**1. Introduction**

For healthcare systems, skin wounds represent a significant burden. Their high rates of morbidity chronification, and relapse, pose a significant strain on healthcare-related aspects of living, elevating both individual and societal expenses as well as posing a problem for healthcare systems across the globe [1–3]. Based on the ageing of the population and a rise in the rates of diabetes, overweight, and heart disease, skin wounds and their potential to become chronic will likely grow more common [2,4]. As a result, managing wounds is a

**Citation:** Mahajan, N.M.; Wanaskar, K.; Ali, N.; Mahapatra, D.K.; Iqbal, M.; Bhat, A.R.; Kaleem, M. Innovative Wound Healing Hydrogel Containing Chicken Feather Keratin and Soy Isoflavone Genistein: In Vivo Studies. *Gels* **2023**, *9*, 462. https://doi.org/10.3390/ gels9060462

Academic Editor: Shige Wang

Received: 17 May 2023 Revised: 30 May 2023 Accepted: 31 May 2023 Published: 5 June 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

major issue that is becoming more and more prevalent globally [4,5]. As such, it is crucial to develop new products that can accelerate the healing process.

When the integrity of any tissue is damaged, a wound develops (such as when the skin or a muscle becomes damaged, a bone is fractured, or burns occur) [6]. A wound could occur from a fall, surgery, an infection, circumstances, or another event [7]. Several antioxidant genes, including catalase (CAT) and glutathione peroxide (GPx), are expressed more frequently during the natural healing of wounds, indicating a possible function for free-radical-scavenging enzymes [8].

In order to promote cellular growth and cell-guided tissue development, the protein layer has a distinct three-dimensional architecture. Hair, nails, and other areas of the skin contain large amounts of the naturally occurring protein keratin [9]. In comparison with the other proteins, it contains greater levels of cysteine [10]. The protein is hard, resilient, and lighter because the sulphur atoms in the remnants of cysteine frequently interconnect with each other [11]. Chicken feathers can serve as a good source of protein due to their elevated keratin protein level [12].

Genistein, which is extracted from soybean, is used as an inhibitor of epidermal growth factor (EGF)-induced proliferation [11,13]. It promotes the synthesis of collagen, which is important for preserving the equilibrium of the skin as well as accelerating wound repair [12]. In human dermal fibroblasts, genistein has a direct influence on the signalling pathways which govern collagen production [14]. T-BHP (t-butylhydroperoxide) induces collagen production in the presence of oxidative stress [15]. Because of its antioxidant effects, this phytochemical guards human dermal fibroblasts from oxidative-stress-related production of collagen suppression [16]. Through changing the inflammatory reaction, it further modulates the healing of wounds [17,18]. Through enhancing antioxidant activity while regulating the expression of cytokines that are pro-inflammatory in the initial stages of wound repair, genistein supplements decrease the risk of oxidative stress [19]. Without proper antioxidant activities, wound healing might be delayed, or severe tissue damage can occur [20]. Isoflavone (genistein) in soybean has been reported to have potent antioxidant properties and also possess pharmaceutical potency with oestrogen-like activity [21].

The current study was performed to isolate keratin protein from chicken feathers with the intention to develop a keratin–genistein-combination-based wound-healing hydrogel. Since hydrogel polymer material can absorb and hold a greater amount of exudates, it was rationally chosen for this study. The pre-formulation aspects, such as protein content, drug–excipient interactions, etc., were determined through standard methods and sophisticated instruments (UV, FT-IR, SEM, and HPTLC). The prepared gel was evaluated comprehensively with respect to the physicochemical parameter (gel strength, viscosity, pH, spreadability, drug content, etc.). An in vivo wound-healing study in experimental animals followed by biochemical assays (ELISA and qRT-PCR) against pro-inflammatory factors (IL-2, IL-6, IL-1β, IL-10, and COX-2) and histopathological (liver, skin, and kidney) studies were performed to determine the possible wound closure and anti-inflammatory effects of the innovative product.

### **2. Results and Discussion**

*2.1. Characterisation of Keratin*

### 2.1.1. Protein Content

The protein content as determined by the Kjeldahl method showed 86.89% *w*/*w* of protein content from 500 mg of keratin sample. This was found to be a good percentage of protein content available in the keratin sample, as it was expected to contain above 80% *w*/*w* [22].
