Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Focus Review
  • Published:

Poly(ethylene glycol)-based biofunctional hydrogels mediated by peroxidase-catalyzed cross-linking reactions

Polymer Journal volume 52, pages 899–911 (2020)Cite this article

Subjects

Abstract

Biofunctional hydrogels prepared by a peroxidase, especially horseradish peroxidase (HRP), serve as an excellent class of materials or platform for the development of cellular scaffolds because their biocompatibility and mild and tunable reaction conditions provide them with desirable properties. In this focus review, we summarize our decade of research into HRP-mediated fabrication of biofunctional hydrogels and their applications, in particular cell culture scaffolds. A brief overview of potential substrates employed in HRP and improvement of the HRP hydrogelation system from the initial step until the hydrogen peroxide removal stage in an effort to meet environmental standards is discussed. We highlight our system and describe its biocompatibility and ability to functionalize molecules to support biofabrication by increasing cellular adhesiveness, retaining growth factor affinity, and finally accelerating the formation of two- and three-dimensional multicellular architectures. In the last section, we outline the adoption of hydrogelation as a self-standing, compartmentalized reaction system, i.e., the use of hydrogel marble to conduct cell-free biosynthesis. We believe that this HRP-mediated hydrogel system offers great potential not only as a cell culture scaffold but also for various biomedical applications.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

References

  1. Green JJ, Elisseeff JH. Mimicking biological functionality with polymers for biomedical applications. Nature. 2016;540:386–94.

    CAS  PubMed  Google Scholar 

  2. Lee SC, Kwon IK, Park K. Hydrogels for delivery of bioactive agents: a historical perspective. Adv Drug Deliv Rev. 2013;65:17–20.

    CAS  PubMed  Google Scholar 

  3. Kopeček J. Swell gels. Nature. 2002;417:389–91.

    Google Scholar 

  4. Hoffman AS. Hydrogels for biomedical applications. Adv Drug Deliv Rev. 2002;54:3–12.

    CAS  PubMed  Google Scholar 

  5. Kang DH, Kim D, Wang S, Song D, Yoon MH. Water-insoluble, nanocrystalline, and hydrogel fibrillar scaffolds for biomedical applications. Polym J. 2018;50:637–47.

    CAS  Google Scholar 

  6. Schneider-Barthold C, Baganz S, Wilhelmi M, Scheper T, Pepelanova I. Hydrogels based on collagen and fibrin - frontiers and applications. BioNanoMaterials. 2016;17:3–12.

    Google Scholar 

  7. Janmey PA, Winer JP, Weisel JW. Fibrin gels and their clinical and bioengineering applications. J R Soc Interface. 2009;6:1–10.

    CAS  Google Scholar 

  8. Hughes CS, Postovit LM, Lajoie GA. Matrigel: a complex protein mixture required for optimal growth of cell culture. Proteomics. 2010;10:1886–90.

    CAS  PubMed  Google Scholar 

  9. Caliari SR, Burdick JA. A practical guide to hydrogels for cell culture. Nat Methods. 2016;13:405–14.

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Spicer CD. Hydrogel scaffolds for tissue engineering: the importance of polymer choice. Polym Chem. 2020;11:184–219.

    CAS  Google Scholar 

  11. Buwalda SJ, Boere KWM, Dijkstra PJ, Feijen J, Vermonden T, Hennink WE. Hydrogels in a historical perspective: from simple networks to smart materials. J Control Release. 2014;190:254–73.

    CAS  PubMed  Google Scholar 

  12. Kuo CK, Ma PX. Ionically crosslinked alginate hydrogels as scaffolds for tissue engineering: Part 1. Structure, gelation rate and mechanical properties. Biomaterials. 2001;22:511–21.

    CAS  PubMed  Google Scholar 

  13. Choi JR, Yong KW, Choi JY, Cowie AC. Recent advances in photo-crosslinkable hydrogels for biomedical applications. Biotechniques. 2019;66:40–53.

    CAS  PubMed  Google Scholar 

  14. Crescenzi V, Cornelio L, Di Meo C, Nardecchia S, Lamanna R. Novel hydrogels via click chemistry: synthesis and potential biomedical applications. Biomacromolecules. 2007;8:1844–50.

    CAS  PubMed  Google Scholar 

  15. Hu BH, Messersmith PB. Rational design of transglutaminase substrate peptides for rapid enzymatic formation of hydrogels. J Am Chem Soc. 2003;125:14298–9.

    CAS  PubMed  Google Scholar 

  16. Mironi-Harpaz I, Wang DY, Venkatraman S, Seliktar D. Photopolymerization of cell-encapsulating hydrogels: crosslinking efficiency versus cytotoxicity. Acta Biomater. 2012;8:1838–48.

    CAS  PubMed  Google Scholar 

  17. Steinhilber D, Haag R. Multifunctional dendritic polyglycerol nano- and microgels for encapsulation and release of functional. Biomacromolecules. 2011;20:7545.

    Google Scholar 

  18. Moreira Teixeira LS, Feijen J, van Blitterswijk CA, Dijkstra PJ, Karperien M. Enzyme-catalyzed crosslinkable hydrogels: emerging strategies for tissue engineering. Biomaterials. 2012;33:1281–90.

    CAS  Google Scholar 

  19. Buwalda SJ, Vermonden T, Hennink WE. Hydrogels for therapeutic delivery: current developments and future directions. Biomacromolecules. 2017;18:316–30.

    CAS  PubMed  Google Scholar 

  20. Kurisawa M, Chung JE, Yang YY, Gao SJ, Uyama H. Injectable biodegradable composed of hyaluronic acid-conjugates for drug delivery and tissue engineering. Chem Chomm. 2005;34:4312–4.

    Google Scholar 

  21. Kim EH, Lim S, Kim TE, Jeon IO, Choi YS. Preparation of in situ injectable chitosan/gelatin hydrogel using an acid-tolerant tyrosinase. Biotechnol Bioprocess Eng. 2018;23:500–6.

    CAS  Google Scholar 

  22. Huber D, Grzelak A, Baumann M, Borth N, Schleining G, Nyanhongo GS, et al. Anti-inflammatory and anti-oxidant properties of laccase-synthesized phenolic-O-carboxymethyl chitosan hydrogels. N. Biotechnol. 2018;40:236–44.

    CAS  PubMed  Google Scholar 

  23. Huber D, Tegl G, Baumann M, Sommer E, Gorji EG, Borth N, et al. Chitosan hydrogel formation using laccase activated phenolics as cross-linkers. Carbohydr Polym 2017;157:814–22.

    CAS  PubMed  Google Scholar 

  24. Yung CW, Bentley WE, Barbari TA. Diffusion of interleukin-2 from cells overlaid with cytocompatible enzyme-crosslinked gelatin hydrogels. J Biomed Mater Res Part A. 2010;95:25–32.

    Google Scholar 

  25. Cambria E, Renggli K, Ahrens CC, Cook CD, Kroll C, Krueger AT, et al. Covalent modification of synthetic hydrogels with bioactive proteins via sortase-mediated ligation. Biomacromolecules. 2015;16:2316–26.

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Yang Z, Gu H, Fu D, Gao P, Lam JK, Xu B. Enzymatic formation of supramolecular hydrogels. Adv Mater. 2004;16:1440–4.

    CAS  Google Scholar 

  27. Bakota EL, Aulisa L, Galler KM, Hartgerink JD. Enzymatic cross-linking of a nanofibrous peptide hydrogel. Biomacromolecules. 2011;12:82–7.

    CAS  PubMed  Google Scholar 

  28. Mosiewicz KA, Johnsson K, Lutolf MP. Phosphopantetheinyl transferase-catalyzed formation of bioactive hydrogels for tissue engineering. J Am Chem Soc. 2010;132:5972–4.

    CAS  PubMed  Google Scholar 

  29. Yang Z, Ho PL, Liang G, Chow KH, Wang Q, Cao Y, et al. Using β-lactamase to trigger supramolecular hydrogelation. J Am Chem Soc. 2007;129:266–7.

    CAS  PubMed  Google Scholar 

  30. Toledano S, Williams RJ, Jayawarna V, Ulijn RV. Enzyme-triggered self-assembly of peptide hydrogels via reversed hydrolysis. J Am Chem Soc. 2006;128:1070–1.

    CAS  PubMed  Google Scholar 

  31. Xie Y, Huang R, Qi W, Wang Y, Su R, He Z. Enzyme-substrate interactions promote the self-assembly of amino acid derivatives into supramolecular hydrogels. J Mater Chem B. 2016;4:844–51.

    CAS  PubMed  Google Scholar 

  32. Hai Z, Li J, Wu J, Xu J, Liang G. Alkaline phosphatase-triggered simultaneous hydrogelation and chemiluminescence. J Am Chem Soc. 2017;139:1041–4.

    CAS  PubMed  Google Scholar 

  33. Yang Z, Liang G, Wang L, Xu B. Using a kinase/phosphatase switch to regulate a supramolecular hydrogel and forming the supramolecular hydrogel in vivo. J Am Chem Soc. 2006;128:3038–43.

    CAS  PubMed  Google Scholar 

  34. Rowe SL, Lee SY, Stegemann JP. Influence of thrombin concentration on the mechanical and morphological properties of cell-seeded fibrin hydrogels. Acta Biomater. 2007;3:59–67.

    CAS  PubMed  Google Scholar 

  35. Sofia SJ, Singh A, Kaplan DL. Peroxidase-catalyzed crosslinking of functionalized polyaspartic acid polymers. J Macromol Sci - Pure Appl Chem. 2002;39 A:1151–81.

    Google Scholar 

  36. Lopes GR, Pinto DCGA, Silva AMS. Horseradish peroxidase (HRP) as a tool in green chemistry. RSC Adv. 2014;4:37244–65.

    CAS  Google Scholar 

  37. Sakai S, Nakahata M. Horseradish peroxidase catalyzed hydrogelation for biomedical, biopharmaceutical, and biofabrication applications. Chem Asian J. 2017;12:3098–109.

    CAS  PubMed  Google Scholar 

  38. Bae JW, Choi JH, Lee Y, Park KD. Horseradish peroxidase-catalysed in situ -forming hydrogels for tissue-engineering applications. J Tissue Eng Regen Med. 2015;9:1225–32.

    CAS  PubMed  Google Scholar 

  39. Guebitz GM, Nyanhongo GS. Enzymes as green catalysts and interactive biomolecules in wound dressing hydrogels. Trends Biotechnol. 2018;36:1040–53.

    CAS  PubMed  Google Scholar 

  40. Lee F, Bae KH, Kurisawa M. Injectable hydrogel systems crosslinked by horseradish peroxidase. Biomed Mater. 2016;11:014101.

    Google Scholar 

  41. Shakya AK, Nandakumar KS. An update on smart biocatalysts for industrial and biomedical applications. J R Soc Interface. 2018;15:20180062.

    PubMed  PubMed Central  Google Scholar 

  42. Krainer FW, Glieder A. An updated view on horseradish peroxidases: recombinant production and biotechnological applications. Appl Microbiol Biotechnol. 2015;99:1611–25.

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Veitch NC. Horseradish peroxidase: a modern view of a classic enzyme. Phytochemistry. 2004;65:249–59.

    CAS  PubMed  Google Scholar 

  44. Rodríguez-López JN, Lowe DJ, Hernández-Ruiz J, Hiner ANP, García-Cánovas F, Thorneley RNF. Mechanism of reaction of hydrogen peroxide with horseradish peroxidase: identification of intermediates in the catalytic cycle. J Am Chem Soc. 2001;123:11838–47.

    PubMed  Google Scholar 

  45. Zakharova GS, Uporov IV, Tishkov VI. Horseradish peroxidase: modulation of properties by chemical modification of protein and heme. Biochem. 2011;76:1391–401.

    CAS  Google Scholar 

  46. Kobayashi S, Uyama H, Kimura S. Enzymatic polymerization. Chem Rev. 2001;101:3793–818.

    CAS  PubMed  Google Scholar 

  47. Wang LS, Chung JE, Lee F. (12) Patent Application Publication (10) Pub. No.: US 2010/0074956 A1. 1, (2010).

  48. Hoang Thi TT, Lee Y, Le Thi P, Park KD. Engineered horseradish peroxidase-catalyzed hydrogels with high tissue adhesiveness for biomedical applications. J Ind Eng Chem. 2019;78:34–52.

    CAS  Google Scholar 

  49. Raia NR, Partlow BP, McGill M, Kimmerling EP, Ghezzi CE, Kaplan DL. Enzymatically crosslinked silk-hyaluronic acid hydrogels. Biomaterials. 2017;131:58–67.

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Kuo KC, Lin RZ, Tien HW, Wu PY, Li YC, Melero-Martin JM, et al. Bioengineering vascularized tissue constructs using an injectable cell-laden enzymatically crosslinked collagen hydrogel derived from dermal extracellular matrix. Acta Biomater 2015;27:151–66.

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Sakai S, Yamada Y, Zenke T, Kawakami K. Novel chitosan derivative soluble at neutral pH and in-situ gellable via peroxidase-catalyzed enzymatic reaction. J Mater Chem. 2009;19:230–5.

    CAS  Google Scholar 

  52. Chen F, Yu S, Liu B, Ni Y, Yu C, Su Y, et al. An injectable enzymatically crosslinked carboxymethylated pullulan/chondroitin sulfate hydrogel for cartilage tissue engineering. Sci Rep. 2016;6:20014.

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Sakai S, Hirose K, Taguchi K, Ogushi Y, Kawakami K. An injectable, in situ enzymatically gellable, gelatin derivative for drug delivery and tissue engineering. Biomaterials. 2009;30:3371–7.

    CAS  PubMed  Google Scholar 

  54. Jin R, Hiemstra C, Zhong Z, Feijen J. Enzyme-mediated fast in situ formation of hydrogels from dextran-tyramine conjugates. Biomaterials. 2007;28:2791–800.

    CAS  PubMed  Google Scholar 

  55. Zhou B, Wang P, Cui L, Yu Y, Deng C, Wang Q, et al. Self-crosslinking of silk fibroin using H2O2-horseradish peroxidase system and the characteristics of the resulting fibroin membranes. Appl Biochem Biotechnol. 2017;182:1548–63.

    CAS  PubMed  Google Scholar 

  56. Sakai S, Kawakami K. Synthesis and characterization of both ionically and enzymatically cross-linkable alginate. Acta Biomater. 2007;3:495–501.

    CAS  PubMed  Google Scholar 

  57. Park KM, Shin YM, Joung YK, Shin H, Park KD. In situ forming hydrogels based on tyramine conjugated 4-Arm-PPO-PEO via enzymatic oxidative reaction. Biomacromolecules. 2010;11:706–12.

    CAS  PubMed  Google Scholar 

  58. Lee SH, Lee Y, Lee SW, Ji HY, Lee JH, Lee DS, et al. Enzyme-mediated cross-linking of Pluronic copolymer micelles for injectable and in situ forming hydrogels. Acta Biomater. 2011;7:1468–76.

    CAS  PubMed  Google Scholar 

  59. Sakai S, Tsumura M, Inoue M, Koga Y, Fukano K, Taya M. Polyvinyl alcohol-based hydrogel dressing gellable on-wound via a co-enzymatic reaction triggered by glucose in the wound exudate. J Mater Chem B. 2013;1:5067–75.

    CAS  PubMed  Google Scholar 

  60. Sun Y, Deng Z, Tian Y, Lin C. Horseradish peroxidase-mediated in situ forming hydrogels from degradable tyramine-based poly(amido amine)s. J Appl Polym Sci. 2013;127:40–8.

    CAS  Google Scholar 

  61. Macdougall LJ, Pérez-Madrigal MM, Arno MC, Dove AP. Nonswelling thiol-yne cross-linked hydrogel materials as cytocompatible soft tissue scaffolds. Biomacromolecules. 2018;19:1378–88.

    CAS  PubMed  Google Scholar 

  62. Ulery BD, Nair LS, Laurencin CT. Biomedical applications of biodegradable polymers. J Polym Sci Part B Polym Phys. 2011;49:832–64.

    CAS  Google Scholar 

  63. Moriyama K, Wakabayashi R, Goto M, Kamiya N. Characterization of enzymatically gellable, phenolated linear poly(ethylene glycol) with different molecular weights for encapsulating living cells. Biochem Eng J. 2014;93:25–30.

    Google Scholar 

  64. Minamihata K, Goto M, Kamiya N. Protein heteroconjugation by the peroxidase-catalyzed tyrosine coupling reaction. Bioconjug Chem. 2011;22:2332–8.

    CAS  PubMed  Google Scholar 

  65. Minamihata K, Goto M, Kamiya N. Site-specific protein cross-linking by peroxidase-catalyzed activation of a tyrosine-containing peptide tag. Bioconjug Chem. 2011;22:74–81.

    CAS  PubMed  Google Scholar 

  66. Moriyama K, Minamihata K, Wakabayashi R, Goto M, Kamiya N. Enzymatic preparation of streptavidin-immobilized hydrogel using a phenolated linear poly(ethylene glycol). Biochem Eng J. 2013;76:37–42.

    CAS  Google Scholar 

  67. Ohya Y. Temperature-responsive biodegradable injectable polymer systems with conveniently controllable properties. Polym J. 2019;51:997–1005.

    CAS  Google Scholar 

  68. Kambe Y, Tokushige T, Mahara A, Iwasaki Y, Yamaoka T. Cardiac differentiation of induced pluripotent stem cells on elastin-like protein-based hydrogels presenting a single-cell adhesion sequence. Polym J. 2019;51:97–105.

    CAS  Google Scholar 

  69. Sakai S, Komatani K, Taya M. Glucose-triggered co-enzymatic hydrogelation of aqueous polymer solutions. RSC Adv. 2012;2:1502–7.

    CAS  Google Scholar 

  70. Singh S, Topuz F, Hahn K, Albrecht K, Groll J. Embedding of active proteins and living cells in redox-sensitive hydrogels and nanogels through enzymatic cross-linking. Angew Chem - Int Ed. 2013;52:3000–3.

    CAS  Google Scholar 

  71. Moriyama K, Minamihata K, Wakabayashi R, Goto M, Kamiya N. Enzymatic preparation of a redox-responsive hydrogel for encapsulating and releasing living cells. Chem Commun. 2014;50:5895–8.

    CAS  Google Scholar 

  72. Obinger C, Burner U, Ebermann R. Generation of hydrogen peroxide by plant peroxidases mediated thiol oxidation. Phyt Ann Rei Bot. 1997;37:219–26.

    CAS  Google Scholar 

  73. Dunford HB, Adeniran AJ. Hammett ϱσ correlation for reactions of horseradish peroxidase compound II with phenols. Arch Biochem Biophys. 1986;251:536–42.

    CAS  PubMed  Google Scholar 

  74. Matsusaki M, Yoshida H, Akashi M. The construction of 3D-engineered tissues composed of cells and extracellular matrices by hydrogel template approach. Biomaterials. 2007;28:2729–37.

    CAS  PubMed  Google Scholar 

  75. Moriyama K, Naito S, Wakabayashi R, Goto M, Kamiya N. Enzymatically prepared redox-responsive hydrogels as potent matrices for hepatocellular carcinoma cell spheroid formation. Biotechnol J. 2016;11:1452–60.

    CAS  PubMed  Google Scholar 

  76. Moriyama K, Wakabayashi R, Goto M, Kamiya N. Enzyme-mediated preparation of hydrogels composed of poly(ethylene glycol) and gelatin as cell culture platforms. RSC Adv. 2015;5:3070–3.

    CAS  Google Scholar 

  77. Jones CJ, Beni S, Limtiaco JFK, Langeslay DJ, Larive CK. Heparin characterization: challenges and solutions. Annu Rev Anal Chem. 2011;4:439–65.

    CAS  Google Scholar 

  78. Tae G, Kim Y-J, Choi W-I, Kim M, Stayton PS, Hoffman AS. Formation of a novel heparin-based hydrogel in the presence of heparin-binding biomolecules. Biomacromolecules. 2007;8:1979–86.

    CAS  PubMed  Google Scholar 

  79. Ramadhan W, Kagawa G, Hamada Y, Moriyama K, Wakabayashi R, Minamihata K, et al. Enzymatically prepared dual functionalized hydrogels with gelatin and heparin to facilitate cellular attachment and proliferation. ACS Appl Bio Mater. 2019;2:2600–9.

    Google Scholar 

  80. Claaßen C, Sewald L, Tovar G, Borchers K. Controlled release of vascular endothelial growth factor from heparin-functionalized gelatin Type A and albumin hydrogels. Gels. 2017;3:35.

    PubMed Central  Google Scholar 

  81. Li Z, Qu T, Ding C, Ma C, Sun H, Li S, et al. Injectable gelatin derivative hydrogels with sustained vascular endothelial growth factor release for induced angiogenesis. Acta Biomater. 2015;13:88–100.

    CAS  PubMed  Google Scholar 

  82. Kamiya N, Ohama Y, Minamihata K, Wakabayashi R, Goto M. Liquid marbles as an easy-to-handle compartment for cell-free synthesis and in situ immobilization of recombinant. Proteins Biotechnol J. 2018;13:1–5.

    Google Scholar 

Download references

Acknowledgements

This work was supported in part by the Japan Society for the Promotion of Science (JSPS) KAKENHI No. JP17K19016. The authors thank the Center of Advanced Instrumental Analysis, Kyushu University, and Nanotechnology Platform Program of the Ministry of Education, Culture, Sports, Science and Technology (MEXT) for facility support as well as Dr Kosuke Minamihata for the discussion and assistance in protein production.

Author information

Authors and Affiliations

  1. Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, Fukuoka, Japan

    Rie Wakabayashi, Wahyu Ramadhan, Masahiro Goto & Noriho Kamiya

  2. Department of Chemical and Biological Engineering, National Institute of Technology, Sasebo College, Nagasaki, Japan

    Kousuke Moriyama

  3. Division of Biotechnology, Center for Future Chemistry, Kyushu University, Fukuoka, Japan

    Masahiro Goto & Noriho Kamiya

Authors
  1. Rie Wakabayashi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Wahyu Ramadhan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Kousuke Moriyama
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Masahiro Goto
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Noriho Kamiya
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Rie Wakabayashi or Noriho Kamiya.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Wakabayashi, R., Ramadhan, W., Moriyama, K. et al. Poly(ethylene glycol)-based biofunctional hydrogels mediated by peroxidase-catalyzed cross-linking reactions. Polym J 52, 899–911 (2020). https://doi.org/10.1038/s41428-020-0344-7

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41428-020-0344-7

Search

Advanced search

Quick links