Papaya Peel Extract-Capped Copper Oxide Nanoparticle-Based Hydrogel: Enhanced Efficacy for Wound Healing

Abstract

Food wastes with relatively lower bioactive components can be enriched by capping onto nanoparticles, capitalizing on the high surface area and positively charged metallic core. Thus, the present investigation was undertaken to cap papaya peel extract onto copper nanoparticles and develop their hydrogel for potential application in wound healing. Green papaya peel aqueous extract was used to synthesize copper nanoparticles with a mean diameter of 18 nm and a zeta potential of −16.33 mV. The effect of extract concentration, volume, temperature, pH, and reaction time was studied and optimized for the synthesis of CuNPs. Furthermore, CuNPs were loaded in HPMC K4M (0.7% w/v) and xanthan gum (0.4% w/v) hydrogel and characterized for applicability, compatibility, and stability. A wound healing study on Wistar albino rats was performed, and wound contraction was recorded. Biopsies were taken on the 10th day of the study for histological examination. This revealed significantly higher efficacy of the PAE-CuNP hydrogel in healing the wounds than the control, standard, and PAE-hydrogel. While it masked the antimicrobial properties of copper, it enhanced the wound healing efficacy, supporting the hypothesis that capping extracts onto metallic cores can potentiate the effectiveness of bioactives. Considering the demand for nonantibiotic wound dressings, this PAE-CuNP hydrogel can be a potential alternative following further validation.

1. Introduction

The process of wound healing is conventionally categorized into (a) hemostasis, (b) inflammation, (c) proliferation, and (d) remodeling phases in a chronological order. Skin restoration, integral to wound healing, is a complex process that involves diverse factors acting in concert. Considering the complex etiologies involved, a holistic approach to wound healing management is desirable. Accordingly, herbal extracts have been widely investigated for wound healing properties. However, scale-up and sustainable use of herbal resources are challenges in enabling herbal product application. Because of the higher cultivation, edible-plant-derived resources are more amenable to scale-up and are sustainable. Also, edibles have generally higher biocompatibility. Thus, there has been a lot of interest in using “food as medicine,″ and functionalization of food waste has been explored as a sustainable strategy for various biomedical applications. This waste utilization strategy also complies with the international goal to achieve “zero waste”. , Thus, it is worthwhile to explore alternative strategies for using food waste in wound healing.

Papaya (Carica papaya) is the most affordable fruit and is popularly used as a vegetable across tropical and subtropical countries. With a global production of approximately 13,822,328 t, it is one of the major sources of nutrition for these developing countries. Its peel accounts for 12% of the fruit weight and is largely considered a bio waste with a relatively low content of flavonoids/polyphenolics. , Nonetheless, the bioactives/polyphenolics of papaya peels (Table S1) can be concentrated through appropriate extraction and nanoformulation techniques for “waste to wealth” generation. Consistent with these developments, efforts have been made to develop wound healing products based on papaya peel extracts (Table S2). Papaya peels (both ripe and raw) have poor antimicrobial properties as they require concentration at the milligram level to produce any significant antimicrobial effect. − Also, these studies do not suggest the effectiveness against a broad spectrum of microbes. To improve the antimicrobial efficacy, papaya peel extract-capped silver nanoparticles have been investigated. − , However, their effect on wound healing is unclear. Interestingly, despite poor antimicrobial properties, papaya peel extract has been shown to promote wound healing. However, about 13–17 days (Table S2) were taken for complete wound healing, and there is scope to improve this. Thus, potentiating wound healing properties can potentially enable its biomedical application while promoting waste utilization.

Polyphenolics play a significant role in the wound healing effects of herbal extracts. Although the polyphenolic content of papaya peel is relatively low, developing their nanoparticles can allow the concentration of polyphenolics on the surface of the nanoparticles to aid efficacy. The pK _a values of the majority of polyphenolics are reported to be in the range of 7–9. Despite the weak acidic nature, aqueous solutions of copper sulfate have been shown to act as Lewis acid catalysts for facilitating the deprotonation of phenolic compounds. This is known to promote interactions between the anionic form of phenolics with the electropositive copper and can be expected to concentrate polyphenolics on the copper surface. Nanoparticles with both copper and silver metallic cores have been widely investigated for their wound healing properties. Although silver nanoparticles have antimicrobial properties, few recent studies have suggested relatively higher wound healing abilities of copper dressings compared to those of silver because of their superior ability to enhance angiogenesis and tissue regeneration. , Moreover, copper can be a safer alternative to silver due to its essentiality for human health in trace amounts. Thus, it is expected to be biocompatible for healthcare applications. Accordingly, copper nanoparticles have been widely reported to promote wound healing. Other than its antimicrobial properties, the role of copper in reducing inflammation, promoting angiogenesis, and increasing the supply of oxygen/nutrients to accelerate wound healing is well documented. These unique properties have attracted research attention to develop herbal extract-capped copper nanomaterials for wound healing application by capitalizing on the functionality of both copper core and phytoconstituents. Nevertheless, each herbal extract is unique in itself and is expected to have a distinct functionality following the capping onto the metallic core. Thus, papaya peel extract-capped copper oxide nanoparticles can have different functionalities that need exploration. Unlike traditional wound dressings like bandages, nanomaterial-based hydrogels can provide a wet milieu like the extracellular matrix while promoting permeation of polyphenolics to effect wound healing.

Nanomaterial functionalized hydrogels have drawn wide attention for wound-dressing applications. Since this combines the biocompatibility and moisture-retaining properties of hydrogels with the unique functionalities of nanomaterials, there has been continued effort to explore new forms of these against wound healing. The papaya peel extract-capped copper oxide nanoparticles are expected to benefit from the wound healing properties of the phytoconstituents of papaya peel extract and copper. This can be further promoted by incorporating them into an optimized hydrogel. With a three-dimensional cross-linked network, the hydrogel can absorb exudates, hold water, and provide a moist environment to promote wound healing. Xanthan gum is a natural polysaccharide that consists of a linear β-1,4-d-glucose backbone with a trisaccharide side chain. With abundant hydroxyl and carboxylic polar groups, it favors the rapid absorption of wound exudates due to its high swelling capacity, supporting wound healing. Hydroxypropyl methylcellulose (HPMC) is a hydrophilic polymer commonly used in formulations due to its swelling and gelling properties, which can form highly stable, bioadhesive hydrogels. These polymers have been used either alone or in combination with other polymers for the formation of hydrogels involving different synthetic and natural active ingredients for wound healing applications (Table S3). Since active ingredients can modulate the hydrogel properties, it is necessary to develop an optimized combination of polymers for fine-tuning the desirable properties. Hence, for papaya peel extract-capped copper nanoparticles, HPMC and xanthan gum composition was optimized for the development of a hydrogel with acceptable compatibility, pH, viscosity, extrudability, and spreadability for efficient wound healing.

2. Experimental Section

2.1. Chemicals

Copper sulfate (CuSO₄·5H2O, 98.5%, molecular weight: 249.68 g/mol) was purchased from Molychem, Maharashtra, India. Anmol Chemicals, Taloja, India, provided the hydroxyl propyl methyl cellulose (HPMC K4M) BP. SRL Pvt. Ltd., Mumbai, India, supplied the xanthan gum USP. Mueller Hinton Broth (Cat. No. M391–500 G) was purchased from Himedia. Lead acetate and hydrochloric acid were purchased from Thermo Fisher Scientific India Pvt Ltd. Sodium hydroxide was purchased from Sisco Research Laboratories Pvt Ltd. The remaining reagents and solvents were obtained from nearby vendors and were of analytical quality.

2.2. Collection and Identification of Plant Material

Papayas were collected 14 weeks after anthesis. They were unripe and firm and used for the experiment after fresh collection (before sunrise) from the Dasapur area (20°19′16.1″N 85°45′22.7″E) of Bhubaneswar, Odisha, India, in November (winter season). Botanist Prof. P.C. Panda authorized it, and the voucher specimen (2406/CBT) was deposited at the Natural Laboratory, SOA Deemed to be University, Bhubaneswar, India.

2.3. Preparation of Extract and Identification of Polyphenolics and Flavonoids

To eliminate dust particles, fresh, unripe papaya fruit was washed twice with double-distilled water. The surface water was then removed by air-drying the fruit at room temperature. The epicarp was peeled using a fruit peeler to obtain a homogeneous thickness. Peels were air-dried in the shade at room temperature until reaching a constant weight (about 2 weeks) and powdered using a grinder (Bajaj Rex DLX 750 W). Uniform-size powder (# 60) was packed in an airtight, sealable glass container and stored in a desiccator until further use.

10 g of the powder was transferred into a 250 mL beaker. Double-distilled water (200 mL) was slowly poured into the beaker with continuous stirring by a glass stirrer to get a homogeneous dispersion. The dispersion was placed in a serological water bath (RS Scientific, LBX-04S) at 60 °C for 1 h with continuous stirring at 100 rpm by using a mechanical stirrer (Neuation, iSTIR320). Upon cooling, Whatman No. 1 paper was used to filter the extract. The filtrate was concentrated using a rotavapor system (Buchi Pvt. Ltd., R-100 system) at 50 °C and 100 Pa and completely dried at 50 °C in a hot air oven (R. S. Scientific, Labx) (Figure S1). The extract was stored in an airtight glass container and labeled PAE (papaya peel aqueous extract) for further use. Further, the extracts were subjected to qualitative tests for the presence of flavonoids and polyphenolics through the alkaline reagent test and the lead acetate test, respectively (Figure S2).

2.4. Synthesis of Copper Nanoparticles (CuNPs)

The synthesis of CuNPs was performed with modification of the method of Phang et al., 2021. PAEs of 0.5%, 1%, and 5% (10–50 mL) were used to synthesize CuNPs while varying the volume (40–90 mL) of 1 mM CuSO₄·5H₂O (Table ). The experiment was performed at 800 rpm and 60 °C for 1–12 h. Briefly, CuSO₄·5H₂O (1 mM) solution and PAE were heated to 60 °C (iSTIR HP320, NeuationTechnologies, India). Then, with constant stirring at 800 rpm, the extract solution was added dropwise to the CuSO₄·5H₂O solution. The stirring was continued at 800 rpm and 60 °C, up to 12 h, and the pH was adjusted to 6.5. Initially, the nanoparticle formation was confirmed by visual color change, followed by UV absorbance (Shimadzu, UV-1900i) at 12 h. Change in color and UV spectrum of the mixture was compared with the control (1 mM CuSO₄·5H₂O and PAE solution, Figure S3). The majority of the compositions showed a relatively higher intensity of color at 3 h with minimum change afterward. To validate this, the UV absorption spectrum of the composition showing higher absorption was followed every hour for 3 h. The synthesized nanoparticles were separated using centrifugation for 15 min at 25 °C and 10,000 rpm. Double-distilled water was used three times to wash the solid portion and dried at ambient temperature. The product was packed in an airtight glass container with labeling and stored in a desiccator for further characterization.

1. PEA and 1 mM CuSO₄·5H₂O in Different Volumes for the Synthesis of CuNPs under Different Conditions.

code	PEA (%)	PEA (mL)	1 mM CuSO4·5H2O (mL)	temperature (°C)	pH
P0.5C10	0.5	10	90	25	5.2
P0.5C11		10	90	60	5.2
P01C10	1	10	90	25	4.1
P01C11		10	90	60	4.1
P05C10	5	20	40	60	4.2
P05C11		25	50	60	4.0
P05C12		25	55	60	4.1
P05C13		25	60	60	4.4
P05C14		30	55	60	3.9
P05C15		50	60	60	3.1

^aThe final pH of the mixture was adjusted to 6.5 with NaOH solution.

2.5. Characterization of CuNPs

2.5.1. Fourier Transform Infrared (FTIR) Analysis

FTIR (JASCO, FT/IR-4600) spectra of PAE, CuSO₄·5H₂O, CuNPs, xanthan gum, HPMC, nanoparticle-loaded hydrogel (PAE-CuNPs), and PAE-loaded hydrogel (PAE-H) formulations were recorded, and functional groups were identified. Briefly, an individual sample (2–4 mg) was placed on zinc selenide ATR of the spectrometer and scanned from 400 to 4000 cm^–1, and the individual spectrum was recorded.

2.5.2. Zeta Potential

The zeta potential of synthesized CuNPs was analyzed by dynamic light scattering through a Zetasizer advance pro (Model No: 10019609, Malvern Instruments Ltd., Malvern, UK) to examine the dispersibility and stability of the nanoparticles. It was equipped with a 10 mW He–Ne laser (633 nm) and operating in the face scattering mode at a temperature of 25 °C for an equilibrium time of 120 s. A sample volume of 1 mL was filled in the disposable folded capillary cell (DTS1070, Malvern Instruments Ltd., Malvern, UK), and the potential was recorded.

2.5.3. Transmission Electron Microscopy (TEM) Analysis

High-resolution TEM with selected area electron diffraction (SAED) recorded by JEOL (model JM2100) was used to evaluate the morphology, particle size, and shape of the biosynthesized CuNPs.

2.5.4. X-ray Diffraction (XRD) Analysis

An XRD instrument (Ultima IV X-ray diffractometer, Rigaku, Japan) was used to verify the crystallinity of the CuNPs and PAE-CuNPs. During the analysis of the samples, the XRD device was operated at 25 °C with a voltage of 40 kV and a current of 30 mA. The diffraction pattern was recorded over a 2θ range of 5–80° using a step size of 0.02° at a scan speed of 1 s/step.

2.5.5. Antimicrobial Study

Minimum inhibitory concentration (MIC) was determined by the microbroth dilution method using Mueller Hinton broth (Cat. No. M391-500 G) following the Clinical and Laboratory Standards Institute guidelines. , Pathogenic bacterial strains of Escherichia coli (MTCC 443) and Bacillus subtilis (MTCC 441) in the log phase (OD₆₀₀ 0.6–0.8) were added to a 96-well plate (10⁴ cells/well). Various concentrations, ranging from 5 to 0.039 mg/mL, were used against both bacteria. The plates were incubated at 37 °C for 16 h and observed for visible growth to determine the MIC. Alamar blue (Invitrogen) was added, and MIC was recorded as the lowest concentration without color change after 1 h of incubation. The wells containing the bacterial inoculum in media served as the positive control, and only media served as the negative control.

2.6. Formulation of Hydrogel and Loading of CuNPs into Hydrogel

Aqueous solutions of HPMC K4M (0.5–1% w/v) and xanthan gum (0.46–1% w/v) at different concentrations were prepared by mixing with double-distilled water using a magnetic stirrer (Model: iSTIR HP320, Neuation technologies) at 25 °C and 300 rpm (Table ). HPMC K4M solution was added slowly to the xanthan gum solution and stirred at 300 rpm until a completely homogeneous gel was formed. The pH and viscosity were examined in the process and compared with those of the marketed gel (Megaheal Hydrogel, Aristo Pharmaceutical Pvt. Ltd., Mumbai) for optimization. PAE-CuNPs were developed with a slight modification. Nanoparticles (1 mg/mL) were uniformly dispersed in distilled water and divided into two equal parts. The optimum concentration of HPMC K4M (0.7% w/v) and xanthan gum (0.4 6% w/v) was slowly added to the nanoparticle dispersion individually with continuous stirring at room temperature, 300 rpm, until complete dispersion. Similarly, HPMC K4M dispersion was added slowly to the xanthan gum dispersion, and the stirring process was continued until a completely homogeneous gel was obtained. The formulation was transferred to a screw cap glass vial with proper labeling and stored at room temperature for characterization. A similar procedure was followed for the comparative evaluation of the PAE-H formulation (1 mg/mL).

2. Formulation of the Gels and Comparison with the Marketed Formulation for the Optimization of HPMC K4M and Xanthan Gum Concentration (% w/v) for the Development of the Nanogel .

formulation code	HPMC K4M (% w/v)	xanthan gum (% w/v)	viscosity (cp at 50 rpm)	pH	spreadability (cm)
HP50XG50	0.5	0.5	1893.85 ± 5.11	7.33 ± 0.24	6.22 ± 0.44
HP50XG100	0.5	1.0	2944.63 ± 7.23	6.20 ± 0.32	5.00 ± 0.31
HP50XG70	0.5	0.7	2483.56 ± 6.77	6.50 ± 0.17	6.00 ± 0.22
HP70XG50	0.7	0.5	2522.83 ± 7.28	6.30 ± 0.25	5.81 ± 0.41
HP100XG50	1.0	0.5	2834.45 ± 5.80	6.80 ± 0.33	5.03 ± 0.30
HP70XG46	0.7	0.46	2243.50 ± 7.39	6.63 ± 0.30	5.10 ± 0.35
PAE-CuNPs	0.7	0.46	2261.33 ± 5.58	6.87 ± 0.33	5.06 ± 0.37
PAE-H	0.7	0.46	2273.30 ± 7.63	6.43 ± 0.52	5.07 ± 0.31
marketed formulation			2208.34 ± 6.54	6.02 ± 0.34	5.30 ± 0.44

^aViscosity, pH, and spreadability data are represented as mean ± S. D. (n = 3).

2.7. Characterization of PAE-CuNPs

2.7.1. Visual Appearance, Homogeneity, and pH

Visual observations were used to assess the physical appearance, homogeneity, and grittiness by placing 0.5 g of PAE-CuNPs between the thumb and index finger. Any presence of coarse particles was noted. After calibration with the approved standard buffer solutions of pH 4.0, 7.0, and 9.2, the pH of the formulations was measured using a digital pH meter (ESICO International, model alpha-01). The calibrated electrode was dipped in the formulation (15 mL) and allowed to equilibrate for 1 min before the recording of pH.

2.7.2. Viscosity

The viscosity of the PAE-CuNPs was determined by using an Anton Paar rotational viscometer (Visco QC 300). The sample (10 mL) was filled in a small-volume adaptor (SC4–27 spindle). The viscosity was measured at 25 °C in the auto mode. The flow behavior of the formulation was studied while plotting a graph.

2.7.3. Extrudability

The reported method was used to measure the extrudability of the PAE-CuNPs. Briefly, the hydrogel was filled in a clean collapsible aluminum tube (1 ounce, 5 mm tip aperture) and sealed by crimping the end. Then, 50 g of the weight was applied at the bottom of the tube, and the release of the hydrogel through the tip was weighed. The % extruded gel was calculated using the following equation:

$$\mathrm{Extrudability}=\frac{\mathrm{Applied}\mathrm{weight}\mathrm{to}\mathrm{extrude}\mathrm{hydrogel}\mathrm{from}\mathrm{the}\mathrm{tube}(\mathrm{g})}{\mathrm{Area}({\mathrm{cm}}^2)}$$

2.7.4. Spreadability

The spreadability was determined by the parallel plate method. , 1 g of the optimized hydrogel was placed between the glass plates (20 × 20 cm). For 1 min, a 100 g weight was applied to the upper glass slide to allow the formation of a uniform film of the gel between the slides. The diameter of the gel was measured.

2.7.5. Stability Study

The accelerated stability of PAE-CuNPs was assessed according to the ICH Q1A­(R2) guidelines. Sample-packed collapsible aluminum tubes in triplicate were stored (40 ± 5 °C; 75 ± 5% RH) in a stability chamber (Hally Instruments, Mumbai, India). Phase separation, pH, viscosity, and spreadability were analyzed after 0, 3, and 6 months of stability.

2.8. In Vivo Study

2.8.1. Skin Irritation Study

This study was conducted in accordance with ARRIVE (Animal Research Reporting of In Vivo Experiments) guidelines and approved by the IAEC (IAEC/SPS/SOA/109/2022) of Siksha O Anusandhan Deemed to be University, Bhubaneswar. Groups of four (n = 6) Sprague–Dawley rats (180–200 g) were taken and studied as per OECD guidelines 404. Group I (control), group II (standard, 0.8% formalin), group III (PAE-H), and group IV (PAE-CuNPs) were received. An area of 2 cm² was shaved with a depilatory cream (Reckitt Benckiser, Inc., UK) for each rat and caged individually. The animals were left undisturbed for 24 h. An aqueous solution of formalin and sterile water soaked in sterile cotton was topically applied as a standard irritant and control. The developed samples (0.5 g) were evenly spread once/day on the shaved areas of the test rats. The sites were visually observed and photographed at 24, 48, and 72 h after application. Erythema and edema were observed and scored using the Draize system. A score of ≥1 in any category indicated skin irritation. ,,

2.8.2. In Vivo Wound Healing Activity

This protocol was compiled with ARRIVE guidelines and approved by the IAEC (IAEC/SPS/SOA/109/2022) of Siksha O Anusandhan Deemed to be University, Bhubaneswar. Twenty-four healthy Wister albino rats weighing 180–200 g were acclimatized in the departmental animal house at 25 ± 2 °C, relative humidity (RH, 44–56%), and a 12 h light/dark cycle for 1 week before and during the experiments. Water and a regular rat pellet diet were provided to the animals. To study the effect of the prepared nanogel in the excision model, animals were divided into groups of four (n = 6)group I: left untreated and considered as the control; group II: standard and treated with povidone iodine ointment USP (5% w/w, 100 mg, once daily topically, Intadine, Intas Pharmaceuticals Ltd., India); group III: PAE-H (100 mg, once daily topically); and group IV: PAE-CuNPs (100 mg, once daily topically). The dorsum part of the animals was shaved using a depilatory cream (Reckitt Benckiser, Inc., UK), and ketamine hydrochloride (50 mg/kg, ip, body weight) was used to induce anesthesia. Making an impression on the shaved dorsal region, the area of the wound was marked. A full-thickness excision circular wound was made along the marking using forceps, a surgical blade, and sharp/pointed scissors. All of the treatments were given once daily for 10 days. Wound contraction and epithelialization at 0, 4, 6, 8, and 10 days were photographed and measured, and the percent contraction was calculated. ,

2.8.3. Histopathological Examination

To study the epithelialization and evidence of granuloma, dysplasia, and growth of granular tissues in the skin under examination, on day 10, under light ketamine anesthesia, tissues were excised from the wounds of the control and PAE-CuNPs groups and fixed in 10% formalin (buffered neutral, Sigma-Aldrich, Michigan, USA), and paraffin sections (5–10 μ, microtome and Leica RM2125 RTS, Leica Biosystems, India) were prepared. Paraffin was removed, and the tissues were stained with hematoxylin and eosin (Sigma-Aldrich, St. Louis, Missouri, USA). Under an electric light microscope (Olympus BH2, Tokyo, Japan), a histological study was performed.

2.9. Statistical Analysis

The data collected were statistically analyzed using the GraphPad Prism version 5.0 (GraphPad Software, San Diego, CA, USA). The obtained data were presented as mean ± standard deviation (S. D.). One-way ANOVA followed by t test was used to test the levels of significance of difference.

3. Results and Discussion

3.1. Synthesis of CuNPs from PAE

Fresh papaya peel was washed with double-distilled water, dried at room temperature, and ground into uniform-sized powder (# 60). PAE was prepared by decoction at 60 °C and dried using a rotavapor system, followed by a hot air oven at 50 °C (Figure S1). The PAE tested positive for the presence of flavonoids and polyphenolics (Figure S2). Because the level of phytocomponents and their physicochemical attributes can modulate the CuNPs, their concentration was varied along with volume, temperature, pH, and reaction time.

No color change was observed with a lower concentration of PAE (0.5%) at 25 and 60 °C, 800 rpm after 12 h. Consequently, it did not show any relevant UV absorption peak (Figure A, P0.5C10 and P0.5C11). Further, an increase in concentration (1%) while keeping the volume of PAE (10 mL) constant at 25 °C also showed no change in color or absorbance (Figure S4, P01C10). Although a light-brown color was observed with a UV absorption peak at 294 nm when the temperature was raised to 60 °C (Figures S4 and A, P01C11), the yield of CuNPs was insignificant. Encouraged by this, subsequent experiments were conducted at 60 °C, while other parameters were varied (Table and Figure S4). Investigations with different combinations revealed that 5% w/v PAE, when used in a ratio of 25:60 (volume of PAE: CuSO₄ solution) for a reaction time of 3 h, resulted in a high peak intensity and a blue shift of the absorption peak (Figures B and ). Further increases in the reaction time failed to improve the absorption properties or yield. Flavonoids and phenolics show a characteristic peak at around 240–295 nm due to the benzoyl ring system. The PAE (Figure S3) showed peaks around the 240–295 nm region, suggesting the presence of flavonoids and phenolics. Copper sulfate is characterized by a broad and weak absorption band around 800 nm. While some studies have shown the surface plasmon resonance (SPR) effect of CuNPs with a band around 562–573 nm, , others have reported the lack of this effect due to the proneness of copper to oxidation. Accordingly, herbal extract-capped copper oxide nanoparticles have been reported without this characteristic band and instead have shown peaks around 234–255 nm. A similar UV–vis spectrum was observed in this study, with characteristic peaks within 235–280 nm. Thus, the SPR effect seems to be minimized in these nanoparticles. Considering that UV nanoparticle absorption is primarily a surface phenomenon, and minimum SPR is observed for these nanoparticles, the absorbance can be predominantly attributed to the flavonoids and phenolics capped on the surface. An increase in the capping agent level can increase nanoparticle absorbance while contributing to stability and efficacy. , Hence, the higher absorption observed in these nanoparticles can be related to the concentrations of flavonoids and phenolics on the surface. Accordingly, the composition showing the highest absorption in these peaks was considered the optimum composition for further investigation.

1.

(A) UV spectra of the CuNPs prepared under different conditions. (B) Time-dependent spectral analysis of synthesized CuNPs at 1, 2, and 3 h of synthesis.

2.

Graphical representation of the green synthesis of CuNPs using PAE.

3.2. FTIR

Possible phytochemicals in PAE responsible for the reduction and stabilization of CuNPs were identified by FTIR. IR spectra of PAE, CuSO₄, and biosynthesized CuNPs are shown in Figure . The IR spectrum of the PAE at 3250 cm^–1 represents broad O–H stretching. Peaks corresponding to aromatic groups were observed at 2927 cm^–1. Also, peaks corresponding to CO (1591 cm^–1) stretching and C–O stretching (1030 cm^–1), which are characteristics of phenolic and acidic groups, were observed for PAE. Although these peaks cannot be assigned to a single phytocomponent, taken together, these peaks reflect characteristic peaks of phenolics and flavonoids. , The IR spectrum of CuSO₄·5H₂O showed a characteristic broad absorption peak for the water molecule at 3104 cm^–1 (O–H stretching) and 1666 cm^–1 (O–H bending). Also, it showed a strong peak at 861 cm^–1, which is characteristic of its sulfate group. This peak disappeared in the FTIR spectra of CuNPs, suggesting replacement of sulfate groups and reduction. Although characteristic peaks of phenolic/flavonoid compounds were also found in the spectrum of CuNPs, peaks corresponding to O–H stretching and C–O stretching in the PAE were shifted to lower frequencies at 3242 and 1007 cm^–1, respectively, suggesting polar interactions between PAE and copper in the CuNPs. Moreover, the intensity of these peaks was reduced in the CuNPs, which supports the capping of flavonoids and phenolics on the metallic core. Further, a medium peak at 920 cm^–1 in the FTIR spectrum of the extract can be attributed to the phenolic OH out-of-plane bending. Interestingly, this was shifted to 955 cm^–1 with an increase in the intensity of the FTIR spectrum of CuNPs. Since this type of shifting is usually attributed to metal–oxygen dative bonds, involvement of coordination interaction in the formation of CuNPs cannot be ruled out. Thus, the FTIR data suggest possible involvement of multiple types of interactions between phytocomponents and the metallic core in the CuNPs.

3.

FTIR spectra of biosynthesized CuNPs from the PAE. FTIR spectra of (A) PAE, (B) pure CuSO₄·5H₂O, and (C) CuNPs. All of the ingredients were scanned with a diamond ATR spectrophotometer from 500 to 4000 cm^–1 to check the development of CuNPs and compatibility with hydrogel excipients.

3.3. Zeta Potential

The zeta potential of CuNPs was determined by a Zetasizer Nano-ZS. The biosynthesized CuNPs showed a zeta potential of −16.33 mV (Figure ). The zeta potential significantly affects the stability of the nanosuspension after suitable dilution with distilled water. The ideal values between +40 and −40 mV guarantee proper dispersibility without aggregation or flocculation. The obtained zeta potential of CuNPs implies a strong repellent force among the particles and prevents aggregation. This indicates the sufficient stability of the CuNPs biosynthesized from PAE. Metallic copper is positively charged, whereas many phenolics/flavonoids rich in hydroxyl and carboxylic groups can be anionic. Thus, the negative potential of the CuNPs can be attributed to concentrated phenolics and flavonoids on the surface of the copper. This finding agrees with the FTIR and UV visible spectra (Figures and ), which suggested the presence of these components on the CuNP surface.

4.

Graph of the zeta potential of the CuNPs. The value −16.33 mV indicates the formation of stable NPs.

3.4. TEM

TEM (JEOL, JM2100) was used to investigate the morphology, particle size, size distribution, and shape of the CuNPs. The particle size of the synthesized CuNPs varied from 13.25 to 22.49 nm, with an average particle size of 18.01 ± 3.55 nm (Figure A). These smaller particle formations may be due to the reducing and capping abilities of the biomolecules in PAE. The narrow SD value indicates the uniformity of particle size formation. The particles are spherical in shape and without any aggregation (Figure A). This is in good agreement with the zeta potential value, confirming the stability of the nanoparticles. The SAED pattern illustrates the amorphous nature of the biosynthesized CuNPs (Figure B).

5.

TEM and SAED images of the biosynthesized CuNPs. (A) TEM image of CuNPs observed at 20 nm showed an average particle size of 18.01 ± 3.55 nm with a spherical shape without aggregations. (B) SAED image confirms the amorphous structure of CuNPs.

3.5. XRD

The XRD analyzes the atomic structure of the material and identifies the size of the crystalline material. , The reported XRD pattern of pure CuSO₄·5H₂O showed sharp, distinct peaks at 2θ: 16.146°, 18.746°, and 48.476° (inset of Figure ). This indicates the crystalline nature of CuSO₄·5H₂O. The identified powder diffraction file (pdf) database number “01-077-1900” of this XRD result indicated the purity of CuSO₄·5H₂O. The CuNPs synthesized from PAE showed two broad peaks in the 2θ range of 10–25° (Figure A). It suggested the amorphous nature of the CuNPs synthesized from PAE. Further, the relatively low percentage of crystallinity (27.43%) supported the transition to the amorphous phase. This is in good agreement with the TEM and SAED pattern data, which indicates the amorphous nature. Based on prior reports, this can be attributed to interference with the regular arrangement of atoms in the CuNP lattice by the extract components, which hinder the ordered crystalline structure. − Similarly, two broad humps were observed in the CuNP loaded hydrogel in the 2θ range of 10°–42°. This suggests that there may be a short-range order of the atoms without a well-defined crystal lattice. ,, This reveals the maintenance of the amorphous state in the hydrogel, which is suitable for the development of aqueous-based formulations for topical application.

6.

XRD diffractograms of (A) biosynthesized CuNPs and (B) PAE-CuNPs. The inset reveals the strong crystalline diffraction pattern of the pure CuSO₄·5H₂O.

3.6. Antibacterial Study

The antibacterial activity of PAE and CuNPs against the pathogenic bacterial strains of E. coli and B. subtilis was determined by a broth dilution assay and confirmed by an Alamar assay (Figure S7). Concentrations (5–0.039 mg/mL) were used against selected pathogenic bacterial strains. Poor antimicrobial activity is indicated by the MIC of >1 mg/mL against both strains. It demonstrated that E. coli and B. subtilis are not sensitive to PAE and CuNPs.

3.7. Formulation of PAE-CuNPs

Hydrogel is an efficient vehicle for topical drug delivery. − The cross-linked three-dimensional structure and hydrophilic polymer network of hydrogels allow the efficient loading of nanoparticles for prolonged action. This prompted us to develop a hydrogel of the CuNPs. FDA-approved HPMC K4M and xanthan gum have been shown to form a stable hydrogel for topical drug delivery. − Their efficient bioadhesiveness and biocompatibility are conducive to the development of an effective hydrogel of CuNPs. Hence, an attempt was made to formulate a topical hydrogel with HPMC K4M (0.5–1% w/v) and xanthan gum (0.46–1% w/v). Out of six formulation trials (Table ), HP70XG46 (HPMC K4M 0.7% w/v and xanthan gum 0.46% w/v) was chosen as it showed optimum viscosity and pH as compared with the marketed product. The CuNPs and PAE were loaded into the optimized hydrogel to achieve the PAE-CuNPs and PAE-H. Further, it was characterized and compared with the marketed hydrogel formulation used for wound healing.

3.8. Characterization of PAE-CuNPs

The FTIR spectra were recorded to analyze the chemical compatibility of CuNPs and excipients (Figure S5). The IR spectra of xanthan gum are shown in Figure S5B. The bands associated with O–H stretching vibrations with intermolecular H-bonding and C–H stretching were observed at 3359 and 2889 cm^–1. The symmetrical CO stretching vibration at 1414 cm^–1 was produced by the carboxylate anion (COO) of xanthan gum. The other peak, measured at 1742 cm^–1, was assigned to the xanthan gum’s free carboxylic acid or ester groups. , The observed O–H group (3398 cm^–1) and C–H group (2921 cm^–1) represented the stretching vibrations of HPMC (Figure S5C). Also, it showed −CH₂ groups (1456 cm^–1), vibrations of the O–H bending groups (1370 cm^–1), and −CO groups (1048 cm^–1), similar to earlier report. The developed PAE-CuNPs showed characteristic peaks of O–H stretching (3338 cm^–1), CC stretching (1633 cm^–1), and −CO stretching (1080 cm^–1). These results indicate a slight shifting of the peaks. These FTIR spectra of the developed formulations confirm the physical cross-linking in hydrogel networks. Other peaks are the characteristic peaks of the excipients. No new peaks and no major shifting of the peaks indicated compatibility with excipients in the formulation.

3.8.1. Visual Appearance, Homogeneity, and pH

The white color of the hydrogel base turned dark-greenish following the loading of the PAE-CuNPs (Figure S6). Absence of any grittiness or lumps suggested homogeneity of the hydrogel. Also, its pH was found to be 6.87 ± 0.33, indicating compatibility for topical application.

3.8.2. Viscosity, Spreadability, and Extrudability

Viscosity has been used as a parameter to optimize the retentive ability of the topical gel during formulation development. Although higher viscosity may aid higher retention on skin, it may compromise the spreadability. Prior studies have suggested a viscosity of <4000 cps for topical gel products. , Accordingly, the marketed product that showed viscosity in the range of 2200 cps and spreadability in the range of 5.3 cm was taken as a control to develop an optimum gel. HP70XG46, showing (Table ) viscosity similar to that of the marketed formulation, was taken as the base or blank hydrogel. Loading of PAE-CuNPs or PAE-H did not alter the viscosity significantly (p < 0.05). Thus, both PAE-CuNPs and PAE-H hydrogels showed optimum viscosity comparable to that of the marketed formulation (Table ). This indicated that the inclusion of NPs did not alter the flow behavior of the gel. The viscosity of PAE-CuNPs varied inversely with the shear rate, resulting in shear thinning or pseudoplastic behavior (Figure A). This behavior demonstrates that the PAE-CuNPs hydrogel is a non-Newtonian fluid. The shear rate and stress plot (Figure B) of PAE-CuNPs represent an increase in shear stress with a change in shear rate. This result indicates higher retention in the target site and promising skin applicability.

7.

Flow behavior of the PAE-CuNP hydrogel. Viscosity of the hydrogel was determined as a function of the shear rate (s^–1) using an Anton Paar viscometer at 25 °C. (A) The viscosity graph of PAE-CuNPs represents the decrease in viscosity with an increase in shear rate. (B) The data of PAE-CuNPs represent an increase in shear stress with a change in shear rate. Values are represented as mean ± S. D. (n = 3).

The optimum viscosity also supported a suitable spreadability. Both PAE-CuNPs and PAE-H hydrogels showed spreadability in the range of 5 cm, which was close to that of the marketed product. This was also in the desirable range for topical gels and shows its suitability for uniform distribution.

Further, the extrudability of the PAE-CuNP was found to be 87.45 ± 2.34%. This complies with the desirable extrudability of the topical gel and suggests ease of removal of products from the tube for its topical application. Thus, the optimum extrudability, spreadability, and viscosity indicate the amenability of the PAE-CuNP hydrogel for convenient administration, retention on the wound surface, and uniform spreading across the affected area.

3.8.3. Stability Study

According to the ICH Q1A­(R2) guidelines, an accelerated stability study was carried out. Phase separation, pH, viscosity, and spreadability were the criteria employed to assess the PAE-CuNP hydrogel. After 0, 3, and 6 months of the stability period of the sample, all of the above parameters were analyzed and are reported in Table . There was no significant difference (p > 0.05) in the pH after the six months stability period as compared with the initial formulation. This indicates no chemical alteration. Because of the accelerated conditions (40 °C) selected for the study, the viscosity decreased to the range of 2000 cps. Expectedly, this increased the spreadability to the range of 6.5 cm. Nonetheless, these parameters are within the acceptable range. However, further long-term stability studies at room temperature can be taken up to estimate the shelf life of the hydrogel. These findings confirm the stable nature of the PAE-CuNP hydrogel.

3. Accelerated Stability Data of the PAE-CuNP Hydrogel at Different Time Points.

parameters	0 month	3rd month	6th month
phase separation	no phase separation	no phase separation	no phase separation
pH	6.87 ± 0.33	6.84 ± 0.12	6.85 ± 0.09
viscosity (cps)	2261.33 ± 5.58	2083.54 ± 6.43	2067.62 ± 5.72
spreadability (cm)	5.06 ± 0.37	6.53 ± 0.37	6.64 ± 0.73

^aNo significant difference (p > 0.05) compared to the initial month. The standard value of viscosity and spreadability for the hydrogel is 2000–4000 cps and 5–7 cm, respectively. Each data point is represented as mean ± S. D. (n = 3).

3.9. In Vivo Study

3.9.1. Skin Irritation Study

A three-day skin irritation test was carried out to assess the risk of skin irritation on direct physical contact with the PAE-CuNPs. In this study, male Sprague–Dawley rats’ shaved skin after application was observed for irritation, which was scored, and photographs were taken (Figure ). In consonance with its irritating properties, formalin treatment produced erythema and edema with a Draize scoring of 1. Meanwhile, PAE-CuNPs and PAE-H showed a Draize score of 0. There were no observable indicators of erythema (redness) or edema (swelling brought on by too much fluid being trapped in the body’s tissues). The results showed that the formulation was nonirritant and safe for topical application.

8.

Skin compatibility studies of the PAE-H and PAE-CuNPs compared with the control and standard (0.8% formalin) using a male Sprague–Dawley rat model. The study was performed for 3 days; inflammation was observed after each application, which was scored, and photographs were taken.

3.9.2. Wound Healing Study

An excision wound model was adopted to determine the efficacy of the PAE-CuNPs. The wound healing study was performed on a rat model (n = 6). The healing potential of the test was determined by physical observation and a histopathological analysis of the wound retraction. The photographs of the wound healing activity of the PAE-CuNPs and PAE-H with the positive control and negative control are shown in Figure A. During the 10 day trial period, the study focused on evaluating the wound healing potential of PAE-CuNPs. The progress was monitored by measuring the percentage of wound contraction, as depicted in Figure B. The wound closure percentage was assessed by comparing the initial wound size. Intriguingly, on the fourth day following the injury, the control group displayed a minor reduction in the incision area, around 29.29%. In contrast, the group treated with the PAE-CuNPs demonstrated rapid healing, with a substantial ∼62.20% (p < 0.01) wound contraction within the same time frame. As the trial progressed, the test group (PAE-CuNPs) exhibited continued improvement. The rats treated with the PAE-CuNP hydrogel showed significant (p < 0.01) wound closure within 10 days as compared with the control and PAE-H. PAE-CuNPs showed 2.51-fold more wound contraction than the negative control and ∼1.75-fold wound contraction than the positive control and PAE-H. By the 10th day, the topical application of PAE-CuNPs resulted in complete wound recovery, while the control group lagged behind with only ∼57% recovery.

9.

Photographs of the wound healing experiment performed using a rat model (A); photographs of the wound healing activities of the control (positive and negative) and test groups. The photographs were taken at 0, 4, 6, 8, and 10 days. (B) The rate of wound contraction of all of the groups was calculated in all reported days (average ± S. D., n = 6). The level of significance is denoted as ***p < 0.01.

The mode of wound healing was also supported by histopathological findings that showed re-epithelization, angiogenesis, collagen deposition, and granulation tissue formation on the wound, resulting in wound contraction and healing (Figure ). The uncovered epithelium on the wound borders with vacuolation was observed in diseased control animals. Hemorrhage and fibrin were indicators of immaturity in the granulation tissue that filled the incision (Figure A). Sections of granulation tissue obtained from the standard drug (povidone iodine ointment)-treated animals showed a significant rise in collagen deposition, a few macrophages, and more fibroblasts (Figure B). However, high collagen deposition and a significant decrease in infiltration of macrophages were observed in the wound tissue section treated with only the extract (Figure C). This was improved by the application of the PAE-CuNP hydrogel, where it showed re-epithelization, angiogenesis, collagen deposition, and mature granulation tissue. Overall, the evaluation of PAE-CuNPs showed a higher healing performance than the control (Figure D). Our results are in agreement with other studies that showed less inflammatory cells, more collagen, and increased angiogenesis in wounds treated with plant extracts.

10.

Microphotograph of hematoxylin/eosin staining of the healed tissue after 10 days: (A) negative control, (B) positive control (standard), (C) PAE-H, and (D) PAE-CuNPs. The black arrows indicate epithelial tissues, green arrows indicate blood capillaries, and orange arrows indicate granular tissues. Magnification is 250×.

The CUNPs have been widely reported to have antimicrobial properties. The antimicrobial effect of copper and products based on this can be ascribed to the interaction with bacterial cell membranes. The electropositive copper is perceived to interact through electrostatic modes with the predominantly electronegative charge of the bacterial cell. At pH above 4, the phosphoryl and carboxylate substituents on the outer cell envelope macromolecules of bacteria exposed to the extracellular environment remain ionized. Thus, the bacterial cell surfaces possess a net negative charge, and electrostatic interaction of copper ions (Cu⁺ and Cu²⁺) with these groups causes membrane rupture and cell death. , Interestingly, CuNP showed poor antimicrobial potency with high MIC (>1 mg/mL, Figure S7), similar to those of PAE. Thus, capping seems to have masked the antimicrobial potency. Since the release of soluble copper from nanoparticles is essential for the bactericidal effect, it is possible that capping with PAE has influenced the CuNP. This is also supported by the negative zeta potential of the PAE-CuNPs, which indicates that phytocomponents (phenolics and flavonoids) capped on the CuNPs have imparted an overall negative electrostatic charge by masking the positively charged copper metallic core of the nanoparticle. Since CuNPs were developed using water extracts (PAE), the hydrophilic flavonoids/phenolics are likely on the surface of these nanoparticles. Many of these have poor direct antimicrobial properties. , Although a detailed structure–activity relationship for antimicrobial activity has not been established, it has been observed that hydrophilic flavonoids have lower potency than lipophilic flavonoids.

Despite the poor antimicrobial properties, CuNP-based gels showed enhanced wound healing properties. Wound healing can occur independent of antimicrobial properties by modulation of factors related to the immune system and tissue generation. Thus, it is likely that the enrichment of polyphenolics and flavonoids on the nanoparticle surface has modulated host factors associated with wound healing. However, further studies are necessary to validate this. Moreover, the contribution of the physical barrier (protective effect) of the hydrogel on the wound surface against infection or wound aggravation cannot be ruled out. Traditionally, antibiotics have been used for wound dressing. However, there is a growing concern that long-term or improper use of antibiotics for wound dressings promotes multidrug resistance in bacteria. Accordingly, wound dressings that do not contain antibiotics and antimicrobials have received the attention of the World Union of Wound Healing Societies. Thus, in the meeting reports, hydrogel has been proposed as one such nonmedicated wound dressing that sequesters bacteria based on physical mechanisms without antibiotics. Accordingly, the PAE-CuNP-based hydrogel can be studied further to validate its utility for wound healing applications.

4. Conclusions

To add value to papaya peel waste, the extract-capped CuNP was synthesized and developed as a hydrogel for application as wound dressings. While demonstrating good skin compatibility, the PAE-CuNP hydrogel was significantly more effective in healing the wounds completely within one and a half weeks. The nanoparticles seem to have contributed to the higher efficacy of the PAE-CuNPs. This study also indicates that the formation of a nanoparticle (18.01 ± 3.55 nm) allowed enrichment of the bioactives on the surface of the CuNP. Thus, despite the poor antimicrobial properties of PAE-CuNPs, the hydrogel showed complete wound healing in relatively less time compared to the positive control. At this stage, any specific mechanism of action or special physicochemical or biological interactions are not known. Also, the roles of dissolved/soluble Cu or CuO nanoparticles are not very clear. Since the phytocomponent composition of each type of plant extract varies, the nanoparticles made with them may not be comparable to each other without a greater understanding of the mechanism of action and quantization of components. Thus, more investigations are required to elucidate the mode of action. Nonetheless, keeping in view the demand for nonantibiotic wound dressings, this PAE-CuNP hydrogel can be a potential alternative following further validation.

All data generated or analyzed during this study are included in this article (and its Supporting Information).

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c04221.

• Table S1, phytoconstituents in papaya peel; Table S2, antimicrobial and wound healing effects of the unripe and ripe peel of Carica papaya; Table S3, comparison of the hydrogel developed using the xanthan gum and HPMC and its application; Figure S1, graphical representation of aqueous extraction by the maceration process from Carica papaya freshly collected unripe fruit peel; Figure S2, phytochemical analysis (flavonoids and phenolics) of the PAE; Figure S3, UV–visible spectra of PAE extract (5%) and 1 mM CuSO₄·5H₂O solution; Figure S4, image of the reaction mixture of the PAE and CuSO4.H₂0 at different conditions (Table ); Figure S5, FTIR spectra of excipients using the formulation of PAE-CuNPs and PAE-H; Figure S6, picture of the formulations of the optimized blank gel (HP70XG46), PAE loaded hydrogel (PAE-H), and CuNP loaded hydrogel (PAE-CuNPs); and Figure S7, antibacterial activity of PAE and CuNPs against pathogenic bacterial strains of E. coli (MTCC 443) and Bacillus subtilis (MTCC 441) (PDF)

D.M.: Methodology, investigation, visualization, data analysis, data curation, writingoriginal draft and review; R.P.S.: conceptualization, methodology, supervision, visualization, data analysis, writingoriginal draft, review, and editing; B.B.S.: supervision, resource, visualization, data analysis, writingoriginal draft, review, and editing. All authors reviewed the manuscript and approved it.

The authors declare no competing financial interest.