Characterization of nanoparticles
Characterization of nanoparticles provides essential information about their size, shape, surface charge, surface area and, dispersity (Jayaseelan et al. 2012). The XRD of the prepared ZnO-NPs samples is shown in Fig. 1. All detectable peaks can be indexed as the ZnO-NPs hexagonal wurtzite-type structure in the standard data (JCPDS, 36-1451) (Ghamsari et al. 2019). The synthesized ZnO-NPs have good crystallinity and are of high purity. The FTIR revealed the asymmetric molecular stretching, vibration, and rotation of chemical bonds when exposed to the designated wavelengths of light. As shown in Fig. 2, the FTIR spectra of the prepared Green / Chemical ZnO nanoparticles showed that the main absorption bands at 3500, 2354, 1720, 1514, and 1220 cm− 1, which correspond to O-H mode, CO2 mode, O-H mode, and asymmetric and symmetric C = O stretching modes, respectively. The intense broadband in the vicinity of 418 cm− 1 is the characteristic absorption peak of Zn–O bond and also authenticates the presence of ZnO the same results were obtained by Kwon et al. (Kwon et al. 2002) Silva and Zainiquelli (Silva and Zaniquelli 2002); Li et al. (Li et al. 2004); Kim et al. (Kim et al. 2005); Liufu et al. (Liufu et al. 2005) and Labuayai et al. (Labuayai et al. 2009). The TEM as shown in Fig. 3, revealed that ZnO-NPs consist of nanowires as the prepared ZnO-NPs and TEM images confirmed the hexagonal structure of the synthesized ZnO-NPs. Green ZnO-NPs have wires length and wires diameter more than the obtained ZnO nanoparticles by chemical method. It is well documented that decreasing particle size increases their functionality as antimicrobial and anticancer agents due to the larger surface-to-volume ratio (Selim et al. 2020). The phase and purity of the prepared samples is unaffected by the preparation methods, indicating that the green method using Lawsonia inermis leaves is an effective method for the preparation of ZnO nanoparticles, which in turn confirms that green technology is superior to physical and chemical methods for producing nanoparticles (Rosi and Mirkin 2005; Gnanajobitha et al. 2013; Vidya et al. 2013; Parveen et al. 2016). Our study has confirmed that the synthesized green ZnO-NPs, possess a smaller particle size, enhanced the wound healing activity due to the large surface area to volume ratio and surface reactivity when compared to the synthesized chemical ZnO-NPs (Gunalan et al. 2012).
Characters of gels
Green and chemically synthesized ZnO-NPs gels are translucent in color as shown in Fig. 4. The gels were highly viscous and homogenous, stable, and spread out easily on the skin surface. The gels pH was 6.5 to 7, so there is no detectable itching or hypersensitivity reaction. The level of water absorption capacity % of gels containing nanoparticles at the 1st, 2nd and 3rd hours are shown in Fig. 5. The potential of green ZnO-NPs gel to absorb water is higher than that of chemically synthesized ZnO-NPs gel at the different periods of the experiment. Carbopol hydrogels loaded with a bioactive agent have been reported to accelerate the healing process, and improve the tissue quality of burn wounds (Singh et al. 2017). It maintains a moist environment within the wound site as a vehicle for topical administration. Maximum swelling of carbomer 940 ® was observed in simulated wound fluid (Singh et al. 2017). Therefore, we used Carbopol 940 ® as a vehicle for our studied nanomaterials. Our study results agreed with Morgado et al. (Morgado et al. 2015), who reported that the ideal wound dressing should be able to absorb exudates from the wound bed where water absorption or hydrophilicity allows the penetration of nutrients, cells, and bioactive molecules.
Clinical picture of the wounds
Grossly as in Fig. 6, swelling and hyperemia of the wound edges were observed on the day of wounding. On the 3rd dpt, wounds of rats in green ZnO-NPs and ZnO-NPs groups were lost their rounded forms and covered with moist reddish-brown scabs, and their wound contraction was increased with less exudate formation. In contrast, the wounds of rats in the control group were covered with moist scabs, and there was clearly noticeable yellowish exudate and edema through the wound edges. On the 7th dpt, wounds of rats in green ZnO-NPs and ZnO-NPs groups were covered with thin dry scabs, and the wound size of rats in green ZnO-NPs group was non significantly narrower than those in rats of ZnO-NPs group. The wounds of the control group were covered with thick moist scab and with less contraction when significantly compared with green ZnO-NPs and ZnO-NPs groups. On the 14th dpt, the wounds were covered with small and dry scab in individual rats in green ZnO-NPs and ZnO-NPs groups. The wounds of rats of the control group were covered with a relatively thick scab. On the 21st dpt, the scab sloughed from wounds of rats in green ZnO-NPs and ZnO-NPs groups leaving scar tissue, which was relatively clearer in rats of group 3 than those of green ZnO-NPs group. Meanwhile, the control group showed a clear red scar.
Wound surface area (WSA) is shown in Fig. 7. Wound surface area percent (WSA %) is shown in Fig. 8, and Wound contraction percent (WC %) is shown in Fig. 9. At 7th dpt, WSA and WSA% significantly decreased in both ZnO-NPs treated groups, compared with the control group. While at the 7th dpt, WC% significantly increased in both ZnO-NPs treated groups (chemical and green) compared with the control group. Furthermore, at the 3rd, 14th and 21st days PT WSA, WSA% and WC% showed no significance between the treated groups or the control group when p ≤ 0.05. As shown in Fig. 10, the healing time of wounds is significantly decreased (p ≤ 0.05) in rats of green ZnO-NPs and ZnO-NPs groups when compared with the control group.
The wound gap of the control group showed large blood clots, necrotic tissue, the appearance of fibroblastic proliferation under the scabs, and there was edema and abundant neutrophils infiltration as well at the 3rd dpt, however at the 7th dpt they showed fibroblastic proliferation and abundant cell infiltration mainly macrophage with newly formed blood vessels perpendicular on the wound edge. At the 14th dpt, the wound gap showed fibroblast proliferation with collagen-rich production associated with extensive inflammatory cells infiltration. At the 21st dpt, the wounds showed granulation tissue covered with epidermis with inflammatory cell infiltration beneath the granulation tissue as shown in Fig. 11. The wound gap of green ZnO-NPs group revealed necrotic tissues with a little blood clot, edema, and heavy infiltration with inflammatory cells, mainly neutrophils at the 3rd dpt. Meanwhile, they showed predominant fibroblast proliferation with collagenous fibers-rich production, high cellular macrophages, and lymphocytes infiltration with newly-formed blood vessel formation at the 7th dpt. At the 14th dpt, wounds showed a noticeable re-epithelization of the granulation tissue with less inflammatory cell infiltration and abundant collagenous fibers. At the 21st dpt, the wounds showed complete re-epithelization and intact epidermis with minimal inflammatory cell infiltration, and neoformed blood vessel formation, and rich mature scar formation as shown in Fig. 11. At the 3rd dpt, wound gaps of ZnO-NPs group were filled with extravasation of RBCs, edema, and aggregation of neutrophils. At the 7th dpt, wounds showed the presence of granulation tissue in the gap with hemorrhage and aggregation of neutrophils and eosinophils. At the 14th dpt, the wound gap decreased in size with complete re-epithelization and proliferation of fibroblasts and collagenous fiber production with a smaller number of inflammatory cells. At the 21st dpt, remarkable epithelization with heavily collage fibers production was observed. Also, minimal fibroblasts and infiltration with neutrophils and macrophages were noticed as shown in Fig. 11.
Wound-healing histological scoring
The score of wound healing (epithelization, vasculature, necrosis, connective tissue formation, and collagen synthesis) was qualitatively evaluated in all animals as shown in Fig. 12. The epithelization was not significantly changed at the 3rd, 7th, and 14th dpt in green ZnO-NPs and ZnO-NPs groups in comparison to those of the control group but was significantly increased (p ≤ 0.05) at the 21st dpt. It was noticeable that the vasculature, necrosis, connective tissue formation, and collagen synthesis at the 3rd, 7th, 14th, and 21st dpt were not significantly changed in green ZnO-NPs and ZnO-NPs groups when compared to the control group.
Lawsonia inermis is one of the plants that accelerates the wound healing process via reducing the epithelialization period and increasing the wound contraction percent (Yassine et al. 2020). The topical application of L. inermis rapidly initiates the inflammatory process by enhancing higher inflammatory cell infiltration and subsequently reduce the inflammatory phase by inhibiting monocyte-to‐macrophage differentiation (Daemi et al. 2019). It is reported that Lawsonia inermis leaves extract to contain flavonoids, alkaloids, and terpenoids, which accelerate the phenomena of wound healing by their astringent and antibacterial activities (James and Friday 2010; Bapat and Mhapsekar 2014), prevention of cell necrosis, improvement of angiogenesis (Fikru et al. 2012), inhibition of prostaglandin synthesis (Jain et al. 2011), and modulation of cytokines expression during the inflammation phase (Antunes-Ricardo et al. 2015). L. inermis methanolic extracts showed antibacterial effects against different bacteria (Elansary et al. 2020; Nigussie et al. 2021) i.e. LI inhibits the growth of gram-positive and gram-negative bacteria (Pasandi Pour and Farahbakhsh 2020). LI has strong antifungal and antioxidant activities (Elansary et al. 2020). ZnO-NPs accelerate the collagen synthesis and wound contraction with a relatively reduced scar (Saremi et al. 2016), regulate the endogenous growth factors and insulin-like growth factor-I, which may increase epithelialization (Ågren et al. 1991; Tarnow et al. 1994; Ågren 1999; Li et al. 2006; Aksoy et al. 2010; Sazegar et al. 2011; Arslan et al. 2012). Microscopically, ZnO-NPs had a beneficial impact on skin repair. ZnO-NPs enhanced angiogenesis, platelet activation re-epithelialization, and debris removal. ZnO-NPs achieve effective wound closure, aesthetic wound closure-healing power, and they can act as antimicrobial adhesives for the tissues (Batool et al. 2021). ZnO enhanced collagen deposition via the migration of a greater number of fibroblasts to the wound site (Kumar et al. 2013). It's attributed that the healing action of ZnO-NPs is due to the zinc ions, which enhance re-epithelization through keratinocyte migration to the wound site (Kumar et al. 2012). Therefore, and as a result of the synergistic effect, the ZnO Nanoparticles were synthesized using Lawsonia leaves methanolic extract to improve the antibiotic and wound healing activities of both. Our study revealed that the ZnO nanoparticles synthesized using Lawsonia inermis methanolic extract are more effective than the chemically synthesized ZnO NPs in the acceleration of wound healing, re-epithelization, fibroblasts stimulation, reducing inflammation and scar formation (Metwally et al. 2020).