C. esculenta leaves were washed with Milli-Q water to detach dust and attached impurities. Leaves were subjected to air drying under shade at room temperature (25 °C) for 3–4 days. Afterward, 40 g of dried leaves was sliced into small pieces and boiled in 500 mL of distilled water while stirring continuously via a magnetic stirrer at 60 °C for 30 min. The resulting mixture was cooled to room temperature (25 °C), filtered through Whatman No. 1 filter paper, and subjected to vacuum filtration assembly to acquire the clear extract. The pH of the prepared extract was recorded (∼6.8–7.0), and it was stored in amber-colored bottles at 4 °C for further use (K. K. P. Kumar et al., 2019).

Silver NPs were prepared according to the method described by Lade and Patil (2017) with small modifications. The aqueous AgNO₃ solution (8 mM) and varying concentrations of aqueous extract of C. esculenta were mixed in Erlenmeyer flasks to synthesize the silver nanoparticles, as shown in Table 1. The pH of the reaction mixture was adjusted to ∼8.0 with 0.1 M NaOH, and reactions were carried out at 70 °C in the water bath for different time intervals (30 min, 45 min, and 60 min) with continuous stirring. The color of the mixture changed from pale yellow to brown, indicating the Ag⁺ reduction to Ag° nanoparticles. The colloidal solution was centrifuged at 10,000 rpm for 10 min. The NP pellet was harvested and rinsed twice with distilled water, followed by freeze-drying in a benchtop freeze dryer (LYO60B-1 PT Bioevopeak, China). The NP pellet was then stored at 4 °C for further use.

Varying concentrations (1%–3%, w/v) of chitosan (CS) were dissolved in 1%–2% acetic acid solution (v/v) individually to make the CS solutions to achieve the desired thickness and mechanical properties of the patch. The solution was stirred under magnetic stirring for several hours (12–24 h) to achieve the complete dissolution of the CS in the solution. Polyethylene glycol (PEG-400, 1%–2% v/v) was added to the CS solution as a plasticizer to enhance the mechanical properties and flexibility of the formulated patch. Similarly, 100–300 mg of PVP-k30 was added to enhance the mechanical strength, skin adhesion, and stability of the prepared patch. Subsequently, 5% (w/v) of C. esculenta-derived AgNP product (lyophilized powder redispersed in distilled water) was mixed into the CS solution. The resulting mixture was poured into the casting molds or Petri dishes to generate the thin layer. The solution was then dried in the drying oven under controlled conditions at 30 °C–40 °C for 24 h. After drying, the formed patches/films from the molds were peeled off carefully (Sabbagh and Kim, 2022). Various formulations of patches manufactured with C. esculenta AgNPs are presented in Table 2. Regarding quantification in the final patch, it contained AgNPs (5% w/v) as per the formulation shown in Table 2. The AgNP concentration was kept uniform based on the controlled batch conditions and the synthesis protocol, ensuring a consistent AgNP concentration across the patches.

All prepared formulations of C. esculenta AgNPs loaded CS patches were characterized for the below mentioned parameters:

Ex vivo investigations of prepared patches and nanoparticles were conducted on the skin of rats. The procedure to extract the rat skin received approval from the Institutional Animal Ethical Committee (Ref. no: PHM.Eth/Lhr-05/08-24). Male Sprague–Dawley rats weighing 200–250 g were sacrificed via cervical dislocation. Hairs of rats were excised from the dorsal area utilizing an electric trimmer, followed by skin excision surgically. Skin was rinsed with a 0.9% sodium chloride solution and stored at −20 °C ± 1 °C. The rat’s skin was subsequently positioned between the donor and receptor compartments of the Franz diffusion cell. The phosphate buffer (pH 7.4) was filled in the recipient compartment, and the temperature was set at 37 °C ± 2 °C. The receiving medium was agitated at 100 revolutions per minute. The donor partition was filled with a nanoparticle patch exhibiting 5 mg of the drug. Subsequently, 2-mL aliquots were collected at designated time intervals (0.5 h, 1 h, 1.5 h, 4 h, 8 h, 12 h, 16 h, and 24 h), and the medium was replenished with fresh buffer to preserve the sink conditions. The samples were subjected to UV–Vis spectrophotometry at a wavelength of 425 nm for further analysis (Nawaz et al., 2022).

The in vitro anti-inflammatory effect was assessed through a protein denaturation test utilizing egg albumin. We performed the egg albumin denaturation assay following the protocol described in previously conducted studies (Abbas et al., 2021; Mizushima and Kobayashi, 1968) with minor modifications. This method involved mixing a test sample (2 mL) with a concentration of 100–400 g/mL, egg albumin (0.2 mL), and phosphate buffer saline, pH = 6.5 (2.8 mL). The reaction mixture was incubated at 37 °C for 20 min, followed by heating at 70 °C for 5 min. When the temperature dropped to room temperature, the absorbance was assessed at 660 nm using a spectrophotometer. Phosphate buffer served as the control, while diclofenac Na was employed as the standard drug. Percentage inhibition was calculated using the following equation: %  Inhibition of denaturation = 100   *   AC − AS / AC where AC is the absorption of the control, and AS is the absorption of the test sample.

All statistical analyses were performed by GraphPad Prism v. 9.0 (GraphPad Software, USA), and data were expressed as mean ± standard deviation (SD). One-way ANOVA followed by Tukey’s multiple comparison test was selected to compare multiple formulations in the evaluation of physicochemical parameters of transdermal patches. In addition, a paired t-test was employed to compare results before and after treatment for Group II, while one-way ANOVA was used for intergroup comparison in the evaluation of biochemical parameters. An unpaired t-test was applied to compare the control with the treatment groups. Results were considered statistically significant at p < 0.05.

FTIR analysis was performed to verify the successful integration of C. esculenta AgNP into CS transdermal patches. The spectral analysis of C. esculenta AgNP, the chitosan patch, and the C. esculenta AgNP-loaded chitosan patch demonstrated notable differences. Significant absorption peaks in C. esculenta AgNP were observed at 596 cm⁻¹, 713 cm⁻¹, 846 cm⁻¹, 1,047 cm⁻¹, 1,390 cm⁻¹, 1,624 cm⁻¹, 3,217 cm⁻¹, and 3,394 cm⁻¹, indicative of metal–ligand vibrations and biomolecule capping. The CS patch displayed peaks at 580 cm⁻¹, 837 cm⁻¹, 1,082 cm⁻¹, 1,647 cm⁻¹, and 3,331–3,468 cm⁻¹, corresponding to the CS and PEG functional groups employed in the patch formation, as summarized in Figure 3A. Upon the incorporation of C. esculenta AgNP into the patch, novel peaks appeared at 569 cm⁻¹, 650 cm⁻¹, 840 cm⁻¹, 945 cm⁻¹, 1,099 cm⁻¹, 1,377 cm⁻¹, 1,639 cm⁻¹, and 2,156 cm⁻¹, signifying Ag-polymer interactions. Displacements in O–H or N–H stretching (3,427 cm⁻¹) and the emergence peaks at 3,716 cm⁻¹, 3,867 cm⁻¹, and 3,992 cm⁻¹ validated the augmentation of hydrogen bonding and molecular reconfiguration. The absence of peaks at 1,251 cm⁻¹, 1,296 cm⁻¹, and 1,357 cm⁻¹ in the CS patch spectrum indicates an interaction with C. esculenta AgNP, altering the polymer matrix. The spectral alterations provide compelling evidence that silver nanoparticles were effectively integrated into the chitosan-PEG patch, establishing stable interactions with the polymer’s functional groups (Alven and Aderibigbe, 2024).

Different concentrations of ingredients were employed to formulate the patches (F1-F6), as mentioned in Table 3. The physical appearance of the formulated chitosan patch and the C. esculenta AgNP-loaded chitosan patch was assessed by visual inspection of the prepared patches (Figures 6a,b). All formulated patches had a non-sticky, homogeneous, opaque, flexible, and smooth nature. In addition, the formulated patches exhibited even distribution of adhesive layer to diminish the lump formation. Furthermore, formulated patches exhibited the acceptable pH range (5.2–5.9). Therefore, the prepared transdermal patches are user friendly, as they will cause no irritation to the skin.

The thickness uniformity of the patches was observed by using the digital calipers, and the average thickness was investigated (Table 3). Chitosan concentration has an impact on the overall thickness of the patch, as it may result in a denser and more robust formulation of the matrix system of the polymer. The results suggest the optimum thickness uniformity in all prepared transdermal patches with the thickness order: F-1 < F-2 < F-3. However, no significant difference in thickness was observed in the formulations F4, F5, and F6.

The moisture absorption profile of the prepared patches was investigated (Table 3) and showed the moisture absorption of the formulated patches in the order of F-3 > F-2 > F-1; however, formulations F-4 to F-6 have comparable moisture content due to similar CS concentrations. All formulations showed the moisture absorption in the acceptable range and retained an appropriate shape and texture.

The weight uniformity of the drug-loaded patches (1 × 1 cm²) was evaluated according to the lower SD values (Table 3). Patch weights were found to be uniform, as the thickness uniformity of the patches ensured the weight uniformity. As the concentration of the CS changes, achieving a uniform layer is difficult, and hence, the uniformity of weight may change accordingly.

All prepared transdermal patches exhibit favorable film properties and folding endurance, as shown in Table 3. The folding endurance of the prepared transdermal patches was found to be in the order of F-1 < F-2 > F-3, with the F2 formulation showing more folding endurance (97 ± 2). In addition, F4 and F6 exhibited the same folding endurance with value 96 ± 2, while F5 showed lesser folding endurance with value 95 ± 3. These results indicate that the folding endurance of the prepared patches increased with the corresponding increase in the CS concentration initially due to an increase in the elasticity and flexibility of the patch. However, as the concentration of CS increases above a certain value, it may decrease folding endurance due to the formation of a very rigid structure that reduces overall flexibility.

The tensile strength of the formulated patches was determined (Table 3) by employing the fabricated apparatus according to previous studies. Tensile strength was measured in triplicate, and the average was calculated. The tensile strength of the prepared patches increased with increased CS proportion, indicating that tensile strength can be increased with a high concentration of the polymer in the formulation.

The surface morphology of the CS transdermal patches and CS patches (Figure 4b) loaded with C. esculenta AgNPs was analyzed using SEM to assess the impact of nanoparticle incorporation. Figure 4c displays the SEM image of the CS-based patch prior to C. esculenta AgNP incorporation, showing a smooth and uniform surface. The lack of particulate matter indicates a uniform polymeric structure, typical of a well-constructed CS-PEG film.

Figure 4c depicts the chitosan patch post-loading with C. esculenta AgNPs, revealing notable surface alterations. The formerly smooth surface now displays dispersed nanoparticle clusters, validating the successful integration of C. esculenta AgNPs into the polymeric matrix. The irregular and diverse morphology indicates significant interactions between C. esculenta AgNPs and CS, potentially affecting release dynamics and mechanical characteristics. These observations validate that nanoparticle incorporation modifies the patch morphology, shifting from a smooth surface to a rougher texture due to AgNP distribution, thereby validating the effective fabrication of a nanoparticle-loaded chitosan patch for prospective biomedical applications.

A skin irritation test was done with the F4 formulation. A transdermal patch was employed on the skin of the experimental subjects, and subjects were examined after 24 h and 72 h. There was no sign of erythema and edema over the skin of experimental subjects. The primary irritancy index (PII) for the control patches and medicated patches was <2, while the PII for the formalin-treated group was 5.67 ± 0.52, indicating high irritation of the skin. Formulations with PII<2 are considered a non-irritant according to the Draize patch test. Therefore, patches formulated in this study were safe and non-irritant when applied to the skin for the specific time of application (Supplementary Figure S1).

These results demonstrate that the formulated transdermal patches (F4) are non-irritant and present good skin compatibility. The absence of any adverse reaction and irritation suggests that these patches are safe and reliable for prolonged application over the skin and are a suitable option for their intended transdermal drug delivery applications.

The hematological parameters were assessed to evaluate the anti-inflammatory potential of the C. esculenta AgNP-loaded CS patches. The notable decrease in WBC count from 11.94 ± 0.92 × 10⁹/L prior to treatment to 6.94 ± 0.19 × 10⁹/L post-treatment signifies a reduction in systemic inflammation, given that elevated WBC levels are generally linked to an inflammatory response. Furthermore, neutrophil levels, essential in acute inflammation, exhibited a significant reduction from 28.11% ± 9.04% to 21.02% ± 7.11% following treatment, thereby corroborating the resolution of inflammation in the treatment group. The RBC count, initially lower before treatment (5.03 ± 0.31 × 10⁶/mm³), rose to 6.15 ± 0.22 × 10⁶/mm³ post-treatment, indicating enhanced hematological stability, presumably attributable to diminished oxidative stress and inflammation-related hemolysis. The platelet count, significantly elevated prior to treatment (1,610.98 ± 6.21 × 10⁹/L), decreased to 1,010.98 ± 4.78 × 10⁹/L following treatment, signifying a reduction in inflammatory and pro-thrombotic activity, given the pivotal role of platelets in immune responses and inflammation. Furthermore, the slight fluctuations noted in MCV, MCH, and mean corpuscular hemoglobin concentration (MCHC) indicate that the treatment did not negatively impact erythrocyte morphology or hemoglobin levels. The hematological parameters are presented in Table 4.

The anti-inflammatory effects of the prepared nanoparticle-loaded patches are illustrated in Supplementary Figure S2. Female Sprague–Dawley rats were employed in this study due to the prevalence of inflammation being 3–5 times higher in females than in males. C. esculenta is extensively documented as a potential anti-inflammatory agent owing to the presence of phenolic and flavonoid compounds (Sudhakar et al., 2020). The anti-inflammatory efficacy of C. esculenta phytochemicals was augmented by their transformation into nanoparticles to improve skin permeation. The results indicated that the highest percentage of inhibition in paw volume was recorded with patches containing nanoparticles (ANOVA; p < 0.05). Patches significantly suppress inflammation and edema relative to the control group. Diclofenac Na-loaded patches also diminish edema volume relative to the control, albeit to a lesser extent than the C. esculenta AgNP-loaded chitosan patches. This resulted from increased penetration of phytochemicals into skin layers due to enhanced skin permeation. The elevated concentration of nanoparticles in the deeper dermal layers aids in mitigating skin infections and inflammation.

The nanoparticle-infused patches administer the requisite quantity of anti-inflammatory agents at the target location with greater efficiency. Consequently, the topical administration of natural compounds via nanoparticles embedded in patches enhances the management of inflammation. The findings of anti-inflammatory studies are encapsulated in Table 5. The findings indicate that the treatment effectively reduced inflammatory responses and aided in restoring hematological homeostasis in the experimental model.

The anti-inflammatory effect found in the study is predominantly due to the silver nanoparticles (AgNPs). Several studies described that AgNPs exhibit anti-inflammatory characteristics as they inhibit the pro-inflammatory cytokines (e.g., IL-6 and TNF-α), modulate the NF-κ signaling pathway, and suppress the reactive oxygen species (ROS) (Gopinath et al., 2010; Liu et al., 2014). AgNPs may release some silver ions (Ag+); they typically act synergistically and may contribute to the anti-inflammatory effect. In our study, C. esculenta-mediated AgNPs incorporated into the CS patches exerted an active anti-inflammatory effect.

In the present study, the CS-based matrix played a pivotal role in the transdermal mechanism as CS is a bioadhesive, biocompatible, and permeation-enhancing polymer, having the ability to open the tight junctions present in the skin, thus facilitating the paracellular transport. CS also promotes the intimate contact between skin and the applied patch, thus enhancing the absorption through the skin.

AgNPs may penetrate the upper layer of the skin more efficiently, owing to their nano-size, further facilitating the skin permeability. These factors demonstrate the efficient and sustained transdermal delivery of the active constituents via a combination of NP-assisted penetration and polymer-aided permeation enhancement.