Green Synthesised Cleome Viscosa Silver Nanoparticles CvAgNPs Their Biological In Vitro and In Vivo Evaluation of Wound Healing Potential

Wound healing is a dynamic and complex biological process that restores tissue integrity following injury [1]. It involves four overlapping phases: hemostasis, inflammation, proliferation, and remodeling. While this process is well-orchestrated, several complications can interfere with effective healing. Factors such as microbial infection, chronic inflammation, poor vascularization, nutritional deficiencies, and systemic conditions like diabetes often delay tissue repair [2]. Non-healing or chronic wounds pose a significant clinical challenge, increasing morbidity and healthcare costs. Moreover, conventional wound management strategies sometimes fail to adequately prevent microbial colonization and support tissue regeneration, highlighting the demand for innovative therapies that can accelerate and enhance wound healing outcomes [3].

Plants produce a wide range of secondary metabolites, such as alkaloids, flavonoids, tannins, terpenoids, and phenolic compounds, which play crucial roles in defence and adaptation [4]. These bioactive molecules possess antimicrobial, anti-inflammatory, antioxidant, and immunomodulatory properties, making them therapeutically significant in the treatment of wounds and related complications. For instance, flavonoids are known to scavenge reactive oxygen species (ROS), thereby reducing oxidative stress within the wound environment, while tannins promote tissue contraction by precipitating proteins at the wound site. Terpenoids and alkaloids contribute to enhanced collagen synthesis and angiogenesis, which are critical for granulation tissue formation and epithelialization. Unlike synthetic drugs, plant-based compounds are biocompatible and generally associated with fewer side effects, which makes them attractive candidates for wound management. Their ability to target multiple pathways simultaneously strengthens their efficacy in complex biological processes like tissue regeneration. Harnessing these phytochemicals through advanced strategies opens new avenues for developing eco-friendly wound healing formulations.

Nanoparticles have emerged as highly effective agents in regenerative medicine, particularly in wound healing applications [5]. Their nanoscale size allows superior interaction with biological molecules, improved cellular uptake, and enhanced penetration into wound tissue [6]. Among these, silver nanoparticles (AgNPs) are especially important due to their broad-spectrum antimicrobial activity, which prevents bacterial colonization and reduces the risk of infections, a major cause of delayed healing. Furthermore, nanoparticles can serve as carriers for plant-derived bioactive compounds, improving their stability, solubility, and controlled release at the wound site [7]. Their ability to modulate inflammatory responses, stimulate fibroblast migration, enhance collagen deposition, and promote angiogenesis significantly contributes to accelerated tissue repair [8]. Green synthesis of nanoparticles using plant extracts combines the natural healing potential of phytochemicals with the advanced therapeutic properties of nanomaterials [9, 10]. This integration offers a sustainable and biocompatible approach, making nanoparticles a vital tool for the development of next-generation wound healing therapies. Green-synthesised Cleome viscosa silver nanoparticles (CvAgNPs) and their biological evaluation for wound healing potential through in vitro and in vivo studies.

MATERIAL AND METHODS

In vitro wound healing activity-scratch assay of Cleoma viscosa silver nanoparticles (CvAgNPs)

Cleome viscosa silver nanoparticles (CvAgNPs) were synthesized, characterized, with their in vitro biological activity published in the first manuscript, and the second manuscript highlighting their in vitro and in vivo wound healing activity. Fibroblast cells from the L6 line were grown in sterile six-well plates until they reached full confluence, creating a continuous cell layer [11]. To initiate a wound, a straight scratch was carefully introduced into the monolayer using a sterile pipette tip, after which the wells were rinsed with sterile phosphate-buffered solution to remove any dislodged cells. The scratched cell layers were then treated under sterile conditions with Cleome viscosa-derived silver nanoparticles (50 µg) or a control solution containing 0.2% fetal bovine serum (FBS). The plates were subsequently placed in a cell culture incubator and maintained at 37°C for 48 hours. Following this incubation period, the migration and regrowth of cells into the scratched area were examined using phase-contrast microscopy to assess the wound healing response.

In-Vivo wound healing property of CvAgNPs.

The wound healing effects of silver nanoparticles synthesized from Cleome viscosa were evaluated in adult male albino rats, each weighing between 200 and 250 grams. All experimental procedures were conducted in accordance with ethical standards and under the oversight of the Committee for Control and Supervision of Experimental Animals (CPCSEA) [12]. The animals, housed in clean cages, were provided with commercial pellet rat chow and clean water daily. Silver nanoparticles are known for their antimicrobial, anti-inflammatory, and collagen-promoting properties, which enhance wound healing by reducing bacterial load, stimulating fibroblast activity, and increasing extracellular matrix deposition. This study highlights the therapeutic potential of biogenic silver nanoparticles for wound management.

Four groups of animals, each containing four animals

The research evaluated the wound healing capabilities of silver nanoparticles derived from Cleome viscosa in a rat excision wound model. Rats were randomly assigned to four groups, each consisting of six animals. The first group received topical treatment with a silver sulfadiazine ointment (10 mg/g), which served as a reference standard. The second group acted as a negative control and received no treatment. In the third group, wounds were treated with Cleome viscosa silver nanoparticles over a period of 16 days. The fourth group served as a positive control for wound healing. Throughout the experimental period, the progression of wound closure was systematically monitored by measuring wound area on days 0, 8, and 16 after injury. Using these measurements, each group's wound contraction percentage was calculated according to a standard formula, allowing for direct comparison of healing rates across all groups. This methodology enabled the researchers to quantitatively assess how effectively each treatment promoted wound closure and tissue regeneration. The comparative analysis provided insights into the potential benefits of Cleome viscosa-derived silver nanoparticles as a topical agent for enhancing wound repair relative to conventional and untreated controls.

Animals were divided into four groups of six each:

• Group G1: Treated topically with 10 mg/g silver sulfadiazine ointment.
• Group G2: Negative control group (no treatment).
• Group G3: Treated with Cleome viscosa silver nanoparticles for 16 days.
• Group G4: Positive wound control group.

Wound size was measured on days 0, 8, and 16 post-wounding. The percentage of wound contraction was calculated using a standard formula. This approach assessed the comparative effectiveness of treatments in promoting wound closure and healing.

Histopathological study

Formaldehyde (10%) was used to fix pancreatic tissue for approximately 48 hours. The tissue was then dehydrated using an ethyl alcohol-water mixture, cleared with xylene, and embedded in paraffin. Using microtomes, sections of 4.5 mm thickness were cut. Each section was stained with hematoxylin and eosin dye and placed in a neutral deparaffinized xylene solution for microscopic examination. This histopathological method effectively preserves tissue structure for detailed analysis, enabling researchers to study cellular morphology and pathological changes in the pancreatic tissue.

Statistical analysis

This statistical strategy strengthens the reliability and validity of the findings by offering a rigorous approach to data interpretation using statistical software.

RESULTS AND DISCUSSION

In vitro inflammatory activity of Cleoma viscosa silver nanoparticles (CvAgNPs)

Cells were cultured, and a scratch wound assay was performed to assess the influence of molecules on skin fibroblasts. Regrowth to close the scratch was measured after 0, 24, and 48 hours in media containing 50µg Cleoma viscosa silver nanoparticles (CvAgNPs). Results showed that fibroblasts in the CvAgNP-treated group achieved faster restoration of cellular density compared to the control group, indicating enhanced cell migration. Specifically, CvAgNPs promoted fibroblast migration at each measured time point. These findings suggest that CvAgNPs possess properties that accelerate wound closure by stimulating fibroblast movement, a crucial process in tissue regeneration and wound healing. The study highlights the potential of CvAgNPs as a bioactive agent for promoting skin repair.

Wound healing is a complex and dynamic process involving a series of coordinated events that restore the integrity of damaged tissue. Central to this process are skin fibroblasts, which are responsible for synthesizing extracellular matrix components, facilitating tissue remodeling, and migrating into the wound bed to close the gap created by injury. The migration of fibroblasts is particularly critical during the proliferative phase of wound healing, as it directly influences tissue regeneration rate and quality. In recent years, nanotechnology has emerged as a promising field for the development of novel wound healing agents, with silver nanoparticles (AgNPs) gaining attention for their antimicrobial, anti-inflammatory, and tissue-regenerative properties. The green synthesis of AgNPs using plant extracts, such as those from Cleome viscosa, offers an eco-friendly and biocompatible approach, potentially enhancing the bioactivity of the nanoparticles due to the presence of phytochemical capping agents. The present study investigates the influence of CvAgNPs on fibroblast migration and wound closure using the scratch wound assay, a well-established in vitro model for studying cell migration and tissue repair.

In-vivo wound healing activity of ethanol extract of CvAgNPs (F2) on Wistar albino rats.

Over 14 days, the wound healing rate was monitored in rats treated with Cleoma viscosa silver nanoparticles (CvAgNPs) ethanol extract, alongside control and standard treatment groups. All rat groups exhibited increased wound healing, with treated groups showing significantly reduced wound areas on days 10 and 14 compared to the control (P ≤ 0.001). Notably, the drug-treated group displayed superior wound healing on day 2 (P ≤ 0.001). While differences between treatment and control groups were less pronounced by day 6, the CvAgNPs F2 formulation significantly outperformed all other groups, including the standard treatment, in promoting wound closure by day 14 (P ≤ 0.001), as evidenced in Figure 16. This demonstrates the potential of CvAgNPs to enhance wound healing compared to both control and conventional treatments. The in vivo wound healing study conducted over 14 days in Wistar albino rats provides a comprehensive evaluation of the efficacy of Cleome viscosa silver nanoparticles (CvAgNPs) ethanol extract compared to both negative control and standard treatments. All experimental groups demonstrated progressive wound closure over the observation period, but the extent and rate of healing varied significantly between groups, especially at key time points. On day 2, the earliest phase post-wounding, the group treated with silver sulfadiazine (the standard reference drug) and the Cleome viscosa ethanolic extract exhibited the highest percentage of wound closure (12.3% and 10.55%, respectively), while the CvAgNPs group (6.52%) and negative control (4.23%) lagged. This early advantage for the standard and extract groups suggests a rapid initial response, possibly due to their established antimicrobial and anti-inflammatory properties, which are critical for controlling infection and modulating the inflammatory phase of wound healing.

By day 6, the differences between groups became less pronounced. The negative control, CvAgNPs, and silver sulfadiazine groups all achieved similar levels of wound closure (30.11%, 29.5%, and 29.7%, respectively), while the Cleome viscosa ethanolic extract group showed a modestly higher value (34.59%). This convergence may reflect the natural progression of wound healing, where the proliferative phase is underway in all groups, and the initial benefits of antimicrobial action have been balanced by the intrinsic regenerative capacity of the rats. However, from day 8 onward, the distinctions between treatment groups and control began to widen again, particularly as the wounds entered the later stages of healing. By day 10, the CvAgNPs group exhibited a marked acceleration in wound closure (72.7%), significantly surpassing the negative control (60.44%) and even outperforming both the Cleome viscosa ethanolic extract (67.5%) and silver sulfadiazine (68.2%) groups. This trend continued through days 12 and 14, with CvAgNPs achieving 85.11% and 95.2% wound closure, respectively, compared to 75.56% and 76.2% in the negative control. Notably, by day 14, the CvAgNPs group displayed a wound closure rate nearly 19 percentage points higher than the control and outperformed the standard treatment by over 4 percentage points.

The statistical analysis supports these findings, with significant differences (P ≤ 0.001) observed between the CvAgNPs group and the control at critical time points, particularly on days 10 and 14. The superior performance of CvAgNPs in the later stages of healing suggests that these nanoparticles not only support the initial phases of wound repair but also promote sustained tissue regeneration and remodeling. The enhanced effect of CvAgNPs compared to both the crude plant extract and the standard silver sulfadiazine may be attributed to several factors. Silver nanoparticles are known for their potent antimicrobial activity, which helps prevent infection a major impediment to wound healing. Additionally, the nanoscale size and large surface area of CvAgNPs facilitate better penetration into the wound bed, allowing for more effective interaction with cellular and molecular targets involved in tissue repair.

Figure 1: In vivo wound healing activity of ethanol extract of CvAgNPs (F2) on Wistar albino rats.

Table 1: In-vivo wound healing activity of ethanol extract of CvAgNPs (F2) on Wistar albino rats.

The provided histological images (G1–G4) and corresponding group descriptions offer a comparative perspective on the wound healing efficacy of different treatments: negative control (G1), Cleome viscosa-mediated silver nanoparticles (CvAgNPs, G2), Cleome viscosa ethanolic extract (G3), and silver sulfadiazine (G4). Careful examination of these tissue sections reveals notable differences in tissue architecture, collagen deposition, and re-epithelialization, key markers of wound healing progression. In the negative control group (G1), the histological section shows incomplete re-epithelialization, disrupted tissue organization, and limited collagen deposition, indicating a delayed or suboptimal healing response. This is consistent with the expected natural course of wound healing in the absence of active therapeutic intervention, where the repair process is slower and more susceptible to infection and prolonged inflammation. Such findings underscore the need for effective wound care strategies, especially in cases where rapid healing and infection control are critical.

Figure 2: G1: Negative Control, G2: CvAgNPs 5mg/g, G3: Cleome viscosa EE 10 mg/g, and G4: Silver sulfadiazine 10 mg/g.