(W1130-09-55) Investigating the Protective Effects of Multifunctional Nano Herbal Gel for Wound Healing

Purpose: Burns are known as one of the most common and destructive forms of injury, when tissue is injured, various inflammatory mediators like histamine, bradykinin, and prostaglandins are released, initiating an inflammatory response. Though the inflammation is a necessary response in wound healing, excessive or prolonged inflammation can delay the healing process and may also increase scarring. Burn wounds are more susceptible to microbial colonization and infection, which may further lead to a chronic wound state or even sepsis. Neutrophil Extracellular Traps (NETs) are released by neutrophils and trap and kill pathogens, resulting in anti-microbial activity, which is essential in wound management. But excessive NET formation can lead to delayed healing and tissue damage. Thus, an ideal therapeutic agent for wound healing must possess anti-microbial, anti-inflammatory, and anti-oxidant properties altogether and also actively promote tissue regeneration. Conventional therapies, although effective to an extent, often fail to minimize scarring, accelerate wound healing. The need for novel, affordable, and biocompatible therapeutic moiety that could enhance the healing process of wounds is growing. Nanoparticle (NPs) like silver and cerium oxide are effective in enhancing wound healing as they possess anti-microbial and anti-inflammatory properties, respectively^1,2,3. Polydopamine (PDA) possesses anti-oxidant properties, which makes it a suitable add-on for a wound dressing. DNase I can cleave and dissolve the excessive NETS and improve tissue perfusion, thereby accelerating wound healing⁴. Concurrently, biocompatible herbal extracts like aloe vera and basil seed mucilage have significant attention in wound management due to their potent anti-inflammatory, antimicrobial, and antioxidant properties⁵. Combining nanotechnology with herbal medicine offers a promising strategy to accelerate tissue repair and skin regeneration.

Methods: Silver nanoparticles (AgNPs) were synthesized using tri-sodium citrate and sodium borohydride as reducing agents¹. Nanoceria (NC) was coated with PDA, followed by conjugation with DNase I to form DNase-studded PDA-coated nanoceria (NPD). The NPDs were isolated by centrifugation. For the gel, aloe juice and basil seed mucilage were mixed with Carbopol 934, and the optimized AgNPs and NPDs were incorporated into this base. The optimized NPs have been extensively characterised using UV-Visible spectroscopy, Fourier transform infrared (FTIR) spectroscopy, hydrodynamic diameter, and Scanning Electron Microscopy (SEM). The coated DNase I was quantified by Bicinchoninic Acid (BCA) assay. Stability studies were conducted over 3 months, assessing pH, appearance, spreadability, and rheology. Cytotoxicity was assessed using a cell viability assay on L929 cells. The herbal gel was topically applied twice daily for 21 days to a Wistar rat burn model. Wound healing was monitored through blistering, redness, swelling, and wound contraction. Oxidative stress was measured by nitric oxide, lipid peroxidation (MDA), and glutathione (GSH) levels in tissue samples. Collagen was assessed via hydroxyproline content, Masson’s trichrome, and Picrosirius red staining. Pro-inflammatory cytokines (IL-6, IL-1β, TNF-α) were measured via ELISA. Histopathological analysis was done using haematoxylin-eosin (H&E)-staining, and immunoblotting was used for inflammation and regeneration markers.⁶

Results: The size of AgNPs was 68.51 ± 6.02 nm, while NC, NC-PDA, and NPD were 134.9 ± 10.02 nm, 187.6 ± 9.61 nm, and 214.5 ± 15.32 nm, respectively. About 60.38 ± 3.51% of DNase I was successfully coated on the PDA-coated nanoceria. TEM imaging confirmed NPD morphology. The formulated herbal gel was clear, smooth, and homogeneous. Gel firmness was 124.8 ± 0.97 g, and stability parameters remained consistent at room and refrigerated conditions, with minor changes at 45°C. In the burn model, treatment with AgNPs & NPD gel resulted in 93% wound closure, significantly higher than the untreated group (Fig. 2). Burn injuries caused elevated nitrite and MDA levels and reduced GSH, indicating oxidative stress. Treatment normalized nitrite and MDA levels and restored GSH, highlighting enhanced antioxidant defense. The burn-injured control group exhibited elevated IL-6, IL-1β, and TNF-α levels (p < 0.0001). Treated animals showed a significant reduction in these cytokines, suggesting anti-inflammatory effects. H&E staining confirmed epidermal regeneration (Fig. 3). Collagen deposition was evident through hydroxyproline assay, Masson’s trichome and Picrosirius staining, indicating enhanced tissue remodelling.

Conclusion: The AgNPs and NPD-loaded herbal gel exhibited strong antioxidant, anti-inflammatory, and regenerative effects. This nano-herbal gel shows strong potential as a safe and effective burn treatment.

References: 1.Yousefpoor, Y., et al., The combined effects of Aloe vera gel and silver nanoparticles on wound healing in rats. 2016.
2.Barkat, M.A., et al., Nanosuspension-based aloe vera gel of silver sulfadiazine with improved wound healing activity. 2017. 18: p. 3274-3285.
3.Chigurupati, S., et al., Effects of cerium oxide nanoparticles on the growth of keratinocytes, fibroblasts and vascular endothelial cells in cutaneous wound healing. 2013. 34(9): p. 2194-2201.
4.Papayannopoulos, V., et al., Neutrophil elastase and myeloperoxidase regulate the formation of neutrophil extracellular traps. 2010. 191(3): p. 677-691.
5. Shahrajabian, M.H., W. Sun, and Q.J.I.J.o.F.P. Cheng, Chemical components and pharmacological benefits of Basil (Ocimum basilicum): A review. 2020. 23(1): p. 1961-1970.
6.Devabattula, G., Bakchi, B., Sharma, A., Sidhartha, N.N., Dikundwar, A.G., Yeddanapudi, V.M. and Godugu, C., 2025. Mitochondria‐Targeting SIRT3 Activator Effectively Controls Bleomycin‐Induced Pulmonary Fibrosis. BioFactors, 51(4), p.e70032.

Figure 1: Particle size (Hydrodynamic diameter) of A) AgNPs, B) NC, C) NC-PDA and D) NPD.

Figure 2: Represents visual wound appearance after treatment on day 0, 3, 14, and 21 respectively

Figure 3: Photomicrographs of H&E-stained tissue sections of different study groups. Normal control group (A), Wound untreated group (B), Treated with herbal gel (C), AgNPs (D), NC (E) NC-PDA (F) NPD (G), AgNPs + NPD (H). EP indicates epithelial layer, HF indicates hair follicle, * indicates neutrophil infiltration.