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

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.

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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.

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Acknowledgements

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

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Authors and Affiliations

Authors

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.

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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 (GOxBPmoc-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 (GOxBPmoc-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)

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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

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