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 Table of Contents 
ORIGINAL ARTICLE
Year : 2021  |  Volume : 44  |  Issue : 1  |  Page : 34-41  

Healing efficacy and dermal toxicity of topical silver nanoparticles-loaded hydrogel in Sprague–Dawley rats


1 CBRN Protection and Decontamination Research Group, Division of CBRN Defence, Institute of Nuclear Medicine and Allied Sciences, Delhi, India
2 Experimental Animal Facility, Institute of Nuclear Medicine and Allied Sciences, Delhi, India
3 Department of Chemistry, Miranda House, University of Delhi, Delhi, India

Date of Submission02-Oct-2020
Date of Decision20-Jan-2021
Date of Acceptance02-Mar-2021
Date of Web Publication07-Jun-2021

Correspondence Address:
Himanshu Ojha
CBRN Protection and Decontamination Research Group, Division of CBRN Defence, Institute of Nuclear Medicine and Allied Sciences, Defence Research and Development Organisation, Brig S K Mazumdar Road, Timarpur, Delhi - 110 054
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/rpe.rpe_51_20

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  Abstract 


Traumatic wounds are the wounds that damage both the skin and the underlying tissues. Bacterial load in wounded tissue triggers elongation of the inflammation phase of wound healing. In case of excessive inflammation, the wound may undergo delayed healing that can lead to complications such as sepsis or amputation. In the present work, a hydrogel using green-synthesized silver nanoparticles (AgNPs) was synthesized and characterized in terms of homogeneity, viscosity, spreadability, excipient compatibility, etc. The hydrogels containing different percentages of AgNPs were tested for healing efficacy in full-thickness excision wound model in adult female Sprague–Dawley (SD) rats. Safety study of hydrogels was performed in SD rats as per the OECD guideline 410. The prepared hydrogels were stable for over 3 months and remain intact on parameters such as homogeneity, pH balance, good spreadability, and extrudability. Healing efficacy study showed that an increased amount of AgNPs in hydrogel enhanced wound contraction over 100% with increased tensile strength and dense aligned collagen fibers in treated wound tissues as compared to standard (silver sulfadiazine), placebo, and sham groups. Dermal toxicity studies showed that there were no signs of irritation, inflammation, and edema on the dorsum of SD rats. Besides, there was no local and systemic toxicity in hydrogel-treated groups.

Keywords: Dermal toxicity, hydrogel, silver nanoparticles, surface healing, traumatic wounds


How to cite this article:
Sharma D, Sharma N, Roy BG, Pathak M, Kumar V, Ojha H. Healing efficacy and dermal toxicity of topical silver nanoparticles-loaded hydrogel in Sprague–Dawley rats. Radiat Prot Environ 2021;44:34-41

How to cite this URL:
Sharma D, Sharma N, Roy BG, Pathak M, Kumar V, Ojha H. Healing efficacy and dermal toxicity of topical silver nanoparticles-loaded hydrogel in Sprague–Dawley rats. Radiat Prot Environ [serial online] 2021 [cited 2021 Jun 24];44:34-41. Available from: https://www.rpe.org.in/text.asp?2021/44/1/34/317949




  Introduction Top


Traumatic wounds are the wounds that damage both the skin and the underlying tissues. These can be scrapes, open cuts, or puncture wounds. Injuries that caused by knife stab, gunshots, road accidents, and animal bites are some common types of traumatic wounds.[1] Skin wound healing progresses through four general phases: hemostasis phase, inflammatory phase, proliferative phase, and remodeling phase. It involves a complex integration of signals between enzymes, growth factors, different types of cells, and cellular microenvironment.[2] Bacterial load in wounded tissue triggers elongation of the inflammation phase of wound healing. In case of excessive inflammation, the wound may undergo delayed healing that can lead to complications such as sepsis or amputation.[3],[4]

Silver and its derivatives show broad-spectrum antibacterial activity against both the Gram-positive and Gram-negative bacteria.[5] It is well established that silver modulates the inflammatory and proliferation phases of wound healing and thus enhances the epidermal repair.[6] However, long-term use of silver and its compounds may lead to both the local and systemic toxicities. Argyria is the most common harmful effect wherein the affected area undergoes irreversible skin pigmentation.[7] In recent times, silver nanoparticles (AgNPs) have emerged as an excellent substitute for silver. AgNPs have been used in the coating of biomedical devices (catheters, dental biomaterials, orthopedic implants, etc.) and wound dressings.[8]

The topical wound-healing formulation should provide a moist environment and accelerate the regeneration of tissue to improve the healing of a wound. Hydrogel consists of a three-dimensional mesh of hydrophilic polymers that can absorb and retain water up to 1000 times their equivalent weight. The mucoadhesive characteristics of hydrogels help immobilize them at the application by forming a connective layer.[9] It is also used as a carrier for delivering the active ingredient compound to the site of injury.[10]

In this background, a hydrogel was formulated using green-synthesized AgNPs. Hydrogels containing different percentages of AgNPs were prepared and their wound-healing efficacy was assessed in full-thickness excision wound model on the dorsal skin of Sprague–Dawley (SD) rats. Finally, the dermal toxicity of hydrogel was assessed as per the OECD guideline 410.


  Materials and Methods Top


Chemicals

Silver nitrate (>99.9%), carbopol R934 was procured from Qualikems Chemicals Pvt. Ltd., India. Phosphatidylcholine, triethylamine, methanol, methyl paraben, potassium dihydrogen phosphate, sodium dodecyl sulfate (SDS), tris hydrochloride, sodium bicinchoninate, sodium tartrate, sodium hydroxide, sodium bicarbonate, cupric sulfate, bovine serum albumin (BSA), direct red 80, fast green (≥85%), formaldehyde (40%), paraffin wax, ethanol, xylene, hematoxylin, eosin, disodium hydrogen phosphate, sodium chloride, and potassium chloride were procured from Merck, Germany. Silver sulfadiazine (1%) was purchased from the local market, New Delhi, India, used as a standard drug in the experiment. The water used for solution preparation was 18 Mega Ohm Milli-Q grade water derived from the Millipore water system (Model Elix 3, Millipore Corp, USA).

The preparation and characterization of AgNPs are given in Supplementary Data.

Hydrogel formulation preparation containing different amount of silver nanoparticles

The hydrogels were prepared by the thin-film hydration procedure suggested by Zhang with a few modifications.[11] Briefly, 1% w/v carbopol gel was prepared in Milli-Q water, and its pH and consistency were adjusted with 1% v/v triethylamine solution. Simultaneously, the liposome mixture was prepared with required amounts of phosphatidylcholine and mixed with required concentrations of AgNPs in a ratio of 2:1 (phosphatidylcholine: AgNPs) with continuous stirring. Four different concentrations of AgNPs, 0.2%, 0.5%, 1%, and 2%, were prepared separately and kept at 65°C for 1 h on a magnetic stirrer. These mixtures were cooled in 4°C and mixed with carbopol gel preparation with slow stirring (~70 rpm). The same procedure was followed for the placebo (vehicle) preparation without adding AgNPs. Finally, 0.2% w/w methyl paraben was added as a preservative in all the freshly prepared formulations. The characterization of AgNPs-loaded hydrogel formulation is given in Supplementary Data.

Animal description

A 2–3-month-old female SD rats, weighing 200 ± 20 g, were procured from the experimental animal facility of the “Institute of Nuclear Medicine and Allied Sciences, DRDO, Delhi,” for this experiment. All the animal experiments were approved by the ethical committee of the institution (IAEC sanction number: INM/IAEC/2018/08). All the animals were housed and cared, and tests were performed as per the CPCSEA guidelines of India.

Wound-healing study

Animals grouping

Animals were divided into seven treatment groups (n = 10) as per the approved protocol of IAEC: Group A: Control (no treatment); Group B: Placebo (vehicle); Group C: Standard drug (silver sulfadiazine 1%); Group D: 0.2% AgNPs hydrogel; Group E: 0.5% AgNPs hydrogel; Group F: 1% AgNPs hydrogel; and Group G: 2% AgNPs hydrogel.

The vehicle, standard, and hydrogel treatments were applied topically daily till healing of a wound.

Preparation of excision wound

The rats were anesthetized before wound creation by intravenous injection of ketamine hydrochloride (91 mg/kg body weight). The dorsal hair of animals was shaved with a sterile blade, and the incision site was sterilized with surgical spirit. A full-thickness excision wound of about 2 cm2 in the area was created with the help of sterile scissors and forceps.[12] The wound was cleaned using surgical spirit to avoid the occurrence of infection by the microbiome.

Rate of wound contraction and re-epithelialization

The margins of excision wound were traced at 2-day intervals on a transparent sheet. The traced wound margins were measured using a Vernier caliper with a precision of 1/20 mm [Figure S10]. The wounds were photographed periodically till the healing of wounds. Rate of wound area contraction was measured as the percentage of wound area that had healed using the following equation (1):[13]



%Rate of wound contraction



Where n denotes the number of the day of measurement.

Tensile strength measurement

The tensile strength of healed tissue was measured by tensiometer. One edge of the healed tissue was fixed with wire attached to tensiometer and weights were applied to the wire tied to the other edge. The weight (kg) required to break the healed tissue was noted to calculate the tensile strength.

Determination of protein content

The wound tissue samples were homogenized in lysis buffer (pH 7.8 containing 2% SDS and 0.1 M Tris HCl) using a ultrasonic homogenizer (Biologics, Virginia). The total protein content of the healed skin tissues was estimated by bicinchoninic acid (BCA) assay as described by Huang et al. with some modifications.[14] BSA stock (2 mg/ml) was serially diluted in the range of 20–2000 μg/ml. BCA working reagent was prepared by adding 50 parts of BCA Reagent A (solution containing 2.6 mM sodium bichinchonate, 8.2 mM sodium tartrate, 0.1 M sodium hydroxide, and 0.11 M sodium bicarbonate [pH 11.25]) in 1 part of BCA Reagent B (4% w/v cupric sulfate in Milli-Q water) (50:1). Ten microliters of each BSA standard and tissue protein sample was pipetted in triplicate into a microplate wells. Milli-Q water was used as blank and BSA standard was used to obtain a standard curve for the comparison and determination of the protein content of the samples. Two hundred microliters of BCA working reagent was added to each well and incubated at 60°C for 1 h. The microplate was kept at 25°C for 10 min and thr absorbance was taken at 562 nm in Multiskan GO microplate reader (Thermo Scientific, India).

Histopathological analysis

Histopathological studies of skin samples were carried out as per the standard protocol for elucidation of any possible adverse effect of the formulation on cellular structure, hair growth, blood vessel, and collagen assembly of the skin.[15] Animals from each group were sacrificed by euthanasia in the CO2 gas chamber after complete healing of wounds. Treated skin was biopsied and immediately washed in ice-chilled phosphate-buffered saline (PBS), weighed, and transferred to 10% neutral-buffered formalin. The tissues were processed in alcohol gradient (25%, 50%, 70%, 90%, and 100%) for their dehydration and fixed in paraffin wax. 5 μm sections of fixed tissues were cut by microtome and stained with H and E stain.

Collagen staining (picrosirius red) was also performed to analyze the reorganization of collagen fibers in the healed skin tissues. The healed skin sections were observed for any type of anatomical change under the Binocular light microscope BM-X (LMI, UK) vis-a-vis control skin section.

Dermal toxicity study of topical silver nanoparticles hydrogel

Animals grouping and treatment

As per the OECD 410 and IAEC-approved protocols, nine female SD rats, aged 2–3 months, were used to perform dermal toxicity of 2% AgNPs-loaded hydrogel. Rats were acclimatized for 7 days prior experiment. Animals were randomly kept into three groups (n = 3): treatment, vehicle, and control. About 10% of the dorsal body surface area of each of the animals was shaved for the treatment by test substance. Animals were caged individually after shaving till the completion of the experiment. The temperature of the animal experimentation room was maintained at 22°C (±2°C) and the relative humidity was 30%–70% with 12-h dark and 12-h light cycle. For feeding, conventional laboratory diets (M/s Golden Feed, Mehrauli, New Delhi, India) were provided with a continuous supply of drinking water.

The respective treatment groups were treated with the 2% AgNPs-loaded hydrogel, vehicle (placebo), and a control (PBS). The test substance was applied uniformly over the shaved dorsal skin. Sign of toxicity and body weights of rats were recorded at a regular period.

Hematological and biochemical parameters

After completion of the test period, approximately 400 μl blood samples were collected from the retro-orbital plexus of each of the animals in heparin-coated tubes with the help of hematocrit capillary. Hematological parameters, including hemoglobin, RBCs, WBCs, and platelets count, were measured with the help of Hematology Analyzer (Nihon Kohden, Celtac α). Biochemical parameters were analyzed by collecting 400 μl of blood in clot activator tubes, allowed it to clot for 30 min on ice, and centrifuged at 2500–3000 rpm for 15 min, and the serum was isolated. Serum samples were analyzed with Biochemistry Analyzer (Randox Monza, UK).

Scanning electron microscopy analysis

Skin samples of the control and 2% AgNPs hydrogel-treated rats were cut into the rectangular size of 15 mm × 20 mm, sputter with gold ions, and set inside the instrument to unveil the topological characteristics of the skin.

Statistical analysis

The results were represented as mean value with standard error. The results were analyzed statistically by one-way ANOVA with Tukey's post hoc test analysis using GraphPad Prism version-5.0 software (GraphPad Software, San Diego, California, USA). A P < 0.05 was considered statistically significant at a 95% confidence level.


  Results Top


Wound-healing studies

Rate of wound contraction and re-epithelialization

The re-epithelialization was found to be significantly higher (P < 0.05) in AgNPs hydrogel-treated wounds [Figure S11] compared to placebo (vehicle) and standard groups. Hydrogel loaded with 0.2% AgNPs produced 99.7% ± 0.2% wound contraction, 0.5% AgNPs produced 97.9% ± 1.6% contraction, and 1% AgNPs and 2% AgNPs produced 100% contraction on the 16th day, and the complete re-epithelialization was observed on the 21st day. The healing response is directly proportional to the concentration of AgNPs in the hydrogel. Maximum wound contraction has appeared in 2% AgNPs-loaded hydrogel in less time interval [Figure 1].
Figure 1: The effect of four hydrogel formulations containing different concentrations of silver nanoparticles (0.25%, 0.5%, 1%, and 2%) on a percentage of wound area contraction compared with control and standard at an interval of 2 days

Click here to view



Wounds treated with 1% and 2% AgNPs-loaded hydrogel show 100% healing of the wound and contraction was 21.3% higher when compared to standard (silver sulfadiazine). [Figure S12] shows the percentage of wound area contraction measured and analyzed by ImageJ software (ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA).



Tensile strength

The tensile strength of healing tissue was significantly higher in 2% AgNPs-loaded hydrogel treated wound as compared to other concentrations of AgNPs hydrogels as well as standard proving the assembly of well-arranged collagen fibers [Figure 2].
Figure 2: Graph depicts the tensile strength of healed wound tissue of control, standard, and silver nanoparticles hydrogel-treated Sprague–Dawley rats. The tensile strength of treated wound tissues was found to be significant at a confidence interval of 95% (P < 0.05). *denotes significant change at a confidence interval of 95 % between the values of tensile strength observed for 1 % AgNP formulation vis-à-vis observed value for control sample. **denotes significant change at a confidence interval of 95 % between the values of tensile strength observed for 2 % AgNP formulation vis-à-vis observed value for standard sample. ***denotes significant change at a confidence interval of 95 % between the values of tensile strength observed for 2 % AgNP formulation vis-à-vis observed value for control sample.

Click here to view


It was 1.14 kg/cm2 higher in 2% AgNPs hydrogel-treated wounds compared to the standard. Healing response of wound seemed to be AgNPs dose dependent.

Protein content measurement

The total protein content of the wound tissues biopsied was measured by BCA assay. Treatment with the topical AgNPs hydrogels increased the total protein level in the tissues significantly (P < 0.05) when compared to standard [Figure 3]. The total protein content in AgNPs hydrogel-treated wound skin was 357 μg/ml higher than the standard.
Figure 3: Graph depicted the total protein content of healed wound tissue of control, standard, and silver nanoparticles hydrogel-treated Sprague–Dawley rats. The protein content in treated wound tissues was found to be significant at a confidence interval of 95% (P < 0.05). *** denotes significant change at a confidence interval of 95 % between the values of Protein content observed for all samples vis-à-vis observed value for control sample

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Histopathological analysis

Skin samples from the healed excision wounds were collected after complete healing for histopathological examination. In AgNPs hydrogel-treated wounds, there was a complete and even re-epithelialization with the generation of many new hair follicles, sebaceous glands, blood vessels, no infiltration of inflammatory cells such as neutrophils, monocytes, and macrophages, higher deposition of granulation tissue, and well-arranged collagen in comparison to naturally healed and vehicle-treated skin [Figure 4]. The standard drug treatment has also shown the generation of a few hair follices an, blood vessels with less dense collagen fibers. There were no alterations in the microstructure of healed skin. In Sirius red-stained healed skin sections, the collagen fibers were pink/red, thick with parallel arrangement compared to control sections [Figure 5].
Figure 4: Representative photomicrographs of H and E-stained slides of wound skin treated with (a) control, (b) vehicle, (c) standard, (d) 0.2% silver nanoparticles hydrogel, (e) 0.5% silver nanoparticles hydrogel, (f) 1% silver nanoparticles hydrogel, and (g) 2% silver nanoparticles hydrogel (red arrows: Blood vessels; yellow arrows: Collagen fibers; black arrows: Hair follicles)

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Figure 5: Representative photomicrographs of Sirius red-stained slides of wound skin treated with (a) control, (b) vehicle, (c) standard, (d) 0.2% silver nanoparticles hydrogel, (e) 0.5% silver nanoparticles hydrogel, (f) 1% silver nanoparticles hydrogel, and (g) 2% silver nanoparticles hydrogel showing thick and parallel arranged collagen fibers (red and pink) in healed tissues

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Dermal toxicity studies of topical silver nanoparticle hydrogel

Physical observation

The AgNPs hydrogel-treated rats were weighed every 7th day, and no significant difference occurred among the groups [Figure S13]. Other parameters such as physical activity, behavior, secretions, tremors, convulsions, posture, and food and water intake were found normal in AgNPs hydrogel-treated rats vis-a-vis rats treated with vehicle. Besides, the formulation did not cause any skin irritation, edema, or inflammation [Figure S14]. The examination further proceeded toward the evaluation of hematological and biochemical parameters, which showed that the values of all the parameters for hydrogel falling within or near the reference range as cited in the literature [Table S1] and [Table S2].[16]



Scanning electron microscopy

The skin morphology of control and AgNPs hydrogel-treated rats was analyzed by scanning electron microscopy (SEM). It is shown in [Figure 6]b; there were no topographical changes seen in hydrogel-treated skin. It was clear that the treatment retains the normal integrity of the skin when compared to control skin [Figure 6]a.
Figure 6: Scanning electron microscopy image of the rat skin (a) control and (b) treated with 2% silver nanoparticle-loaded hydrogel

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  Discussion Top


Injury to the skin tissue can cause trauma to the affected person. These traumatic injuries can damage both the skin and the underlying tissues. In war areas, traumatic injuries are very common and could cause delay in wound healing. The main focus of this study was to find out the better wound-healing agent that not only heals the wound but also provides an extracellular matrix (ECM)-like cushion to the wound tissue, which can absorb moisture from the wound tissue and provide nutrients and oxygen to it. The liposomal hydrogels containing different concentrations of AgNPs were prepared to determine their wound-healing efficacy in full-thickness excision wound model in SD rats. The liposomal hydrogels have a great ability to retain a significant amount of water so that they can provide an artificial ECM after swelling. The drugs are loaded in the matrix of gel inside the pores and allowed to release it to the wound site.[17] The prepared AgNPs-loaded hydrogel formulations were physically evaluated for their homogeneity, pH, spreadability, and extrudability and found suitable for dermal applications [Table S3].

Wound-healing efficacy of prepared AgNPs hydrogels against full-thickness excision wound in SD rats was found better than the standard treatment group. Wound contraction and re-epithelialization enhanced in the AgNPs hydrogel treatment groups and reached to 100% in 1% and 2% AgNPs-loaded hydrogel on the 16th day, implying the enhanced migration and proliferation of keratinocytes from the wound margins for the formation of new epithelial tissue formation.[18] Parveen et al. in their study also showed a significant wound contraction and re-epithelialization in AgNPs cream-treated excision wounds.[19] There were a few reported studies that showed the efficacy of AgNPs in animal models in terms of wound contraction parameter. All these studies reported the monitoring of wound contraction after the continued application of AgNPs formulation for 13-15 days. In these studies the wound closure was observed around 90%; however, in none of these studies, the complete re-epithelialization of the wound was not observed.[15],[20],[21] On the other hand, some studies had conducted similar wound efficacy studies of AgNPs-loaded hydrogel formulation in the animal model over 21 days and showed the wound closure completed by 20–21 days with complete re-epithelialization.[22],[23] The similar results were found in the present study. Comparing the wound contraction in the present study for AgNPs-loaded hydrogels with the reported studies, it indicated that AgNPs-loaded hydrogels were found better. The assembly of type I collagen in the extracellular space increases the tensile strength of skin tissues significantly.[24] It plays a vital role in wound healing as it is the main component of connective tissue and provides a structural framework to the regenerating healing tissues.[25] The tensile strength of the AgNPs-loaded hydrogel-treated skin tissues was higher compared to the standard's treatment. The total protein amount is a marker that tells about the level of protein and proliferation of cells in the wound tissue. Total protein measurement is an indirect method to find out about the collagen and elastin levels in wound tissues.[26] In this study, AgNPs-loaded hydrogel topical treatment increases the entire protein content in the healed wound tissues, which signify that it increases the synthesis of proteins and hence proliferation and migration of cells to the wound site.

Histological examination revealed the complete re-epithelialization of wound tissues in the standard and AgNPs hydrogels treatment groups. However, wound tissue of untreated control group showed thin, degraded, and immature epithelial lining. Besides, in treatment groups, the collagen fibers were well arranged and cross-linked. Ye et al. have studied the effect of AgNPs dressing on infected wounds and showed that histopathological analysis of treated skin showed the formation of new, thick epithelial tissue layers in comparison to control groups where the epithelial layer was uneven.[27] Mohseni et al. in their study demonstrated the effect of AgNPs dressing on an excision wound model.[28] The results of histopathological analysis of the treated skin showed an increase in angiogenesis in the tissues, well-arranged collagen fibers, and fibroblast cells in the dermis layer of skin and intact thick epithelial layer.[28] These kinds of results were also found in other studies that performed time-bound wound healing and proved that formulations containing AgNPs accelerated the formation of new hair follicles with angiogenesis and arrangement of dense collagen fibers. However, there was an infiltration of a low number of inflammatory cells in the dermis of healed skin tissues.[15],[22] The present study is in agreement with the reported studies of Ye et al. and Mohseni et al., with no presence of inflammatory cells.[27],[28] The Sirius red stain was used in this study because it does not stain the reticular fibers and the basement membranes.[29] In the literature, it is shown that Sirius red stains the collagen fibers red. The presence of thick, elongated, and parallelly arranged collagen fibers in the healed tissue imples the significant better healing.[30] These results support the histopathological findings as obtained in the present study. The 2% AgNPs hydrogel formulation was taken into consideration for dermal toxicity testing, due to its higher wound healing efficacy. The dermal toxicity study of AgNPs-loaded hydrogel showed no toxic effects on application.


  Conclusions Top


The prepared AgNPs hydrogel was homogeneous and had physiological pH, better spreadability, extrudability, and viscosity. These hydrogel formulations were tested for their wound-healing efficacy and toxicity. The topical administration of AgNPs hydrogel has beneficial results on wound healing in the full-thickness excision wound model. The 2% AgNPs hydrogel has shown a better wound healing than the other AgNPs hydrogels, vehicle, and standard treatments, and there were no alterations in the microstructure of healed skin. The toxicity studies have shown no signs of inflammation, edema, and irritation on the skin. The above studies have proved that the developed formulations can have better wound-healing potential against combat wounds with no dermal toxicity.

Acknowledgments

The authors are thankful to Director INMAS, DRDO, for supporting this research work. One of the authors Ms. Deepti Sharma thanks UGC for providing senior research fellowship. The authors are thankful to Ms. Nidhi Darmwal and Ms. Pooja Gupta for helping sincerely in this work during their internship.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.


  Supplementary Methods Top


Preparation and characterization of silver nanoparticles

Silver nanoparticles (AgNPs) were synthesized using clove extract as per the procedure suggested by Pathak et al.[1] In brief, cloves were washed with Milli-Q water and dried in an oven. Subsequently, they were crushed finely and 1% w/v clove extract was prepared by boiling in Milli-Q water. The prepared extract was filtered using Whatman filter paper No. 1 and 22 μ syringe filter. 1 mM solution of aqueous silver nitrate (AgNO3) was prepared and stirred continuously on a magnetic stirrer with a hot plate (Tarsons SPINOT) at 60°C. After 2–3 min, 1 ml of 1% clove extract was pipetted drop-wise to the AgNO3 solution. AgNPs preparation was monitored by the change in color, Tyndall effect, and ultraviolet visible (UV-Vis) absorbance measurement on Specord 250 plus UV-Vis spectrophotometer (Analytik Jena, Germany). Size evaluation of AgNPs was carried out on the Zetasizer analyzer (Zetasizer 3600, Malvern Instruments, UK) at 25°C with a 90° scattering angle (Nanosizer/Zetasizer ZS). Transmission electron microscope (TEM, Morgagni 268D, FEI, Netherlands) and single-crystal X-ray diffraction (XRD, Oxford diffraction, XCalibur-Single Crystal XRD, UK) were used to examine the size and morphological features of freshly prepared AgNPs.

Evaluation of hydrogel formulations

The evaluation of the physical parameters of freshly prepared hydrogels before their application was assessed using the following procedures:

Physical evaluation

The developed hydrogel formulations were inspected visually for color, homogeneity, and presence of lumps before their application.[2] The pH and spreadability of the prepared AgNPs-loaded hydrogel formulations were assessed according to the procedure given by Zakaria et al.[2]

Extrudability

The extrudability of hydrogel formulations was determined by using the procedure of Rana et al.[3]

Viscosity and rheological studies

Viscosity and rheological flow of the hydrogel formulations were determined at 25°C using a rheometer of cone and plate structure with spindle CPE-41 and CP-5221(Physica MCR 101 Anton Paar, Graz, Austria). A run was started comprising changes in the angular velocity from 0.0 to 100 rpm.[4] The average readings were used to compute the viscosity. All parameters of the physical evaluation were performed in triplicate.

In vitro drug diffusion study

In vitro diffusion of AgNPs from prepared hydrogel was carried out by the dialysis membrane diffusion technique suggested by Salerno et al. with few modifications.[5] Briefly, the receptor medium (phosphate-buffered saline [PBS], 0.1 M, pH 7.4)-soaked dialysis membrane was placed on Franz's diffusion cell assembly. The formulation sample was positioned in the donor chamber under the conditions of 37°C ± 2°C and constantly stirred at 100 rpm. 1 ml aliquots of solution were pipetted out from the receptor chamber at different time intervals (0.5, 1, 2, 4, 6, 8, 10, 12, and 24 h), and instantly, the same volume of fresh medium was added to it. The aliquots were diluted suitably with PBS and measured by ultraviolet visible (UV-Vis) spectrophotometer at 430 nm.

Transmission electron microscope of prepared hydrogel

TEM imaging of formulated hydrogel was performed on TEM (Morgagni 268D, FEI, Netherlands) to check the distribution of AgNPs and the physical nature of the hydrogel.

Stability study of hydrogel

The stability study of hydrogel was conducted as per the ICH guidelines.[6] The hydrogel formulations were packed in the closed tubes and kept at differential humidity and temperature gradients, viz., 25°C ± 2°C/60% ± 5% relative humidity (RH), 30°C ± 2°C/65% ± 5% RH, 40°C ± 2°C/75% ± 5% RH for up to 3 months with periodic monitoring of its appearance, spreadability, extrudability and pH.


  Supplementary Results Top


Characterization of silver nanoparticles

Ultraviolet visible spectroscopy

UV-Vis spectroscopy is an extensive and simple method for the characterization of any metal-based nanoparticles based on their surface plasmon resonance (SPR). AgNPs electrons in the conduction band exhibit SPR due to their collective oscillations, which results in their absorption in the visible region.[7] An absorption peak between the wavelength range of 350 and 600 nm validates the presence of AgNPs.[8] UV-Vis absorption spectra of synthesized AgNPs showed a broad SPR band at 430 nm [Figure S1].



The SPR peak in the wavelength range of 400–450 nm confirmed the preparation of AgNPs.[7] In the literature, it is also reported that the absorption peak at this wavelength range indicates the spherical shape of nanoparticles.[9]

Transmission electron microscope and single-crystal X-ray diffraction

TEM is used for the characterization of the size, shape, and surface morphology of the AgNPs. It is reported in the literature that generally nanomaterials up to 100 nm have therapeutic potential.[10] In the present study, [Figure S2] confirms the size of synthesized AgNPs in the range from 35 to 52 nm and the shape of the AgNPs is spherical.



The investigations of the crystalline nature of synthesized AgNPs were performed using a single XRD. The spectra of synthesized nanoparticles were recorded from 10° to 60° [Figure S3], and it was observed that the two peaks were recorded at 38.2° and 44.5°.



On comparing the observed values with the reported XRD studies of synthesized AgNPs, it was confirmed that the synthesized AgNPs are crystalline with face-centered cubic planes 111 and 200.[11],[12] It was confirmed further by comparing with standard silver values as reported in JCPDS-PDF card 04-0783.

Zeta potential and dynamic light scattering

Zeta potential is the surface potential of nanoparticles indicated by the long-term stability of the same. The zeta potential value of prepared AgNPs was measured at −16 mV [Figure S4]. The negative value of zeta potential suggests that the synthesized nanoparticles are stable.[13] The particle size distribution of synthesized AgNPs and size were determined using the poly-dispersed nature [Figure S4]. The size varies from 4 to 550 nm. The average diameter of the AgNPs synthesized was recorded at 55.59 nm. The average diameter value as obtained in the present study was found in agreement with the reported value (60 nm) of AgNPs as reported by Thirumurugan et al.[14] Therefore, the observations were found in agreement with the reported facts.



Evaluation of silver nanoparticles hydrogel

Physical appearance

The prepared AgNPs-loaded hydrogel was yellowish in color, translucent in appearance and showed suitable property of homogeneity with no lumps [Figure S3] and [Figure S5].



pH measurements

The pH of the prepared hydrogel was measured throughout the study period and beyond it for up to 3 months. There was no significant change in the pH values over 3 months, implying the long-term stability of the prepared formulation [Table S3].

Spreadability

The spreadability of different hydrogels in the time interval of 3 months has been shown in [Table S3]. The 2% AgNPs-loaded hydrogel showed maximum spreadability followed by 1% AgNPs-loaded hydrogel and so on. It can be inferred that the prepared hydrogels fulfilled the requirements of gel-based formulations for dermatological use.



Extrudability

The extrudability of the hydrogel formulations was found in the range between 0.2 and 0.3 g that entails the ease of application of the hydrogel formulations [Table S3].

Viscosity and rheological measurement

The shear rate was investigated between 0/s and 100/s and the yield stress was (τ0) found to be 20. The flow index was found to be 0.01, indicating the pseudoplastic/shear-thinning flow of developed hydrogel. From the rheogram, it can be observed that with the increase in shear rate, the gel can be spread uniformly without applying friction [Figure S6]. The developed hydrogel formulation shows a decrease in viscosity with an increasing rate of shear strain [Figure S7].



In vitro drug diffusion study

In vitro drug diffusion study was performed for all the four formulations in phosphate buffer (pH 7.4) for 24 h, and the results are shown in [Figure S8]. It is shown in [Figure S8] that more than 80% AgNPs was released from the hydrogel for 24 h.



Transmission electron microscope imaging of prepared hydrogel

[Figure S9] shows the TEM imaging of prepared AgNPs-loaded hydrogel. It is evident from [Figure S9] that AgNPs were distributed uniformly within the formulation. The formulation was homogeneous as there were no clots observed in the TEM imaging.



Stability studies

A stability study of all the developed hydrogel formulations was carried out. The results revealed that all the hydrogel formulations were stable throughout 3 months study with uniform spreadability, pH, color, and homogeneity [Table S3].

Dermal toxicity

Body weight [Figure S13].

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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]



 

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