Engineering an Anaerobic Microenvironment to Empower Hydrogenase-Catalyzed Hydrogen Therapy for Diabetic Wound Healing

Abstract

The inherent oxygen sensitivity of hydrogenases has limited their biomedical use. We report a hybrid peptide–nanocluster hydrogel that establishes a self-sustained anaerobic microenvironment, enabling hydrogenase-catalyzed hydrogen therapy under aerobic conditions. The Fmoc-KYF peptide network traps O₂ in hydrophobic pockets, while photoexcited silver nanoclusters rapidly scavenge residual oxygen, ensuring stable hydrogen evolution. In vitro, the generated hydrogen mitigates oxidative stress and inflammation. In diabetic mice, the light-activated system accelerates wound closure, promotes angiogenesis, and drives macrophage polarization toward a reparative phenotype. This study introduces a bioengineering strategy that integrates material design, enzyme catalysis, and photodynamics to overcome oxygen limitation and advance hydrogenase-based therapeutic applications.

Keywords

diabetic wound healing / hybrid hydrogel / hydrogen therapy / hydrogenase / reactive oxygen species / silver nanocluster

References

[1] W. Lubitz, H. Ogata, O. Rudiger, and E. Reijerse, “Hydrogenases,” Chemical Reviews 114 (2014): 4081–4148.

[2] P. M. Vignais, B. Billoud, and J. Meyer, “Classification and Phylogeny of Hydrogenases,” FEMS Microbiology Review 25 (2001): 455–501.

[3] R. K. Thauer, A. K. Kaster, M. Goenrich, M. Schick, T. Hiromoto, and S. Shima, “Hydrogenases From Methanogenic Archaea, Nickel, a Novel Cofactor, and H₂ Storage,” Annual Review of Biochemistry 79 (2010): 507–536.

[4] D. S. Horner, P. G. Foster, and T. M. Embley, “Iron Hydrogenases and the Evolution of Anaerobic Eukaryotes,” Molecular Biology and Evolution 17 (2000): 1695–1709.

[5] L. F. Wu and M. A. Mandrand, “Microbial Hydrogenases: Primary Structure, Classification, Signatures and Phylogeny,” FEMS Microbiology Review 104 (1993): 243–270.

[6] B. L. Greene, C.-H. Wu, P. M. McTernan, M. W. W. Adams, and R. B. Dyer, “Proton-Coupled Electron Transfer Dynamics in the Catalytic Mechanism of a [NiFe]-Hydrogenase,” Journal of the American Chemical Society 137 (2015): 4558–4566.

[7] K. A. Vincent, A. Parkin, O. Lenz, et al., “Electrochemical Definitions of O₂ Sensitivity and Oxidative Inactivation in Hydrogenases,” Journal of the American Chemical Society 127 (2005): 18179–18189.

[8] H. Ji, L. Wan, Y. Gao, et al., “Hydrogenase as the Basis for Green Hydrogen Production and Utilization,” Journal of Energy Chemistry 85 (2023): 348–362.

[9] J. Koo, T. Schnabel, S. Liong, N. H. Evitt, and J. R. Swartz, “High-Throughput Screening of Catalytic H₂ Production,” Angewandte Chemie International Edition 56 (2017): 1012–1016.

[10] R. M. Evans, N. Krahn, B. J. Murphy, H. Lee, F. A. Armstrong, and D. Soll, “Selective Cysteine-to-Selenocysteine Changes in a [NiFe]-Hydrogenase Confirm a Special Position for Catalysis and Oxygen Tolerance,” Proceeding of the National Academy of Sciences of the United States of America 118 (2021): e2100921118.

[11] N. Plumere, O. Rudiger, A. A. Oughli, et al., “A Redox Hydrogel Protects Hydrogenase From High-Potential Deactivation and Oxygen Damage,” Nature Chemistry 6 (2014): 822–827.

[12] A. A. Oughli, F. Conzuelo, M. Winkler, et al., “A Redox Hydrogel Protects the O₂-Sensitive [FeFe]-Hydrogenase From Chlamydomonas reinhardtii From Oxidative Damage,” Angewandte Chemie International Edition 54 (2015): 12329–12333.

[13] V. Fourmond, S. Stapf, H. Li, et al., “Mechanism of Protection of Catalysts Supported in Redox Hydrogel Films,” Journal of the American Chemical Society 137 (2015): 5494–5505.

[14] H. Li, D. Buesen, S. Dementin, C. Leger, V. Fourmond, and N. Plumere, “Complete Protection of O₂-Sensitive Catalysts in Thin Films,” Journal of the American Chemical Society 141 (2019): 16734–16742.

[15] J. Szczesny, J. A. Birrell, F. Conzuelo, W. Lubitz, A. Ruff, and W. Schuhmann, “Redox-Polymer-Based High-Current-Density Gas-Diffusion H₂-Oxidation Bioanode Using [FeFe] Hydrogenase From Desulfovibrio desulfuricans in a Membrane-Free Biofuel Cell,” Angewandte Chemie International Edition 59 (2020): 16506–16510.

[16] O. Ben-Zvi, I. Grinberg, A. A. Orr, et al., “Protection of Oxygen-Sensitive Enzymes by Peptide Hydrogel,” ACS Nano 15 (2021): 6530–6539.

[17] Y. Shomura, M. Taketa, H. Nakashima, et al., “Structural Basis of the Redox Switches in the NAD⁺-Reducing Soluble [NiFe]-Hydrogenase,” Science 357 (2017): 928–932.

[18] K. P. Sokol, W. E. Robinson, J. Warnan, et al., “Bias-free Photoelectrochemical Water Splitting With Photosystem II on a Dye-Sensitized Photoanode Wired to Hydrogenase,” Nature Energy 3 (2018): 944–951.

[19] L. Zhang, G. Morello, S. B. Carr, and F. A. Armstrong, “Aerobic Photocatalytic H₂ Production by a [NiFe] Hydrogenase Engineered to Place a Silver Nanocluster in the Electron Relay,” Journal of the American Chemical Society 142 (2020): 12699–12707.

[20] G. A. Hutton, B. Reuillard, B. C. Martindale, et al., “Carbon Dots as Versatile Photosensitizers for Solar-Driven Catalysis With Redox Enzymes,” Journal of the American Chemical Society 138 (2016): 16722–16730.

[21] L. Zhang, S. E. Beaton, S. B. Carr, and F. A. Armstrong, “Direct Visible Light Activation of a Surface Cysteine-Engineered [NiFe]-Hydrogenase by Silver Nanoclusters,” Energy & Environmental Science 11 (2018): 3342–3348.

[22] T. Sakai, D. Mersch, and E. Reisner, “Photocatalytic Hydrogen Evolution With a Hydrogenase in a Mediator-Free System Under High Levels of Oxygen,” Angewandte Chemie International Edition 52 (2013): 12313–12316.

[23] C. A. Caputo, M. A. Gross, V. W. Lau, C. Cavazza, B. V. Lotsch, and E. Reisner, “Photocatalytic Hydrogen Production Using Polymeric Carbon Nitride With a Hydrogenase and a Bioinspired Synthetic Ni Catalyst,” Angewandte Chemie International Edition 53 (2014): 11538–11542.

[24] E. E. Moore, V. Andrei, S. Zacarias, I. A. C. Pereira, and E. Reisner, “Integration of a Hydrogenase in a Lead Halide Perovskite Photoelectrode for Tandem Solar Water Splitting,” ACS Energy Letters Journal 5 (2020): 232–237.

[25] V. Jayawarna, M. Ali, T. A. Jowitt, et al., “Nanostructured Hydrogels for Three-Dimensional Cell Culture through Self-Assembly of Fluorenylmethoxycarbonyl–Dipeptides,” Advanced Materials 18 (2006): 611–614.

[26] A. Mahler, M. Reches, M. Rechter, S. Cohen, and E. Gazit, “Rigid, Self-Assembled Hydrogel Composed of a Modified Aromatic Dipeptide,” Advanced Materials 18 (2006): 1365–1370.

[27] A. M. Smith, R. J. Williams, C. Tang, et al., “Fmoc-Diphenylalanine Self Assembles to a Hydrogel via a Novel Architecture Based on π–π Interlocked β-Sheets,” Advanced Materials 20 (2008): 37–41.

[28] M. Ghosh, S. Bera, S. Schiffmann, L. J. W. Shimon, and L. Adler-Abramovich, “Collagen-Inspired Helical Peptide Coassembly Forms a Rigid Hydrogel With Twisted Polyproline II Architecture,” ACS Nano 14 (2020): 9990–10000.

[29] A. J. Engler, S. Sen, H. L. Sweeney, and D. E. Discher, “Matrix Elasticity Directs Stem Cell Lineage Specification,” Cell 126 (2006): 677–689.

[30] N. Gjorevski, N. Sachs, A. Manfrin, et al., “Designer Matrices for Intestinal Stem Cell and Organoid Culture,” Nature 539 (2016): 560–564.

[31] D. R. Griffin, M. M. Archang, C. H. Kuan, et al., “Activating an Adaptive Immune Response From a Hydrogel Scaffold Imparts Regenerative Wound Healing,” Materials 20 (2021): 560–569.

[32] Y. Wang, T. Pan, J. Li, et al., “Developing Isomeric Peptides for Mimicking the Sequence–Activity Landscapes of Enzyme Evolution,” ACS Applied Materials & Interfaces 16 (2024): 22369–22378.

[33] B. Hess, C. Kutzner, D. van der Spoel, and E. Lindahl, “GROMACS 4: Algorithms for Highly Efficient, Load-Balanced, and Scalable Molecular Simulation,” Journal of Chemical Theory and Computation 4 (2008): 435–447.

[34] A. K. Malde, L. Zuo, M. Breeze, et al., “An Automated Force Field Topology Builder (ATB) and Repository: Version 1.0,” Journal of Chemical Theory and Computation 7 (2011): 4026–4037.

[35] Z. Wu and R. Jin, “On the Ligand's Role in the Fluorescence of Gold Nanoclusters,” Nano Letters 10 (2010): 2568–2573.

[36] E. Eruslanov and S. Kusmartsev, “Identification of ROS Using Oxidized DCFDA and Flow-Cytometry,” in Advanced Protocols in Oxidative Stress II, ed. D. Armstrong (Humana Press, 2010), 57–72.

[37] W. Li, L. Wan, Y. Dong, et al., “A Coupled Enzyme-Photocatalytic System for Efficient NADH Regeneration and l-Glutamate Production,” Chemical Engineering Journal 517 (2025): 164375.

[38] M. Cao, S. Wang, J. H. Hu, B. H. Lu, Q. Y. Wang, and S. Q. Zang, “Silver Cluster-Porphyrin-Assembled Materials as Advanced Bioprotective Materials for Combating Superbacteria,” Advanced Science 9 (2021): 2103721.

[39] I. Ohsawa, M. Ishikawa, K. Takahashi, et al., “Hydrogen Acts as a Therapeutic Antioxidant by Selectively Reducing Cytotoxic Oxygen Radicals,” Nature Medicine 13 (2007): 688–694.

[40] G. Zhou, E. Goshi, and Q. He, “Micro/Nanomaterials-Augmented Hydrogen Therapy,” Advanced Healthcare Materials 8 (2019): e1900463.

[41] M. Cortes, A. Brischetto, M. C. Martinez-Campanario, et al., “Inflammatory Macrophages Reprogram to Immunosuppression by Reducing Mitochondrial Translation,” Nature Communications 14 (2023): 7471.

[42] M. Chang and T. T. Nguyen, “Strategy for Treatment of Infected Diabetic Foot Ulcers,” Accounts of Chemical Research 54 (2021): 1080–1093.

[43] T. Xiang, Q. Guo, L. Jia, et al., “Multifunctional Hydrogels for the Healing of Diabetic Wounds,” Advanced Healthcare Materials 13 (2024): 2301885.

[44] S. Chen, Y. Zhu, Q. Xu, et al., “Photocatalytic Glucose Depletion and Hydrogen Generation for Diabetic Wound Healing,” Nature Communications 13 (2022): 5684.

[45] L. C. Seefeldt, B. M. Hoffman, J. W. Peters, et al., “Energy Transduction in Nitrogenase,” Accounts of Chemical Research 51 (2018): 2179–2186.

[46] V. C. Wang, M. Can, E. Pierce, S. W. Ragsdale, and F. A. Armstrong, “A Unified Electrocatalytic Description of the Action of Inhibitors of Nickel Carbon Monoxide Dehydrogenase,” Journal of the American Chemical Society 135 (2013): 2198–2206.

[47] A. Bassegoda, C. Madden, D. W. Wakerley, E. Reisner, and J. Hirst, “Reversible Interconversion of CO₂ and Formate by a Molybdenum-Containing Formate Dehydrogenase,” Journal of the American Chemical Society 136 (2014): 15473–15476.

[48] H. Chen, Y. Guo, Z. Zhang, et al., “Symbiotic Algae–Bacteria Dressing for Producing Hydrogen to Accelerate Diabetic Wound Healing,” Nano Letters 22 (2022): 229–237.

[49] L. Chen, S.-F. Zhou, L. Su, and J. Song, “Gas-Mediated Cancer Bioimaging and Therapy,” ACS Nano 13 (2019): 10887–10917.

[50] X. Qi, E. Cai, Y. Xiang, et al., “An Immunomodulatory Hydrogel by Hyperthermia-Assisted Self-Cascade Glucose Depletion and ROS Scavenging for Diabetic Foot Ulcer Wound Therapeutics,” Advanced Materials 35 (2023): 2306632.

[51] C. Xu, S. Wang, H. Wang, et al., “Magnesium-Based Micromotors as Hydrogen Generators for Precise Rheumatoid Arthritis Therapy,” Nano Letters 21 (2021): 1982–1991.

[52] N. Kim, H. Lee, G. Han, et al., “3D-Printed Functional Hydrogel by DNA-Induced Biomineralization for Accelerated Diabetic Wound Healing,” Advanced Materials 10 (2023): 2300816.

[53] C. H. Moon, H. G. Ko, H. Lee, et al., “Mesenchymal Stem Cell-Inspired Microneedle Platform for NIR-Responsive Immunomodulation and Accelerated Chronic Wound Healing,” Advanced Materials (2025): e14081, https://doi.org/10.1002/adma.202514081.

[54] M. Luo, Q. Wang, G. Zhao, et al., “Solid-State Atomic Hydrogen as a Broad-Spectrum RONS Scavenger for Accelerated Diabetic Wound Healing,” National Science Review 11 (2024): nwad269.

RIGHTS & PERMISSIONS

2026 The Author(s). Aggregate published by SCUT, AIEI, and John Wiley & Sons Australia, Ltd.