Multi-enzyme active temperature-sensitive hydrogel with reactive oxygen species scavenging and antimicrobial capacity for diabetic wound repair

Abstract

Background: Diabetic wound healing is often impaired due to the high-glucose microenvironment in patients. Among the relevant factors, bacterial infection and overproduction of reactive oxygen species (ROS) have critical roles, and sustained oxidative stress further impairs angiogenesis and increases apoptosis, thereby hindering wound repair. To reduce these effects, we aimed to develop an injectable temperature-sensitive cellulose hydrogel exhibiting anti-apoptotic, oxidative stress-attenuating, antimicrobial, and multi-species enzymatic activities. Methods: By simulating the dual active sites of natural copper–zinc superoxide dismutase (CuZn–SOD), a bimetallic mimetic nanoenzyme [Cu/Zn-metal–organic framework (MOF)] was synthesized. Subsequently, Cu/Zn-MOF was incorporated into a hydroxypropyl methylcellulose hydrogel, and the gelation temperature was adjusted to enable a sol-to-gel transition near physiological temperature. A rheometer was used to measure the gelation temperature, and scanning electron microscopy was performed to characterize the surface morphology. The hydrogels were evaluated for multiple enzyme-like activities, including those of SOD, glutathione peroxidase (GPx), thiol peroxidase (TPx), and ascorbate peroxidase (APx). Mouse fibroblasts (L929 cells) and human umbilical vein endothelial cells were used to assess antioxidant, pro-migratory, pro-angiogenic, and anti-apoptotic properties. Antimicrobial activity was assessed against Escherichia coli and Staphylococcus aureus. Western blotting was performed to verify potential anti-inflammatory mechanisms. Finally, wound and infected-wound models were established in diabetic mice to evaluate the hydrogel’s effects on wound repair. Results: The hydrogel exhibited a sol-to-gel transition at 37°C and demonstrated favorable injectability and hydrophilicity, providing a moist healing environment. The Cu/Zn-MOF nanoenzymes demonstrated four enzyme-like activities (SOD, GPx, TPx, and APx), enabling cascade ROS scavenging, which was further confirmed in cellular experiments. The Cu/Zn-MOF nanoenzymes also modulated Sirt1/nuclear factor-κ beta expression to influence inflammatory factor release, thereby exhibiting strong anti-inflammatory activity. The hydrogel also exerted cell migration, angiogenesis, and anti-apoptotic effects. Antimicrobial assays showed kill rates of 99.39% and 99.67% against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus), respectively. In a diabetic mouse wound model, the hydrogel significantly enhanced pro-healing effects by promoting neovascularization and collagen deposition through ROS and bacterial clearance, thereby reducing the inflammatory response. Conclusions: Biomimetic nanoenzymes were synthesized and incorporated into temperature-sensitive injectable hydrogels, which exhibited strong antioxidant and antimicrobial activities that have considerable potential for diabetic wound therapy.

Keywords

Diabetic wounds / Bionic nanoenzymes / Temperature-sensitive hydrogels / Reactive oxygen species / Angiogenesis / Antimicrobial

References

[1] Cheng S, Wang H, Pan X, Zhang C, Zhang K, Chen Z, et al. Dendritic hydrogels with robust inherent antibacterial properties for promoting bacteria-infected wound healing. ACS Appl Mater Interfaces 2022; 14:11144-55. https://doi.org/10.1021/acsami.1c25014.

[2] Cai Y, Chen K, Liu C, Qu X. Harnessing strategies for enhancing diabetic wound healing from the perspective of spatial inflammation patterns. Bioact Mater 2023; 28:243-54. https://doi.org/10.1016/j.bioactmat.2023.04.019.

[3] Saeedi P, Petersohn I, Salpea P, Malanda B, Karuranga S, Unwin N, et al. Global and regional diabetes prevalence estimates for 2019 and projections for 2030 and 2045: results from the international diabetes federation diabetes atlas, 9th edition. Diabetes Res Clin Pract 2019; 157:107843. https://doi.org/10.1016/j.diabres.2019.107843.

[4] Cho H, Blatchley MR, Duh EJ, Gerecht S. Acellular and cellular approaches to improve diabetic wound healing. Adv Drug Deliv Rev 2019; 146:267-88. https://doi.org/10.1016/j.addr.2018.07.019.

[5] Deng L, Du C, Song P, Chen T, Rui S, Armstrong DG, et al. The role of oxidative stress and antioxidants in diabetic wound healing. Oxidative Med Cell Longev 2021; 2021:8852759. https://doi.org/10.1155/2021/8852759.

[6] Yuan T, Yang T, Chen H, Fu D, Hu Y, Wang J, et al. New insights into oxidative stress and inflammation during diabetes mellitus-accelerated atherosclerosis. Redox Biol 2019; 20:247-60. https://doi.org/10.1016/j.redox.2018.09.025.

[7] Zhao X, Wang LY, Li JM, Peng LM, Tang CY, Zha XJ, et al. Redox-mediated artificial non-enzymatic antioxidant MXene nanoplatforms for acute kidney injury alleviation. Adv Sci 2021; 8:e2101498. https://doi.org/10.1002/advs.202101498.

[8] Rizwan H, Pal S, Sabnam S, Pal A. High glucose augments ROS generation regulates mitochondrial dysfunction and apoptosis via stress signalling cascades in keratinocytes. Life Sci 2020; 241:117148. https://doi.org/10.1016/j.lfs.2019.117148.

[9] Sabnam S, Rizwan H, Pal S, Pal A. CEES-induced ROS accumulation regulates mitochondrial complications and inflammatory response in keratinocytes. Chem Biol Interact 2020; 321:109031. https://doi.org/10.1016/j.cbi.2020.109031.

[10] Fu J, Chen X, Liu X, Xu D, Yang H, Zeng C, et al. ELABELA ameliorates hypoxic/ischemic-induced bone mesenchymal stem cell apoptosis via alleviation of mitochondrial dysfunction and activation of PI3K/AKT and ERK1/2 pathways. Stem Cell Res Ther 2020; 11:541. https://doi.org/10.1186/s13287-020-02063-1.

[11] Morse PT, Arroum T, Wan J, Pham L, Vaishnav A, Bell J, et al. Phosphorylations and Acetylations of Cytochrome c Control Mitochondrial Respiration, Mitochondrial Membrane Potential, Energy, ROS, and Apoptosis. Cells. 2024; 13:493. https://doi.org/10.3390/cells13060493.

[12] Li F, Zhang C, Zhong X, Li B, Zhang M, Li W, et al. A 3D radially aligned nanofiber scaffold co-loaded with LL37 mimetic peptide and PDGF-BB for the management of infected chronic wounds. Mater Today Bio. 2024; 28:101237. https://doi.org/10.1016/j.mtbio.2024.101237.

[13] Holl J, Kowalewski C, Zimek Z, Fiedor P, Kaminski A, Oldak T, et al. Chronic Diabetic Wounds and Their Treatment with Skin Substitutes. Cells. 2021; 10:655. https://doi.org/10.3390/cells10030655.

[14] Xu Z, Liu Y, Ma R, Chen J, Qiu J, Du S, et al. Thermosensitive hydrogel incorporating Prussian blue nanoparticles promotes diabetic wound healing via ROS scavenging and mitochondrial function restoration. ACS Appl Mater Interfaces 2022; 14:14059-71. https://doi.org/10.1021/acsami.1c24569.

[15] Liu T, Xiao B, Xiang F, Tan J, Chen Z, Zhang X, et al. Ultrasmall copper-based nanoparticles for reactive oxygen species scavenging and alleviation of inflammation related diseases. Nat Commun 2020; 11:2788. https://doi.org/10.1038/s41467-020-16544-7.

[16] Zhang C, Wang X, Du J, Gu Z, Zhao Y. Reactive oxygen species-regulating strategies based on nanomaterials for disease treatment. Adv Sci 2020; 8:2002797. https://doi.org/10.1002/advs.202002797.

[17] Nguyen NH, Tran GB, Nguyen CT. Anti-oxidative effects of superoxide dismutase 3 on inflammatory diseases. J Mol Med 2020; 98:59-69. https://doi.org/10.1007/s00109-019-01845-2.

[18] Kumar S, Bhardwaj VK, Guleria S, Purohit R, Kumar S. Improving the catalytic efficiency and dimeric stability of Cu, Zn superoxide dismutase by combining structure-guided consensus approach with site-directed mutagenesis. Biochim Biophys Acta Bioenerg 2022; 1863:148505. https://doi.org/10.1016/j.bbabio.2021.148505.

[19] Shang L, Yu Y, Jiang Y, Liu X, Sui N, Yang D, et al. Ultrasound-augmented multienzyme-like nanozyme hydrogel spray for promoting diabetic wound healing. ACS Nano 2023; 17:15962-77. https://doi.org/10.1021/acsnano.3c04134.

[20] Lu X, Gao S, Lin H, Tian H, Xu D, Shi J. Bridging oxidase catalysis and oxygen reduction electrocatalysis by model single-atom catalysts. Natl Sci Rev 2022; 9:nwac022. https://doi.org/10.1093/nsr/nwac022.

[21] Mu J, Li C, Shi Y, Liu G, Zou J, Zhang DY, et al. Protective effect of platinum nano-antioxidant and nitric oxide against hepatic ischemia-reperfusion injury. Nat Commun 2022; 13:2513. https://doi.org/10.1038/s41467-022-29772-w.

[22] Jiang B, Guo Z, Liang M. Recent progress in single-atom nanozymes research. Nano Res 2023; 16:1878-89. https://doi.org/10.1007/s12274-022-4856-7.

[23] Singh R, Umapathi A, Patel G, Patra C, Malik U, Bhargava SK, et al. Nanozyme-based pollutant sensing and environmental treatment: trends, challenges, and perspectives. Sci Total Environ 2023; 854:158771. https://doi.org/10.1016/j.scitotenv.2022.158771.

[24] Jin GX, Liu J, Wang C, Gu WX, Ran GX, Liu B, et al. Ir nanoparticles with multi-enzyme activities and its application in the selective oxidation of aromatic alcohols. Appl Catal B Environ 2020; 267:118725. https://doi.org/10.1016/j.apcatb.2020.118725.

[25] Yang D, Chen Z, Gao Z, Tammina SK, Yang Y. Nanozymes used for antimicrobials and their applications. Colloids Surf B Biointerfaces 2020; 195:111252. https://doi.org/10.1016/j.colsurfb.2020.111252.

[26] Liu X, Huang D, Lai C, Qin L, Zeng G, Xu P, et al. Peroxidase-like activity of smart nanomaterials and their advanced application in colorimetric glucose biosensors. Small 2019; 15:e1900133. https://doi.org/10.1002/smll.201900133.

[27] Chen Z, Yu Y, Gao Y, Zhu Z. Rational design strategies for nanozymes. ACS Nano 2023; 17:13062-80. https://doi.org/10.1021/acsnano.3c04378.

[28] Ali A, Ovais M, Zhou H, Rui Y, Chen C. Tailoring metal-organic frameworks-based nanozymes for bacterial theranostics. Biomaterials 2021; 275:120951. https://doi.org/10.1016/j.biomaterials.2021.120951.

[29] Li QJ, Xing F, Wu WT, Zhe M, Zhang WQ, Qin L, et al. Multifunctional metal-organic frameworks as promising nanomaterials for antimicrobial strategies. Burns Trauma 2025; 13:tkaf008. https://doi.org/10.1093/burnst/tkaf008.

[30] Wang J, Zhou Y, Zeng M, Zhao Y, Zuo X, Meng F, et al. Zr(IV)-based metal-organic framework nanocomposites with enhanced peroxidase-like activity as a colorimetric sensing platform for sensitive detection of hydrogen peroxide and phenol. Environ Res 2022; 203:111818. https://doi.org/10.1016/j.envres.2021.111818.

[31] Dong W, Yang L, Huang Y. Glycine post-synthetic modification of MIL-53(Fe) metal-organic framework with enhanced and stable peroxidase-like activity for sensitive glucose biosensing. Talanta 2017; 167:359-66. https://doi.org/10.1016/j.talanta.2017.02.039.

[32] Li S, Liang L, Tian L, Wu J, Zhu Y, Qin Y, et al. Enhanced peroxidase-like activity of MOF nanozymes by co-catalysis for colorimetric detection of cholesterol. J Mater Chem B 2023; 11:7913-9. https://doi.org/10.1039/D3TB00958K.

[33] Wang J, Bao M, Wei T, Wang Z, Dai Z. Bimetallic metal-organic framework for enzyme immobilization by biomimetic mineralization: constructing a mimic enzyme and simultaneously immobilizing natural enzymes. Anal Chim Acta 2020; 1098:148-54. https://doi.org/10.1016/j.aca.2019.11.039.

[34] Maleki A, He J, Bochani S, Nosrati V, Shahbazi MA, Guo B. Multifunctional photoactive hydrogels for wound healing acceleration. ACS Nano 2021; 15:18895-930. https://doi.org/10.1021/acsnano.1c08334.

[35] Wang Y, Wu H, Pan Y, Xiao Y, Chen Y, Yang S, et al. Innovations in hydrogel therapies for diabetic wound healing: bridging the gap between pathophysiology and clinical application. Burns Trauma 2025; 13:tkaf025. https://doi.org/10.1093/burnst/tkaf025.

[36] Klouda L. Thermoresponsive hydrogels in biomedical applications: a seven-year update. Eur J Pharm Biopharm 2015; 97:338-49. https://doi.org/10.1016/j.ejpb.2015.05.017.

[37] Alven S, Aderibigbe BA. Chitosan and cellulose-based hydrogels for wound management. Int J Mol Sci 2020; 21:9656. https://doi.org/10.3390/ijms21249656.

[38] Kim MH, Park H, Nam HC, Park SR, Jung JY, Park WH. Injectable methylcellulose hydrogel containing silver oxide nanoparticles for burn wound healing. Carbohydr Polym 2018; 181:579-86. https://doi.org/10.1016/j.carbpol.2017.11.109.

[39] Xu L, Jiao G, Huang Y, Ren P, Liang M, Wei D, et al. Laponite nanoparticle-crosslinked carboxymethyl cellulose-based injectable hydrogels with efficient underwater-specific adhesion for rapid hemostasis. Int J Biol Macromol 2024; 255:128288. https://doi.org/10.1016/j.ijbiomac.2023.128288.

[40] Perez-Robles S, Carotenuto C, Minale M. HPMC hydrogel formation mechanisms unveiled by the evaluation of the activation energy. Polymers 2022; 14:635. https://doi.org/10.3390/polym14030635.

[41] Wang L, Song D, Wei C, Chen C, Yang Y, Deng X, et al. Telocytes inhibited inflammatory factor expression and enhanced cell migration in LPS-induced skin wound healing models in vitro and in vivo. J Transl Med 2020; 18:60. https://doi.org/10.1186/s12967-020-02217-y.

[42] Amanzholkyzy A, Zhumagaliyeva S, Sultanova N, Abilov Z, Ongalbek D, Donbayeva E, et al. Hydrogel delivery systems for biological active substances: properties and the role of HPMC as a carrier. Molecules 2025; 30:1354. https://doi.org/10.3390/molecules30061354.

[43] An Y, Xu BT, Wan SR, Ma XM, Long Y, Xu Y, et al. The role of oxidative stress in diabetes mellitus-induced vascular endothelial dysfunction. Cardiovasc Diabetol 2023; 22:237. https://doi.org/10.1186/s12933-023-01965-7.

[44] Kang T, Kim YG, Kim D, Hyeon T. Inorganic nanoparticles with enzyme-mimetic activities for biomedical applications. Coord Chem Rev 2020; 403:213092. https://doi.org/10.1016/j.ccr.2019.213092.

[45] Liu Y, Cheng Y, Zhang H, Zhou M, Yu Y, Lin S, et al. Integrated cascade nanozyme catalyzes in vivo ROS scavenging for anti-inflammatory therapy. Sci Adv 2020; 6:eabb2695. https://doi.org/10.1126/sciadv.abb2695.

[46] Liu W, Zhang Y, Wei G, Zhang M, Li T, Liu Q, et al. Integrated cascade nanozymes with antisenescence activities for atherosclerosis therapy. Angew Chem Int Ed Engl 2023; 62:e202304465. https://doi.org/10.1002/anie.202304465.

[47] Caligiuri G. CD31 as a therapeutic target in atherosclerosis. Circ Res 2020; 126:1178-89. https://doi.org/10.1161/CIRCRESAHA.120.315935.

[48] Peña OA, Martin P. Cellular and molecular mechanisms of skin wound healing. Nat Rev Mol Cell Biol 2024; 25:599-616. https://doi.org/10.1038/s41580-024-00715-1.

[49] Wang R, Li B, Dong M, Zhu H, Jin P, Zou Y. Targeting oxidative damage in diabetic foot ulcers: integrative strategies involving antioxidant drugs and nanotechnologies. Burns Trauma 2025; 13:tkaf020. https://doi.org/10.1093/burnst/tkaf020.

[50] Anilkumar S, Wright-Jin E. NF-κB as an Inducible Regulator of Inflammation in the Central Nervous System. Cells. 2024; 13:485. https://doi.org/10.3390/cells13060485.

[51] Zhang P, He L, Zhang J, Mei X, Zhang Y, Tian H, et al. Preparation of novel berberine nano-colloids for improving wound healing of diabetic rats by acting Sirt1/NF-κB pathway. Colloids Surf B Biointerfaces 2020; 187:110647. https://doi.org/10.1016/j.colsurfb.2019.110647.

[52] Rousselle P, Braye F, Dayan G. Re-epithelialization of adult skin wounds: cellular mechanisms and therapeutic strategies. Adv Drug Deliv Rev 2019; 146:344-65. https://doi.org/10.1016/j.addr.2018.06.019.

[53] Zhang Y, Li M, Wang Y, Han F, Shen K, Luo L, et al. Exosome/metformin-loaded self-healing conductive hydrogel rescues microvascular dysfunction and promotes chronic diabetic wound healing by inhibiting mitochondrial fission. Bioact Mater 2023; 26:323-36. https://doi.org/10.1016/j.bioactmat.2023.01.020.

[54] Qi X, Cai E, Xiang Y, Zhang C, Ge X, Wang J, et al. An immunomodulatory hydrogel by hyperthermia-assisted self-cascade glucose depletion and ROS scavenging for diabetic foot ulcer wound therapeutics. Adv Mater 2023; 35:e2306632. https://doi.org/10.1002/adma.202306632.

[55] Vasileiou PVS, Evangelou K, Vlasis K, Fildisis G, Panayiotidis MI, Chronopoulos E, et al. Mitochondrial homeostasis and cellular senescence. Cells 2019; 8:686. https://doi.org/10.3390/cells8070686.

[56] De Gaetano A, Gibellini L, Zanini G, Nasi M, Cossarizza A, Pinti M. Mitophagy and oxidative stress: the role of aging. Antioxidants 2021; 10:794. https://doi.org/10.3390/antiox10050794.

[57] Boardman NT, Trani G, Scalabrin M, Romanello V, Wüst RCI. Intracellular to interorgan mitochondrial communication in striated muscle in health and disease. Endocr Rev 2023; 44:668-92. https://doi.org/10.1210/endrev/bnad004.

[58] Ge H, Wang M, Wei X, Chen XL, Wang X. Copper-based nanozymes: potential therapies for infectious wounds. Small 2025; 21:e2407195. https://doi.org/10.1002/smll.202407195.

[59] Li X, Liu Y, Qi X, Xiao S, Xu Z, Yuan Z, et al. Sensitive activatable nanoprobes for real-time ratiometric magnetic resonance imaging of reactive oxygen species and ameliorating inflammation in vivo. Adv Mater 2022; 34:e2109004. https://doi.org/10.1002/adma.202109004.

[60] Khivantsev K, Pham H, Engelhard MH, Aleksandrov HA, Kovarik L, Bowden M, et al. Transforming ceria into 2D clusters enhances catalytic activity. Nature 2025; 640:947-53. https://doi.org/10.1038/s41586-025-08684-x.

[61] Andrabi SM, Majumder S, Gupta KC, Kumar A. Dextran based amphiphilic nano-hybrid hydrogel system incorporated with curcumin and cerium oxide nanoparticles for wound healing. Colloids Surf B Biointerfaces 2020; 195:111263. https://doi.org/10.1016/j.colsurfb.2020.111263.

[62] Xia Y, Li X, Huang F, Wu Y, Liu J, Liu J. Design and advances in antioxidant hydrogels for ROS-induced oxidative disease. Acta Biomater 2025; 194:80-97. https://doi.org/10.1016/j.actbio.2025.01.057.

[63] Ye J, Xiao Z, Gao L, Zhang J, He L, Zhang H, et al. Assessment of the effects of four crosslinking agents on gelatin hydrogel for myocardial tissue engineering applications. Biomed Mater 2021; 16:045026. https://doi.org/10.1088/1748-605X/abfff2.