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.

  • Protocol
  • Published:

Preparation of supramolecular hydrogel–enzyme hybrids exhibiting biomolecule-responsive gel degradation

Nature Protocols volume 11, pages 1744–1756 (2016)Cite this article

Subjects

Abstract

Hydrogelators are small, self-assembling molecules that form supramolecular nanofiber networks that exhibit unique dynamic properties. Development of supramolecular hydrogels that degrade in response to various biomolecules could potentially be used for applications in areas such as drug delivery and diagnostics. Here we provide a synthetic procedure for preparing redox-responsive supramolecular hydrogelators that are used to create hydrogels that degrade in response to oxidizing or reducing conditions. The synthesis takes ∼2–4 d, and it can potentially be carried out in parallel to prepare multiple hydrogelator candidates. This described solid-phase peptide synthesis protocol can be used to produce previously described hydrogelators or to construct a focused molecular library to efficiently discover and optimize new hydrogelators. In addition, we describe the preparation of redox-responsive supramolecular hydrogel–enzyme hybrids that are created by mixing aqueous solutions of hydrogelators and enzymes, which requires 2 h for completion. The resultant supramolecular hydrogel–enzyme hybrids exhibit gel degradation in response to various biomolecules, and can be rationally designed by connecting the chemical reactions of the hydrogelators with enzymatic reactions. Gel degradation in response to biomolecules as triggers occurs within a few hours. We also describe the preparation of hydrogel–enzyme hybrids arrayed on flat glass slides, enabling high-throughput analysis of biomolecules such as glucose, uric acid, lactate and so on by gel degradation, which is detectable by the naked eye. The protocol requires ∼6 h to prepare the hydrogel–enzyme hybrid array and to complete the biomolecule assay.

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

Figure 1: Molecular design of a supramolecular hydrogelator (Xmoc peptide; several chemical structures are shown in Fig. 3) showing stimulus-responsive macroscopic gel degradation (gel-to-sol transition).
Figure 2: Schematic representation of a supramolecular hydrogel–enzyme hybrid showing stimulus-responsive macroscopic gel degradation (gel-to-sol transition) through coupling of the chemical reactions of hydrogelators and enzymatic reactions, allowing the integration of biomolecules (expanded stimuli) into common chemical stimulus.
Figure 3: Molecular design of a hydrogelator consisting of chemically reactive groups (Xmoc) and oligopeptides.
Figure 4: Chemical amplification system using an amplifier molecule (BP-(sarcosine)2) to develop a stimulus-responsive supramolecular hydrogel with greater sensitivity.
Figure 5: Solid-phase (two-step) synthesis of Xmoc peptide derivative (Xmoc-F(4-R1)F(4-R2)).
Figure 6: Synthesis of Xmoc-OSu.
Figure 7
Figure 8: Stimulus-responsive gel degradation of hybrid gels.

Similar content being viewed by others

References

  1. Tu, Y. et al. Mimicking the cell: bio-inspired functions of supramolecular assemblies. Chem. Rev. 116, 2023–2078 (2016).

    Article  CAS  Google Scholar 

  2. Burdick, J.A. & Murphy, W.L. Moving from static to dynamic complexity in hydrogel design. Nat. Commun. 3, 1269 (2012).

    Article  Google Scholar 

  3. Estroff, L.A. & Hamilton, A.D. Water gelation by small organic molecules. Chem. Rev. 104, 1201–1217 (2004).

    Article  CAS  Google Scholar 

  4. de Loos, M., Feringa, B.L. & van Esch, J.H. Design and application of self-assembled low molecular weight hydrogels. Eur. J. Org. Chem. 2005, 3615–3631 (2005).

    Article  Google Scholar 

  5. Hirst, A.R., Escuder, B., Miravet, J.F. & Smith, D.K. High-tech applications of self-assembling supramolecular nanostructured gel-phase materials: from regenerative medicine to electronic devices. Angew. Chem. Int. Ed. 47, 8002–8018 (2008).

    Article  CAS  Google Scholar 

  6. Du, X., Zhou, J., Shi, J. & Xu, B. Supramolecular hydrogelators and hydrogels: from soft matter to molecular biomaterials. Chem. Rev. 115, 13165–13307 (2015).

    Article  CAS  Google Scholar 

  7. Dasgupta, A., Mondal, J.H. & Das, D. Peptide hydrogels. RSC Adv. 3, 9117–9149 (2013).

    Article  CAS  Google Scholar 

  8. Ryan, D.M. & Nilsson, B.L. Self-assembled amino acids and dipeptides as noncovalent hydrogels for tissue engineering. Polym. Chem. 3, 18–33 (2012).

    Article  CAS  Google Scholar 

  9. Segarra-Maset, M.D., Nebot, V.J., Miravet, J.F. & Escuder, B. Control of molecular gelation by chemical stimuli. Chem. Soc. Rev. 42, 7086–7098 (2013).

    Article  CAS  Google Scholar 

  10. Yang, Z., Liang, G. & Xu, B. Enzymatic control of the self-assembly of small molecules: a new way to generate supramolecular hydrogels. Soft Matter 3, 515–520 (2007).

    Article  CAS  Google Scholar 

  11. Ulijn, R.V. Enzyme-responsive materials: a new class of smart biomaterials. J. Mater. Chem. 16, 2217–2225 (2006).

    Article  CAS  Google Scholar 

  12. Sáez, J.A., Escuder, B. & Miravet, J.F. Supramolecular hydrogels for enzymatically triggered self-immolative drug delivery. Tetrahedron 66, 2614–2618 (2010).

    Article  Google Scholar 

  13. Shigemitsu, H. & Hamachi, I. Supramolecular assemblies responsive to biomolecules toward biological applications. Chem. Asian J. 10, 2026–2038 (2015).

    Article  CAS  Google Scholar 

  14. Holtz, J.H. & Asher, S.A. Polymerized colloidal crystal hydrogel films as intelligent chemical sensing materials. Nature 389, 829–832 (1997).

    Article  CAS  Google Scholar 

  15. Gajanayake, T. et al. A single localized dose of enzyme-responsive hydrogel improves long-term survival of a vascularized composite allograft. Sci. Transl. Med. 6, 249ra110 (2014).

    Article  Google Scholar 

  16. Zhang, S. et al. An inflammation-targeting hydrogel for local drug delivery in inflammatory bowel disease. Sci. Transl. Med. 7, 300ra128 (2015).

    Article  Google Scholar 

  17. Lutolf, M.P. et al. Repair of bone defects using synthetic mimetics of collagenous extracellular matrices. Nat. Biotechnol. 21, 513–518 (2003).

    Article  CAS  Google Scholar 

  18. Silva, G.A. et al. Selective differentiation of neural progenitor cells by high-epitope density nanofibers. Science 303, 1352–1355 (2004).

    Article  CAS  Google Scholar 

  19. Haines-Butterick, L. et al. Controlling hydrogelation kinetics by peptide design for three-dimensional encapsulation and injectable delivery of cells. Proc. Natl Acad. Sci. USA 104, 7791–7796 (2007).

    Article  CAS  Google Scholar 

  20. Ikeda, M., Tanida, T., Yoshii, T. & Hamachi, I. Rational molecular design of stimulus-responsive supramolecular hydrogels based on dipeptides. Adv. Mater. 23, 2819–2822 (2011).

    Article  CAS  Google Scholar 

  21. Ikeda, M. et al. Installing logic-gate responses to a variety of biological substances in supramolecular hydrogel-enzyme hybrids. Nat. Chem. 6, 511–518 (2014).

    Article  CAS  Google Scholar 

  22. Yoshii, T., Ikeda, M. & Hamachi, I. Two-photon-responsive supramolecular hydrogel for controlling materials motion in micrometer space. Angew. Chem. Int. Ed. 53, 7264–7267 (2014).

    Article  CAS  Google Scholar 

  23. Yoshii, T., Onogi, S., Shigemitsu, H. & Hamachi, I. Chemically reactive supramolecular hydrogel coupled with a signal amplification system for enhanced analyte sensitivity. J. Am. Chem. Soc. 137, 3360–3365 (2015).

    Article  CAS  Google Scholar 

  24. Jayawarna, V. et al. Nanostructured hydrogels for three-dimensional cell culture through self-assembly of fluorenylmethoxycarbonyl–dipeptides. Adv. Mater. 18, 611–614 (2006).

    Article  CAS  Google Scholar 

  25. Mahler, A., Reches, M., Rechter, M., Cohen, S. & Gazit, E. Rigid, self-assembled hydrogel composed of a modified aromatic dipeptide. Adv. Mater. 18, 1365–1370 (2006).

    Article  CAS  Google Scholar 

  26. Frederix, P.W.J.M. et al. Exploring the sequence space for (tri-)peptide self-assembly to design and discover new hydrogels. Nat. Chem. 7, 30–37 (2015).

    Article  CAS  Google Scholar 

  27. Ryan, D.M., Doran, T.M., Anderson, S.B. & Nilsson, B.L. Effect of C-terminal modification on the self-assembly and hydrogelation of fluorinated Fmoc-phe derivatives. Langmuir 27, 4029–4039 (2011).

    Article  CAS  Google Scholar 

  28. Buerkle, L.E., Li, Z., Jamieson, A.M. & Rowan, S.J. Tailoring the properties of guanosine-based supramolecular hydrogels. Langmuir 25, 8833–8840 (2009).

    Article  CAS  Google Scholar 

  29. Ryan, D.M., Anderson, S.B. & Nilsson, B.L. The influence of side-chain halogenation on the self-assembly and hydrogelation of Fmoc-phenylalanine derivatives. Soft Matter 6, 3220–3231 (2010).

    Article  CAS  Google Scholar 

  30. Bertolani, A. et al. Supramolecular amplification of amyloid self-assembly by iodination. Nat. Commun. 6, 7574 (2015).

    Article  Google Scholar 

  31. Chen, L., Revel, S., Morris, K., Serpell, L.C. & Adams, D.J. Effect of molecular structure on the properties of naphthalenedipeptide hydrogelators. Langmuir 26, 13466–13471 (2010).

    Article  CAS  Google Scholar 

  32. Wang, H. et al. A structure–gelation ability study in a short peptide-based 'Super Hydrogelator' system. Soft Matter 7, 3897–3905 (2011).

    Article  CAS  Google Scholar 

  33. Sella, E. & Shabat, D. Dendritic chain reaction. J. Am. Chem. Soc. 131, 9934–9936 (2009).

    Article  CAS  Google Scholar 

  34. Roth, M.E., Green, O., Gnaim, S. & Shabat, D. D, oligomeric, and polymeric self-immolative molecular amplification. Chem. Rev. 116, 1309–1352 (2015).

    Article  Google Scholar 

  35. Weingarten, A.S. et al. Self-assembling hydrogel scaffolds for photocatalytic hydrogen production. Nat. Chem. 6, 964–970 (2014).

    Article  CAS  Google Scholar 

  36. Wang, Q. et al. A supramolecular-hydrogel-encapsulated hemin as an artificial enzyme to mimic peroxidase. Angew. Chem. Int. Ed. 46, 4285–4289 (2007).

    Article  CAS  Google Scholar 

  37. Yoshimura, I. et al. Molecular recognition in a supramolecular hydrogel to afford a semi-wet sensor chip. J. Am. Chem. Soc. 126, 12204–12205 (2004).

    Article  CAS  Google Scholar 

  38. Ikeda, M., Ochi, R. & Hamachi, I. Supramolecular hydrogel-based protein and chemosensor array. Lab Chip 10, 3325–3334 (2010).

    Article  CAS  Google Scholar 

  39. Kiriya, D. et al. Meter-long and robust supramolecular strands encapsulated in hydrogel jackets. Angew. Chem. Int. Ed. 51, 1553–1557 (2012).

    Article  CAS  Google Scholar 

  40. Cornwell, D.J. & Smith, D.K. Expanding the scope of gels – combining polymers with low-molecular-weight gelators to yield modified self-assembling smart materials with high-tech applications. Mater. Horiz. 2, 279–293 (2015).

    Article  CAS  Google Scholar 

  41. Heeres, A. et al. Orthogonal self-assembly of low molecular weight hydrogelators and surfactants. J. Am. Chem. Soc. 125, 14252–14253 (2003).

    Article  CAS  Google Scholar 

  42. Brizard, A. et al. Preparation of nanostructures by orthogonal self-assembly of hydrogelators and surfactants. Angew. Chem. Int. Ed. 47, 2063–2066 (2008).

    Article  CAS  Google Scholar 

  43. Wickremasinghe, N.C., Kumar, V.A., Shi, S. & Hartgerink, J.D. Controlled angiogenesis in peptide nanofiber composite hydrogels. ACS Biomater. Sci. Eng. 1, 845–854 (2015).

    Article  CAS  Google Scholar 

  44. Patil, A.J., Kumar, R.K., Barron, N.J. & Mann, S. Cerium oxide nanoparticle-mediated self-assembly of hybrid supramolecular hydrogels. Chem. Commun. 48, 7934–7936 (2012).

    Article  CAS  Google Scholar 

  45. Wada, A., Tamaru, S.-I., Ikeda, M. & Hamachi, I. MCM-enzyme-supramolecular hydrogel hybrid as a fluorescence sensing material for polyanions of biological significance. J. Am. Chem. Soc. 131, 5321–5330 (2009).

    Article  CAS  Google Scholar 

  46. Ikeda, M. et al. Montmorillonite-supramolecular hydrogel hybrid for fluorocolorimetric sensing of polyamines. J. Am. Chem. Soc. 133, 1670–1673 (2011).

    Article  CAS  Google Scholar 

  47. Kiyonaka, S. et al. Semi-wet peptide/protein array using supramolecular hydrogel. Nat. Mater. 3, 58–64 (2004).

    Article  CAS  Google Scholar 

  48. Guler, M.O., Pokorski, J.K., Appella, D.H. & Stupp, S.I. Enhanced oligonucleotide binding to self-assembled nanofibers. Bioconjugate Chem. 16, 501–503 (2005).

    Article  CAS  Google Scholar 

  49. Patil, S.P., Jeong, H.S. & Kim, B.H. A low-molecular-weight supramolecular hydrogel of riboflavin bolaamphiphile for VEGF-siRNA delivery. Chem. Commun. 48, 8901–8903 (2012).

    Article  CAS  Google Scholar 

  50. Poolman, J.M. et al. Variable gelation time and stiffness of low-molecular-weight hydrogels through catalytic control over self-assembly. Nat. Protoc. 9, 977–988 (2014).

    Article  CAS  Google Scholar 

  51. Chan, W.C. & White, P.D. Fmoc Solid Peptide Synthesis (Oxford University Press, New York, 2000).

  52. Slavici, T., Darvasi, D., Mnerie, A. & Cechin-Crista, P. Development of economical and efficient mixed reactive carbonates for peptide synthesis through scientific planning. Rev. Chim. 64, 425–429 (2013).

    CAS  Google Scholar 

  53. Raeburn, J., Zamith Cardoso, A. & Adams, D.J. The importance of the self-assembly process to control mechanical properties of low molecular weight hydrogels. Chem. Soc. Rev. 42, 5143–5156 (2013).

    Article  CAS  Google Scholar 

  54. Morris, K.L. et al. Chemically programmed self-sorting of gelator networks. Nat. Commun. 4, 1480 (2013).

    Article  Google Scholar 

  55. Komatsu, H. et al. Supramolecular hydrogel exhibiting four basic logic gate functions to fine-tune substance release. J. Am. Chem. Soc. 131, 5580–5585 (2009).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We gratefully acknowledge the financial support from the CREST program of the JST.

Author information

Authors and Affiliations

  1. Department of Synthetic Chemistry and Biological Chemistry,

    Hajime Shigemitsu, Takahiro Fujisaku, Shoji Onogi, Tatsuyuki Yoshii & Itaru Hamachi

  2. , Graduate School of Engineering, Kyoto University, Kyoto, Japan

    Hajime Shigemitsu, Takahiro Fujisaku, Shoji Onogi, Tatsuyuki Yoshii & Itaru Hamachi

  3. Department of Chemistry and Biomolecular Science, Faculty of Engineering, Gifu University, Gifu, Japan

    Masato Ikeda

  4. Department of Biomolecular Science, Graduate School of Engineering, Gifu University, Gifu, Japan

    Masato Ikeda

  5. United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, Gifu, Japan

    Masato Ikeda

  6. Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), Tokyo, Japan

    Itaru Hamachi

Authors
  1. Hajime Shigemitsu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Takahiro Fujisaku
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Shoji Onogi
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Tatsuyuki Yoshii
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Masato Ikeda
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Itaru Hamachi
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

H.S., T.F., S.O. and T.Y. performed the experiments and analyzed the data; H.S., T.Y., M.I. and I.H. designed the experiments; and H.S., M.I. and I.H. wrote the paper. All authors contributed to discussion of results and editing of the manuscript.

Corresponding authors

Correspondence to Masato Ikeda or Itaru Hamachi.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Gelation tests of NPmoc-FF derivatives (NPmoc-F(4-R1)F(4-R2)).

Photographs showing the gelation ability of NPmoc-FF derivatives (NPmoc-F(4-R1)F(4-R2)). Conditions: 200 mM MES (pH 7.0), 37 ºC.

Supplementary information

Supplementary Text and Figures

Supplementary Figure 1 (PDF 243 kb)

The method for the preparation of GOx-encapsulating supramolecular gels (GOx⊂BPmoc-FFF hybrid gels) in a screw vial and their glucose-responsive gel degradation.

Conditions: [BPmoc-FFF] = 0.15 wt% (100 mM MES (pH 7.0), 100 μl), [GOx] = 50 mg/mL (10 mM HEPES (pH 7.0), 1 μL). (MP4 6305 kb)

The method for the preparation of enzymes (GOx, SOx, UOx and COx) encapsulating supramolecular gels (GOx, Sox, UOx or COx ⊂BPmoc-FFF hybrid gels) and checking of gel–sol transitions on a glass slide.

In the video, we added glucose solution to the gels. The GOx-encapsulating supramolecular gel (GOx⊂BPmoc-FFF hybrid gel) changed to a sol >4 h from the addition of aqueous glucose solution (1 μl, 24 mM). The corresponding spot was easily washed off by water. Conditions: [BPmoc-FFF] = 0.15 wt% (100 mM MES (pH 7.0), 10 μl), [GOx] = 2.7 μM, [SOx] = 6.7 μM, [UOx] = 3.6 μM, [COx] = 4.6 μM (10 mM HEPES (pH 7.0), 0.5 μl). (MP4 5070 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Shigemitsu, H., Fujisaku, T., Onogi, S. et al. Preparation of supramolecular hydrogel–enzyme hybrids exhibiting biomolecule-responsive gel degradation. Nat Protoc 11, 1744–1756 (2016). https://doi.org/10.1038/nprot.2016.099

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2016.099

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research