Wound healing activities of PVA–urea composites from Ormocarpum cochinchinense

Abstract The aim of this work is to prepare ternary blended polyvinyl alcohol (PVA)–urea hydrogels containing Ormocarpum cochinchinense, Cinnamomum zeylanicum and antibiotic cephalexin by freezing-thawing method in order to assess the wound healing qualities. In addition to being a synthetic polymer, PVA is a recyclable and biocompatible artificial polymer blend that has attracted a lot of interest in biological applications. The freezing-thawing process with PVA–urea blend is used to make hydrogel film. Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), scanning electron microscopy (SEM) and swelling investigations were carried out for the composite membranes. Biological studies involving antibacterial, antifungal, cytotoxicity and wound healing activities were also carried out for the composite membranes. The composite membrane developed has a lot of potential for wound dressing and other applications. Graphical Abstract


Introduction
The composition of a synthetic dressing should be safe, mechanically stable, biodegradable, as well as provide a suitable atmosphere for tissue repair.Polymeric membrane materials for wound dressings have recently become extremely popular.Recently PVA bio-nanocomposites developed possess (Zhou et al. 2010) extraordinary wound dressing properties particularly when used with natural extracts.Urea is a powerful solution has been reported to have certain bacterial qualities when applied for wound dressing (Wohler 1828).Blending two or more components is now an increasingly essential strategy for generating novel biomaterials with desirable properties.Elum Botti (Ormocarpum cochinchinense) is a tiny herb found in Tamilnadu, India, that belongs to the Fabaceae family.It is the most beneficial medicinal plant and locals have historically used it to mend fractures (Dinesh Kumar et al. 2013).Cinnamomum zeylanicum is the botanical name for cinnamon.It is an anti-viral, antioxidant, antibacterial and anti-fungal properties, and it is used for wound healing (Shipra et al. 2020).Cephalexin enhanced the membranes' stability by forming a thin film layer.The present study involves combining PVA-urea hydrogels blend with natural leaf extract of Ormocarpum cochinchinense, Cinnamomum zeylanicum and drug cephalexin, prepared by the freezing-thawing method to form a thin biofilm.The focus of this research was to evaluate the synthesized composites for wound dressing behavior.Studies on swelling and wound healing were carried out to discover non-toxic techniques to reduce healing behavior (Adeli et al. 2018).

FTIR (Fourier transform infrared spectroscopy) analysis
Figure S1 shows the FTIR analysis of the PVA-urea composite samples.The FTIR spectra of the samples revealed the various distinct bands corresponding to the presence of several absorption groups.Figure S1a shows the FTIR spectra of PE composites.The O-H stretching vibrations of PVA-urea/Ormocarpum cochinchinense are shown by the broad peaks at 3226 cm −1 , C-H stretching bands occur in the range 3084 cm −1 which is due to the presence of alkene group, C = O stretching peaks appear in the range 1771 cm −1 which is shown by weak bands (Gonzalez et al. 2014).Stretching vibrations at 1599 cm −1 corresponds to α, β unsaturated ketone.C-H bending frequency appears at 1413 and 723 cm −1 which is attributed to the C = C bending vibrations of the alkene moiety.Figure S1b shows the FTIR spectra of PCn composites.The O-H stretching vibrations of PVA-Urea/Cinnamomum zeylanicum are shown by the broad peaks at 3135 cm −1 and the C = C stretching vibrations at 1639 cm −1 corresponds to the alkene group.The C-H bending frequency appears at 1450 and 799 cm −1 , which corresponds to the C = C bending vibrations of the alkene moiety.Figure S1c shows the FTIR spectra of PCp composites.The O-H stretching vibrations of PVA-urea/ Cephalexin are shown by the broad peaks at 3368 cm −1 .The C-H stretching bands appearing in the range at 3077 cm −1 is well marked to the alkene group, C = O stretching bands appearing in the range at 1770 cm −1 are shown by weak peaks.Stretching vibrations due to unsaturated ketone appear at 1599 cm −1 and C-H bending frequencies appear at 1418 cm −1 , and bands at 771 cm −1 corresponds to C = C bending vibrations of the alkene moiety.Figure S1d shows the FTIR spectra of PECnCp composites.Absorption peaks of PVA-urea, Ormocarpum cochinchinense, Cinnamomum zeylanicum and cephalexin occurs in the same region as shown previously, and the absorption peak values for the ternary blended PECnCp composites show broader peaks in the range around 3477 and 3409 cm −1 which corresponds to the presence of O-H stretching vibration.The peaks between 1838 and 1601 cm −1 signify the presence of C = O and C = C bond.The sharp and broad peak further validates the presence of a wide array of both C = O and C = C double bonds.The C-H bending frequency appears at 1452 and 1157 cm −1 (Pal et al. 2007).This demonstrates that both composite membranes are made up of PVA-urea composites.

XRD (X-ray diffraction) studies
Table S2 and Figure S2 shows the XRD analysis for the PVA-urea composites.X-ray broad diffraction patterns of Figure S2a shows the PE composites appear in the range 2Ѳ = 18.95-39.91,Figure S2b shows the PCn composites occur at 2Ѳ = 22.95-40.02and Figure S2c shows the (PCp) composites occur at 2Ѳ = 18.87-41.55The XRD patterns show the absence of peaks which is due to the large steric hindrance of quarterly amino groups, which in turn disturbs the crystal domains, thereby showing amorphous nature.Figure S2d shows the three-part (PECnCp) composites values occurring at 2Ѳ = 19.53-35.48 corresponds to the semi-crystalline phase as that of the quartz phase appearing at 26.38, which is due to the less steric hindrance of amorphous composites changing into the semi-crystalline form (Dongying et al. 2017).

Swelling studies
Table S3(a-d) and Figure S3(a-d) show the swelling percentage curve of PVA-urea composites.The sample time intervals are 1, 2, 4, 6, 8 and 10 h.After 4 h the swelling ratio increased.The sample PECnCp composite has the largest proportion of the swelling 384.87 when compared to the samples PE, PCn and PCp.As a result, the natural and drug-loaded composites improve water absorption capacity.The chemical structure of the polymer chain in an additional repeating unit alters the ratio of hydrophilic to hydrophobic groups, which controls the swelling ratio.As degradation occurs, the chains diminish their transparency, polymer hydrogels containing hydrophilic groups swell far more than hydrophobic polymer hydrogels.Due to the presence of the water molecule, the swelling ratio is reduced (Abbas et al. 2017).Hydrophilic groups in the network, such as per -OH, -CONH 2 , -CONH, and -SO 3 H, absorb water, yet cross-linking across network chains gives disintegration resistance.Due to the presence of water within the polymeric hydrogel, specific solute molecules can easily diffuse.Urea and cephalexin are a carbamide, hence it increases the porous nature of the composites and as a result swelling ratio is also increased.Table S4 shows the swelling study of literature survey when compared to the ternary blended PECnCp composites.

HRSEM EDS analysis
Figure S4a depicts the PE development of composite particle in Ormocarpum cochinchinense.These composites have been clumped together.Figure S4(b, c) of PCn, PCp shows a creation of group composites of Cinnamomum zeylanicum and cephalexin.HRSEM micrographs of Figure S4d due to PECnCp blended composites shows needle like structures which are even with pores ranging to five microns.All HRSEM images of PVA show dense, homogenous, smooth and continuous surfaces.These surfaces of the dense hydrogel composite prevent bacteria from passing through the wound and the size of the pores and how well they connect would help cell migration and angiogenesis, which are both important for wound healing and tissue engineering (Afshin et al. 2020).Figure S4(a-d) shows the EDS of PE, PCn, PCp and PECnCp composites, these composites show the presence of the elements O, C, N, an additional peak shows the presence of the metal compounds Ni, Al, Cl and S (Anuar et al. 2011).The percentage of C, N and O peak is high in the case of ternary blended PECnCp composites when compared to other composites.

Antimicrobial study
Antibacterial activity is critical in the hydrogel composites, which is required for wound healing.Cephalexin acts by interfering with the production of the bacteria's cell wall, causing it to burst and the bacteria to die (Rafik et al. 2015).However, the results showed that cephalexin concentration to be about 30 mg in a blended PVA/urea hydrogel composite.It shows excellent antibacterial activity.Table S5 and Figure S5 show the evidence of antimicrobial action (Altaf et al. 2021).PECnCp composite displays the good antifungal activity is due to the Ormocarpum cochinchinense/ Cinnamomum zeylanicum has naturally observed antifungal properties and they are found to be very effective against Aspergillus niger and Candida albicans.Good antifungal properties are important for wound healing (Bhusnure et al. 2021).

In vitro cytotoxicity study
Biocompatibility, particularly minimal or no cytotoxicity, is a critical consideration in determining whether a material is appropriate for use as a medical device.It is viable if it complies with ISO 10993-5:2009 standards for biodegradable materials or does not produce harmful or toxic substances.Cell viability decrease of 30% is measured in medical devices which is found to have cytotoxic properties (I.O.f.S. (ISO) 2009).Medically used are several numbers of cell lines 1. A549 (Human, Lung cancer cell line), 2. MDAMB231 (Human, Breast cancer cell line), 3. MG-63 (Human, Bone Osteosarcoma cancer cell line), 4. HepG2 (Human, Liver cancer cell line) and 5. L929 (Mice, Fibroblast cell line).Elumbotty (Ormocarpum cochinchinense) leaf is a traditional bone scaffold wound reconstructed, consequently making use of (MG-63) cell line.The ternary PECnCp blended composites were analyzed in the Human Bone Osteosarcoma cell line (MG-63) with various cell viability concentrations and incubation times of 4 and 24 h for each cell line, as shown in Table S6. Figure S6 shows that IC50 = 126.84.07 g/ml for 24 h of incubation via different absorption of ternary PECnCp composites (25, 50, 100, 250 and 500).concentrations (100, 250 and 500) were less-cytotoxic to MG-63 cell lines when compared with the (50, 25) concentration which has no toxic effect and the cell viability is good, implying that the generated ternary blended PECnCp films were non-cytotoxic and suitable for further use in wound healing activities.

Wound healing test
Wound healing is crucial in the treatment of skin lesions, with natural chemicals receiving special attention as shown in the graph illustrated in Figure S7.The maximum cell migration rate was seen at all times, and wound healing was completed with success rate of 90% within 48 h.As a result, the suture containing 30% medication increased cell proliferation, migration, as well as MG-63 cells' ability to seal bone scaffold wounds by growing of keratinocytes in the injured area (Xiaoxuan et al. 2021).The ternary PECnCp blended composites scratch assay indicated that 48 h is the best formulation with the best biocompatibility, as it closed 90% of wounds.MG-63 cells migration indicate that they are effective in applied bone scaffold wound closure and reliving bone pain.

Conclusion
In this study, PVA-urea blend with Ormocarpum cochinchinense, Cinnamomum zeylanicum and cephalexin drug composites were properly synthesized by freezing-thawing method for wound dressing application.The HRSEM morphology upheld the needle like structure.FTIR studies shows the existence of important functional groups of the components.Swelling studies evaluated the wound characteristics.The composites proved that the no-toxicity is being utilized in vitro wound healing test.Scratch assay offered excellent cell growth and migration to fill in the wound break.The PVA-urea blended Ormocarpum cochinchinense, Cinnamomum zeylanicum and cephalexin drug composites (PECnCp) created could effectively defend the wound from bacterial infections.In future, we intend to conduct a more in-depth analysis for wound dressing making use of synthesized nanofibers.