The particle size of synthesized BR–PolyET–NC was measured using Malvern Zetasizer instrument (Nano ZSP, Malvern Instruments, Worcestershire, United Kingdom). The working of the instrument was based on the dynamic light scattering (DLS) technique. When a beam of light passed through the particle of the samples, it scattered due to the Brownian movement or the zigzag motion of sample particles. The scattering phenomenon was observed at a scattering angle of 90° at room temperature. The NPs were extracted from the gel formulation by diluting in Milli-Q grade water at a ratio of 1:100, vortexed, and sonicated to reach homogeneous consistency. The particle size was measured in triplicate (n = 3). The surface charge on the BR–Ach–NC was also measured.

The BR-loaded nanocomposite, BR–PolyET–NC, was centrifuged at 12,000 rpm for 20 min. The aliquot of suspension was pulled out and analyzed at λmax of 422 nm in the UV spectrophotometer for free BR concentration. The amount of BR entrapped and BR loading in the nanocomposite was determined using equations given below.

A total of 5–10 mg of the BR–PolyET–NC sample was used for FTIR spectral analysis (BRUKER Corporation, Billerica, MA, United States). The sample was kept in contact with a beam of light that emerged from the FTIR instrument, and the sample was scanned over the wave number range of 4,000–500 cm⁻¹. XRD was performed for BR, alginate, chitosan, and physical mixture and optimized gel applying diffractometer (PANalytical X’pert PRO, Netherland). The measures of electric current from the x-ray tube of the XRD were 40 kV and 100 mA during the process of Cu Kb reduction using a nickel-filter. The sample scanned at 2θ angle ranged from 10 to 50°, at a speed limit of 10°/min.

Transmission electron microscope further evaluated the particle size with a TEM instrument (Techni TEM 200 Kv, Fei, Electron optics). For electron microsope examination, a drop of the diluted sample was disseminated onto the copper grid (carbon-coated) and the grid was coated with phosphotungstic acid. Finally, the developed sample was examined under the microscope.

BR release from the BR–PolyET–NC nanocmoposite was carried out in simulated intestinal fluid (SIF) along with PBS with pH of 6.8 and then compared with BR dispersion. Then, the study nanocomposite was passed through the activated dialysis membrane in PBS. The measured quantity of BR–PolyET–NC was 10 mg, and the BR gel was enwrapped in a dialysis bag with a PBS volume of 95 mL (MW; 8–12 kDa; Repligen, Waltham, MA, United States) with its ends closed and transferred into dissolution medium. The dissolution medium was maintained at a physiological temperature of 37 ± 0.5°C with uninterrupted stirring (40, 41). The requisite sample volume of 1 mL in various time frames, that is, 0, 8, 16, 24, 32, 40, and 48 h, were withdrawn, and the exact volume of fresh buffer was put back in dissolution medium. The collected samples were suitably treated (i.e., filtered and diluted) and quantified using UV-spectrophotometry. BR release from the carrier was interpreted through plotting data between %drug release versus time. Furthermore, the incurred drug release data fitted into various mathematical models, and a good model fit for BR release from the nanocomposite was predicted. The mathematical equations expressing different models are shown below.

where, K represents a kinetic rate constant, Mt./Mo indicates the fraction of drug released at time t, and n is the diffusion exponent. The n-value expresses the mechanism of drug release, herein, n ≤ 0.5 (Fickian diffusion), 0.5 < n < 1.0 (Anomalous, non-Fickian transport), and n = 1.0 (relaxation Case-II).

The BR-loaded nanocomposite, i.e., BR–PolyET–NC gel, was developed by compounding and mixing using a propeller-type mixer 1% BR–PolyET–NC in previously dissolved 1% Carbopol 940 in distilled water, which is followed by the addition of 1% propylene glycol and preservative, 0.01% methyl paraben, and, thereafter, few drops of triethanolamine to the mixture. The mixing process was continued until a clear, transparent gel has not been formed, and its pH was adjusted to make it compatible with the physiological skin pH. The development of the chitosan–alginate nanocomposite gel using polymer Carbopol 940 grades was carried out in accordance with our previously reported study with some modifications (36).

Wound contraction percentage (%woundcontraction) was determined after measuring the diameter with a scale and, thereafter, using the following formula:

The swabs excluded from the wound part of the skin on every 5th, 10th, and 15th days of wound dressing change. Using the serial dilution technique for quantitative investigation of bacterial colon count, swabs were diluted by 10-fold using normal saline. A total of 500 μL of each diluted specimen was kept on 2% agar medium spread on petri plates and incubated at physiological temperature for 24 h. After incubation time, the colony count of bacterial generation was investigated.

On 5th, 10th, and 15th days, mice from different groups were captured after removing the dead tissues from the wound site, and these tissues were taken for microscopic analysis to investigate the pattern of re-epithelization, fibrogenesis, and collagen formation.

For this analysis, after euthanizing the animals, the wounded area was carefully removed and rinsed with saline water, and the skin tissue was dissected and preserved in formalin (10%). Thereafter, sections of approximately 5 μm thickness were paraffinized using a microtome with prior treatment with xylene and ethanol. Then, the specimens were treated to remove a paraffin layer, hydrated, and stained using hematoxylin and eosin (43). The prepared slides were examined by a pathologist who was blindled to the origin of the specimens using a CX31 microscope (Olympus Corporation, Tokyo, Japan) connected with a NanoZoomer-SQ Digital slide scanner C13140-01 (Hamamatsu Photonics, Shizuoka, Japan).

The particle size of BR–PolyET–NC observed to be 71 ± 3.5 nm, and the particle population in the formulation was uniform and consistent and unimodal. The PI value of <0.5 could be considered as the monodispered system. The polydispersity index of formulation was low, that is, 0.45, which suggested homogeneity and monodispered nanosize system (Figure 2A). The surface charge on BR–PolyET–NC was reported to be +22 mV using Malvern Zetasizer Nano ZSP. The positive surface charge on the chitosan NPs was due to the presence of the cationic functional group, and it provides the colloidal stability to the NPs. The surface charges help to integrate the cell membrane in vivo (49). The high percentage of BR entrapment (91 ± 1.6%) and loading (12.5 ± 0.91%) was estimated.

Percentage release of BR from BR–PolyET–NC and BR-dispersion at predefined time points has been investigated, as shown in Figure 3. The drug release characteristics follows the biphasic pattern, where it appears that an abrupt release of the drug ahead of time abide by the sustained release model from the nanocomposite mentioned at the end of the study. In the initial 2 h of injection, a fast drug release was noticed, followed by a slow and sustained release in a controlled way for a period of 72 h. The burst release was due to adsorbed or weakly held drug particle on to theNP surface, and BR-entrapment and dispersion were estimated to be 38 ± 5.19% and 13 ± 3% in the initial 2 h at room temperature in pH 6.8. The poor release of BR from BR dispersion may be ascribed to the hydrophobicity of the drug, resulting in low aqueous solubility and dissolution. The maximum BR release from the nanocomposite after the completion of 72 h was observed to be 89.50 ± 6.9% at pH 6.8 compared to 46 ± 10% the BR release from BR dispersion. Overall, % cumulative release from BR–PolyET–NC was apparently high over drug dispersion. A higher release from the nanocomposite may be attributed to the straightaway dissolution and then drug diffusion at pH 6.8. The dissolution study reported herein is in concurrence with previous studies in the literature (50, 51).

The polysaccharide nanocomposite of alginate–chitosan, bearing Contrary surface charge or opposite surface charge, has been widely explored in drug delivery and biomedical application (52). Owing to the swelling property of polysaccharides, chitosan is more repulsive in an environment of positive charge, which thereby initiates the movement of positive ions from chitosan and negative ions from alginate in their combined complexes with calcium, with Ca²⁺ ion triggering improved drug release. The PolyNC formed primarily due to the ionic interaction of chitosan with alginate in the presence of divalent cations encapsulate protection of the surface by reducing pores in the complex and thus enhancing the controlled release of BR. The prompt solubility of chitosan at slightly acidic pH and contrarily high solubility of alginate at neutral pH in the nanocomposite concomitantly led to the sustained release of BR, which is an ideal feature for improved therapy at the wound site. Despite the good sustainability of BR through the gel system, the bioadhesive nature of chitosan prolonged the abidance of BR at the wound site, minimized rapid clearance of the drug, and thereby improved the efficacy of BR at the target (44, 53). The alginate fraction of the complexes showed sustained release and improved longer-acting BR release. Furthermore, BR release from the alginate–chitosan nanocomposite complex was enhanced by aqueous penetration into the polyelectrolyte complex, causing gel swelling, matrix erosion, and, therefore, drug diffusion into the circumferential medium. Additionally, drug release from the nanocomposite also relies on pH of the dissolution medium, physicochemical properties of the drug and nanocarriers, and nanocarrier interaction with the biological system at the wound surface (41).

The BR release of the nanocomposite was fitted to kinetic models of first order, and Higuchi, Korsmeyer–Peppas, and Hixson–Crowell were screened out as the best models of good fit. The coefficient of correlation (R2) of the selected kinetic model was estimated, and it indicatded that the model of good fit for BR release from the nanocomposite was Korsmeyer–Peppas, with highest value of R2 being 0.9632. Furthermore, the n exponent value was determined to be 0.398 (0.5 < n < 1), revealing Fickian diffusion of BR from the polymeric composite, BR–PolyET–NC (52).

XRD is an important analytical parameter to illustrate the molecular state of drug encapsulated in the nanoparticles. The physical state of the drug indicates the extent of solubility, dissolution, absorption and thus reflects the bioavailability of the drug from the formulation. The XRD peaks of BR-loaded nanocomposite clarified that some of the berberine peaks could be noticed in XRD of the nanocomposite (Figure 4A). These peaks were observed in reduced intensity at 2Θ of 14.81, 21.26°, 23.54° and 29.72°. Based on the diffraction pattern of the compound, BR has been greatly reduced in the nanocomposite, stating the amorphization or conversion into molecular BR in the formulation (37, 54, 55). All the characteristics peaks of berberine appeared in the formulation, although their intensity slashed to zero level or reduced, which affirmed the encapsulation of the alcohol and carboxylate groups or other groups of the compound berberine in the chitosan–alginate nanocomposite. Although some reduced characteristic peaks appeared at 2,293.36 cm⁻¹ due to the C=C bond stretching, at 1,639.49 cm⁻¹ due to C=O stretching, at 1,346.31 cm⁻¹ due to C–C stretching, and at 1,166.93 cm⁻¹ due to N-H stretching. The peak shown at 1,037.70 cm⁻¹ is due to C–O stretching (alcoholic and carboxylic) (Figure 4B). Thus, the FTIR spectrum of nanocomposite substantiated the drug excipients’ stability in the formulation.

Particle size and their distribution in the bulk of nanosize preparation are shown in Figure 2A. The TEM image shows that particle size is uniform, and the spherical, scattered, and agreeable size distribution was uniform and monodisperse with prior investigation with Malvern Zetasizer. A TEM study revealed the particle distribution was 71 ± 3.5 nm (Figure 2B). NP in this range is assorted with antimicrobial activity at the wound site. The SEM image revealed a spherical morphology of uniform consistency and was non-porous (Figure 2C).

The macroscopic features of various treated groups of animals with BR–PolyET–NC gel, BR gel, blank gel, and untreated/positive control for the wound healing progress and closure are shown in Figure 6A. As shown in the Figure 6B, among the entire treated group of the wound, the BR–PolyET–NC gel treated group had their wound closed at approximately the 15th day. On 5th, 10th, and 15th days, the wound closure percentage was reported to be 42 ± 5%; 85.55 ± 3.5%; and 98.22 ± 1%, respectively, in the animals in the BR–PolyET–NC gel treated group. Similarly, in the BR gel and blank gel treated groups, wound closure percentage was determined to be 68 ± 9% and 55 ± 9% on the final day of treatment. In the case of the control group (untreated), only 35 ± 8% wound closure was measured on the 15th day. The BR–PolyET–NC gel reduced the wound gap significantly compared to the BR, blank, and control gels on every 5th, 10th and 15th days (Figure 6B).

It is believed that berberine exert the anti-inflammatory action in wound healing process, which primarily relies on the signalling pathway of NF-кB, that is, silent information regulator 1 (Sirt1). The wound healing mechanism of berberine involves upregulation of the NF-κB protein Sirt1 and thus lead to decreased expression of NF-κB, inhibiting TNF-a and IL-6 expression and decreasing inflammatory protein expression. The reduced inflammatory response altered the wound microenvironement by increasing the expression of VEGF and CD31 and eventually wound healing progressed. Sirt1 also directly involves reversal of the inflammatory cells and anti-oxidant stress (58). Berberine as a wound healing agent has been recommended by various researchers. A few illustrative examples have been indicated in this study. Panda et al. recently developed BR–loaded lecithin–chitosan nanosystem for wound healing in diabetic rats. The findings of these authors suggested that combining chitosan and berberine together in a carrier gave synergistic effect toward wound healing. These authors further observed that optimum formulation worked by eliminating inflammatory cells and increasing mature collagen fibers (44).

Yin et al. developed a berberine-decorated zinc oxide colloid nanohydrogel (ZnO-Ber/H) for the wound healing activity in diabetic rats. The developed gel had an excellent wound closure rate of 92.9% by the end of the 15th day. The developed preparation ZnO-Ber/H downregulated inflammatory factors, upregulated vascular factors, and improved the re-epithelization in the wound (59).

Zhang et al. developed BR nanohydrogel of alginate. The molecular mechanism of healing relied on BR-mediated activation of Sirt1, which, in turn, promotes wound healing by reducing inflammatory factors and progressing angiogenesis (58). In addition, Samadian et al. developed a berberine-loaded electrospun cellulose acetate/gelatin mat in treating diabetic foot ulcers (60). The nanofiber produced a mean diameter of 502 ± 150 nm. In vivo studies disclosed that CA/Gel bandage dressing exhibited their antibacterial potential and improved collagen density by 8.8 ± 6.7% and received an angiogenesis score of 19.8 ± 3.8. Thus, an electrospun mat bearing BR proved to be helpful in wound healing activity in animals. Moreover, Amato et al. developed a nanogel consisted of BR, hyaluronan, and poly-L-lysine (61). The results expressed that a nanogel encapsulating BR reduced the fibroblast gap after 42 h. Zhou et al. proved that TrxR1 plays an important role in governing redox homeostasis in different pathologic conditions. The BBR works on TrxR1 was elucidated. BBR outstandingly promotes the synthesis of the extracellular matrix and remarkably damages HaCaT cells in the wound healing process. Furthermore, BBR activated TrxR1, leading to the suppression of downstream JNK signal and preventing oxidative stress and apoptosis, and thus raised cell proliferation, growth factor, amd tissue inhibitors and later lowered matrix metalloproteinase, thus promoting wound healing in diabetic rats (62). Remarkably, we observed that, compared to the BR gel, the BR–PolyET–NC gel enabled the replacement of the damaged tissues at the wound site and complete reversal of the wound area with normal tissues on the 15th day of treatment.

The antibacterial effect of BR–PolyET–NC at the wound site was compared to BR gel, blank gel, and control (untreated) is shown in Figure 6C. The bacterial colony was measured lowest for the BR–PolyET–NC gel, followed by BR gel and blank gel. The colony counts for a group of animals treated with BR–PolyET–NC gel, BR gel, and blank gel and negative control were noted. The highest and lowest numbers of colony counts on the 15th day were found to be for negative control and BR–PolyET–NC, respectively. The bacterial colony counts of the BR–PolyET–NC gel on 5th, 10th, and 15th days were recorded to be 15, 17, and 6 CFU, respectively compared to 26, 35, and 20 CFU for the BR gel. The bacterial count on these days at the wound site in the case of the BR–PolyET–NC gel was significantly lower than the BR gel (p < 0.05), indicating the potential of therapeutic use in wound healing therapy (61).

Histopathology is an important parameter for investigating the wound microenvironment and the healing process. Histolopathogical images of the wound site are expressed in Figure 7. The microscopy assessment of wound tissue calls for the measurement of macrophagic cells, neutrophil cells, fibroblast, fibrocytes, and collagen cells in the animal groups included for wound study. The microscopic evaluation of the histopathology in various treated groups are shown in Figure 6. The observations revealed that the BR–PolyET–NC treated group of animals had the best re-epithelization in the wound healing process and achieved the highest percentage of healing compared to the BR gel treated group in the entire treatment schedule of 15 days in BALB/c mice. The BR–gel treated group of animal showed better result in healing process compared to placebo gel and untreated group. The process of re-epithelization was observed to begin on 5th day and, consequently, wound closure completed on 15th day. The BR–PolyET–NC treated group showed statistically significant difference in the wound closure rate, i.e., >99% of the wound closed on the 15th day, followed by the BR gel and blank gel treated groups and the untreated group.

Microscopic studies demonstrated that the treatment outcome of the BR–PolyET–NC gel treated group was excellent and statistically significantly different compared to other groups, including the BR gel treated group. The haematoxylin and eosin (H and E) stained slides of various treated groups illustrate the re-epithelization and normalization of the wound treated skin. It was further disclosed that the number of neutrophils and macrophages significantly increased on 4th day and then their number significantly decreased on 7th day onwards for the entire group, but the decline was observed to higher in the BR–PolyET–NC treated group. The inflammatory infiltrate greatly reduced in the early phase in the BR–PolyET–NC treated animals compared to the BR gel treated group and other groups. Furthermore, an increased number of macrophages was observed until 7th day, and thereafter, it decreased on 14th day in all the groups, and the decrease was more pronounced in the BR–PolyET–NC treated group than other groups. Moreover, a steep increase in fibroblasts and fibrocytes was observed on 7th day, indicating re-epithelization, and then a sharp decline on 14th day of treatment in the BR–PolyET–NC treated group compared to other groups (63).

The epidermal layer of the control group shows evidence of stern acanthosis and follicular hyperkeratosis, as depicted in Figure 7. On the other hand, the dermal layer bearing an ample number of fibers from the extracellular matrix such as collagen fibers, disperse blood vessels, and large chronic inflammatory infiltrates such as macrophages, lymphocytes, and B and T cells. These characteristics of hispathological alteration skin tissues are a clear indicative of chronic wound. In the BR–PolyET–NC gel treated group, as shown in Figure 7, the epidermal, dermal, and hypodermal tissues showed normal characteristics after treatment compared to the control group.

Acanthosis and follicular hyperkeratosis were absent in the epidermis layer. The dermal tissue bears bundle fibers of collagen, muscle fibers, and skin appendages. Fatty tissues and blood vessels are shown in the hypodermal layer. In the BR gel and blank gel treatment groups (Figure 7), the epidermis layer indicated mild acanthosis as well as follicular hyperkeratosis. The dermal layer showed muscle fibers, bundle fibers of collagen, skin appendages, less inflammatory infiltrates, and lymphocytes. The hypodermis layer depicted subcutaneous fatty tissues and blood vessels (42).

In the early inflammatory phase of the healing process after 5th day, polymorphonuclear cells such as neutrophil infiltration trigger the recruitment of monocytes on the wound site, which later leads to the formation of macrophages. Following this, the proliferative phase begins with the formation of the granulation tissues, fibroblasts, and angiogenesis cells. The appearance of different cells such as lymphocytes, monocytes, angiogenesis, and fibroblast cells at the wound site supports the healing progress with the formation of new blood vessels and capillaries.

The numbers of monocytes and lymphocytes were largely reduced once the healing progression reached a peak level and disappeared completely at the end of the 15th day. The fibroblast cell number significantly reduced after the 10th day and the percentage of re-epithelization increased after the 7th day, leading to the formulation of epidermal cells around wound edges. As shown in the macroscopic structure of the wound covered almost completely on the 15th day. Collagen deposition in the later stage of wound healing is an important stage toward the normalization of cells, and the deposition was more pronounced in the advanced phase of healing. Overall, the results suggested that animals in the BR–PolyET–NC treated groups showed rapid recovery in the healing process involving different transition phases such as inflammatory and proliferative ones, and re-epithelization of, collagen deposition at, neovascularization of, and normalization of the wound site indicates wound closure (64, 65).

Generally, wound healing is a complex physiological process mediated through various cells such asgrowth factors, cytokines, and chemokines (1). These cells may directly or indirectly be involved in the healing process, which is triggered by various imbricating phases, i.e., hemostasis, inflammation, proliferation, and remodeling of tissues to achieve proper healing of the intended site (38). The first response to the injury site starts with hemostasis, which leads to the arresting of bleeding and hemorrhage. Thereafter, an aggregation of platelets and inflammatory cells accumulates at this site and binds with newly formed collagen tissues in the extracellular matrix (ECM). Platelets secrete proteins such as vitronectins, fibronectins, and Sphingosine-1-Phosphate which help in fibrin clots, accelerates vasocontriction, and stops bleeding. They further assist in supplying inflammatory cells, which harbor cell scaffolds that produce chemokines and cytokines that orchestrate the early phase of wound repair (66, 67).