Enhanced in vivo wound healing efficacy and excellent antibacterial, antifungal, antioxidant and anticancer activities via AgNPs@PCS

2.1 Materials

Silver nitrate (AgNO₃, 99.99%) was obtained from Merck, while methanol and DMSO were purchased from Sigma-Aldrich (St Louis, MO, USA). Gram-negative and gram-positive microorganisms were obtained from the Pasteur Institute of Iran. The Zantox kit, used to evaluate antioxidant capacity, was purchased from Kavosh Arian Azma Company. Ketamine (10%) and xylazine (2%) were obtained from Alfasan Company. Dulbecco's modified Eagle's medium (DMEM), trypsin-EDTA solution, 1% antibiotic–antimycotic solution, penicillin–streptomycin solution, and fetal bovine serum (FBS) were prepared by BIO IDEA in Iran. All experiments were conducted in triplicate, and deionized water was used as the solution in all processes.

2.2 Preparation of Petroselinum crispum seeds extracts

The extraction process was conducted according to the following procedure. Initially, Petroselinum crispum seeds were collected from Bagheran Mountains in the South Khorasan province of Iran. The collected seeds were then washed with sterile deionized water and dried. Subsequently, the prepared seeds were ground into a powder form. A quantity of 100 g of Petroselinum crispum was macerated in 1000 ml of methanol, serving as the solvent. The mixture was agitated at 150 rpm for duration of 72 h. Afterward, the solution was filtered, and the solvent was separated from the extract using a rotary vacuum. The resulting product was stored at a temperature of 4 °C.

2.3 Green synthesis of silver nanoparticles (AgNPs@PCS)

The fabrication of AgNPs was carried out using the following procedure. Initially, an aqueous solution of silver nitrate (AgNO₃) was prepared by dissolving 24 mg of silver nitrate in 10 ml of deionized water, resulting in a concentration of 15 mM. The solution was vigorously stirred during the preparation. Simultaneously, an aqueous solution of Petroselinum crispum extract was prepared by adding 0.1 g of the extract to 10 ml of deionized water, adjusting the pH to 12. Next, the aqueous solution of Petroselinum crispum extract was added to the silver nitrate solution, and the mixture was stirred for 60 min. During this process, the Ag⁺ ions were reduced to Ag⁰ nanoparticles. The fabricated AgNPs@PCS were then subjected to centrifugation at 5000 rpm for 6 min and washed with double distilled water. Finally, the AgNPs@PCS were dried in an oven for 24 h. Scheme 1 provides a schematic representation of the biosynthesis of the nanoparticles.

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Scheme 1

The stages of formation of silver nanoparticles using Petroselinum crispum seed extract.

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2.4 Antibacterial and antifungal activities of AgNPs@PCS

2.5 Antioxidant capacity of AgNPs@PCS

The in vitro antioxidant capacity of the AgNPs@PCS (sample no. 14) and Petroselinum crispum seeds extract was calculated according to the 2,2-Diphenyl-1-Picrylhydrazyl (DPPH) scavenging free radicals method using Zantox kits (Kavosh Arian Azma Co.) (Brand-Williams et al., 1995). In this method, 250 μl of DPPH was added after addition of 10 μl of sample to each well. After shaking for 30 s, plate incubated at room temperature for 20 min. Exposing DPPH radicals to reducing agents or hydrogen donors, leads to change the primary purple to yellow color. These changes can be measured at 517 nm. To calculate the percentage of DPPH radical inhibition, the following formula was utilized (Oryan et al., 2019): Scavenging rate (%) = (1 − A_s/A₀) × 100 (1); Here, A_s represents the absorbance of the sample, while A₀ represents the absorbance of the blank. By substituting the appropriate values into the formula, the percentage of DPPH radical inhibition can be determined.

2.6 Anticancer activity

Anticancer activity was done using the report of Zare-Bidaki and her colleagues (Zare-Bidaki et al., 2022). Human breast cancer cells (MCF-7) were obtained from the Pasteur Institute in Tehran, Iran. The MCF-7 cells were cultured in 75 ml flasks with Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% FBS and 1% penicillin/streptomycin. The cell culture was maintained in a 37 °C incubator with 5% CO₂. To evaluate the cytotoxic influence of the fabricated sample (designated as sample no. 14), the MTT assay was performed. Briefly, 10,000 cancer cells were seeded in each well. Following this, the medium in the wells was replaced with fresh medium containing different concentrations of AgNPs (3.25–200 µg/ml). After the incubation period, 10 µl of MTT solution was added to each well. Subsequently, the MTT solution was removed, and DMSO was added, followed by 10 min incubation. The cell viability percentage was calculated using the following formula: Cell viability (%) = (sample absorption/control absorption) × 100 (2). By substituting the appropriate values into equation (2), the cell viability percentage for each sample concentration can be determined.

2.7 Preparation of Vaseline ointment containing AgNPs@PCS

To prepare Vaseline ointment containing AgNPs (sample no. 14), 30 g of pure Vaseline is melted in a bain-marie water bath at 60 °C and 0.3 g of AgNPs@PCS were added (1% w/w). Finally, the prepared ointment was sonicated at 60 °C for 30 min to form uniform.

2.8 In vivo experimental procedure in rat

2.8.1

2.8.1 Burn wound model

Twelve Wistar rats (males, 7–9 weeks old, 210 ± 10 g) were chosen and kept in individual cages and all parameters such as diet, temperature, and light were in standard and controlled for a week before the study. All surgical procedures and experiments conducted in this study were carried out in compliance with the protocols approved by the local Ethics Committee in the Student Research Committee of Birjand University of Medical Sciences. Prior to creating the burn wounds, all animals were anesthetized by intramuscular injection of ketamine (10%, 75 mg/kg body weight) and xylazine (2%, 10 mg/kg body weight). This anesthesia protocol was followed for ensuring the well-being and minimizing any discomfort or pain experienced by the animals during the procedure. Then, the hair of the dorsum of the rats was shaven, and the aluminum bar, which was heated for 20 s in the flame, applied vertically to the skin surface on the back of rats for 10 s without any pressure immediately. Two full-thickness burns, each measuring 10 mm in diameter, were intentionally created on the skin of each rat. The wounds of each rat were divided randomly into four groups as follows:

Group A: The group in which, the wounds were remained untreated (n = 6, negative control group).

Group B: The group in which, the treated wounds by topical use of 1% silver sulfadiazine (n = 6, positive control group).

Group C: The group in which, the treated wounds by topical use of pure Vaseline (n = 6, vehicle control group).

Group D: The group in which, the wounds were treated with 1% w/w Vaseline ointment containing sample no. 14 (n = 6, AgNPs@PCS group).

Each treatment was applied once a day for 21 days. Two of six rats were euthanized on the days of 7, 14, and 21 post-injuries, and the biopsy specimens were gathered after imaging from the wounded area for analysis. In the Scheme 2, the scheme of the in vivo experimental methodology has been shown.

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Scheme 2

Scheme of the in vivo experimental methodology.

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2.9 Histopathology

Histological examinations were conducted at three different time points following the injury: 7, 14, and 28 days. To accomplish this, samples of the skin were obtained. All samples were fixed in a 10% neutral buffered formalin solution, dehydrated using various concentrations of ethanol, clarified in xylol, embedded in paraffin, and then sliced into sections measuring 5 μm in thickness. These prepared tissue samples were subjected to staining using hematoxylin and eosin (H&E) and subsequently examined under a standard light microscope. The assessment primarily focused on comparing the degree of epithelialization, infiltration of inflammatory cells, formation of granulation tissue, and the density of blood vessels among the different groups.

2.10 Data analysis

The statistical analysis was conducted using SPSS 19.0 software (Chicago, IL, USA). One-Way ANOVA or Kruskal-Wallis test (for non-parametric variables) was employed, and if the results were found to be significant, Tukey HSD multiple comparisons or Mann-Whitney U test (for non-parametric variables) was performed as a post hoc test.

2.11 Characterization

To verify the synthesis of the biofabricated nanoparticles (NPs), various analytical techniques were employed. UV–Vis spectroscopy was performed using a NanoDrop system (BioTek model Epoch, USA) in the wavelength range of 300–500 nm. Fourier infrared spectroscopy (FT-IR) was conducted using a PerkinElmer Spectrum TwoTM IR spectrometer (Model L160000U) in the wave number range of 400–4000 cm⁻¹ at room temperature. For further characterization, field emission scanning electron microscopy (FESEM) was performed using a TESCAN BRNO-Mira3 LMU instrument, while transmission electron microscopy (TEM) was performed using a Zeiss-EM10C-100 KV microscope. The crystalline phase and crystallite size of the AgNPs@PCS (sample no. 14) were determined using an X-ray diffractometer (Philips PW 1800) with Cu Kα radiation. The particle size and surface charge of the AgNPs@PCS (sample no. 14) were assessed using dynamic light-scattering technique (DLS) with a NanoBrook 90Plus instrument (Brookhaven Instruments, model 18051; USA).

3.1 Fabrication of silver nanoparticles (AgNPs@PCS)

UV–Vis spectroscopy was conducted to validate the synthesis of AgNPs, specifically focusing on sample number 14. The process of AgNPs formation can be identified by the alteration in color, transitioning from a pale yellow shade to a deep brown hue, allowing for easy visual observation. The absorbance of different AgNPs samples fabricated under varying conditions was recorded using UV–Vis spectroscopy in the wavelength range of 300–500 nm. The observed color changes in the suspensions resulted in the appearance of a surface plasmon resonance (SPR) absorption band, which can be attributed to the oscillation of free electrons within the metal nanoparticles that resonates with the incident light wave (Ebrahimzadeh et al., 2020; Anandalakshmi et al., 2016). In this study, the reaction parameters that were important for synthesizing the nanoparticles were optimized at different experimental conditions.

3.1.1

3.1.1 Effect of silver nitrate concentration on AgNPs@PCS

The influence of different concentrations of silver nitrate on AgNPs@PCS (sample number 14) was examined using UV/Vis spectra, as depicted in Fig. 1a. It is evident that the molar ratio of silver nitrate concentration is a significant parameter affecting the synthesis of AgNPs. The peaks observed in the UV/Vis spectra indicate that the absorption increases with an increase in the concentration of silver nitrate up to 15 mM, after which it starts to decrease. The decrease in the intensity of the absorption band can be attributed to the adhesion and aggregation of nanoparticles (Sharma et al., 2020). Consequently, a concentration of 15 mM was chosen as the optimal concentration due to the maximum absorption associated with it.

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Fig. 1

UV–Vis spectra of the fabricated silver nanoparticles for optimization at various concentration (a), time (b), temperature (c).

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3.2 XRD analysis

The crystalline structure of the fabricated nanoparticles was assessed through XRD analysis, as depicted in Fig. 2. The XRD results displayed four distinct peaks at 2θ angles of 38.2°, 44.22°, 64.92°, and 77.5°, corresponding to the (1 1 1), (2 0 0), (2 2 0), and (3 1 1) Bragg planes, respectively. These findings indicate the formation of a cubic crystal structure at the core of the AgNPs. Moreover, the obtained results were consistent with the reference data from the Joint Committee on Powder Diffraction Standards (JCPDS 01–087-0717), confirming the high purity of the fabricated nanoparticles as no additional peaks were observed. The outcomes of this study aligned with the findings reported by Ghoshal et al. (Ghoshal and Singh, 2022). The average crystal size of the fabricated nanoparticles was determined using the Debye-Scherr equation (D = 0.9λ/βcosθ (4)), where D represents the average crystal size, λ represents the wavelength, β represents the peak width at half maximum intensity, and θ represents the Bragg angle. According to this calculation, the average crystal size of the fabricated nanoparticles was estimated to be 38.7 nm.

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Fig. 2

XRD pattern of AgNPs@PCS (sample no. 14).

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3.3 EDS, FESEM and TEM analysis

The EDS diagram of the fabricated nanoparticles, as illustrated in Fig. 3, exhibited a prominent peak at 3 KeV, primarily associated with AgNPs. Also, small amount of carbon and oxygen were observed in the graph, which is associated to the elements in the extract of Petroselinum crispum on the surface of the fabricated nanoparticles. The results of this analysis were in consistent with the results of Rautela et al. study (Rautela et al., 2019). FESEM images of fabricated nanoparticles showed the formation of spherical, uniform and homogeneous nanoparticles (Fig. 4). According to these tests, the average size of the fabricated nanoparticles was approximately 30–50 nm. As it is clear, irregular structure that was observed in some areas was related to the aggregation of nanoparticles which can be attributed to the adhesion of the extract on the surface of the NPs. Dinparvar et al., reported the fabricated AgNPs using Cuminum cyminum L. seed extract and the SEM images of the fabricated nanoparticles showed that the size of the fabricated nanoparticles was about 100 nm. Our data indicated that use of Petroselinum crispum extract smaller AgNPs nanoparticles can be fabricated (Dinparvar et al., 2020). Furthermore, TEM spectroscopy was used to obtain the precise size and morphology of the fabricated nanoparticles (Fig. 5). The images clearly showed the spherical and uniform shape of the sample no. 14 with the size around 30–50 nm. The existence of a dark shadow around the fabricated nanoparticles in the TEM images confirmed the placement of Petroselinum crispum plant extract on the surface of the fabricated nanoparticles. Ragupathi and his colleagues utilized Petroselinum crispum plant extract to synthesize silver nanoparticles (Ragupathi et al., 2016). However, their research resulted in nanoparticles with a rod-like morphology and significant agglomeration. In contrast, our study achieved nanoparticles with more suitable size and morphology, offering an improvement over the previous research.

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Fig. 3

EDS spectrum of AgNPs@PCS (sample no. 14).

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Fig. 4

FESEM images of sample no. 14.

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Fig. 5

TEM images of sample no. 14.

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3.4 DLS and zeta potential analysis

The hydrodynamic diameter of NPs measured by DLS analysis. The hydrodynamic diameter measured the size of the nanoparticles with the various biological components of the extract that are located on the surface of the nanoparticles. For this reason, the size of the NPs in DLS analysis shows a greater value than the results of FESEM and TEM analysis (Kouhbanani et al., 2019). The results of DLS analysis (Fig. 6a) showed the average hydrodynamic diameter of the fabricated nanoparticles about 150 nm. Ravichandran et al., fabricated the AgNPs with the average hydrodynamic diameter about 155.3 nm (Ravichandran et al., 2019). Zeta potential measurement is a method to determine the surface charge of fabricated nanoparticles. The results of zeta potential showed (Fig. 6b) that the fabricated sample no. 14 had a surface charge of –23.2 mV. Negative charge of the fabricated nanoparticles indicated the presence of extract compounds on the surface (Ravichandran et al., 2019). Other benefit of using plants extract as a capping agent is due to the charge on the surface of nanoparticles which cause the repulsion between particles and then, high stability of the suspension obtained. The high surface charge of sample no. 14 indicated the high stability of the fabricated nanoparticles. Sharma et al. reported the negative charge on the surface of fabricated sample no. 14 that was the same result with our study, (Sharma et al., 2018).

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Fig. 6

DLS and zeta potential analysis of images of sample no. 14.

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3.5 FT-IR

The formation of nanoparticles and the involvement of functional groups were examined through FT-IR analysis. The FT-IR spectrum of Petroselinum crispum extract revealed absorption bands at specific wavenumbers: 3384.1, 2912.6, 1712.3, 1237.7, 1055.2, and 814.5 cm⁻¹ (Fig. 7). The absorption peak at 3384.1 cm⁻¹ indicated strong stretching vibrations of the O—H functional group in alcoholic and phenolic groups (Isaac et al., 2013). The peak observed at 2912.6 cm⁻¹ was attributed to the stretching of the C—H group in alkenes (Dhand et al., 2016). The peak at 1712.3 cm⁻¹ could be associated with the stretching or bending of the C⚌O group present in amides (Mahadevan et al., 2017). The peak at 1237.7 cm⁻¹ was related to the C—O stretching of the ester group (Chinnasamy et al., 2019). The observed peak at 1055.2 cm⁻¹ indicated the C⚌O stretching of the alcoholic group (Chinnasamy et al., 2019). The absorption peak at 814.5 cm⁻¹ was related to the C—H bending of the alkynes group (Paulkumar et al., 2014). Fig. 7 depicted the fabricated AgNPs using Petroselinum crispum extract. It can be observed that the FT-IR spectrum of the fabricated nanoparticles was similar to the FT-IR spectrum of Petroselinum crispum extract, albeit with lower displacement and intensity. This similarity indicates the influence of functional groups present in Petroselinum crispum extract in reducing Ag⁺ ions to Ag⁰ nanoparticles (Khodadadi et al., 2021).

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Fig. 7

FT-IR of sample no. 14 and extract.

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3.6 Antibacterial and antifungal activity

The antibacterial properties of AgNPs@PCS (sample number 14) were evaluated against both gram-negative (Escherichia coli and Klebsiella pneumoniae) and gram-positive bacteria (Staphylococcus aureus, Streptococcus mutans, Streptococcus mitis and Enterococcus faecalis), as presented in Table. 2. It was observed that the effectiveness of AgNPs@PCS against gram-negative bacteria was higher compared to gram-positive bacteria, consistent with the well-known trend in which silver nanoparticles exhibit greater antimicrobial activity against gram-negative strains. Escherichia coli showed the greatest effect of AgNPs@PCS (sample no. 14) on gram-negative bacteria with MIC about 31.25 μg/ml, and Streptococcus mitis and Enterococcus faecalis showed the lower effect on gram-positive bacteria with MIC around 250 μg/ml. The difference between gram-positive and gram-negative bacteria is the existence of a thicker peptidoglycan layer in gram-positive cell wall and maybe the main reason influencing the more effect of AgNPs@PCS on gram-negative bacteria (Monedeiro et al., 2019). Furthermore, gram-negative bacteria have a lipopolysaccharide layer that has a positive charge, but AgNPs@PCS has a negative surface charge, which causes more accumulation of nanoparticles on the surface of gram-negative bacteria (Sharifi-Rad et al., 2020). AgNPs fabricated in Behravan et al. study using Berberis vulgaris aqueous extract showed more antibacterial effects against gram-negative bacteria same as in our study (Behravan et al., 2019). Haq Khan and his colleagues successfully produced silver nanoparticles using an extract derived from the Petroselinum crispum plant. The antibacterial activity of these nanoparticles was evaluated against Escherichia coli and Staphylococcus aureus bacteria, and the results indicated that the MIC for Escherichia coli was 235 µl/ml, while for Staphylococcus aureus, it was 120 µl/ml. Notably, our antibacterial activity was more effective against Escherichia coli (MIC value of 31.25 µl/ml) compared to their study (Khan et al., 2020). This can be attributed to achieving the optimal conditions for nanoparticle synthesis, ensuring the nanoparticles possessed the appropriate size and morphology. In this study, the antifungal effect of AgNPs@PCS against Candida albicans strain was investigated. The results showed (Table. 3) that the strong effect of fabricated nanoparticles on antifungal with the MIC = 15.62 μg/ml. Rizwana and colleagues, reported the fabricated AgNPs that had significant antifungal properties (Rizwana et al., 2021). Studies claimed that shape and size of the nanoparticles directly affect the antimicrobial activity, so that the smaller size of the NPs and the higher the surface-to-volume ratio, shows the stronger antimicrobial activity (Mitiku and Yilma, 2018). One of the possible mechanisms of the antimicrobial effects of silver nanoparticles is nanoparticles binding to the bacterial cell wall and the creation of free radicals. Then, disturbance in the permeability of the membrane and the entry of silver ions into the bacteria occur (Pirtarighat et al., 2019). Ag⁺ ions disrupt the function of the bacteria by binding to phosphorus and sulfur in DNA, RNA or proteins and eventually destroy it (Ahmad et al., 2022, 2022).

3.7 Analysis of total antioxidant properties (TAC)

Free radicals are cell metabolisms that have one or more unpaired electrons in their outer electron orbit. The presence of these unpaired electrons makes them highly reactive that make instability by transferring the electron and damaging the cell and eventually cell death happen, so-called oxidative stress (Gonca et al., 2021). In order to prevent this damage, cells have antioxidant molecules that neutralize them by giving electrons to free radicals and thus prevent damage to cells (Liguori et al., 2018). Oxidative stress causes various diseases such as Alzheimer's, cancer, arteriosclerosis, and chronic lung obstruction. The use of antioxidants can significantly reduce the risks of oxidative stress (Forman and Zhang, 2021). The DPPH scavenging free radicals was employed to examine the antioxidant capabilities of AgNPs@PCS and Petroselinum crispum. The results of examining the antioxidant properties of AgNPs@PCS and Petroselinum crispum in Fig. 8 are remarkable. As can be seen, Petroselinum crispum extract has been able to increase the percentage of DPPH radical inhibition from 11 to 83% by increasing the concentration from 3.12 to 25 mg/ml. Also, the results revealed that the use of Petroselinum crispum extract for the synthesis of AgNPs significantly increased the antioxidant capacity of AgNPs, therefore the AgNPs@PCS could inhibit free radicals about 47% at the 31.25 µg/ml concentration. Also, the percentage of inhibition increasing with the increasing in concentration so that at a concentration of 250 µg/ml, it inhibited 96% of DPPH. The results of the present study are similar to previous studies (Khorrami et al., 2018). Lalitha et al., published the fabricated AgNPs by Azhadirachta indica extract that inhibited DPPH radical about 48% at 250 µg/ml concentration, which showed a lower antioxidant activity compared to the nanoparticles fabricated in our study (Lalitha et al., 2013). As a result, AgNPs@PCS can be considered as nanoparticles with high antioxidant capacity.

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Fig. 8

DPPH inhibition percentage at different concentrations of AgNPs@PCS (sample no. 14) (a) and PCS extract (b).

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3.8 Anticancer activity

The cytotoxicity of the prepared nanoparticles was assessed using the MTT assay at various concentrations of AgNPs (3.25, 6.25, 12.5, 25, 50, 100, 200 µg/ml) on MCF-7 cell lines, as shown in Fig. 9. It can be observed that the viability of cancer cells decreased as the concentration of AgNPs increased. At the lowest concentration, 100% cell viability was observed, while at concentrations of 100 µg/ml and 200 µg/ml, cell viability decreased to approximately 69% and 49%, respectively. In this study, the IC50 value of the AgNPs was determined to be 200 µg/ml. In a study conducted by Farah et al. (Farah et al., 2016), IC50 value of 217 µg/ml was found for AgNPs fabricated using A. obesum leaves extract against MCF-7 cells. On the other hand, Moteriya et al. (Moteriya and Chanda, 2020) fabricated AgNPs using leaf extract of Caesalpinia pulcherrima for the treatment of human breast cancer cells (MCF-7), and the IC50 value was reported to be approximately 70 µg/ml after 24 h.

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Fig. 9

The results of the MTT assay in MCF-7 Cell treated with AgNPs after 24 h. Results were reported as survival percentage compared to control samples.

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3.9 Evaluation of wound closure percentage

The rate of wound closure was assessed by calculating the area of the unclosed wound in Fig. 10. The data revealed a significant increase in the percentage of wound closure in the nanoparticles group compared to both the negative control group and the Vaseline group after 7 and 14 days (Fig. 11). By the 21st day, the wounds in the nanoparticles group were completely closed, and a significant difference was observed compared to the Vaseline group at this time point. In a related study, Oryan et al. {Oryan, 2018 #79} (Oryan et al., 2018) investigated the therapeutic effect of AgNPs coated with chitosan. Their results demonstrated that the group treated with a high dose of nanoparticles exhibited significantly higher wound closure percentages than the control groups on the seventh day after injury. These findings indicate the promising wound healing properties of AgNPs, especially in combination with chitosan or other wound healing agents, which could potentially accelerate the wound closure process and promote better recovery. Further investigation showed that in both treatment groups using high and low doses of AgNPs coated with chitosan, the percentage of wound closure were in agreement with the results obtained in our study. Also, Tian & co-workers followed the appearance of healed wounds, and it was found that the healed wounds using AgNPs group were similar to normal skin. This study showed that wounds healing in the group treated with AgNPs was about 60%, while it was about 40% in the control groups (Tian et al., 2007). The results of our study and previous studies showed that the use of AgNPs plays a significant role in accelerating the healing of skin wounds. Also, in addition to accelerating the formation of granulation tissue and re-epithelialization, AgNPs play a role in reducing the size of the wound more rapidly and forming the normal appearance of the skin.

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Fig. 10

Effect of AgNPs@PCS group (Silver NP), pure Vaseline (Vaseline), silver sulfadiazine (Positive control) and Negative control on the wound closure rate in rats.

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Fig. 11

Percentage of wound closure at 7, 14 and 21 days post-injury (mean ± SD) (p < 0.001 ***, p < 0.01 **, p < 0.05 *).

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3.10 Histopathological assays

Histological analysis of the wounds on days 7, 14, and 21 was conducted and presented in Fig. 12. The results indicated that re-epithelialization was completed after 14 days in the group treated with nanoparticles, and the damaged region was entirely covered with a thick and mature epidermis. Notably, the number of blood vessels in the group treated with nanoparticles ointment did not show a significant difference compared to the other groups (p > 0.05). Furthermore, after 7 days of treatment with AgNPs@PCS ointment (sample number 14), a significant decrease in the number of inflammatory cells was observed compared to the group treated with Vaseline (p < 0.05), as shown in Fig. 13. Similarly, the results indicated a significant decrease in the number of inflammatory cells in the nanoparticles ointment-treated group compared to the negative control group and Vaseline after 14 days of treatment (p < 0.05) (Fig. 13). Interestingly, wounds treated with AgNPs@PCS ointment exhibited a significant increase in the number of fibroblasts and fibrocytes after 7 and 14 days of treatment compared to the other groups (p < 0.05) (Fig. 13). However, after 21 days, the group treated with AgNPs@PCS ointment showed a significant decrease in the number of fibroblasts and fibrocytes compared to the other groups (p < 0.05) (Fig. 13). The results obtained after 21 days of treatment showed the regular structure of connective tissue bundles in the dermis in the group treated with AgNPs@PCS ointment. Generally, the group treated with AgNPs@PCS ointment showed a decrease in the number of inflammatory cells and increase in fibroblasts and fibrocytes, and regular organization of collagen fibers compared to Vaseline and negative control groups. Results support our hypothesis that the use of AgNPs@PCS ointment can be used as a favorable option for the reconstruction of damaged areas and wounds. Based on the results obtained by histopathological evaluations, AgNPs@PCS ointment led to a significant reduction of inflammatory cells. Also, data confirmed the improvement and acceleration of granulation tissue formation and re-epithelialization by AgNPs@PCS ointment. The histopathology results of our study showed that Vaseline ointment containing AgNPs@PCS prevented inflammation in the wound area and increased the number of fibroblast cell production and migration that lead to the acceleration of wound healing. Moreover, prevention from infection at the wound site is the other benefit of AgNPs@PCS (sample no. 14) due to its potential antimicrobial property. According to the study conducted by Tian & co-workers, the most benefit of AgNPs in wound healing process in mice was to increase the speed of wound healing. In addition, green fabricated AgNPs have the ability to prevent wound infection due to their antibacterial effect, which prevents secondary damage into wounds during the healing period. On the other hand, the ability to reduce wound inflammation by reducing the infiltration of mast cells and lymphocytes and modulating fibrogenic cytokines such as IL-6 showed satisfactory results. The results of this study demonstrated that the use of silver nanoparticles in burn wounds can be a new and effective treatment to achieve scar-free wound healing at the bedside (Tian et al., 2007). Also, in a study by Liu & co-workers, it was shown that AgNPs can differentiate fibroblasts into myofibroblasts, thus wound contraction can be promoted. Moreover, the restorative nature of AgNPs was shown by increasing the production of keratinocytes and their movement into the wound site (Liu et al., 2010). Also, Lakkim & co-workers revealed that the use of AgNPs in the treatment of burn wounds prevented microbial growth, bleeding, or pus formation during treatment. Also, this study showed that the use of Vaseline ointment containing AgNPs accelerated the reduction of wound size, which was due to the anti-microbial effect of green fabricated AgNPs around the wound area (Lakkim et al., 2020).

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Fig. 12

Histopathology of wounds on the 7th, 14th, and 21th days after injury (hematoxylin and eosin, ×40 and × 400).

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Fig. 13

histological analysis of wounds after 7, 14, and 21 days of injuring (p < 0.001 ****, p < 0.005 ***, p < 0.01 **, p < 0.05 *).

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