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Shear-thinning and self-healing hydrogels as injectable therapeutics and for 3D-printing

Nature Protocols volume 12, pages 15211541 (2017) | Download Citation

Abstract

The design of injectable hydrogel systems addresses the growing demand for minimally invasive approaches for local and sustained delivery of therapeutics. We developed a class of hyaluronic acid (HA) hydrogels that form through noncovalent guest–host interactions, undergo disassembly (shear-thinning) when injected through a syringe and then reassemble within seconds (self-healing) when shear forces are removed. Its unique properties enable the use of this hydrogel system for numerous applications, such as injection in vivo (including with cells and therapeutic molecules) or as a 'bioink' in 3D-printing applications. Here, we describe the functionalization of HA either with adamantanes (guest moieties) via controlled esterification or with β-cyclodextrins (host moieties) through amidation. We also describe how to modify the HA derivatives with methacrylates for secondary covalent cross-linking and for reaction with fluorophores for in vitro and in vivo imaging. HA polymers are rationally designed from relatively low-molecular-weight starting materials, with the degree of modification controlled, and have matched guest-to-host stoichiometry, allowing the preparation of hydrogels with tailored properties. This procedure takes 3–4 weeks to complete. We detail the preparation and characterization of the guest–host hydrogels, including assessment of their rheological properties, erosion and biomolecule release in vitro. We furthermore demonstrate how to encapsulate cells in vitro and provide procedures for quantitative assessment of in vivo hydrogel degradation by imaging of fluorescently derivatized materials.

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References

  1. 1.

    & Designing hydrogels for controlled drug delivery. Nat. Rev. Mater. 1, 16071 (2016).

  2. 2.

    & Moving from static to dynamic complexity in hydrogel design. Nat. Commun. 3, 1269 (2012).

  3. 3.

    & Hydrogels for tissue engineering: scaffold design variables and applications. Biomaterials 24, 4337–4351 (2003).

  4. 4.

    & Review: photopolymerizable and degradable biomaterials for tissue engineering applications. Tissue Eng. 13, 2369–2385 (2007).

  5. 5.

    & Photopolymerizable hydrogels for tissue engineering applications. Biomaterials 23, 4307–4314 (2002).

  6. 6.

    et al. Injectable hydrogel properties influence infarct expansion and extent of postinfarction left ventricular remodeling in an ovine model. Proc. Natl. Acad. Sci. USA 107, 11507–11512 (2010).

  7. 7.

    et al. Synthesis and characterization of hyaluronic acid–poly(ethylene glycol) hydrogels via Michael addition: an injectable biomaterial for cartilage repair. Acta Biomater. 6, 1968–1977 (2010).

  8. 8.

    Perspectives in supramolecular chemistry—from molecular recognition towards molecular information processing and self-organization. Angew. Chem. Int. Ed. Engl. 29, 1304–1319 (1990).

  9. 9.

    , , & Progress in material design for biomedical applications. Proc. Natl. Acad. Sci. USA 112, 14444–14451 (2015).

  10. 10.

    & Adaptable hydrogel networks with reversible linkages for tissue engineering. Adv. Mater. 27, 3717–3736 (2015).

  11. 11.

    , , & Supramolecular biomaterials. Nat. Mater. 15, 13–26 (2016).

  12. 12.

    , & Shear-thinning hydrogels for biomedical applications. Soft Matter 8, 260–272 (2012).

  13. 13.

    & Multifunctional materials through modular protein engineering. Adv. Mater. 24, 3923–3940 (2012).

  14. 14.

    , , & Injectable shear-thinning hydrogels engineered with a self-assembling Dock-and-Lock mechanism. Biomaterials 33, 2145–2153 (2012).

  15. 15.

    , & Supramolecular guest-host interactions for the preparation of biomedical materials. Bioconjug. Chem. 26, 2279–2289 (2015).

  16. 16.

    et al. Self-healing polymer materials constructed by macrocycle-based host-guest interactions. Soft Matter 11, 1242–1252 (2015).

  17. 17.

    & Biomedical applications of supramolecular systems based on host–guest interactions. Chem. Rev. 115, 7794–7839 (2015).

  18. 18.

    , & Recent advances in hyaluronic acid hydrogels for biomedical applications. Curr. Opin. Biotechnol. 40, 35–40 (2016).

  19. 19.

    & Synthesis of novel supramolecular assemblies based on hyaluronic acid derivatives bearing bivalent β-cyclodextrin and adamantane moieties. Macromolecules 40, 1147–1158 (2007).

  20. 20.

    , & Specific interactions in model charged polysaccharide systems. J. Phys. Chem. B 107, 8248–8254 (2003).

  21. 21.

    , , & Rheological properties of binary associating polymers. Rheol. Acta 46, 541–568 (2007).

  22. 22.

    , & Rational design of network properties in guest-host assembled and shear-thinning hyaluronic acid hydrogels. Biomacromolecules 14, 4125–4134 (2013).

  23. 23.

    et al. Injectable shear-thinning hydrogels used to deliver endothelial progenitor cells, enhance cell engraftment, and improve ischemic myocardium. J. Thorac. Cardiovasc. Surg. 150, 1268–1276 (2015).

  24. 24.

    et al. Shear-thinning supramolecular hydrogels with secondary autonomous covalent crosslinking to modulate viscoelastic properties in vivo. Adv. Funct. Mater. 25, 636–644 (2015).

  25. 25.

    et al. Injectable shear-thinning hydrogels for minimally invasive delivery to infarcted myocardium to limit left ventricular remodeling. Circ. Cardiovasc. Interv. 9, e004058 (2016).

  26. 26.

    , , , & Local immunotherapy via delivery of interleukin-10 and transforming growth factor β antagonist for treatment of chronic kidney disease. J. Control Release 206, 131–139 (2015).

  27. 27.

    et al. Delivery of interleukin-10 via injectable hydrogels improves renal outcomes and reduces systemic inflammation following ischemic acute kidney injury in mice. Am. J. Physiol. Renal Physiol. 311, F362–F372 (2016).

  28. 28.

    , & Direct 3D printing of shear-thinning hydrogels into self-healing hydrogels. Adv. Mater. 27, 5075–5079 (2015).

  29. 29.

    , , , & 3D printing of shear-thinning hyaluronic acid hydrogels with secondary cross-linking. ACS Biomater. Sci. Eng. 2, 1743–1751 (2016).

  30. 30.

    , , , & Secondary photocrosslinking of injectable shear-thinning dock-and-lock hydrogels. Adv. Healthc. Mater. 2, 1028–1036 (2013).

  31. 31.

    , & Injectable hydrogels with in situ double network formation enhance retention of transplanted stem cells. Adv. Funct. Mater. 25, 1344–1351 (2015).

  32. 32.

    , , , & Supramolecular hydrogels formed by β-cyclodextrin self-association and host–guest inclusion complexes. Soft Matter 6, 187–194 (2010).

  33. 33.

    et al. Injectable, guest-host assembled polyethylenimine hydrogel for siRNA delivery. Biomacromolecules 18, 77–86 (2017).

  34. 34.

    , , , & Immunotherapy with injectable hydrogels to treat obstructive nephropathy. J. Biomed. Mater. Res. A 102, 2173–2180 (2014).

  35. 35.

    et al. Antibody to transforming growth factor-beta ameliorates tubular apoptosis in unilateral ureteral obstruction. Kidney Int. 58, 2301–2313 (2000).

  36. 36.

    , & Sustained small molecule delivery from injectable hyaluronic acid hydrogels through host-guest mediated retention. J. Mater. Chem. B Mater. Biol. Med. 3, 8010–8019 (2015).

  37. 37.

    et al. Comparison of biomaterial delivery vehicles for improving acute retention of stem cells in the infarcted heart. Biomaterials 35, 6850–6858 (2014).

  38. 38.

    , & To serve and protect: hydrogels to improve stem cell-based therapies. Cell Stem Cell 18, 13–15 (2016).

  39. 39.

    et al. Stiffening hydrogels for investigating the dynamics of hepatic stellate cell mechanotransduction during myofibroblast activation. Sci. Rep. 6, 21387 (2016).

  40. 40.

    , , & Injectable and cytocompatible tough double-network hydrogels through tandem supramolecular and covalent crosslinking. Adv. Mater. 28, 8419–8424 (2016).

  41. 41.

    , , , & Dynamic display of bioactivity through host–guest chemistry. Angew. Chem. Int. Ed. Engl. 52, 12077–12080 (2013).

  42. 42.

    , & Modulation of stem cell adhesion and morphology via facile control over surface presentation of cell adhesion molecules. Biomacromolecules 15, 43–52 (2014).

  43. 43.

    , , , & Selective proteolytic degradation of guest–host assembled, injectable hyaluronic acid hydrogels. ACS Biomater. Sci. Eng. 1, 277–286 (2015).

  44. 44.

    , , , & Controlled degradation and mechanical behavior of photopolymerized hyaluronic acid networks. Biomacromolecules 6, 386–391 (2005).

  45. 45.

    & Photocrosslinkable polysaccharides for in situ hydrogel formation. J. Biomed. Mater. Res. A 54, 115–121 (2001).

  46. 46.

    , , & Hydrolytically degradable hyaluronic acid hydrogels with controlled temporal structures. Biomacromolecules 9, 1088–1092 (2008).

  47. 47.

    , , , & Cyclodextrin-derived diorganyl tellurides as glutathione peroxidase mimics and inhibitors of thioredoxin reductase and cancer cell growth. J. Med. Chem. 47, 233–239 (2004).

  48. 48.

    & Synthesis and characterization of physical crosslinking systems based on cyclodextrin inclusion/host-guest complexation. J. Polym. Sci. A Polym. Chem. 48, 581–592 (2010).

  49. 49.

    , & Solid-phase peptide synthesis: from standard procedures to the synthesis of difficult sequences. Nat. Protoc. 2, 3247–3256 (2007).

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Acknowledgements

The authors are grateful for financial support from the National Science Foundation (DMR Award 1610525), an NIH/NIAMS training grant (T32-AR007132; M.H.C.), a postdoctoral fellowship (C.L.) from the IBSA Foundation, Switzerland, and predoctoral fellowships (C.B.R., M.H.C.) and an Established Investigator Award (J.A.B) from the American Heart Association.

Author information

Author notes

    • Christopher B Rodell

    Present address: Center for Systems Biology, Massachusetts General Hospital, Boston, Massachusetts, USA.

    • Claudia Loebel
    •  & Christopher B Rodell

    These authors contributed equally to this work.

Affiliations

  1. Department of Bioengineering, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

    • Claudia Loebel
    • , Christopher B Rodell
    • , Minna H Chen
    •  & Jason A Burdick

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Contributions

C.L., C.B.R. and M.H.C. performed the experiments and analyzed data. All authors wrote the manuscript, and J.A.B. supervised the research.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Jason A Burdick.

Integrated supplementary information

Supplementary figures

  1. 1.

    Rheological characterization of a representative CD-MeHA and Ad-MeHA hydrogel before and after covalent crosslinking (20% modified CD-MeHA mixed with 20% modified Ad-MeHA, stoichiometric ratio 1:1, 3.5% (wt/vol) polymer concentration).

  2. 2.

    Preparation of agarose well-in-well molds for culturing cellular guest-host hydrogels.

  3. 3.

    Reaction setup for synthesis of 6-o-monotosyl-6-deoxy-β-cyclodextrin (CD-Tos) and 6-(6-aminohexyl)amino-6-deoxy-β-cyclodextrin (CD-HDA).

  4. 4.

    Rheological characterization of GH hydrogel replicates (20% modified CD-HA mixed with 20% modified Ad-HA, stoichiometric ratio 1:1, 7.5% (wt/vol) polymer concentration).

  5. 5.

    Erosion and molecule release characteristics of GH hydrogels.

  6. 6.

    In vivo fluorescence imaging of GH hydrogels pre- and post injection subcutaneously in the right flank of mice.

  7. 7.

    Confocal microscopy (calcein and ethidium staining) of encapsulated human MSCs.

  8. 8.

    1H NMR spectrum of methacrylated hyaluronic acid in D2O.

  9. 9.

    1H NMR spectra of tetrabutyl ammonium salt of hyaluronic acid (HA-TBA) and methacrylated hyaluronic acid (MeHA-TBA) in D2O.

  10. 10.

    1H NMR spectrum of 6-o-monotosyl-6-deoxy-β-cyclodextrin (CD-Tos) in DMSO-d6.

  11. 11.

    1H NMR spectrum of 6-o-monotosyl-6-deoxy-β-cyclodextrin (CD-Tos) in DMSO-d6.

  12. 12.

    1H NMR spectrum of 6-(6-aminohexyl)amino-6-deoxy-β-cyclodextrin (CD-HDA) in DMSO-d6.

  13. 13.

    1H NMR spectra of β-cyclodextrin modification of hyaluronic acid (CD-HA) and methacrylated hyaluronic acid (MeHA-CD) in D2O.

  14. 14.

    1H NMR spectra of adamantane functionalization of hyaluronic acid (Ad-HA) and methacrylated adamantane hyaluronic acid (Ad-MeHA) in D2O.

  15. 15.

    Confocal microscopy (calcein and ethidium staining) of rat endothelial progenitor cells (EPCs) encapsulated in GH hydrogels (5% (wt/vol)).

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–15.

Zip files

  1. 1.

    Supplementary Data (zip file).

    Stereolithography file for plastic mold.

Videos

  1. 1.

    Supplementary Video 1.

    Mixing of CD-HA and Ad-HA in a tube.

  2. 2.

    Supplementary Video 2.

    Loading of a syringe with preformed GH hydrogels and subsequent injection.

  3. 3.

    Supplementary Video 3.

    Injection of GH hydrogels into customized acrylamide molds.

  4. 4.

    Supplementary Video 4.

    Mixing of CD-HA and Ad-HA in the back of a syringe.

About this article

Publication history

Published

DOI

https://doi.org/10.1038/nprot.2017.053

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