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

Nature Protocols volume 12, pages 1521–1541 (2017)Cite this article

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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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Figure 1: Overview of the guest–host hydrogel platform.
Figure 2: Application of GH hydrogels for local delivery of therapeutics.
Figure 3: Application of GH hydrogels as injectable cell carriers.
Figure 4: Direct 3D-bioprinting of GH hydrogels and secondary stabilization for fabrication of complex 3D structures.
Figure 5: Guest and host polymer synthesis schemes.
Figure 6: Guest–host hydrogel preparation and injection.
Figure 7: Guest–host assembly and recovery mechanism.

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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.

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Author notes

  1. Christopher B Rodell

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

  2. Claudia Loebel and Christopher B Rodell: These authors contributed equally to this work.

Authors and 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.

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Correspondence to Jason A Burdick.

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Integrated supplementary information

Supplementary Figure 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).

(a) Frequency dependence of GH hydrogels presents an increasing storage modulus (G’) at higher frequencies. (b) Photocrosslinking in the presence of UV light and a radical photoinitiator (0.05% Irgacure 2959) induced mechanical stabilization of the material as shown by an increased G’ and reduced frequency dependence with a lack of re-arrangement (i.e. no cross-over of the G’ and G’’).

Supplementary Figure 2 Preparation of agarose well-in-well molds for culturing cellular guest-host hydrogels.

(a) Customized plastic mold made of Acrylonitrile-Butadiene-Styrene (ABS). (b) The mold is designed to fit into a 24-well plate with inserts of 8 mm length and 5 mm diameter. (c) After solidification of 2 mL agarose, molds can be used to contain injected guest-host hydrogels for culture.

Supplementary Figure 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).

Setup for (a) CD-Tos and (b) CD-HDA synthesis under anhydrous conditions.

Supplementary Figure 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).

Frequency sweeps with G’ (filled symbols) and G’’ (open symbols) show the crossover of G’ and G’’ and the reproducibility of the measurement (n=3).

Supplementary Figure 5 Erosion and molecule release characteristics of GH hydrogels.

Examples for (a) cumulative hydrogel erosion profile and (b) FITC-BSA (0.1% (wt/vol)) release for 5% (wt/vol) GH hydrogels over 60 days (n=3, mean ± SD).

Supplementary Figure 6 In vivo fluorescence imaging of GH hydrogels pre- and post injection subcutaneously in the right flank of mice.

Representative overlay of near-infrared and black and white images taken after subcutaneous injection of 25 μl GH hydrogels (3.5% (wt/vol) and subsequently over 14 days illustrate degradation behavior in vivo (Pearl® Impulse, LI-COR, λex/λem = 785/820 nm).

Supplementary Figure 7 Confocal microscopy (calcein and ethidium staining) of encapsulated human MSCs.

Human MSCs (5x106/mL) were encapsulated in GH hydrogels (5% (wt/vol)) and high viability (95%) was observed after 24 h, scale bar 200 μm.

Supplementary Figure 8 1H NMR spectrum of methacrylated hyaluronic acid in D2O.

Methacrylate modification (26% shown) is determined by integration of the vinyl singlets (1H each, shaded green) relative to the sugar ring of hyaluronic acid (HA, 10H, shaded gray).

Supplementary Figure 9 1H NMR spectra of tetrabutyl ammonium salt of hyaluronic acid (HA-TBA) and methacrylated hyaluronic acid (MeHA-TBA) in D2O.

Modification of (a) HA with TBA salt is determined by the integration of the TBA methyl-groups (12H, shaded brown) relative to the N-acetyl group of HA (3H, shaded gray). Modification of (b) methacrylation and TBA salt is determined by the integration of the TBA methyl-groups (3H, shaded brown) and the vinyl singlets (1H each, shaded green) relative to the sugar ring of hyaluronic acid (HA, 10H, shaded gray).

Supplementary Figure 10 1H NMR spectrum of 6-o-monotosyl-6-deoxy-β-cyclodextrin (CD-Tos) in DMSO-d6.

Tosylation of CD is confirmed by integration of the methyl (3H, shaded red) and aromatic hydrogens (2H each, shaded orange), relative to the secondary hydroxyls of CD (14H, shaded gray). Anticipated minor impurities in the spectra may include trace acetone (δ = 2.10), ammonium chloride (δ = 6.98, 7.12, 2.27 (s)), ethyl ether (δ = 3.42 (q), 1.13 (t, 3H)), and water (see Supplementary Figure 11).

Supplementary Figure 11 1H NMR spectrum of 6-o-monotosyl-6-deoxy-β-cyclodextrin (CD-Tos) in DMSO-d6.

The product was not fully dried, which causes combination of the HOD peak with the hydroxyl groups of the CD ring (purple shaded), prohibiting reliable integration of the CD necessary to confirm mono-tosylation.

Supplementary Figure 12 1H NMR spectrum of 6-(6-aminohexyl)amino-6-deoxy-β-cyclodextrin (CD-HDA) in DMSO-d6.

Modification of CD with HDA is determined by integration of the hexane linker (12H, shaded red) relative to the secondary hydroxyls of CD (14H, shaded gray). Anticipated minor impurities in the spectra may include trace dimethlyformamide (δ = 7.98, 2.92, 2.08), acetone (δ = 2.10), and ethyl ether (δ = 3.42 (q), 1.13 (t, 3H)).

Supplementary Figure 13 1H NMR spectra of β-cyclodextrin modification of hyaluronic acid (CD-HA) and methacrylated hyaluronic acid (MeHA-CD) in D2O.

(a) Modification of HA with pendant CD (30.2%) is determined by integration of the hexane linkers (12H, shaded red) relative to the N-acetyl singlet of HA (3H, shaded gray). (b) CD functionalization of MeHA shows an overlap of the spectra of the modifications with the sugar ring and the N-acetyl group of HA. Therefore, to determine the modification of MeHA with CD (30.3%), the hexane linker (12H, shaded red) is integrated relative to the methacrylate modification, which is assumed to be conserved throughout the reaction (25%, 1H each, shaded green).

Supplementary Figure 14 1H NMR spectra of adamantane functionalization of hyaluronic acid (Ad-HA) and methacrylated adamantane hyaluronic acid (Ad-MeHA) in D2O.

(a) Modification of HA with pendant Ad (32.5%) is determined by integration of the ethyl multiplet (12H, shaded blue) relative to the sugar ring of HA (10H, shaded gray). (b) Modification of MeHA with pendant Ad (32.8%) is determined by integration of the ethyl multiplet (12H, shaded blue) relative to the sugar ring of HA (10H, shaded gray). Integration of the vinyl singlets (1H each, shaded green) relative to the sugar ring of hyaluronic acid (HA, 10H, shaded gray) confirms the conservation of the methacrylate modification.

Supplementary Figure 15 Confocal microscopy (calcein and ethidium staining) of rat endothelial progenitor cells (EPCs) encapsulated in GH hydrogels (5% (wt/vol)).

EPCs were not homogenously mixed with the GH hydrogel, which caused poor cell viability (45.5%) after injection through a 25G ¼ needle, scale bar 200 μm.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–15. (PDF 1700 kb)

Supplementary Data (zip file).

Stereolithography file for plastic mold. (ZIP 118 kb)

Supplementary Video 1.

Mixing of CD-HA and Ad-HA in a tube. (MP4 2536 kb)

Supplementary Video 2.

Loading of a syringe with preformed GH hydrogels and subsequent injection. (MP4 3289 kb)

Supplementary Video 3.

Injection of GH hydrogels into customized acrylamide molds. (MP4 2381 kb)

Supplementary Video 4.

Mixing of CD-HA and Ad-HA in the back of a syringe. (MP4 2002 kb)

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Loebel, C., Rodell, C., Chen, M. et al. Shear-thinning and self-healing hydrogels as injectable therapeutics and for 3D-printing. Nat Protoc 12, 1521–1541 (2017). https://doi.org/10.1038/nprot.2017.053

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