at 2930 and 1740 cm⁻¹, respectively. The -OH bending vibrations exhibited an absorption peak at 1647 cm⁻¹, while symmetrical COO⁻ stretching appeared at 1373 cm⁻¹. TSP shows characteristics peak at 1149 cm⁻¹ for -C-O-C asymmetric stretching vibration of glucopyranosyl and xylopyranosyl units, at 1056 cm⁻¹ for -CHO asymmetric stretching, at 893 cm⁻¹ for β-glycosidic bond, and at 756 cm⁻¹ for bending vibrations of pyranose ring. Similar IR spectra of TSP are noted in the literature.^45,46

FTIR spectra of the hydrogel film displayed different absorption peaks (Figure 3(b)). Upon the addition of borax, it was observed that the absorption peak of -OH at 3264 cm⁻¹ weakened. This may be due to the hydroxyl groups of TSP involved in the formation of borate ester linkages. The similar peaks were observed at 2927 cm⁻¹ for stretching vibrations of C-H, while a band for OH-bending at 1651 cm⁻¹ was observed. The peaks were observed at 1403 and 1343 cm⁻¹ for asymmetric stretching of B-O-C, at 1030 cm⁻¹ for C-O stretching, at 813 cm⁻¹ for B-O stretching vibrations from free borate ions, and at 650 cm⁻¹ for bending of B-O-B linkages.^24,47 Thus, the FTIR analysis showed the cross-linking of TSP with borax through the formation of a borate ester bond.

Scanning electron microscopy (SEM) analysis of NCL and cross-linked hydrogel film is represented in Figure 4. The NCL film exhibited a rough texture due to the agglomeration of large and small globular structures (indicated by dotted circles), which were unevenly dispersed throughout the matrix (Figure 4(a)). These visible agglomerates formed as a result of the self-aggregation behavior of TSP molecules.⁴⁸ It has been reported that upon hydration, TSP polysaccharide chains interact with water through non-covalent interactions, including hydrophobic forces and strong hydrogen bonding, leading to the clustering of polymer chains into bundles.

Furthermore, numerous irregular pores and fissures were observed on the surface of the NCL film (indicated by solid circles), resulting from weak interactions between the saccharide units of TSP. Similar findings were reported by Majeed et al., who observed porous holes within the rough texture of native TSP through SEM analysis.⁴⁹ These non-uniform porous structures were attributed to weak intermolecular interactions between the sugar molecules within the polymer matrix. Thus, the presence of rough, irregular pores led to the formation of a highly porous and loosely packed network structure within the polysaccharide matrix. The high porosity of the NCL film also enhanced water entrapment within the matrix, which in turn compromised its mechanical integrity.

Conversely, upon cross-linking with borax, the formation of a borate ester complex between free borate ions and the cis-1,2-OH groups of TSP causes the polymeric chains to become more rigid and densely packed (Figure 4(b)). As a result, the chains come closer together and become more uniform, leading to the formation of a film with a smooth and continuous texture (indicated by the dotted box). Moreover, a rigid and denser network (indicated by the arrow) was formed upon borax cross-linking. This may be attributed to the disruption of hydrogen bonding networks due to increased borate–diol complexation in the hydrogels.⁵⁰ Furthermore, the enhanced cross-linking of borate ions with the hydroxyl groups of TSP effectively reduces chain mobility and free volume within the TSP network, thereby contributing to the overall mechanical integrity of the material.^51,52 Liu et al. showed that borax cross-linked hydrogels have denser networks compared to pure gum hydrogels due to enhanced cross-linking of xyloglucan/okra with borax.²⁴

The thickness of the prepared films in dried form is illustrated in Table 2. The ideal thickness for a wound dressing material is considered less than the thickness of the dermis of human skin, which ranges from 0.5 to 2.0 mm. It was reported from the literature that film thicknesses smaller than 0.5 mm are appropriate for wound healing.³⁵

Table 2.

Thickness of the hydrogel film.

Formulation code	Thickness of film (mm; mean ± SD, n = 3)
F1	0.247 ± 0.004
F2	0.262 ± 0.046
F3	0.353 ± 0.025
F4	0.449 ± 0.026
F5	0.473 ± 0.012
F6	0.472 ± 0.021
F7	0.476 ± 0.052

The thickness of the film varies with changes in polymeric concentration as well as change in borax concentration, ranging from 0.247 ± 0.005 to 0.476 ± 0.052 mm. The prepared NCL film (F1) with 2% w/v polymer has a thickness of 0.247 ± 0.004 mm. While cross-linking with borax, the thickness of the film was increased to 0.262 ± 0.046 mm. However, adding borax led to no significant increase in the thickness of the hydrogel film (p > 0.05). Polymer concentrations at 2%, 2.5%, and 3% w/v in formulations F2, F3, and F4, the thickness of the film was 0.262 ± 0.046, 0.353 ± 0.025, and 0.449 ± 0.026 mm, respectively. The thickness of the film was significantly increased by increasing the concentration of polymer from 2 to 3% w/v (p < 0.05). It was reported that the concentration of TSP significantly influences its self-aggregation behavior in an aqueous solution.⁵³ At low concentrations, TSP forms loose networks, resulting in a reduced ability to retain water within the matrix and thin films upon evaporation. However, at higher concentrations, the enhanced self-aggregation property of TSP promotes stronger gel formation and more porous network structures that retain larger volumes of water. The higher polymeric content retaining high water content within the matrix contributes to a greater film thickness after evaporation. Further, TSP concentration was fixed at 3% w/v and borax concentration increased from 5 to 20 wt% of TSP depicted as F5 (5 wt% of TSP), F4 (10 wt% of TSP), F6 (15 wt% of TSP) and F7 (20 wt% of TSP). The formulations F5 and F4 possess thicknesses of 0.473 ± 0.012 mm and 0.449 ± 0.026 mm, respectively. On increasing the borax content to 15 wt% of TSP (F6) the thickness of the film was 0.472 ± 0.021 mm. No significant change was observed in the thickness of the film when the borax concentration was increased to 15 wt% of TSP (p > 0.05). Similarly, with a further increment in borax concentration to 20 wt% of TSP in formulation F7, a negligible change in thickness (0.476 ± 0.052 mm) was observed (p > 0.05). Patel et al. and Rezvanian et al. developed a hydrogel film for wound healing, and the optimized hydrogel film showed a thickness of 0.452 and 0.188 mm, respectively.^54,55

Light transmission is a vital property for observing the wound-healing process without removing the film. UV rays (200–400 nm) are harmful and cause degradation of wounds. It is necessary for the films for wound healing to have low transparency, which is favorable for protecting wounds from UV radiation and thereby reducing the generation of reactive oxygen species (ROS) and promoting wound healing.³⁰ Light transmission of ideal hydrogel film should be low in the UV region (200–400 nm) and higher in the visible region (400–800 nm). A schematic representation of the percentage transparency of hydrogel films at various wavelengths (200–800 nm) was recorded in Figure 5.

The NCL film (F1) exhibited a maximum transparency of 2.709 ± 0.171% at 400 nm and 10.62 ± 0.310% at 800 nm. With the addition of borax (F2), the transparency of the film has been significantly increased (p < 0.05) in both UV and visible regions, exhibiting 2.858 ± 0.076% at 400 nm and 12.18 ± 0.138% at 800 nm, respectively. The low transparency of NCL films was due to hydrogen bonding between the polysaccharide chains, forming an irregular three-dimensional structure. However, upon the addition of borax, the interconnection of borate ions and free hydroxyl groups resulted in a decrease in hydrogen bonding between polymers. This leads to the creation of a uniform pore structure, thereby enhancing transparency.

As the polymeric concentration augmented from 2% to 3%, the transparency of the film decreased significantly (p < 0.05) in both UV and visible regions. At 400 nm, formulations F2, F3 and F4 have a transparency of 2.858 ± 0.076%, 2.519 ± 0.116% and 2.164 ± 0.035%. While at 800 nm, they possess a transparency of 12.18 ± 0.138%, 10.056 ± 1.03% and 9.268 ± 0.042%, respectively. With the increase in polymer concentration, the formation of a bigger crystal structure enhances the opacity of the hydrogel film, thus hindering light transmission significantly.⁵⁶ Increasing the borax concentration from 5 to 20 wt% in formulations F5, F4, F6, and F7 increases the transparency of the film significantly (p < 0.05). Formulation F4 exhibited transparency of 2.179 ± 0.035% at 400 nm and 11.544 ± 0.213% at 800 nm, which were higher than those of formulation F5 having transparency of 2.164 ± 0.035% at 400 nm and 9.268 ± 0.042% at 800 nm (p < 0.05). On further increasing the concentration of borax to 15 wt% (F6) and 20 wt% (F7), transparency of hydrogel films increased significantly (p < 0.05) and at 400 nm was 2.426 ± 0.022% and 2.769 ± 0.065%, whereas at 800 nm was 12.331 ± 0.028% and 13.307±0.37, respectively.

The incorporation of borax facilitated the formation of borate ester complexes, which in turn disrupted the hydrogen bonding within the matrices, leading to the formation of less crystalline polysaccharide structure, resulting in enhanced transparency of the hydrogel film.⁵⁷ Geng et al. showed that dissociation of borax generates boric acid, which increases the solubilization of insoluble particles and increases the intramolecular space within the polymeric matrix, resulting in an enhancement of light transmission through the film.⁵⁸ The formulation F6 and F7 demonstrated the highest transparency suggesting optimal arrangement of a polymeric matrix for light transmission. However, formulation F7 exhibited high transparency compared to other formulations, which allowed more UV rays to penetrate the wound. Therefore, it is recommended that the optimal borax concentration range of 5–15 wt% be considered to limit UV rays from penetrating the wound. Pereira et al. demonstrated that their optimized hydrogel film, incorporating alginate and aloe vera, exhibited a transparency of 1.60 ± 0.16% at 600 nm, indicating relatively low transparency.³⁰ Similarly, Drápalová et al. observed that the prepared gum karaya/chitosan-based hydrogel film displayed transparency in the range of 40%–60% at 600 nm.⁵⁹

Following an injury, the wound surface may lose evaporative water up to twenty times faster than healthy skin.⁶⁰ The WVTR of the hydrogel film plays a crucial role in assessing the ability of water loss from the dressing. Therefore, moisture in the wounded area can be regulated by selecting the wound dressings with varying WVTRs. A wound may become dehydrated as a result of an abnormally high WVTR, whereas an excessively low WVTR may result in a buildup of wound fluid exudates. To establish the ideal moist environment, a dressing with an appropriate WVTR is necessary for spontaneous wound healing. The WVTR for normal skin was reported at 204 g m⁻² day⁻¹. However, in injured skin, may WVTR increase from 279 g m⁻² day⁻¹ (in first-degree burns) to 5138 g m⁻² day⁻¹(in granulating wounds).⁶¹ The ideal WVTR for commercial wound dressing was reported in the range of 100–3300 g m⁻² day⁻¹.⁵⁵ Xu et al. reported the WVTR of commercially available film dressings, Tegaderm and Opsite, from both external (outside-in) and internal (inside-out) environments.⁶⁰ The study found that the Tegaderm film dressing had WVTR values of 846 g m⁻² day⁻¹ h for the external environment and 794 g m⁻² day⁻¹ for the internal environment. The Opsite dressing exhibited WVTR values of 862 g m⁻² day⁻¹ and 839 g m⁻² day⁻¹ for the external and internal environments, respectively. The WVTR of prepared hydrogel films is represented in Figure 6. Polymer concentration and cross-linking density greatly affect the WVTR of hydrogel film. The NCL film (F1) demonstrated a higher WVTR of 849.88 ± 69.555 g m⁻² day⁻¹. The WVTR of the cross-linked film (F2) showed 744.82 ± 65.166 g m⁻² day⁻¹. The addition of borax resulted in a significant decrease in the WVTR of the hydrogel film (p < 0.05). The reduction in WVTR of F2 was attributed to the formation of cross-linked network structures that restrict chain mobility and limit the diffusion of water across the film. When the TSP content in the formulation was increased to 2.5% w/v (F3), the WVTR of the film was decreased significantly (p < 0.05) and the value was 535.34 ± 59.719 g m⁻² day⁻¹. Surprisingly, upon increasing the polymer concentration to 3%w/v, the WVTR of the film (F4) increased significantly (p < 0.05) and reached 1130.45 ± 18.87 g m⁻² day⁻¹, despite the increased polymer concentration and thickness. At this concentration, the available –OH groups exceeded the cross-linking capacity of borax, resulting in incomplete cross-linking with the formation of microchannels and higher free volume, which facilitated WVTR.

Film thickness influences the WVTR, and the WVTR values for F1–F4 have been explained with their respective thicknesses. The thickness of the film increases from F1 to F4 (Table 2). The WVTR decreases from F1 to F3 and then increases in F4. Increased film thickness extends the diffusion path for water vapor, thereby reducing WVTR (F1–F3), as expected in dense, polymer-rich films. However, in formulation F4, despite thicker films typically offering greater resistance to vapor diffusion, the increased polymer content led to incomplete cross-linking, resulting in the formation of microchannels and higher free volume, which facilitated WVTR.

Increasing the borax concentration from 5 to 15 wt% in formulations F5, F4, and F6 significantly increased the WVTR of the films (p < 0.05), as the polymer network transitioned from an under-cross-linked (F5) to an intermediate cross-linked state (F4 and F6). Formulations F5, F4, F6 showed the WVTR of 721.55 ± 7.85, 1130.45 ± 18.87, and 1175.74 ± 7.54 g m⁻² day⁻¹ respectively (p < 0.05). The lower WVTR of F5 compared to F4 is attributed to dominant polysaccharide–polysaccharide hydrogen bonding and chain entanglement, which limit water vapor diffusion. The higher WVTR of F4 and F6 compared to F5 is attributed to intermediate, incomplete cross-linking, resulting in the formation of microchannels and higher free volume. Wang et al. found that with an increase in borax content, the formation of borate ester linkages would increase macroscopic pores and increase the gap between the polymeric chains, increasing the diffusivity of the film.⁵⁰ Further, with the increase in concentration of borax to 20% (F7), the WVTR of the film was significantly (p<0.05) reduced to 1059.68 ± 33.36 g m⁻² day⁻¹. The higher loading of borax leads to a denser, over-cross-linked network with reduced free volume and restricted chain mobility, which in turn limits vapor diffusion and causes a decrease in WVTR.

The formulation F6 showed an optimal WVTR of 1175.74 ± 7.54 g m⁻² day⁻¹, positioning the range comparable to the commercial dressings. These results indicated that the hydrogel film possesses adequate moisture retention, thereby preventing maceration and fluid accumulation. The excellent WVTR of the hydrogel film was due to the formation of uniform borate cross-linked network structures for enhancing permeability. Additionally, the presence of glycerol and free hydroxyl groups provided sufficient hydrophilicity to the hydrogel film.⁶² These components allow the film to maintain moisture within the wound. Increasing borax concentration from 5 to 10 wt% (F5–F4) decreases film thickness and increases WVTR, as thinner films provide a shorter path for vapor diffusion. From 10 to 15 wt% (F4–F6), both thickness and WVTR increase, likely due to uneven crosslinking that creates microchannels and higher free volume, facilitating vapor transport. Further increasing borax to 20 wt% (F6–F7) increases thickness but decreases WVTR, possibly due to excessive crosslinking forming a denser, less permeable network with reduced free volume.

Zhu et al. developed alginate-based dressings using CaCl₂ and citric acid as cross-linkers.⁶³ It was found that a pure alginate hydrogel dressing showed a maximum WVTR of 821.44 g m⁻² day⁻¹ while on treatment with CaCl₂ and citric acid, the WVTR of the cross-linked film decreased to 715.39 and 675.37 g m⁻² day⁻¹. Pitpisutkul and Prachayawarakorn fabricated hydroxypropyl methylcellulose/carboxymethyl starch based hydrogel film using succinic acid as a cross-linking agent.⁶⁴ Uncross-linked films exhibited a WVTR of 426.91 ± 6.13 g m⁻² day⁻¹ while the cross-linked films exhibited a WVTR of 407.38 ± 4.40 g m⁻² day⁻¹.

For wound dressing, the viscosity of the hydrogel should be high to minimize the flow behavior during application on the wound. The viscosity of the prepared TSP-based hydrogels decreased gradually with the increase in shear rate (s⁻¹), demonstrating the pseudoplasticity or shear-thinning behavior of hydrogels (Figure 7). With increasing shear rate, the resistance to the flow decreased. At rest, the polymers exist as tangled chains and exhibit high viscosity. At a high shear rate, the chain structure disentangles, causing a lowering of internal resistance and the systems exhibit shear thinning behavior.

Formulation F1 and F2 at a shear rate of 100 s⁻¹ exhibited a viscosity of 0.0499and 0.0764 Pa.s, respectively (Table 3). Thus, the NCL hydrogel exhibits lower viscosity due to the high water content inside the polymeric matrix. In formulation F2, the introduction of a borax cross-linker restricts molecular movement and reduces free space, resulting in increased hydrogel viscosity.

Table 3.

Viscosity profile of hydrogels at a shear rate of 100 s⁻¹.

Formulation code	Viscosity (Pa.s; mean ± SD, n = 3)
F1	0.049 ± 0.001
F2	0.078 ± 0.001
F3	0.133 ± 0.003
F4	0.257 ± 0.001
F5	0.157 ± 0.006
F6	0.559 ± 0.001
F7	0.510 ± 0.002

Increasing the TSP content from 2 to 3% w/v in formulations F2, F3, and F4 displayed the viscosities of 0.078 ± 0.001, 0.133 ± 0.003, and 0.257 ± 0.001 Pa.s, respectively. An increase in the viscosity of hydrogels was observed on increasing the concentration of TSP. The reason was attributed to the self-aggregation property of TSP and the high entanglement between the polymeric chains that led to higher viscosity of hydrogels. Increasing the borax concentration from 5 to 15 wt% in formulations F5, F4, and F6 displayed the viscosities of 0.157 ± 0.006, 0.257 ± 0.001, and 0.559 ± 0.001 Pa.s, respectively. An increase in borax content causes the creation of a highly cross-linked network, resulting in more viscous hydrogels. However, on further increasing the borax concentration (20 wt% of TSP) in formulation F7 showed a significant decrease in viscosity of hydrogels (0.510 ± 0.002 Pa.s). The high cross-link density locks polymer chains in place, reducing chain mobility and causing the material to lose its viscoelasticity—its ability to flow or stretch—leading to decreased viscosity.

The mechanical properties of hydrogel films are generally related to the structural integrity of the film.⁶⁵ Protecting the wound and preventing it from rupturing while enduring the pressures of application, handling, or break-in storage is essential to their therapeutic success. To follow skin motions, the hydrogel film should thus combine ductility, elasticity, and a reasonably high strength. However, because of hydrolysis and enzymatic degradation, a sharp decline in these qualities is anticipated to occur during application.⁶⁶ Tensile stress and % tensile strain are the typical characteristics of the hydrogel film’s mechanical properties. Tensile stress is the highest tension at which the film breaks.⁶⁷ While percentage tensile strain determines the stretchability of the film before breaking. The suggested values of tensile stress and percentage strain for skin are 2.5–16 MPa and 70%, respectively.⁶⁸ It is necessary to determine the mechanical strength of a film both in dry and wet conditions. The film may suffer wear and tear during its application, as the mechanical and structural integrity of the film changes after absorbing wound fluid.⁶⁹

The tensile stress of prepared hydrogel films (Figure 8) suggested a marked improvement with the addition of borax and an increase in the concentration of TSP up to 3% w/v. It was shown that the tensile stress of films gradually decreases from the dry state to the wet state, suggesting that the mechanical and structural integrity of the film has been substantially lowered due to the swelling of polymeric chains. In wet and dry states, formulation F1 exhibited tensile stress of 2.023 ± 0.033 MPa and 11.431 ± 0.019 MPa, respectively. This suggested that non cross-linked film exhibited the lowest tensile strength due to loose networks of polymeric chains. On cross-linking with borax, formulation F2 exhibited a significant increase (p < 0.05) in tensile stress of 4.281 ± 0.031 and 16.666 ± 0.379 MPa in wet and dry states respectively, which was higher compared to F1. The reason for this was due to the cross-linking of TSP with borax resulted in increased tensile strength. The cross-linked hydrogel exhibited higher viscosity, resulting in higher mechanical strength (Table 3). Increasing the concentration of a polymer, formulation F3 exhibited a tensile stress of 8.17 ± 0.029 and 18.709 ± 0.349 MPa, and formulation F4 exhibited a tensile stress of 9.37 ± 0.058 and 30.65 ± 0.302 MPa, in wet and dry conditions, respectively. Notably, the formulations F2–F4 possess similar ratios of TSP, borax, and glycerol, with the only difference in the total mass of hydrogel. Despite the similar ratio, the variation in tensile stress of the film was due to a change in the viscosity of the hydrogel. An increase in the viscosity of the hydrogels was observed as the TSP concentration increased from F2 to F4 (Table 3). A more viscous TSP solution typically indicates that the polysaccharide chains are more entangled, resulting in a stronger, more cohesive polymer network and, consequently, greater tensile stress. A viscous system also has more interaction points between polymer chains (e.g. hydrogen bonds, van der Waals forces), which allow stress to be distributed more efficiently throughout the material during stretching, thereby leading to higher tensile stress. Additionally, as the TSP concentration increases in the hydrogel, more hydrogen bonding and entanglement occur between polymer chains. These interactions contribute to mechanical strength, making the hydrogel stiffer and more resistant to tensile forces. Moreover, a higher TSP concentration allows for more cross-linking points, which tighten the hydrogel structure. This enhanced network resists deformation, resulting in higher tensile stress.

The tensile stress of the film increased significantly (p<0.05) with increasing the concentration of borax from 5 to 15 wt% of TSP. The tensile stress values for formulations F5, F4 and F6 under dry conditions were reported as 28.1 ± 0.096, 30.65 ± 0.302, and 38.15 ± 0.795 MPa, respectively. In contrast, the tensile stress values in a wet state were measured at 8.83 ± 0.674, 9.37 ± 0.058, and 10.75 ± 0.620 MPa, respectively.

Increasing the borax concentration to 15 wt% of TSP leads to notable improvements in the mechanical properties of the film. This enhancement can be attributed to the increased formation of borate ions, which serve as effective cross-linkers. Consequently, on increasing the concentration to 20 wt% of TSP (F7), the tensile strength of the film decreased significantly (p < 0.05). The tensile stress of formulation F7 in a dry and wet state was reported at 35.38 ± 3.14 and 7.08 ± 1.34 MPa, respectively. The decrease in tensile stress can be explained by a single, unified mechanism, namely over-cross-linking simultaneously suppresses viscoelastic flow and promotes brittleness. Excessive borax produces a densely cross-linked structure that locks polymer chains in place, resulting in a loss of viscoelastic behavior. The hydrogel is unable to dissipate applied stress through molecular rearrangement due to restricted chain mobility. Accordingly, the hydrogel film becomes brittle, and under high stress, the brittle network undergoes fracture and cracking because it is unable to dissipate applied stress due to restricted chain mobility. Similar results were obtained by Yin et al. and Geng et al. demonstrating high borax content led to reduced mechanical strength of the hydrogel film.^58,70

The % tensile strain increases with the increase in the concentration of polymer as well as the addition of borax both in dry and wet states (Figure 9). In the wet and dry states, % tensile strain of formulation F1 was 27.3% and 19.33%, while that of formulation F2 was 50.66% and 30%. This is due to the formation of rigid cross-linked networks. An increase in polymeric concentration from 2 to 3 % w/v (F2–F4), and an increase in the tensile strain of the film significantly (p < 0.05) were observed. The tensile strength was 50.66% and 30%, 52.33% and 34.6%, 65.66% and 37.53%, in wet and dry conditions, respectively. With the increase in polymeric concentration, the interconnection of polymeric chains results in more complexation of molecules, and tougher hydrogels are formed.

Increasing borax content significantly affects the % tensile strain of the films. It was observed that formulation F5 exhibited a % strain of 53.33% in the wet state and 35.77% in the dry state, while formulation F4 showed 65.66% and 37.53% in the wet and dry states, respectively. Increasing the borax content to 15 wt% of TSP (F6) resulted in a slight increase in tensile strain to 68.66% in wet states and 38.66% in dry states. Further increase in borax concentration to 20%, tensile strain decreased significantly (p < 0.05). Formulation F7 (20% borax) exhibited the lowest strain in both states (18.66% in the wet state and 2% in the dry state). In wet and dry conditions, the high brittleness of the film failed to retain water and reduced the mechanical ability of the hydrogel due to the formation of a rigid hydrogel film lacking flexibility, respectively showing poor tensile strain. Formulation F6 exhibited optimum % tensile strain both in wet and dry conditions.

Pereira et al. found that the tensile stress of an alginate-aloe vera based NCL film ranged between 40.44 and 28.66 MPa, and a % tensile strain of 5.94%–13.27% in dry conditions.³⁰ Following cross-linking, the tensile stress and strain of the hydrogel film in a dry state ranged from 42.36 to 50.91 MPa and 7.86%–13.56%, respectively. While in a wet state, the hydrogel films exhibited tensile stress and strain of 5.70–6.58 MPa and 30.19%–46.66%.

The swelling of the prepared hydrogel films is an essential criterion since the absorption of wound exudates influences drug release behavior and bio-adhesion.⁷¹ A % swelling ratio that falls within the range of 121.58%–483.25% exhibits favorable attributes to ideal dressings.⁷² Excessive swelling (>500%) tends to absorb excessive fluid, thereby compromising the maintenance of optimal moisture levels and leading to maceration. Conversely, less swelling (<100%), characterized by insufficient fluid absorption, causes a delay in the wound healing process. The swelling study of hydrogel films was performed in SWF (Figure 10). All the hydrogel films swelled immediately to various extents in SWF. The swelling eventually reached a steady state rate after about 6 h. Thereafter, the swelling progressively reduced over time, indicating apparent degradation in the hydrogel matrix.

NCL film (F1) and cross-linked film (F2) exhibited the maximum swelling of 511.29 ± 7.374% and 485.06 ± 8.633%, respectively at 4 h. On cross-linking with borax, the % swelling ratio of the film was significantly reduced (p < 0.05). NCL films demonstrated a higher swelling in comparison to cross-linked films due to the availability of more free -OH ions on the polysaccharide to interact with water. Higher viscosity and reduced free space in the cross-linked films lead to decreased water molecule diffusion and, consequently, lower swelling compared to the NCL film (Table 3). The % swelling ratio of formulations increased significantly (p < 0.05) and F2, F3, and F4 at 6 h exhibited a maximum % swelling ratio of 442.87 ± 2.644%, 551.23 ± 3.857%, and 584.17 ± 3.765% respectively. With the increase in TSP concentration, the % swelling ratio of the hydrogel matrix increased. The presence of xylose, fucose, and galactose in TSP is responsible for a hydrophilic character in the polysaccharide (Figure 2(a)).⁷³ Thus, an increase in the polysaccharide concentration resulted in the availability of these groups, inducing a more hydrophilic nature to the polysaccharide. Furthermore, with the enhancement of these groups, the availability of more free-OH groups to interact with water was also increased.

However, on increasing the borax content from 5 to 20 wt% of TSP, the % swelling ratio of films was decreased significantly (p < 0.05). The % swelling ratio of formulations F5, F4, F6, and F7 in 6 h was recorded at 648.61 ± 3.97%, 584.17 ± 3.765%, 506.04 ± 2.093%, and 439.64 ± 2.634%, respectively. It was due to the formation of stable di-diol borate complexes with the hydroxyl groups of polysaccharides, which resulted in higher cross-linking density. Due to the increase in cross-linking density, strong interactions between polymer and borax ions reduce the availability of vacant hydroxyl (-OH) groups present on the TSP to interact with water and exhibit reduced swelling. SEM analysis revealed that cross-linking with borax leads to the formation of a rigid hydrogel network (Figure 4(b)), which restricts the entry of fluid into the matrix and consequently reduces the swelling of hydrogel films. The optimal % swelling ratio shown by formulation F6 near 500% lies in the ideal range for the absorption of wound fluid. Tantiwatcharothai and Prachayawarakorn fabricated a basil seed mucilage-based hydrogel by cross-linking with varying borax concentrations.⁵¹ It was demonstrated that NCL hydrogel dressings exhibited a maximum % swelling ratio of 109% while on cross-linking with a higher content of borax, the % swelling ratio was reduced to 65%. These results showed the impact of borax cross-linking on the swelling of hydrogel films.

In vitro degradation of hydrogel films is crucial for determining their biodegradability in human tissue.⁷⁴ The degradation profile of the films is illustrated in Table 4. NCL film (F1) at 2% w/v of TSP exhibited a degradation rate of 84.9 ± 2.27%, while a similar concentration on cross-linking with borax (F2), decreased significantly (p < 0.05) and possessed a degradation rate of 81.5 ± 2.865% on day one. F1, formulated without cross-linking degraded more rapidly than F2. This was because the increased water adsorption capacity of NCL film weakened the polymeric networks of the film resulting in increased degradation. Conversely, with the introduction of borax, the formation of borate ester bonds increased the stability of the film, resulting in a lower degradation rate of cross-linked film in comparison to NCL film. It was observed that increasing the polymer concentration slows down the degradation. This behavior can be attributed to the self-aggregation property of TSP and reduced water retention capacity within the polymeric matrix, thus significantly delaying the degradation rate. The viscosity was increased with increasing the polymer concentration (Table 3) resulting in a more rigid structure which slowed down the degradation of the hydrogels.

Table 4.

In vitro degradation of hydrogel films.

No. of days	F1 (mean ± SD, n = 3)	F2 (mean ± SD, n = 3)	F3 (mean ± SD, n = 3)	F4 (mean ± SD, n = 3)	F5 (mean ± SD, n = 3)	F6 (mean ± SD, n = 3)	F7 (mean ± SD, n = 3)
0	0	0	0	0	0	0	0
1	84.9±2.27	81.5 ± 2.865	38.9±6.161	23.7 ± 1.468	35.9 ± 1.869	20.4 ± 1.834	49.1 ± 2.632
3	98.3 ± 0.566	99.7 ± 0.215	90.3 ± 2.194	60 ± 3.52	67.9 ± 3.128	50.9 ± 5.452	68.6 ± 2.719
5	98.7 ± 1.065	99.9 ± 0.005	98.7 ± 2.138	97.1 ± 1.3	96.5 ± 3.005	92.5 ± 5.557	97.3 ± 1.003
7	99.9 ± 0.56	99.9 ± 0.05	100 ± 0.004	100 ± 0.003	100 ± 0.005	98.8 ± 1.111	100 ± 0.007

The addition of borax up to certain concentrations into the polysaccharide showed a remarkable decrease in the degradation rate of the hydrogel film. It was observed that TSP concentration at 3% w/v on cross-linking with varying concentrations of borax (5–20 wt% of TSP) showed the complete degradation of film on the seventh day. It was noted that F5 (5 wt% of TSP) and F4 (10 wt% of TSP) exhibited a degradation rate of 67.9 ± 3.128% and 60 ± 3.52%, respectively, on the third day. Similarly, on increasing the borax content in formulation F6 (15 wt% of TSP), the degradation rate of 50.9 ± 5.452% was observed on the third day. Thus, an increase in the content of borax significantly (p < 0.05) slows down the degradation of hydrogel. The addition of borax to the polysaccharide led to an increase in cross-linking density, and hence, the stability of the film was enhanced. The cross-linking of borax resulted in a significant enhancement in the viscosity (Table 3) of hydrogels, leading to the slowdown of hydrogel film degradation. Further, by increasing the borax content to 20 wt% of TSP (F7), the degradation of the film was increased significantly (p < 0.05) to 68.6 ± 2.719% on the third day. Over-crosslinking can also create a rigid and brittle network prone to developing micro-cracks or fractures under stress or swelling. Once these cracks form, water can penetrate easily, accelerating degradation.

TSP has excellent antioxidant activity, which offers the added advantage of utilizing it to develop a hydrogel film to treat wounds. It was observed that TSP has approximately 80% radical scavenging efficiency which is favorable for eliminating reactive oxygen species (ROS) in wounds.⁴⁰ The scavenging rates of the pure TSP and ascorbic acid increased significantly (p < 0.05) from 23.48 ± 1.587% to 79.10 ± 3.541% and 68.83 ± 4.32% to 85.33 ± 2.88%, respectively with increasing concentrations from 2 to 10 µg/ml (Table 5). The EC₅₀ value of TSP and ascorbic acid was noted to be 6.086 mg/ml and 2.476 mg/ml. Ascorbic acid (standard) showed higher DPPH radical scavenging activity than TSP.

Table 5.

Scavenging activity of TSP and ascorbic acid.

Concentration (µg/ml)	% scavenging activity of TSP (mean ± SD, n = 3)	% scavenging activity of ascorbic acid (mean ± SD, n = 3)
0	0	0
2	23.48 ± 1.58	68.83 ± 4.32
4	39.37 ± 3.78	73.72 ± 0.86
6	46.45 ± 0.73	75.45 ± 1.58
8	63.03 ± 6.59	80.24 ± 6.33
10	79.1 ± 3.54	85.33 ± 2.88

DPPH radical scavenging of hydrogel films (Figure 11) demonstrated that all the films exhibited antioxidant activity. However, the TSP content greatly influences the antioxidant activity of the prepared films. Formulation F2, incubated at 30 min, 1, 2, 4, and 6 h shows antioxidant activity of 1.589%, 2.631%, 16.255%, 23.711%, and 26.123%, respectively. Similarly, with an increase in TSP content, formulation F3 has antioxidant efficiency of 1.864%, 5.783%, 17.214%, 26.864%, and 37.637%, respectively at 30 min, 1 h, 2 h, 4 h, and 6 h of incubation. Following this, formulation F4 exhibited a radical scavenging of 16.34%, 17.40%, 19.3%, 28.54%, and 38.34% at 30 min, 1 h, 2 h, 4 h, and 6 h, respectively. It was demonstrated from the results that an increase in TSP concentration from 2 to 3% w/v significantly increased the radical scavenging activity of the hydrogel films (p < 0.05). Similar results were obtained when varying concentrations of borax were cross-linked with TSP. It can be seen that after incubation of 6 h, the prepared films F5, F4, F6, and F7 undergo % DPPH scavenging activity of 38.34%, 43.24%, 43.64%, and 53.39%, respectively. The increase in borax content demonstrated a significant (p < 0.05) increase in the antioxidant activity of hydrogel films. More availability of boric acid (H₃BO₃) resulted in donating the H⁺ ions for the scavenging of DPPH showing the antioxidant ability of the hydrogel to a higher extent.⁷⁵ Thus, the resultant hydrogel films favor eliminating ROS generated in wounds. Emmanuel Adedeji et al. investigated the antioxidant activity of cellulose microfibrils (CMF) and guar gum-based hydrogels cross-linked with borax.⁷⁵ It was revealed that borax-treated CMF/guar gum-based hydrogels displayed higher antioxidant activity compared to the NCL CMF/guar gum hydrogel.

Self-healing hydrogels have reversible dynamic connections within the network, and the self-healing property in hydrogels allows the restoration of the original structure after an injury without compromising its functions. TSP based borax cross-linked hydrogel film was fabricated, where the di-diol borax linkages in the hydrogel have a dynamic nature that significantly improves their flexibility and self-healing properties. One of the most essential self-healing characteristics of borax cross-linked hydrogels depending on reversible borate ester linkage, undergoes rapid self-healing ability in ambient conditions, without any requirement of external triggers such as temperature and pH.⁷⁶

Results obtained from the gel-block fusion test demonstrated that the prepared cross-linked hydrogel film exhibited excellent self-healing properties. Visual observations revealed that in the presence of SWF, the segments of the films were rapidly joined. It was further validated by the diffusion of dyes observed between the interfaces of the hydrogel film after complete self-healing (Figure 12(a)). The segments began to join in the 30 s, while arrows marked in Figure 12(b) demonstrated the initiation of self-healing of the hydrogel film through weak borate ester interaction. Further, the fusion of films was progressed for 1–10 min, and complete fusion was observed at 20 min, leaving no gaps between the cut interfaces (Figure 12(b)).

It was also noted that the healing efficacy of the film increases with time. The healing efficiency of films after 12 h was significantly (p < 0.05) higher than for 30 min, 1 h, and 6 h.⁷⁷ All the cross-linked films (F2–F7) exhibited excellent self-healing on the addition of borax (Figure 12). The prepared cross-linked hydrogel films underwent outstanding self-healing due to dynamic borate ester linkage formation with the free -OH groups of the TSP (Figure 13). The complexation of borate ions and hydroxyl groups on the adjacent polysaccharide chain is very fast (30 s) and the hydrogel film starts to heal when free cis-1,2-hydroxyl groups form a complex with free borate ions.⁷⁸ The prepared hydrogel film with the addition of borax started to self-heal immediately and complete fusion of the film was observed in 20 min (Figure 12).

SEM images of self-healed hydrogel film after damage provided evidence of the self-healing ability of borax cross-linked hydrogel film (Figure 14). This remarkable property was attributed to the reversible covalent borate ester bond formation between the hydroxyl groups of the TSP and borax, facilitating dynamic reversible bonding and repair at the damaged site (Figure 13). The cross-sectional image of the hydrogel film at lower magnification demonstrated the joining of the interfaces of the hydrogel film. The visible borderline indicating assembly of interfaces was further amplified at higher magnification to observe morphological changes in the hydrogel film.

Thus, the SEM images at higher magnification highlighted the fusion of two interfaces (marked with arrows), demonstrating inter-diffusion and overlapping of polymeric networks, which contributed to the rigid and dense network formation. While the other regions exhibited a smoother appearance similar to cross-linked films. The results obtained from the SEM images revealed that the morphology of the film remained unchanged after the self-healing process, with no observable defects.

Beyond qualitative analysis through visual inspection, quantitative analysis via rheological characterization was employed to validate the self-healing behavior of the borax cross-linked hydrogel. The macroscopic interfacial joining of the hydrogel film within 20 min, as observed through visual inspection, was used as a reference time point for the quantitative assessment of self-healing via rheological characterization. An oscillatory amplitude sweep was performed on the pre-dried hydrogel to quantify the network recovery of borate-diol linkages, as observed in the film.

The oscillatory strain sweep tests at a % strain (γ = 0.1–1000%) were performed on the original cross-linked hydrogel and self-healed hydrogel after 20 min of complete healing, defined by visual observation (Figure 15). It was demonstrated that the intersection points of storage modulus (G′) and loss modulus (G″) indicated the critical strain of the hydrogel. As illustrated in Figure 15(a) and (b), the intersection point of G′ and G″ of the original cross-linked hydrogel samples was observed at γ = 110% (Figure 15(a)). Contrarily, the intersection point of G′ and G″ of self-healed hydrogel samples was observed at γ = 101% (Figure 15(b)). Thus, there was no discernible alteration at the intersection points for the self-healed hydrogel.

Based on the obtained critical strain between 100% and 200%, continuous alternative strain sweep measurements of hydrogel samples through the following strain and time sequence of 1% (20 s) → 200% (20 s) → 1% (20 s), with three consecutive cycles, were performed to evaluate the self-healing property (Figure 16(a)). It was illustrated that the linear viscoelastic range (LVER) of the hydrogel was maintained for 20 s as the G′ was higher than G″ at 1% strain, indicating a gel state of hydrogel. However, on switching the strain to 200%, the substantial decrease in G’ from ~940 to ~74.6 Pa, which was significantly lower than G″ (~168 Pa). This decline of G′ to such an extent demonstrated the gel-to-sol transition due to the disruption of the hydrogel network. When the strain was further returned to 1% after a recovery period of 20 s, the G′ and G″ were recovered close to their original values, confirming the hydrogel network reformation. This step-strain analysis was continued for three consecutive cycles, demonstrating the rapid self-healing action of the borax cross-linked network structure.²⁵

To further elaborate on the quantitative parameter for the self-healing property of hydrogel, the same step-strain protocol was followed for 200 s (Figure 16(b)). It was demonstrated that at 1% strain, the G′ was higher than G″, thus maintaining constant LVER. Conversely, at 200% strain, the G′ rapidly declined, indicating structural breakdown. On switching back to 1% strain, the fragmented hydrogel at 160 s began to recover, and within 20 s, G′ and G″ were rapidly returned to the original value at 160 s. Thus, it was observed from the results that the hydrogel rapidly recovers itself within seconds.

As a result, the characteristics of self-healing hydrogels remained predominantly unaffected, highlighting the exceptional capacity of hydrogels to heal while preserving their original characteristics. This indicates that the property of borax cross-linked hydrogel films did not alter significantly after self-healing.

Liu et al. prepared borax cross-linked hydrogel using PVA and carboxymethyl chitosan.²³ It was reported that the original hydrogel’s intersection points, G′ and G″ are clearly defined to be at γ = 0.23%. Subsequently, after 10 min of self-healing, intersection points of the self-healed hydrogel are at γ = 0.14%, indicating that the critical transition point from the gel-sol state was not altered significantly. Additionally results of continuous alternative strain sweep obtained from Nam et al. indicated that the prepared NaCl-treated (PVA), hyaluronic acid (HA), borax, and tannic acid (TA; PBHT-N) hydrogel exhibited rapid disruption of the hydrogel network at a strain of 700%.⁷⁹ Conversely, after a recovery interval, the G′ and G″ regain their initial values depicting self-healing capability of the prepared hydrogel.

The healing efficiency of the film was evaluated by tensile stress measurements (Table 6). The concentration of borax and polysaccharides greatly impacts the healing efficiency of the hydrogel film. With the increase in TSP concentration, the healing efficiency decreased. The healing efficiency of hydrogel films at 6 h of self-healing of F2, F3, and F4 was 48.207 ± 1.205%, 47.442 ± 2.012%, and 34.861 ± 2.012%, respectively. This was because the formation of denser networks and rigid structures at higher concentrations made it challenging for polymeric chains to diffuse and rejoin after damage.⁸⁰ An increase in the borax concentration improves the healing efficiency of films. The healing efficiency of formulations F5, F4, and F6 in 6 h was recorded at 34.861 ± 2.012%, 36.512 ± 0.745, and 43.949 ± 0.566%, respectively. This was attributed to the availability of more cross-linking points in the hydrogel film. While, in formulation F7 (20% borax), the healing efficiency of the film was significantly reduced to 12.892 ± 0.552%. At high borax concentrations, over-crosslinking makes the structure dense and rigid, reducing chain mobility. This decreased chain mobility leads to a reduction in self-healing ability.

Table 6.

Percentage of healing efficiency.

Time (min)	F2 (mean ± SD, n = 3)	F3 (mean ± SD, n = 3)	F4 (mean ± SD, n = 3)	F5 (mean ± SD, n = 3)	F6 (mean ± SD, n = 3)	F7 (mean ± SD, n = 3)
0	0	0	0	0	0	0
30	7.546 ± 2.454	6.56 ± 0.731	4.872 ± 0.57	4.246 ± 0.731	6.482 ± 0.138	2.526 ± 0.192
60	21.137 ± 3.839	18.127 ± 0.408	13.437 ± 0.122	12.254 ± 0.408	17.796 ± 1.009	5.637 ± 4.035
360	48.207 ± 1.205	47.442 ± 2.012	36.512 ± 0.745	34.861 ± 2.012	43.949 ± 0.566	12.892 ± 0.552
720	72.295 ± 0.728	69.559 ± 1.451	52.573 ± 1.639	49.229 ± 1.451	60.7 ± 0.7	14.894 ± 2.61

The optimization of the hydrogel film was carried out in two steps. First, the concentration of TSP was varied from 2% to 3% w/v (F2–F4), while keeping the borax concentration fixed at 10 wt% of TSP. Second, the concentration of borax was varied from 5% to 20 wt% of TSP (F4 and F5–F7), while maintaining a fixed TSP concentration of 3% w/v. Among the three formulations (F2–F4), the F4 formulation exhibited the highest WVTR and viscosity, measured at 1130.45 ± 18.87 g/m²/24 h and 0.259 Pa·s, respectively. F4 also demonstrated improved tensile stress of 9.37 ± 0.058 and 30.65 ± 0.302 MPa under wet and dry conditions, respectively, as well as tensile strain of 52.33% and 37.53% under wet and dry conditions, respectively, compared to F2 and F3. In addition, the F4 formulation showed an enhanced % swelling ratio and radical scavenging activity relative to F2 and F3. However, F4 exhibited lower healing efficiency compared to F2 and F3. Considering the WVTR, viscosity, tensile stress, tensile strain, % swelling ratio, and radical scavenging activity, F4 was selected as the optimized formulation. Subsequently, the F5–F7 formulations were developed using a fixed concentration of TSP (3% w/v) while varying the concentration of borax from 5% to 20% for further optimization.

Among the four formulations (F4 and F5–F7), the F6 formulation exhibited the highest WVTR and viscosity, measured at 1175.74 ± 7.54 g/m²/24 h and 0.558 Pa-s, respectively. F6 also demonstrated improved tensile stress of 10.75 ± 0.620 and 38.15 ± 0.795 MPa under wet and dry conditions, respectively, as well as tensile strain of 68.66% and 38.66% under wet and dry conditions, respectively, compared to F4, F5 and F7. The % swelling ratio decreased with increasing borax concentration from 5 to 20 wt%, and F6 showed a value of 506.04 ± 2.093%, which is close to 500% and lies within the ideal range for wound fluid absorption. The radical scavenging activity and healing efficiency increased with increasing borax concentration. Considering the WVTR, viscosity, tensile stress, tensile strain, and % swelling ratio, F6 was selected as the optimized formulation.

The hemolysis rate of cross-linked film was determined to assess their potential to restrict the release of hemoglobin during hemolysis. Results obtained from blood compatibility assay indicated that the positive control exhibited a 100% hemolysis rate, while the negative control possessed 0% hemolysis. The cross-linked film exhibited a 0.33 ± 0.16% hemolysis rate. The results indicated that cross-linked films were below the toxic level of hemolysis, as per international standards.⁸¹ Samples with a hemolysis rate of 0–2% are considered as non-hemolytic (safe); at a hemolysis rate of 2%–5% are slightly hemolytic; while samples with a hemolysis rate >5% are considered as hemolytic (toxic).⁸¹ Similar observations based on hemolysis test obtained by Sharma et al. demonstrated that the prepared borax cross-linked polysaccharide hydrogel exhibited the % hemolysis rate of 3.59%, demonstrating its blood compatibility in wound healing applications.⁸²

Conclusion

In the present study, a novel self-healing hydrogel film for the treatment of wounds was prepared by using TSP cross-linked with borax by the solvent-casting technique. The FTIR spectra of the hydrogel film confirmed the cross-linking of borax with TSP through boric ester bond formation. The borax cross-linked TSP based self-healing hydrogel film was characterized and optimized based on various parameters including thickness, transparency, WVTR, mechanical strength, self-healing efficiency, % swelling ratio, in vitro degradation, viscosity, and antioxidant activity. The optimized formulation comprised TSP at 3%w/v and borax (15 wt% of TSP). The hydrogel film was examined for its self-healing capacity via visual inspection, and its healing efficiency was evaluated through mechanical and rheological characterization. The results demonstrated a remarkable self-healing ability of the hydrogel film attributed to the formation of reversible borate ester bonds. Based on the biological evaluations, pure TSP and hydrogel film had remarkable DPPH scavenging activity. In conclusion, the incorporation of self-healing properties serves to enhance the mechanical and structural integrity of hydrogel films derived from polysaccharides, thereby presenting significant potential for their application in wound healing.

Footnotes

Acknowledgements

The author acknowledges Hindustan Gum & Chemical Ltd., Bhiwani, India for supplying tamarind kernel powder as a gift sample.

ORCID iD

Tapan Kumar Giri

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Data availability statement

All data and materials are included in the manuscript.

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