Dynamic Supramolecular Hydrogels Spanning an Unprecedented

Letter Cite This: ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Dynamic Supramolecular Hydrogels Spanning an Unprecedented Range of Host−Guest Affinity Lei Zou, Adam S. Braegelman, and Matthew J. Webber* Department of Chemical & Biomolecular Engineering, University of Notre Dame, Notre Dame, Indiana 46556, United States

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ABSTRACT: Cucurbit[7]uril (CB[7]) macrocycles exhibit a broad range of host− guest binding affinity. Attaching pendant CB[7] and complementary guests on 8-arm PEG macromers affords supramolecular hydrogels with cross-link affinity spanning more than 5 orders of magnitude (1.5 × 107 to 5.4 × 1012 M−1) without changing network topology. Cross-link affinity translates directly to bulk dynamic properties; hydrogels with high-affinity cross-linking behave like covalent gels with limited ability to relax or self-heal. Cross-link affinity furthermore dictates the release rate of encapsulated macromolecules, as well as cell infiltration and material clearance in vivo. This work thus informs a role for affinity in dictating supramolecular hydrogel properties by quantifying and isolating this feature over an unprecedented range.

KEYWORDS: molecular engineering, bio-inspired materials, hydrogels, biomaterials, drug delivery

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explored.15 The binding affinity between cyclodextrin and its guests has been shown to directly alter the strength and selfhealing ability of the resulting hydrogels.16 Among these motifs, cyclodextrin-adamantane forms the basis for many supramolecular hydrogels used. Yet, the typical binding affinity (Keq) for such an interaction is on the order of ∼1 × 105 M−1;17 this is among the highest affinities possible from cyclodextrin macrocycles and as such is the effective upper limit to the range of affinities most commonly applied in supramolecular hydrogels. The affinity of host−guest recognition in supramolecular networks dictates, in large part, the cross-link dynamics in the resulting materials. Given the relatively modest affinities achievable with most motifs used to create supramolecular hydrogels, recognition by cucurbit[7]uril (CB[7]) was explored here. CB[7] achieves a broad range of affinity in binding to many different guest chemistries,18 and has shown the highest binding affinity ever reported for any host−guest interaction with Keq of 7.2 × 1017 M−1.19 CB[7] binds with high affinity to guests in which the hydrophobic or aliphatic unit that inserts into the portal is adjacent to a protonating group, leveraging a combination of hydrophobic and electrostatic interactions which stabilize the complex.20 A key feature of binding by CB[7] and related macrocycles is that complex formation typically occurs at or near to the diffusion limit (kon ≈ 1 × 108 M−1 s−1). With kon diffusion-governed, altering Keq translates directly to the lifetime of the interaction by changing

ioinspired materials are designed so as to recapitulate properties of natural materials with synthetic analogues. Accordingly, there have been efforts to capture the mechanical properties,1 hierarchical organization,2,3 and shape or geometry4,5 of natural materials. In one common example, percolated network structures are designed to mimic the properties of native extracellular matrix (ECM), typically using hydrogels prepared from covalently cross-linked polymers.6 Although these materials recreate the hydrated mesh-like architectures of natural ECM,7 their typically static cross-links do not capture the highly dynamic character of ECM.8 One feature of dynamic matrices is an ability to relax and dissipate stress by reorganizing physical cross-links or entanglements. Toward capturing native matrix dynamics, routes to tune stress relaxation in hydrogel materials have altered the molecular weight and packing of polymeric building blocks,9 or tuned the affinity between interacting domains in recombinant protein networks.10 Yet, mimicking the full range of stress relaxation observed in natural matrices with a tunable platform remains a challenge. Supramolecular chemistry affords specific, directional, tunable, and reversible noncovalent interactions that may be used to create organized systems across length-scales.11,12 As an example, host−guest motifs are attached pendant from polymeric or macromeric building blocks to facilitate physical cross-linking in preparing percolated hydrogel materials.13,14 The dynamic and reversible underlying supramolecular interactions typically lead to emergent properties in the resulting materials of shear-thinning flow, relaxation under strain, and self-healing following mechanical perturbation or cutting. In this body of work, recognition motifs from cyclodextrin macrocycles have been the most commonly © XXXX American Chemical Society

Received: December 19, 2018 Accepted: January 30, 2019

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DOI: 10.1021/acsami.8b22151 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Letter

ACS Applied Materials & Interfaces

Figure 1. (a) Schematic illustrating the use of host−guest physical cross-links to prepare hydrogel networks from 8-arm macromers bearing complementary host and guest motifs, with complexes of low affinity (low Keq) exhibiting rapid exchange of cross-links, whereas those of high affinity (high Keq) form stable and slowly exchanging cross-links. (b) An 8-arm PEG macromer (PEG8a-CB[7]) was used as the building block for these hydrogels, and modified completely with CB[7] macrocycles through “click” chemistry. (c) 1H NMR confirmed complete modification of the macromer with CB[7], using p-xylylenediamine (protons ‘h’ and ‘i’) as a guest probe to verify CB[7] content.

xylylenediamine (PEG8a-diN-Xyl), with its binding measured at Keq of 1.3 × 109 M−1. To increase affinity, another macromer was modified with O-linked adamantane (PEG8a-O-Ada) and its binding affinity measured at Keq of 2.6 × 1010 M−1. Finally, a high-affinity macromer was synthesized with an N-linked adamantane (PEG8a-N-Ada), and its binding affinity measured at Keq of 5.4 × 1012 M−1. Hydrogels resulting from host−guest physical cross-linking by mixing each of these five guest macromers with PEG8a-CB[7] will henceforth be abbreviated according to the magnitude of their Keq values: E7, E8, E9, E10, or E12. Upon stoichiometric mixing (1:1 ratio of guest:CB[7]) of PEG8a-CB[7] with the five guest-modified macromers at 5% (w/v) total solids in water, a self-supporting hydrogel formed in all cases (Figure 2c). Dynamic oscillating rheology at 2% strain was then performed on each hydrogel to assess viscoelastic properties. In this study, Keq values for each guest translated to a shift in crossover between the storage modulus (G′) and loss modulus (G′′), a point known as the bulk relaxation rate. Hydrogel E7, E10, and E12 did not have G′− G′′ crossover within the observed frequency range. Specifically, the expected E7 crossover would occur at a frequency beyond the point where measurements break down due to inertia (>300 rad/s), while the expected G′−G′′ crossover frequency of E10 and E12 are below reasonable time constraints for experiments ( 0.5) had release governed by anomalous mechanisms (e.g., swelling and erosion) instead of pure diffusion. The impact of cross-link affinity was also explored in the context of applying these hydrogels as injectable biomaterials. For these studies, E7 hydrogels were compared to E10 hydrogels, as this was the maximum range possible in order to explore the impact of affinity with materials that reform into D

DOI: 10.1021/acsami.8b22151 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Letter

ACS Applied Materials & Interfaces

Figure 4. (a) Release over time of encapsulated 70 kDa FITC-dextran from E9, E10, and E12 hydrogels; data was fit to the Korsmeyer-Peppas equation and the fitting parameters for each hydrogel are shown. E9 data at 144 h was excluded from fitting as hydrogels had dissipated completely by this time. (b) Representative images from the release studies illustrating hydrogel erosion and macromolecule release from E9, E10, and E12 hydrogels. (c) H&E histology over 30 days for E7 and E10 hydrogels injected subcutaneously into mice, with the hydrogel outlined in blue dots on the underside of the skin. A corresponding photograph of one sample at 30 days is included to show the relative hydrogel volume remaining for each sample with the hydrogel outlined in white.

Importantly, even though these hydrogels were not infiltrated or cleared, there was no sign of the typical fibrotic foreign body reaction seen for many nondegradable materials. Taken together, more dynamic host−guest cross-linking in these materials corresponded to more extensive cell infiltration and accelerated hydrogel clearance, whereas materials with slowly dynamic cross-links primarily excluded infiltrating cells and were not substantially cleared from the implant bed over 30 days of implantation. The impact of affinity on the network properties of supramolecular hydrogels is of great interest, yet the vast majority of supramolecular interactions explored to date do not enable the study of a broad range of affinity, nor study of particularly high affinity interactions. The remarkable range of affinities possible from CB[7] recognition of different guest chemistries has been leveraged here to achieve hydrogels with nearly identical composition and network topology, yet with dynamic properties spanning over 5 orders of magnitude. The range of dynamic relaxation rates achieved by this approach surpasses that reported for methods to create tunably dynamic polymeric (∼1−2 orders of magnitude)9,30 and recombinant (∼4 orders of magnitude)10 materials. Altering affinity, and by extension relaxation rates, furthermore leads to interesting properties in the application of these materials. Specifically, controlled macromolecular release, cell infiltration, and hydrogel clearance are all significantly slowed as a result of increased affinity and slower cross-link dynamics. Further

continuous bulk hydrogels following injection. Mice were injected subcutaneously with both E7 and E10 hydrogels, one on each side of the dorsal flank. The skin and implant bed were examined serially by necropsy, and tissue was harvested for histological assessment (Figure 4c). Both E7 and E10 hydrogels persisted in the tissue for the duration of the 30 day study, though over time the apparent volume of E7 hydrogels decreased. The E7 hydrogel also became increasingly opaque, suggesting cellular infiltration. The apparent volume of the E10 hydrogel did not change noticeably with implant time, and the hydrogel remained transparent for the full 30 day study. Hydrogels were then analyzed by histology. Cell infiltration into E7 hydrogel began by the first time evaluated, 5 days, and by 10 days the hydrogel was completely infiltrated with cells. The cell-infiltrated gel bed decreased in volume over the remainder of the study. During the early stages following implant, the gel was primarily infiltrated with neutrophils. At later times, the fraction of macrophages within the hydrogel increased and many of these cells were found to have internalized portions of the E7 hydrogel. The E10 hydrogel, in contrast, excluded cells at early times; neutrophils were found at very high density at the gel margins at 5 days, indicating mild inflammation of the implant bed. By 10 days, signs of inflammation subsided. The E10 hydrogel had a much larger volume apparent by histology, and remained completely uninfiltrated by cells until 30 days when the margins showed signs of some infiltration into the hydrogel network. E

DOI: 10.1021/acsami.8b22151 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Letter

ACS Applied Materials & Interfaces

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studies using these materials, or more generally this design approach, to create surrogate ECM materials may be able to elucidate the importance of network dynamics on cell behavior and stem cell fate. As such, this method to create dynamic hydrogel materials offers a useful tool in the pursuit of biomimetic and bioinspired synthetic materials.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.8b22151.

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Synthetic and experimental procedures, characterization data, supplemental data (PDF)

AUTHOR INFORMATION

Corresponding Author

*Email: [email protected]. ORCID

Matthew J. Webber: 0000-0003-3111-6228 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS M.J.W. acknowledges funding support from the University of Notre Dame through the “Advancing our Vision” initiative. The authors are grateful to the ND Energy Materials Characterization Facility for use of the rheometer and to the Notre Dame Integrated Imaging Facility for histology services.

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REFERENCES

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DOI: 10.1021/acsami.8b22151 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX