Topical red algal-mediated zinc oxide nanoparticles infused in gel as a therapeutic strategy for oral mucositis wound healing

Topical red algal-mediated zinc oxide nanoparticles infused in gel as a therapeutic strategy for oral mucositis wound healing | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Topical red algal-mediated zinc oxide nanoparticles infused in gel as a therapeutic strategy for oral mucositis wound healing Thoshi Tarun K, Ramanathan Snega, Muthupandian Saravanan This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9340679/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 9 You are reading this latest preprint version Abstract Oral mucositis is a debilitating inflammatory condition requiring effective topical therapeutics with enhanced healing potential. Algae-mediated zinc oxide nanoparticles (ZnO NPs) were infused into the sodium alginate gel and then characterised by UV–visible spectroscopy, which confirmed the presence of ZnO-NPs with a peak at 321 nm, while FT-IR analysis identified functional groups including C-Br, C-F, OH, N-H, and NH stretches. The morphology of the ZnO-NPs-infused gel exhibited irregular shapes with an average size of 60–70 nm. EDX analysis showed elemental compositions of Zn (41.48%), O (19.7%), and C (38.7%), along with 64.9% crystallinity. The physical stability of the gel was characterized by the appearance, pH, texture, and phase separation. ZnO-NPs-infused gel showed antioxidant activity, with 74.3% scavenging at 100 µg/ml. Additionally, it showed significant antimicrobial activity against oral pathogens, including Streptoccus mutans, E. coli , and Candida albicans . These findings suggest that the ZnO-NPs–infused gel is a potential therapeutic candidate for managing oral mucositis by reducing oxidative stress, inflammation, and microbial load, thereby promoting improved wound healing. Topical gel Wound Healing Red algae Oral Mucositis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Topical drug delivery systems have gained significant attention in recent years for the effective management of oral mucositis, a inflammatory condition commonly related to chemotherapy and radiotherapy [ 1 ]. Oral mucositis is characterised by epithelial damage, ulceration, pain, and increased susceptibility to secondary infections, thereby severely affecting patients' quality of life and treatment outcomes [ 2 ]. Conventional therapies often provide only symptomatic relief and may lead to undesirable side effects, necessitating the development of safer and more effective therapeutic strategies. Nanotechnology has emerged as a cutting-edge field with widespread applications across various pharmaceutical industries. It also plays a pivotal role in computing, environmental science, drug delivery, and pharmaceutical properties[ 3 ]. Numerous nanoscale devices have been developed using physical, chemical, and green synthesis techniques as nanotechnology a advances. Among these, green synthesis has gained significant attention due to its simplicity, adaptability, and environmental compatibility[ 4 ]. Conventional nanoparticle synthesis methods often pose challenges, including lengthy processing times, high costs, complex steps, and the use of hazardous chemicals. Consequently, research efforts have increasingly focused on developing eco-friendly, rapid-synthesis techniques for nanoparticle production [ 5 ]. For more than a century, metallic nanoparticles have intrigued researchers and are now widely utilised in the biomedical sciences and engineering. With sizes ranging from 1 to 200nm and properties influenced by their shape, these nanoparticles find diverse applications and thus make their synthesis a prominent area of focus in nanotechnology research [ 6 ]. As a result, green synthesis is considered a vital strategy for mitigating the adverse effects of nanoparticles produced by conventional laboratory and industrial methods. Algae, as primitive microscopic plants, offer distinct benefits as cell factories for nanoparticle synthesis compared to larger plants and other microorganisms. Algae, classified in the kingdom Plantae, are aquatic, filamentous, photosynthetic organisms [ 7 ]. Algae possess a remarkable ability to synthesize nanoparticles either intracellularly or extracellularly, driven by their metabolic processes, secondary metabolites, or extracts derived from algal biomass[ 8 ]. In a preliminary phytochemical screening of Gracilaria foliifera , key bioactive compounds, including terpenoids, phenols, tannins, steroids, saponins, and flavonoids, were identified [ 9 ]. These phyoconstituents play a crucial role in the bioreduction and stabilization of metal ions, enabling the transformation of zinc acetate dihydrate into zinc oxide nanoparticles. This green synthesis approach yields environmentally friendly, functionally versatile nanoparticles with a wide range of applications[ 10 ]. Algae are crucial for the reduction and stabilization of nanoparticles at room temperature, which makes them well-suited for numerous applications that promote sustainable practices through green nanoscience [ 11 ]. ZnO-NPs exhibit biocompatible and multifunctional properties, and the U.S. Food and Drug Administration (FDA) classifies them as generally recognised as safe (GRAS) compounds. These properties make them highly suitable for biomedical applications, particularly in wound healing and topical drug delivery systems[ 12 ]. Incorporating ZnO-NPs into a Sodium alginate gel matrix offers a novel therapeutic approach to managing oral mucositis. The gel-based system ensures prolonged retention at the site of application, enhanced bioavailability, and sustained nanoparticle release, thereby improving therapeutic efficacy[ 13 ]. Additionally, the synergistic effect of algal bioactive compounds and ZnO-NPs may further enhance antioxidant and anti-inflammatory responses, promoting faster wound healing. Our ongoing research involves biosynthesized Zinc oxide nanoparticles (ZnO-NPs) incorporated into Sodium alginate gel matrix. These nanoparticles and gel are characterised using UV, FT-IR, XRD, SEM, and EDX to assess their antioxidant, anti-inflammatory, and Therapeutic properties in oral mucositis wound healing. Materials and Methods Analytical-grade Zinc acetate dihydrate was obtained from Sisco Research Laboratory. Nutrient agar and Mueller-Hinton (MH) agar were purchased from Hi-Media, Mumbai, India. A549 Cells were purchased from NCCS, Pune, India. 2.1 Preparation of Sample Gracilaria foliifera used in this study was collected from Mandapam, Ramanad, Tamil Nadu, India, and authenticated by Dr Babu, Assistant Professor, Department of Botany, Madras Christian College, Chennai. The samples were washed, shade-dried at room temperature, and ground into powder. For extraction, 5 g of dried algal powder was mixed with 100 mL of sterile distilled water and shaken for 24 hours. After incubation, 25 mL of 25 mM zinc acetate dihydrate was added dropwise to the extract, followed by overnight shaking—a colour change from dark brown to yellow indicated nanoparticle formation. The mixture was then centrifuged at 4,500 rpm for 30 minutes, and the resulting pellet was dried and stored. 2.1 Preparation of Gel 3g of sodium alginate was mixed with 100ml of sterile distilled water. 2% w/v ZnO-NPs was added dropwise to the extracted gel matrix, followed by overnight shaking. After mixing, the gel was stored for further characterisation and biomedical applications. The preparation of the ZnO-infused gel is shown in the Fig. 1 2.2 Characterization Methods The sodium alginate gel was measured using a Double Beam UV-Vis spectrophotometer (LMSPUV1900S, India) using 1 cm quartz cuvettes, spanning a wavelength range of 200–1000 nm. The functional biomolecules present in the ZnO-NPs infused gel were identified using FT-IR spectroscopy (Bruker Alpha II, Germany) over a spectral range of 500–3500 cm⁻¹. X-ray diffraction (XRD) was used to distinguish the crystalline from the amorphous nature of the gel, providing insights into their phase characteristics, degree of crystallinity, and structural details. The surface morphology of the ZnO-NPs-infused gel was analysed using SEM (JSM-7001F, JEOL, Japan) at 20 kV. The elemental composition was assessed using an EDX spectrometer (OXFORD X-Plor-30/C-Swift) under a nitrogen atmosphere with a heating rate of 10°C/min. 2.3 Stability analysis of ZnO-NPs infused Gel: The stability of the ZnO-NPs-infused Gel was assessed based on various physicochemical properties, including Colour, pH, odour, and microbial load [ 13 ]. 2.4 Antimicrobial activity The antimicrobial activity of ZnO-NPs-infused gel was evaluated using a well diffusion method against oral isolates. Bacterial cultures were subcultured in Mueller-Hinton broth and incubated for 14 hours at 37 ͦ C. Sterile cotton swabs were used to inoculate petri dishes containing Mueller-Hinton agar. Wells with a diameter of 6mm were created in the agar plates, into which various concentrations of ZnO-NPs-infused gel (20,40,60,80 µg/ml) were added. Streptomycin acts as a positive control, while distilled water serves as a negative control. After incubation, the zones of inhibition (ZOI) were measured. 2.5 Anti-inflammatory activity To analyse the anti-inflammatory activity of the ZnO-NPs-infused gel, an albumin denaturation assay was performed in a microtiter plate. Add a 1% BSA to each well. 50–400 µg/mL concentrations were added to the wells, with a nanoparticle serving as a blank. Furthermore, another well contains 1% BSA, and diclofenac sodium serves as a standard. The solution was kept in the incubator at room temperature for 15 min, then incubated for another 20 min at 55°C. After complete incubation, the absorbance was measured at 660 nm, and the percentage of inhibition was calculated. 2.6 Antioxidant activity The antioxidant activity of ZnO-NPs-infused gel was assessed using the DPPH assay. Various concentrations (20–80 µg/mL) were mixed with 1 mL of 1 mM DPPH solution in methanol and incubated in the dark at room temperature for 30 minutes. Absorbance was measured at 520 nm. L-ascorbic acid served as the standard, and 0.1 mM DPPH was used as the control. All tests were conducted in triplicate, and the results were expressed as mean ± standard deviation, calculated using SPSS version 21. Antioxidant activity was reported as a percentage of DPPH radical scavenging. Statistical analysis Each experiment was conducted in triplicate, and the mean ±standard deviation is used to represent the results. GraphPad Prism software version 8 was used for analysis. The Student’s t-test was used to assess statistical significance, with a threshold of p < 0.05. Results The formulated ZnO-NPs infused gel preparation was described in the pictorial representation in Fig. 1 . Stability of Gel The Physical stability of the ZnO-NPs-infused gel was assessed over a period of 14 days, including colour, pH, texture, and phase separation analysis, as demonstrated in Table 1 . The formulation exhibited a light yellow, translucent colour that remained unchanged throughout the study, indicating no chemical degradation or interactions that would affect its visual appearance. The texture of the gel was smooth and homogenous, suggesting uniform distribution of ZnO-NPs within the gel matrix and good formulation integrity. The pH of the gel was 5.03 ± 0.2, within the acceptable physiological range for topical application, indicating that the formulation is skin- and mucosa-compatible and non-irritating. Importantly, no phase separation was observed during the study period, even after storage, demonstrating excellent physical stability and strong polymer network integrity in the gel system. Table 1 S.No Parameters Observation 1 Colour r Light yellow 2 Texture Smooth gel 3 pH 5.03 ± 0.2 4 Phase separation No phase separation 3.1 UV- visible spectroscopy analysis Further, UV-Visible spectra were analysed, revealing the presence of ZnO-NPs. This was affirmed by the identification of peaks detected in the 321nm range Fig. 2 . 3.2 FT-IR The FT-IR spectrum of G.F ZnO-NPs showed in Fig. 3 that the absorption bands at 610.95cm − 1, representing C-Br stretch (Aliphatic bromo compounds), 1099.47cm- 1 indicating C-F stretch (Aliphatic fluoro compounds), 1410.14cm- 1 indicating OH bend (Phenol or tertiary alcohol), 1623.53cm − 1 indicating N-H bend (Secondary amine), 3339.4cm − 1 representing NH stretch (Aliphatic primary amine). The peaks observed at 1099.47 cm⁻¹ (C–F stretching) and 610.95 cm⁻¹ (C–Br stretching) may correspond to minor functional groups or structural vibrations within the complex gel system. 3.3 SEM analysis Scanning electron microscopy is used to analyse the surface morphology of the ZnO-NPs. The SEM images indicated that the ZnO-NPs exhibited a spherically irregular shape with some rough aggregation, as depicted in Fig. 4 . The SEM findings for ZnO-NPs revealed sizes ranging from 60 to 70nm, with an average size of 65nm. 3.4 EDX The elemental composition of ZnO-NPs was quantitatively analysed by EDX, as depicted in Fig. 5 . The EDX spectrum of ZnO-NPs, synthesised using zinc oxide as precursors, exhibited three prominent peaks: zinc at 1 keV and oxygen at 0.5 keV, indicating the presence of ZnO-NPs. The EDX analysis of ZnO-NPs prepared from zinc oxide salt precursors revealed the elemental composition and mass percentages of zinc and oxygen: Zn (41.48%), O (19.7%), and C (38.7%), respectively. Additionally, the EDX graph showed a carbon peak at 0.3 keV with a mass percentage of 38.7%, possibly indicating the presence of carbon atoms in the sodium alginate. The dominant red peaks at 2-8.6 keV correspond to the L and K emission lines of Zinc, confirming that zinc is the main constituent of the nanoparticles. 3.5 XRD XRD analysis provides significant information about the crystalline structure and particle size. In Fig. 6 , the XRD patterns of the ZnO-NPs are depicted. The data reveal diffraction peaks at theta angles of 28.4°, 31.6°, 36.1°, 40.5°, 45.6°, 50.0°, and 66.3 °. These peaks indicate a Crystallinity of 64.9%, with the remaining 35.1% representing an amorphous structure. Antimicrobial activity The antimicrobial activity of the ZnO-NPs-infused gel was significant against oral bacterial isolates, including E. coli, Candida albicans , and S. mutans , as illustrated in the Fig. 7 . The ZnO-NPs-infused gel showed higher antimicrobial activity against Candida albicans (14, 14, and 19mm), and the higher concentrations of the gel (50 and 100 µl) showed higher activity (18mm and 19mm) against S. mutans ; the lower concentration of 25 µl showed no detectable activity. In the case of E. coli , the gel exhibited moderate antimicrobial activity with a dose-dependent activity of 50 and 100 µg/ml, producing zones of inhibition of 14mm and 14mm. Anti-inflammatory activity The anti-inflammatory activity of ZnO-NPs-infused Gel demonstrated a concentration-dependent effect, as shown in the Fig. . 8 A higher concentration (400µg/ml) of ZnO-NPs infused gel showed a higher percentage of albumin denaturation 65.07% and the lower concentration of 50µg/ml showed a lower percentage of albumin denaturation 28.41% comparatively, standard Diclofenac sodium showed a higher percentage of denaturation than the ZnO-NPs infused gel. Antioxidant activity The antioxidant activity of ZnO-NPs infused-Gel showed dose-dependent significant activity in Fig. 9A Maximum concentration of 100µg/ml showed higher radical scavenging activity(25.4%), whereas the minimum concentration of 20µg/ml showed comparatively lower radical scavenging activity(74.3%). In comparison, the standard ascorbic acid showing 80.5% and 27.7% scavenging activity at corresponding concentrations. Intermediate concentrations 40–80µg/ml of the formulation exhibited 35.7%, 48.2% and 66.5%, which were lower than the standard values of 39.2, 49.6, and 78.2%. Discussion In our current study, the synthesis of G.F ZnO-NPs was visually confirmed by a colour transition from brown to yellowish-white after 24 hours of incubation. The aqueous Zn 2+ solution was converted into Zn 0 nanoparticles using the algal extract. The Gracilaria foliifera algal extract contains various bioactive compounds, such as terpenoids, phenols, tannins, steroids, and flavonoids, which act as reducing agents, facilitating the reduction of metal ions to form ZnO-NPs [ 14 ][ 15 ]. The UV–Vis spectroscopic analysis of the ZnO-NPs exhibited a prominent absorption peak at 321 nm, indicating successful nanoparticle formation. This peak is slightly blue-shifted compared with previous studies, such as ZnO-NPs synthesized using Hibiscus sabdariffa , which showed a peak at 377 nm [ 16 ], and Lavandula prunonioides -ZnO/NPs, which exhibited a peak at 370 nm [ 17 ]. This shift may be attributed to the formation of smaller-sized nanoparticles, as reduced particle size often leads to quantum confinement effects, resulting in absorption at lower wavelengths. Furthermore, the gel matrix itself could contribute to this shift by affecting nanoparticle dispersion and preventing aggregation, resulting in a more uniform, smaller-particle distribution. These factors collectively indicate that the synthesis approach and biomaterial environment play a crucial role in determining the physicochemical characteristics of ZnO nanoparticles. The FT-IR spectrum of the ZnO-NPs exhibited characteristic absorption bands corresponding to functional groups present in the polymeric matrix and their interaction with the incorporated nanoparticles[ 18 ]. Overall, the FT-IR analysis confirms the successful incorporation of commercially obtained ZnO nanoparticles into the sodium alginate gel matrix. The interaction between ZnO-NPs and functional groups, such as hydroxyl and carboxyl groups, in alginate enhances nanoparticle stability and prevents aggregation. This improved dispersion is likely to enhance biological activities, including antimicrobial and wound-healing efficacy, making the formulation suitable for applications in oral mucositis. The XRD pattern results indicated a crystallinity of 64.9% and an amorphous fraction of 35.1%. The 2ѳ angles of the observed diffraction peaks were 28.4 ͦ, 31.6 ͦ, 36.1 2 ͦ, 40.5 ͦ and 45.6 ͦ, 50.0 ͦ, 66.3 ͦ. These peaks correspond to those reported in other studies [19] and to the formation of the hexagonal structure of the ZnO-NPs. Furthermore, the XRD pattern showed only the distinctive ZnO peaks, confirming the purity of the synthesised ZnO-NPs. The ZnO-NPs' ideal crystalline characteristics are further supported by the existence of a strong and narrow diffraction peak [20]. The XRD pattern results indicated a crystallinity of 64.9% and an amorphous structure of 35.1%. The 2ѳ angles of the observed diffraction peaks were 28.4 ͦ, 31.6 ͦ, 36.1 2 ͦ, 40.5 ͦ and 45.6 ͦ, 50.0 ͦ, 66.3 ͦ. These peaks correspond to those reported in other studies [19] and to the formation of the hexagonal structure of the ZnO-NPs. Furthermore, the XRD pattern showed only the distinctive ZnO peaks, confirming the purity of the synthesised ZnO-NPs. The ZnO-NPs' ideal crystalline characteristics are further supported by the existence of a strong and narrow diffraction peak [20]. The SEM analysis of the biosynthesised ZnO nanoparticles revealed an irregular morphology with noticeable surface roughness and aggregation. The particle sizes ranged from 60 to 70 nm, with an average diameter of approximately 65 nm. This nanoscale size range supports successful ZnO-NP synthesis and is consistent with previous reports on green-synthesised nanoparticles. The irregular shape and aggregation may be attributed to organic residues from the Gracilaria foliifera extract, which can act as both reducing and capping agents during synthesis, thereby influencing nanoparticle morphology. Furthermore, EDX analysis provided insight into the elemental composition of the synthesized nanoparticles. The spectrum confirmed the presence of zinc (41.48%) and oxygen (19.7%), validating the formation of ZnO. The presence of carbon (38.7%) likely originates from the phytoconstituents of the algal extract, which may have remained adsorbed on the nanoparticle surface. This carbon content supports the role of biological molecules in stabilizing the nanoparticles, thereby contributing to their bioactivity. Overall, the SEM and EDX results corroborate the successful green synthesis of ZnO-NPs using G. foliifera and highlight the role of algal biomolecules in defining the structural and elemental characteristics of the ZnO-NPs[21]. The antimicrobial activity of ZnO-NPs infused gel showing potetially strong activity for the oral isolates such as C.albigans, S.mutans and E.coli. This enhanced efficacy can be attributed to the intrinsic antimicrobial properties of ZnO nanoparticles, which generate reactive oxygen species and disrupt microbial cell membranes. The gel matrix further aids in sustained release and uniform dispersion of nanoparticles, improving contact with microbial cells. The observed activity highlights the formulation’s potential as an effective therapeutic agent for managing oral infections and promoting wound healing in oral mucositis[22]. The antioxidant activity of the ZnO-NPs-infused gel exhibited a significant, dose-dependent effect, with an IC₅₀ of approximately 47.6 µg/mL for the biosynthesised nanoparticles. A comparable study found that the DPPH scavenging activity of ZnO-NPs was greater than that of the traditional reference, L-ascorbic acid. This enhanced activity may be attributed to the higher levels of bioactive compounds, such as phenolics and flavonoids, found in the algae, which may explain the stronger activity [23]. The ZnO-NPs–infused gel exhibited notable anti-inflammatory activity in a concentration-dependent manner, as evidenced by the inhibition of albumin denaturation. The higher concentration (400 µg/ml) showed maximum inhibition (65.07%), while the lower concentration (50 µg/ml) demonstrated comparatively reduced activity (28.41%). This trend suggests that increasing the concentration of ZnO nanoparticles enhances their ability to stabilize protein structures and prevent denaturation, a key mechanism in inflammation control. The anti-inflammatory effect of ZnO nanoparticles may be attributed to their ability to modulate inflammatory mediators and reduce oxidative stress[24]. Additionally, the sodium alginate gel matrix may facilitate sustained release and improved bioavailability of nanoparticles, thereby enhancing activity. Although the standard drug diclofenac sodium exhibited superior inhibition, the ZnO-NPs–infused gel still showed promising anti-inflammatory potential. Overall, these findings indicate that the formulation could serve as an effective alternative or adjunct for managing inflammation, particularly in the treatment of oral mucositis. Conclusion The ZnO-NPs–infused sodium alginate gel demonstrated promising multifunctional biological properties, including antioxidant, anti-inflammatory, and antimicrobial activities. The formulation exhibited significant dose-dependent antioxidant activity, with a notable IC₅₀ value, indicating effective free-radical scavenging potential, possibly enhanced by the presence of bioactive compounds in the gel system. In addition, the gel exhibited appreciable anti-inflammatory activity by inhibiting protein denaturation, suggesting it can mitigate inflammatory responses. The antimicrobial studies further confirmed strong activity against key oral pathogens, highlighting its effectiveness in controlling infection. Collectively, these findings suggest that the ZnO-NPs–infused gel is a potential therapeutic candidate for oral mucositis management by reducing oxidative stress, inflammation, and microbial load, thereby promoting improved wound healing. Declarations Conflict of interest No conflict of interest is declared by all authors. Consent for Publication Not applicable. Ethics Approval and Consent to Participate Not applicable. Funding No funding. Author Contribution Conceptualisation, T.T and R.S.; methodology, R.S.; software, R.S.; validation, S.M., T.T and S.R; formal analysis, S.M and R.S.; investigation, T.T.; resources,; data curation, R.S.; writing—original draft preparation, R.S and T.T; writing—review and editing, T.T.; visualisation, R.S.; supervision, R.S and S.M; project administration, T.T.; funding acquisition, T.T and R.S. All authors have read and agreed to the published version of the manuscript. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9340679","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":634203041,"identity":"66cd08b4-7aba-4b8f-8183-6a6465cbf81e","order_by":0,"name":"Thoshi Tarun K","email":"","orcid":"","institution":"Saveetha Institute of Medical And Technical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Thoshi","middleName":"Tarun","lastName":"K","suffix":""},{"id":634203042,"identity":"968c1de4-4b76-41e5-a830-dcdefc87391b","order_by":1,"name":"Ramanathan Snega","email":"data:image/png;base64,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","orcid":"","institution":"Saveetha Dental College and Hospitals (SIMATS)","correspondingAuthor":true,"prefix":"","firstName":"Ramanathan","middleName":"","lastName":"Snega","suffix":""},{"id":634203043,"identity":"f9088f60-d077-4afc-b938-3845c03c358b","order_by":2,"name":"Muthupandian Saravanan","email":"","orcid":"","institution":"Saveetha Dental College and Hospitals (SIMATS)","correspondingAuthor":false,"prefix":"","firstName":"Muthupandian","middleName":"","lastName":"Saravanan","suffix":""}],"badges":[],"createdAt":"2026-04-07 06:53:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9340679/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9340679/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108527931,"identity":"074c68ae-68b3-4c68-9bc6-d4fc5e3739b0","added_by":"auto","created_at":"2026-05-05 15:28:17","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":296936,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverview of the Gel preparation\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9340679/v1/5beae021586535ff333b3d77.png"},{"id":108805168,"identity":"b70cf2af-441d-4f98-a7b1-39b5297f9dd8","added_by":"auto","created_at":"2026-05-08 15:25:03","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":32325,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUV-spectroscopy analysis of G.F ZnO-NPs\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9340679/v1/43a632e76782ca1a0ed77d01.png"},{"id":108527935,"identity":"f2e8fb0c-fbde-4e68-b141-e1785f5bba34","added_by":"auto","created_at":"2026-05-05 15:28:17","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":71223,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFT-IR analysis of G.F ZnO-NPs\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9340679/v1/525c6882b78eb2744dcce77c.png"},{"id":108527933,"identity":"23e53c43-5389-498e-9f87-d807b15ddda9","added_by":"auto","created_at":"2026-05-05 15:28:17","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":466865,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSEM analysis of ZnO-NPs-infused Gel (A) 5µm scale (B) 1 µm scale (C) 500nm scale\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-9340679/v1/652fc7d880cf783e1633c48f.png"},{"id":108805132,"identity":"873de5ad-48e0-4a11-9af6-c9f827460e3e","added_by":"auto","created_at":"2026-05-08 15:24:55","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":218656,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEDX analysis of ZnO-NPs-infused Gel\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-9340679/v1/c1c1e291add250a73d43903d.png"},{"id":108803968,"identity":"c9d2f43d-bbfc-47f6-b9ec-d2c87a7b1108","added_by":"auto","created_at":"2026-05-08 15:13:02","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":55404,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eXRD analysis of ZnO-NPs-infused Gel\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-9340679/v1/f507c8a6891cc443eef06beb.png"},{"id":108527937,"identity":"75a300ac-d8b5-4bb5-9cd1-a1435eabc7ad","added_by":"auto","created_at":"2026-05-05 15:28:17","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":221087,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAntimicrobial activity of the ZnO-NPs infused Gel\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-9340679/v1/bd18c9788f1a96a8085d3bb8.png"},{"id":108527940,"identity":"42388175-ae1a-402d-bc94-422bc80ec580","added_by":"auto","created_at":"2026-05-05 15:28:17","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":41585,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-9340679/v1/45997bf67ddff0260782d353.png"},{"id":108527939,"identity":"01905b56-2c1c-4c33-befb-5ef0f47d5894","added_by":"auto","created_at":"2026-05-05 15:28:17","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":39684,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-9340679/v1/0a3d67faecb4c7e1bc3fa539.png"},{"id":108809486,"identity":"edffebbb-ee25-47c3-8c31-06ab9ad3d4a3","added_by":"auto","created_at":"2026-05-08 15:53:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1772512,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9340679/v1/302582ab-c60f-44b3-95e9-1deab0985450.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Topical red algal-mediated zinc oxide nanoparticles infused in gel as a therapeutic strategy for oral mucositis wound healing","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTopical drug delivery systems have gained significant attention in recent years for the effective management of oral mucositis, a inflammatory condition commonly related to chemotherapy and radiotherapy [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Oral mucositis is characterised by epithelial damage, ulceration, pain, and increased susceptibility to secondary infections, thereby severely affecting patients' quality of life and treatment outcomes [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Conventional therapies often provide only symptomatic relief and may lead to undesirable side effects, necessitating the development of safer and more effective therapeutic strategies. Nanotechnology has emerged as a cutting-edge field with widespread applications across various pharmaceutical industries. It also plays a pivotal role in computing, environmental science, drug delivery, and pharmaceutical properties[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Numerous nanoscale devices have been developed using physical, chemical, and green synthesis techniques as nanotechnology a advances. Among these, green synthesis has gained significant attention due to its simplicity, adaptability, and environmental compatibility[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Conventional nanoparticle synthesis methods often pose challenges, including lengthy processing times, high costs, complex steps, and the use of hazardous chemicals. Consequently, research efforts have increasingly focused on developing eco-friendly, rapid-synthesis techniques for nanoparticle production [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. For more than a century, metallic nanoparticles have intrigued researchers and are now widely utilised in the biomedical sciences and engineering. With sizes ranging from 1 to 200nm and properties influenced by their shape, these nanoparticles find diverse applications and thus make their synthesis a prominent area of focus in nanotechnology research [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. As a result, green synthesis is considered a vital strategy for mitigating the adverse effects of nanoparticles produced by conventional laboratory and industrial methods. Algae, as primitive microscopic plants, offer distinct benefits as cell factories for nanoparticle synthesis compared to larger plants and other microorganisms. Algae, classified in the kingdom Plantae, are aquatic, filamentous, photosynthetic organisms [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Algae possess a remarkable ability to synthesize nanoparticles either intracellularly or extracellularly, driven by their metabolic processes, secondary metabolites, or extracts derived from algal biomass[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In a preliminary phytochemical screening of \u003cem\u003eGracilaria foliifera\u003c/em\u003e, key bioactive compounds, including terpenoids, phenols, tannins, steroids, saponins, and flavonoids, were identified [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. These phyoconstituents play a crucial role in the bioreduction and stabilization of metal ions, enabling the transformation of zinc acetate dihydrate into zinc oxide nanoparticles. This green synthesis approach yields environmentally friendly, functionally versatile nanoparticles with a wide range of applications[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Algae are crucial for the reduction and stabilization of nanoparticles at room temperature, which makes them well-suited for numerous applications that promote sustainable practices through green nanoscience [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. ZnO-NPs exhibit biocompatible and multifunctional properties, and the U.S. Food and Drug Administration (FDA) classifies them as generally recognised as safe (GRAS) compounds. These properties make them highly suitable for biomedical applications, particularly in wound healing and topical drug delivery systems[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Incorporating ZnO-NPs into a Sodium alginate gel matrix offers a novel therapeutic approach to managing oral mucositis. The gel-based system ensures prolonged retention at the site of application, enhanced bioavailability, and sustained nanoparticle release, thereby improving therapeutic efficacy[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Additionally, the synergistic effect of algal bioactive compounds and ZnO-NPs may further enhance antioxidant and anti-inflammatory responses, promoting faster wound healing. Our ongoing research involves biosynthesized Zinc oxide nanoparticles (ZnO-NPs) incorporated into Sodium alginate gel matrix. These nanoparticles and gel are characterised using UV, FT-IR, XRD, SEM, and EDX to assess their antioxidant, anti-inflammatory, and Therapeutic properties in oral mucositis wound healing.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eAnalytical-grade Zinc acetate dihydrate was obtained from Sisco Research Laboratory. Nutrient agar and Mueller-Hinton (MH) agar were purchased from Hi-Media, Mumbai, India. A549 Cells were purchased from NCCS, Pune, India.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e2.1 Preparation of Sample\u003c/b\u003e\u003c/h2\u003e \u003cp\u003e \u003cem\u003eGracilaria foliifera\u003c/em\u003e used in this study was collected from Mandapam, Ramanad, Tamil Nadu, India, and authenticated by Dr Babu, Assistant Professor, Department of Botany, Madras Christian College, Chennai. The samples were washed, shade-dried at room temperature, and ground into powder. For extraction, 5 g of dried algal powder was mixed with 100 mL of sterile distilled water and shaken for 24 hours. After incubation, 25 mL of 25 mM zinc acetate dihydrate was added dropwise to the extract, followed by overnight shaking\u0026mdash;a colour change from dark brown to yellow indicated nanoparticle formation. The mixture was then centrifuged at 4,500 rpm for 30 minutes, and the resulting pellet was dried and stored.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Preparation of Gel\u003c/h2\u003e \u003cp\u003e3g of sodium alginate was mixed with 100ml of sterile distilled water. 2% w/v ZnO-NPs was added dropwise to the extracted gel matrix, followed by overnight shaking. After mixing, the gel was stored for further characterisation and biomedical applications. The preparation of the ZnO-infused gel is shown in the Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Characterization Methods\u003c/h2\u003e \u003cp\u003eThe sodium alginate gel was measured using a Double Beam UV-Vis spectrophotometer (LMSPUV1900S, India) using 1 cm quartz cuvettes, spanning a wavelength range of 200\u0026ndash;1000 nm. The functional biomolecules present in the ZnO-NPs infused gel were identified using FT-IR spectroscopy (Bruker Alpha II, Germany) over a spectral range of 500\u0026ndash;3500 cm⁻\u0026sup1;. X-ray diffraction (XRD) was used to distinguish the crystalline from the amorphous nature of the gel, providing insights into their phase characteristics, degree of crystallinity, and structural details. The surface morphology of the ZnO-NPs-infused gel was analysed using SEM (JSM-7001F, JEOL, Japan) at 20 kV. The elemental composition was assessed using an EDX spectrometer (OXFORD X-Plor-30/C-Swift) under a nitrogen atmosphere with a heating rate of 10\u0026deg;C/min.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Stability analysis of ZnO-NPs infused Gel:\u003c/h2\u003e \u003cp\u003eThe stability of the ZnO-NPs-infused Gel was assessed based on various physicochemical properties, including Colour, pH, odour, and microbial load [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Antimicrobial activity\u003c/h2\u003e \u003cp\u003eThe antimicrobial activity of ZnO-NPs-infused gel was evaluated using a well diffusion method against oral isolates. Bacterial cultures were subcultured in Mueller-Hinton broth and incubated for 14 hours at 37 ͦ C. Sterile cotton swabs were used to inoculate petri dishes containing Mueller-Hinton agar. Wells with a diameter of 6mm were created in the agar plates, into which various concentrations of ZnO-NPs-infused gel (20,40,60,80 \u0026micro;g/ml) were added. Streptomycin acts as a positive control, while distilled water serves as a negative control. After incubation, the zones of inhibition (ZOI) were measured.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Anti-inflammatory activity\u003c/h2\u003e \u003cp\u003eTo analyse the anti-inflammatory activity of the ZnO-NPs-infused gel, an albumin denaturation assay was performed in a microtiter plate. Add a 1% BSA to each well. 50\u0026ndash;400 \u0026micro;g/mL concentrations were added to the wells, with a nanoparticle serving as a blank. Furthermore, another well contains 1% BSA, and diclofenac sodium serves as a standard. The solution was kept in the incubator at room temperature for 15 min, then incubated for another 20 min at 55\u0026deg;C. After complete incubation, the absorbance was measured at 660 nm, and the percentage of inhibition was calculated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Antioxidant activity\u003c/h2\u003e \u003cp\u003eThe antioxidant activity of ZnO-NPs-infused gel was assessed using the DPPH assay. Various concentrations (20\u0026ndash;80 \u0026micro;g/mL) were mixed with 1 mL of 1 mM DPPH solution in methanol and incubated in the dark at room temperature for 30 minutes. Absorbance was measured at 520 nm. L-ascorbic acid served as the standard, and 0.1 mM DPPH was used as the control. All tests were conducted in triplicate, and the results were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation, calculated using SPSS version 21. Antioxidant activity was reported as a percentage of DPPH radical scavenging.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eEach experiment was conducted in triplicate, and the mean \u0026plusmn;standard deviation is used to represent the results. GraphPad Prism software version 8 was used for analysis. The Student\u0026rsquo;s t-test was used to assess statistical significance, with a threshold of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eThe formulated ZnO-NPs infused gel preparation was described in the pictorial representation in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eStability of Gel\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe Physical stability of the ZnO-NPs-infused gel was assessed over a period of 14 days, including colour, pH, texture, and phase separation analysis, as demonstrated in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The formulation exhibited a light yellow, translucent colour that remained unchanged throughout the study, indicating no chemical degradation or interactions that would affect its visual appearance. The texture of the gel was smooth and homogenous, suggesting uniform distribution of ZnO-NPs within the gel matrix and good formulation integrity. The pH of the gel was 5.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2, within the acceptable physiological range for topical application, indicating that the formulation is skin- and mucosa-compatible and non-irritating. Importantly, no phase separation was observed during the study period, even after storage, demonstrating excellent physical stability and strong polymer network integrity in the gel system.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e\u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS.No\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eParameters\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eObservation\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eColour\u003c/b\u003e\u003cb\u003er\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLight yellow\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eTexture\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSmooth gel\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003epH\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003ePhase separation\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo phase separation\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.1 UV- visible spectroscopy analysis\u003c/h2\u003e \u003cp\u003eFurther, UV-Visible spectra were analysed, revealing the presence of ZnO-NPs. This was affirmed by the identification of peaks detected in the 321nm range Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.2 FT-IR\u003c/h2\u003e \u003cp\u003eThe FT-IR spectrum of G.F ZnO-NPs showed in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e that the absorption bands at 610.95cm\u003csup\u003e\u0026minus;\u0026thinsp;1,\u003c/sup\u003e representing C-Br stretch (Aliphatic bromo compounds), 1099.47cm-\u003csup\u003e1\u003c/sup\u003e indicating C-F stretch (Aliphatic fluoro compounds), 1410.14cm-\u003csup\u003e1\u003c/sup\u003e indicating OH bend (Phenol or tertiary alcohol), 1623.53cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e indicating N-H bend (Secondary amine), 3339.4cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e representing NH stretch (Aliphatic primary amine). The peaks observed at 1099.47 cm⁻\u0026sup1; (C\u0026ndash;F stretching) and 610.95 cm⁻\u0026sup1; (C\u0026ndash;Br stretching) may correspond to minor functional groups or structural vibrations within the complex gel system.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.3 SEM analysis\u003c/h2\u003e \u003cp\u003eScanning electron microscopy is used to analyse the surface morphology of the ZnO-NPs. The SEM images indicated that the ZnO-NPs exhibited a spherically irregular shape with some rough aggregation, as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. The SEM findings for ZnO-NPs revealed sizes ranging from 60 to 70nm, with an average size of 65nm.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.4 EDX\u003c/h2\u003e \u003cp\u003eThe elemental composition of ZnO-NPs was quantitatively analysed by EDX, as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. The EDX spectrum of ZnO-NPs, synthesised using zinc oxide as precursors, exhibited three prominent peaks: zinc at 1 keV and oxygen at 0.5 keV, indicating the presence of ZnO-NPs. The EDX analysis of ZnO-NPs prepared from zinc oxide salt precursors revealed the elemental composition and mass percentages of zinc and oxygen: Zn (41.48%), O (19.7%), and C (38.7%), respectively. Additionally, the EDX graph showed a carbon peak at 0.3 keV with a mass percentage of 38.7%, possibly indicating the presence of carbon atoms in the sodium alginate. The dominant red peaks at 2-8.6 keV correspond to the L and K emission lines of Zinc, confirming that zinc is the main constituent of the nanoparticles.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.5 XRD\u003c/h2\u003e \u003cp\u003eXRD analysis provides significant information about the crystalline structure and particle size. In Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, the XRD patterns of the ZnO-NPs are depicted. The data reveal diffraction peaks at theta angles of 28.4\u0026deg;, 31.6\u0026deg;, 36.1\u0026deg;, 40.5\u0026deg;, 45.6\u0026deg;, 50.0\u0026deg;, and 66.3 \u0026deg;. These peaks indicate a Crystallinity of 64.9%, with the remaining 35.1% representing an amorphous structure.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eAntimicrobial activity\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe antimicrobial activity of the ZnO-NPs-infused gel was significant against oral bacterial isolates, including \u003cem\u003eE. coli, Candida albicans\u003c/em\u003e, and \u003cem\u003eS. mutans\u003c/em\u003e, as illustrated in the Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e. The ZnO-NPs-infused gel showed higher antimicrobial activity against \u003cem\u003eCandida albicans\u003c/em\u003e (14, 14, and 19mm), and the higher concentrations of the gel (50 and 100 \u0026micro;l) showed higher activity (18mm and 19mm) against \u003cem\u003eS. mutans\u003c/em\u003e; the lower concentration of 25 \u0026micro;l showed no detectable activity. In the case of \u003cem\u003eE. coli\u003c/em\u003e, the gel exhibited moderate antimicrobial activity with a dose-dependent activity of 50 and 100 \u0026micro;g/ml, producing zones of inhibition of 14mm and 14mm.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eAnti-inflammatory activity\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe anti-inflammatory activity of ZnO-NPs-infused Gel demonstrated a concentration-dependent effect, as shown in the \u003cb\u003eFig.\u003c/b\u003e.\u003cb\u003e8\u003c/b\u003e A higher concentration (400\u0026micro;g/ml) of ZnO-NPs infused gel showed a higher percentage of albumin denaturation 65.07% and the lower concentration of 50\u0026micro;g/ml showed a lower percentage of albumin denaturation 28.41% comparatively, standard Diclofenac sodium showed a higher percentage of denaturation than the ZnO-NPs infused gel.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAntioxidant activity\u003c/h3\u003e\n\u003cp\u003eThe antioxidant activity of ZnO-NPs infused-Gel showed dose-dependent significant activity in \u003cb\u003eFig.\u0026nbsp;9A\u003c/b\u003e Maximum concentration of 100\u0026micro;g/ml showed higher radical scavenging activity(25.4%), whereas the minimum concentration of 20\u0026micro;g/ml showed comparatively lower radical scavenging activity(74.3%). In comparison, the standard ascorbic acid showing 80.5% and 27.7% scavenging activity at corresponding concentrations. Intermediate concentrations 40\u0026ndash;80\u0026micro;g/ml of the formulation exhibited 35.7%, 48.2% and 66.5%, which were lower than the standard values of 39.2, 49.6, and 78.2%.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn our current study, the synthesis of G.F ZnO-NPs was visually confirmed by a colour transition from brown to yellowish-white after 24 hours of incubation. The aqueous Zn\u003csup\u003e2+\u003c/sup\u003e solution was converted into Zn\u003csup\u003e0\u003c/sup\u003e nanoparticles using the algal extract. The \u003cem\u003eGracilaria foliifera\u003c/em\u003e algal extract contains various bioactive compounds, such as terpenoids, phenols, tannins, steroids, and flavonoids, which act as reducing agents, facilitating the reduction of metal ions to form ZnO-NPs [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e][\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The UV\u0026ndash;Vis spectroscopic analysis of the ZnO-NPs exhibited a prominent absorption peak at 321 nm, indicating successful nanoparticle formation. This peak is slightly blue-shifted compared with previous studies, such as ZnO-NPs synthesized using \u003cem\u003eHibiscus sabdariffa\u003c/em\u003e, which showed a peak at 377 nm [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], and \u003cem\u003eLavandula prunonioides\u003c/em\u003e-ZnO/NPs, which exhibited a peak at 370 nm [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. This shift may be attributed to the formation of smaller-sized nanoparticles, as reduced particle size often leads to quantum confinement effects, resulting in absorption at lower wavelengths. Furthermore, the gel matrix itself could contribute to this shift by affecting nanoparticle dispersion and preventing aggregation, resulting in a more uniform, smaller-particle distribution. These factors collectively indicate that the synthesis approach and biomaterial environment play a crucial role in determining the physicochemical characteristics of ZnO nanoparticles. The FT-IR spectrum of the ZnO-NPs exhibited characteristic absorption bands corresponding to functional groups present in the polymeric matrix and their interaction with the incorporated nanoparticles[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Overall, the FT-IR analysis confirms the successful incorporation of commercially obtained ZnO nanoparticles into the sodium alginate gel matrix. The interaction between ZnO-NPs and functional groups, such as hydroxyl and carboxyl groups, in alginate enhances nanoparticle stability and prevents aggregation. This improved dispersion is likely to enhance biological activities, including antimicrobial and wound-healing efficacy, making the formulation suitable for applications in oral mucositis. The XRD pattern results indicated a crystallinity of 64.9% and an amorphous fraction of 35.1%. The 2ѳ angles of the observed diffraction peaks were 28.4 ͦ, 31.6 ͦ, 36.1 2 ͦ, 40.5 ͦ and 45.6 ͦ, 50.0 ͦ, 66.3 ͦ. These peaks correspond to those reported in other studies [19] and to the formation of the hexagonal structure of the ZnO-NPs. Furthermore, the XRD pattern showed only the distinctive ZnO peaks, confirming the purity of the synthesised ZnO-NPs. The ZnO-NPs' ideal crystalline characteristics are further supported by the existence of a strong and narrow diffraction peak [20]. The XRD pattern results indicated a crystallinity of 64.9% and an amorphous structure of 35.1%. The 2ѳ angles of the observed diffraction peaks were 28.4 ͦ, 31.6 ͦ, 36.1 2 ͦ, 40.5 ͦ and 45.6 ͦ, 50.0 ͦ, 66.3 ͦ. These peaks correspond to those reported in other studies [19] and to the formation of the hexagonal structure of the ZnO-NPs. Furthermore, the XRD pattern showed only the distinctive ZnO peaks, confirming the purity of the synthesised ZnO-NPs. The ZnO-NPs' ideal crystalline characteristics are further supported by the existence of a strong and narrow diffraction peak [20]. The SEM analysis of the biosynthesised ZnO nanoparticles revealed an irregular morphology with noticeable surface roughness and aggregation. The particle sizes ranged from 60 to 70 nm, with an average diameter of approximately 65 nm. This nanoscale size range supports successful ZnO-NP synthesis and is consistent with previous reports on green-synthesised nanoparticles. The irregular shape and aggregation may be attributed to organic residues from the \u003cem\u003eGracilaria foliifera\u003c/em\u003e extract, which can act as both reducing and capping agents during synthesis, thereby influencing nanoparticle morphology. Furthermore, EDX analysis provided insight into the elemental composition of the synthesized nanoparticles. The spectrum confirmed the presence of zinc (41.48%) and oxygen (19.7%), validating the formation of ZnO. The presence of carbon (38.7%) likely originates from the phytoconstituents of the algal extract, which may have remained adsorbed on the nanoparticle surface. This carbon content supports the role of biological molecules in stabilizing the nanoparticles, thereby contributing to their bioactivity. Overall, the SEM and EDX results corroborate the successful green synthesis of ZnO-NPs using \u003cem\u003eG. foliifera\u003c/em\u003e and highlight the role of algal biomolecules in defining the structural and elemental characteristics of the ZnO-NPs[21]. The antimicrobial activity of ZnO-NPs infused gel showing potetially strong activity for the oral isolates such as \u003cem\u003eC.albigans, S.mutans\u003c/em\u003e and \u003cem\u003eE.coli.\u003c/em\u003e This enhanced efficacy can be attributed to the intrinsic antimicrobial properties of ZnO nanoparticles, which generate reactive oxygen species and disrupt microbial cell membranes. The gel matrix further aids in sustained release and uniform dispersion of nanoparticles, improving contact with microbial cells. The observed activity highlights the formulation\u0026rsquo;s potential as an effective therapeutic agent for managing oral infections and promoting wound healing in oral mucositis[22]. The antioxidant activity of the ZnO-NPs-infused gel exhibited a significant, dose-dependent effect, with an IC₅₀ of approximately 47.6 \u0026micro;g/mL for the biosynthesised nanoparticles. A comparable study found that the DPPH scavenging activity of ZnO-NPs was greater than that of the traditional reference, L-ascorbic acid. This enhanced activity may be attributed to the higher levels of bioactive compounds, such as phenolics and flavonoids, found in the algae, which may explain the stronger activity [23]. The ZnO-NPs\u0026ndash;infused gel exhibited notable anti-inflammatory activity in a concentration-dependent manner, as evidenced by the inhibition of albumin denaturation. The higher concentration (400 \u0026micro;g/ml) showed maximum inhibition (65.07%), while the lower concentration (50 \u0026micro;g/ml) demonstrated comparatively reduced activity (28.41%). This trend suggests that increasing the concentration of ZnO nanoparticles enhances their ability to stabilize protein structures and prevent denaturation, a key mechanism in inflammation control. The anti-inflammatory effect of ZnO nanoparticles may be attributed to their ability to modulate inflammatory mediators and reduce oxidative stress[24]. Additionally, the sodium alginate gel matrix may facilitate sustained release and improved bioavailability of nanoparticles, thereby enhancing activity. Although the standard drug diclofenac sodium exhibited superior inhibition, the ZnO-NPs\u0026ndash;infused gel still showed promising anti-inflammatory potential. Overall, these findings indicate that the formulation could serve as an effective alternative or adjunct for managing inflammation, particularly in the treatment of oral mucositis.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe ZnO-NPs\u0026ndash;infused sodium alginate gel demonstrated promising multifunctional biological properties, including antioxidant, anti-inflammatory, and antimicrobial activities. The formulation exhibited significant dose-dependent antioxidant activity, with a notable IC₅₀ value, indicating effective free-radical scavenging potential, possibly enhanced by the presence of bioactive compounds in the gel system. In addition, the gel exhibited appreciable anti-inflammatory activity by inhibiting protein denaturation, suggesting it can mitigate inflammatory responses. The antimicrobial studies further confirmed strong activity against key oral pathogens, highlighting its effectiveness in controlling infection. Collectively, these findings suggest that the ZnO-NPs\u0026ndash;infused gel is a potential therapeutic candidate for oral mucositis management by reducing oxidative stress, inflammation, and microbial load, thereby promoting improved wound healing.\u003c/p\u003e"},{"header":"Declarations","content":" \u003cp\u003e \u003cstrong\u003eConflict of interest\u003c/strong\u003e \u003cp\u003eNo conflict of interest is declared by all authors.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for Publication\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eEthics Approval and Consent to Participate\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eNo funding.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualisation, T.T and R.S.; methodology, R.S.; software, R.S.; validation, S.M., T.T and S.R; formal analysis, S.M and R.S.; investigation, T.T.; resources,; data curation, R.S.; writing\u0026mdash;original draft preparation, R.S and T.T; writing\u0026mdash;review and editing, T.T.; visualisation, R.S.; supervision, R.S and S.M; project administration, T.T.; funding acquisition, T.T and R.S. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eAuthors would like to acknowledge White Lab, SDC, SIMATS, Chennai, India, for providing the characterization facilities.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data and materials of the study are available and can be provided on request. The corresponding author can be contacted anytime to get the data of the study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAl-Taie A, Al-Shohani AD, Albasry Z, Altaee A (2020) Current topical trends and novel therapeutic approaches and delivery systems for oral mucositis management. 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BioMed research international, 2023, 3280708. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1155/2023/3280708\u003c/span\u003e\u003cspan address=\"10.1155/2023/3280708\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"oral-and-maxillofacial-surgery","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"omfs","sideBox":"Learn more about [Oral and Maxillofacial Surgery](http://link.springer.com/journal/10006)","snPcode":"10006","submissionUrl":"https://submission.nature.com/new-submission/10006/3","title":"Oral and Maxillofacial Surgery","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Topical gel, Wound Healing, Red algae, Oral Mucositis","lastPublishedDoi":"10.21203/rs.3.rs-9340679/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9340679/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eOral mucositis is a debilitating inflammatory condition requiring effective topical therapeutics with enhanced healing potential. Algae-mediated zinc oxide nanoparticles (ZnO NPs) were infused into the sodium alginate gel and then characterised by UV–visible spectroscopy, which confirmed the presence of ZnO-NPs with a peak at 321 nm, while FT-IR analysis identified functional groups including C-Br, C-F, OH, N-H, and NH stretches. The morphology of the ZnO-NPs-infused gel exhibited irregular shapes with an average size of 60–70 nm. EDX analysis showed elemental compositions of Zn (41.48%), O (19.7%), and C (38.7%), along with 64.9% crystallinity. The physical stability of the gel was characterized by the appearance, pH, texture, and phase separation. ZnO-NPs-infused gel showed antioxidant activity, with 74.3% scavenging at 100 µg/ml. Additionally, it showed significant antimicrobial activity against oral pathogens, including \u003cem\u003eStreptoccus mutans, E. coli\u003c/em\u003e, and \u003cem\u003eCandida albicans\u003c/em\u003e. These findings suggest that the ZnO-NPs–infused gel is a potential therapeutic candidate for managing oral mucositis by reducing oxidative stress, inflammation, and microbial load, thereby promoting improved wound healing.\u003c/p\u003e","manuscriptTitle":"Topical red algal-mediated zinc oxide nanoparticles infused in gel as a therapeutic strategy for oral mucositis wound healing","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-05 15:28:12","doi":"10.21203/rs.3.rs-9340679/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-05-16T13:38:28+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-15T07:23:41+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-26T17:51:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"75671646778835906365045283323156475221","date":"2026-04-26T14:31:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"290057980350657448353772453659437592340","date":"2026-04-24T17:16:54+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-24T13:48:45+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-10T03:58:33+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-10T03:58:21+00:00","index":"","fulltext":""},{"type":"submitted","content":"Oral and Maxillofacial Surgery","date":"2026-04-07T06:36:35+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"oral-and-maxillofacial-surgery","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"omfs","sideBox":"Learn more about [Oral and Maxillofacial Surgery](http://link.springer.com/journal/10006)","snPcode":"10006","submissionUrl":"https://submission.nature.com/new-submission/10006/3","title":"Oral and Maxillofacial Surgery","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"08334147-faf3-4ce9-b07e-f2d53e7a8e72","owner":[],"postedDate":"May 5th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-05-16T13:38:28+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-15T07:23:41+00:00","index":21,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-05-16T13:54:42+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-05 15:28:12","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9340679","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9340679","identity":"rs-9340679","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}