Secondary Photocrosslinking of Click Hydrogels To Probe Myoblast

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Secondary photocrosslinking of click hydrogels to probe myoblast mechanotransduction in three dimensions Tobin E. Brown, Jason S. Silver, Brady T. Worrell, Ian A. Marozas, F. Max Yavitt, Kemal Arda Günay, Christopher N. Bowman, and Kristi S. Anseth J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b07551 • Publication Date (Web): 05 Sep 2018 Downloaded from http://pubs.acs.org on September 5, 2018

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Journal of the American Chemical Society

Secondary photocrosslinking of click hydrogels to probe myoblast mechanotransduction in three dimensions Tobin E. Brown,1,2,‡ Jason S. Silver,1,2,‡ Brady T. Worrell,1 Ian A. Marozas,1,2 F. Max Yavitt,1,2 Kemal Arda Günay,1,2 Christopher N. Bowman,1,2,3 and Kristi S. Anseth*1,2 1

Department of Chemical and Biological Engineering, University of Colorado, Boulder, CO 80303 BioFrontiers Institute, University of Colorado, Boulder, CO 80303 3 Department of Materials Science and Engineering, University of Colorado, Boulder, CO 80303 2

ABSTRACT: Muscle cells sense the mechanical properties of their microenvironment, and these properties can change in response to injury or disease. Hydrogels with dynamic material properties can be used to study the effect of such varying mechanical signals. Here, we report the ability of azadibenzocyclooctyne (DBCO) to undergo a cytocompatible, photoinitiated crosslinking reaction. This reaction is exploited as a strategy for on-demand stiffening of three-dimensional cell scaffolds formed through an initial strain-promoted azide alkyne cycloaddition. Myoblasts encapsulated in these networks respond to increased matrix stiffness through decreased cell spreading and nuclear localization of Yes-associated protein 1 (YAP). However, when the photocrosslinking reaction is delayed to allow cell spreading, elongated myoblasts display increased YAP nuclear localization.

Synthetic hydrogels have a wide range of uses in regenerative medicine from drug delivery to scaffolding for threedimensional cell culture and transplantation.1 Many strategies exist to crosslink and functionalize these water-swollen networks, including physical, ionic, and covalent linkages. In the complex milieu associated with biological systems, bioorthogonal click reactions proceed efficiently and selectively to yield stable networks.2–4 Such hydrogels are highly tunable and can often be matched to the mechanical properties of various soft tissues. However, the in vivo mechanical environment is not static. For instance, muscle tissue stiffens in response to exercise,5 aging,6 and disease.7 In dystrophic mice, muscle has an increased stiffness (18 kPa) compared to healthy mice (12 kPa).7 Such changes in substrate elasticity affect the migratory phenotype and regenerative potential of muscle progenitor cells.6,8,9 Therefore, cytocompatible, on-demand stiffening hydrogels offer a powerful tool to explore stiffness-associated changes in the cellular behavior. While the strain-promoted azide-alkyne cycloaddition (SPAAC) reaction is routinely used to synthesize polymer networks,10 here, we report the ability of azadibenzocyclooctyne (DBCO) to undergo a photocrosslinking reaction. This dual reactivity is exploited to formulate hydrogels that can be stiffened on-demand to study the effect of dynamic stiffness associated with muscle pathology. Specifically, the SPAAC reaction is used to create a dual-cure network, where an initial reaction between DBCO- and azide-functionalized macromers forms a soft network, and subsequent photocrosslinking of pendant DBCO groups serves to further stiffen the network (Figure 1). This approach is analogous to (meth)acrylates reacting in both Michael-type additions and subsequent chain homopolymerization,11–13 but the favorable kinetics of the SPAAC reaction at physiological conditions make this platform useful for cell encapsulation. In the current work, C2C12 mouse myoblasts are encapsulated in three-

dimensional hydrogels and subjected to dynamic stiffening of the extracellular environment. These cells sense and respond through differential localization of the mechanoregulatory Yes-associated protein (YAP).

Figure 1. Strategy for consecutive reactions in a click hydrogel. Strain-promoted azide-alkyne cycloaddition occurs spontaneously upon mixing hydrogel precursors to form an initial network. Subsequent light illumination (10 mW cm-2, 365 nm, 120 s) with a photoinitiator (LAP, 2 mM) crosslinks the unreacted cyclooctyne functionalities.

To characterize the photocrosslinking reaction, hydrogels were formed through the SPAAC reaction with varying stoichiometries to yield networks that conditionally contained unreacted cyclooctynes, and network formation was monitored using a rheometer with a light curing accessory. After completion of the SPAAC reaction (10 min), hydrogels were exposed to light (2 min, 365 nm, 10 mW cm-2) in the presence of the photoinitiator LAP14 (2 mM). In hydrogels formed with an excess of cyclooctyne (relative to azide), this approach results in a rapid, stable increase in the storage modulus. Hydrogels with 2 or 3 molar equivalents of cyclooctyne stiffened after

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light exposure to 230% or 680% of their initial values, respectively (Figure 2a,b). Furthermore, compositions entirely lacking the azide macromer could be photocrosslinked to a final shear storage modulus of 3.7 kPa. In contrast, when stoichiometric or excess azide-functionalized macromer is included, the photostiffening reaction is abolished. The small increase observed in the stoichiometric networks is likely attributed to a low concentration of cyclooctyne remaining after 10 min of reaction or side reactions involving abstraction and termination. Consumption of pendant cyclooctynes was also visualized by exposing the off-stoichiometric SPAAC hydrogel to light through a striped photomask before placing the gel in a bath containing azide-functionalized AlexaFluor-594 (Figure 2c). The light-exposed regions of the gel show decreased fluorescence, indicating a depletion of azide-reactive cyclooctynes. The stiffened regions also displayed decreased swelling, which was visualized at the edge of the gel (Figure S1).

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in conjunction with the radical inhibitor 2,2,6,6tetramethylpiperidine-1-oxyl (TEMPO) stiffen only to a small degree over a 120 s exposure (Figure 3). Interestingly, these conditions were observed to stiffen on a much longer time scale (Figure S2), indicating that a small amount of sensitization or a radical-independent reaction occurs in the absence of LAP. Gel permeation chromatography using small molecules confirmed the presence of an oligomerized product when DBCO is photocrosslinked (Figure S3a), and a change in the visible light absorption spectrum, possibly suggesting the formation of a conjugated species (Figure S3b). Finally, photocrosslinking of small molecule DBCO under similar conditions led to the consumption of 40% of the starting material (Figure S3c). Possible mechnamisms may include oligomerization such as encountered during the preparation of activated cycloalkynes,15 or dimer/trimerization as observed in the case of difluorobenzocyclooctyne (DIFBO).16

Figure 3. Effect of photoinitiator concentration and the radical inhibitor TEMPO on the photopolymerization reaction. Hydrogels were formed using a 2:1 (DBCO:azide) stoichiometric ratio in the macromer solution and illuminated (10 mW cm-2, 365 nm, 120 s) after SPAAC gel formation. G’ values normalized to the plateau modulus after SPAAC.

Figure 2. Photocrosslinking of unreacted cyclooctynes leads to an increase in matrix stiffness in SPAAC hydrogels. a) Rheological traces of SPAAC gel evolution (600 s), followed by light exposure (10 mW cm-2, 365 nm, 120s), causing cleavage of the radical photoinitiator LAP. In hydrogels with unreacted DBCO (i.e. 3:1 or 2:1 excess), this led to an increase in storage modulus (G’) upon light exposure at 600 s. For 5wt% macromer solutions, hydrogels formed from PEG-8DBCO alone (green) when exposed to continuous irradiation after 600 s. b) Final shear storage moduli of hydrogels formed with various stoichiometries show that excess alkyne is necessary for the photocrosslinking (PC) reaction. Error bars represent standard deviation. c) A striped photomask was used to photopattern hydrogel stiffness, followed by reaction with AlexaFluor594-azide. Dark regions correspond to depleted cyclooctyne in the illuminated (stiffened) area. Scale bar 100 µm.

In support of the hypothesized photoinitiated reaction of DBCO, hydrogels exposed to light in the absence of LAP, or

The magnitude of network stiffening provided by secondary photocrosslinking accesses a relevant range for muscle biology. Adherent myoblasts have distinct mechanical responses to substrates with elastic moduli ~1-4 kPa (soft) compared to ~10 kPa (stiff)7,8,17 (i.e. shear storage modulus values of ~1 and 3.3 kPa assuming E=2(1+ν)G with a Poisson ratio, ν, of 0.5). Changes in ECM stiffness are relayed to the cell through cytoskeletal machinery in a process known as mechanotransduction, and the transcriptional coactivator Yes-associated protein-1 (YAP) plays a critical role in mechanosensing.18 In a stiffness-dependent manner, cytoskeletal tension causes dephosphorylation of YAP at Ser127 that allows nuclear shuttling.18 In the nucleus, YAP promotes early myogenesis and proliferation in myoblasts.19 In contrast, soft materials or confined cell spreading promote phosphorylation of YAP to sequester it in the cytoplasm. In 3D environments, however, other factors such as network degradability and crosslinking density combine to affect cell shape, which affects the cellular response.20 We therefore sought to determine the mechanotransduction response of encapsulated myoblasts to a dynamic change in ECM stiffness. For myoblast encapsulation, networks that allow cell spreading through integrin binding and enzyme-mediated degradation were employed.21 Therefore, an enzymaticallydegradable peptide was substituted as the azide macromer (Fig

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Journal of the American Chemical Society 4a), and an azide-functionalized adhesive peptide (azido-RGD, 1 mM), was included. Using this peptide crosslinker rather than PEG-diazide, a pre- and post-stiffening modulus of 710 and 5050 Pa, respectively, were achieved at a stoichiometry of 2:1 (Figure S4). One of the key advantages of the DBCO-azide bioorthogonal reaction is the ability to proceed selectively in the presence of cells and culture medium, resulting in high cytocompatibility. We first sought to determine whether the photocrosslinking reaction would be similarly benign. Other photoinitiated reactions, notably the radical polymerization of (meth)acrylates and the thiol-ene reaction, are routinely employed in the presence of cells with high viability.22,23 Indeed, 24 h post-encapsulation, cell viability was 87±4% in click hydrogels, and only slightly decreased (81±4%, p=0.10) in hydrogels stiffened following gelation, confirming the cytocompatibility of the photocrosslinking reaction (Figure 4b-d).

turally intact (Figure S5). Cell-mediated matrix degradation has been shown to be closely linked to 3D mechanotransduction and cell behavior.24 These results for C2C12 myoblasts are similar to those seen with mesenchymal stem cells (MSCs) and heart valve fibroblasts encapsulated in hydrogels that were stiffened. MSCs in stiff hydrogels display rounded morphology, tendency towards adipogenesis over osteogenesis,24 and decreased YAP nuclear localization,20 whereas valve fibroblasts show decreased activation to their myofibroblast phenotype.25 This behavior is contrary to what has historically been observed in 2D experiments, where stiff substrates cause increased spreading and mechanical activation of adherent cells, further highlighting the importance of dimensionality and cell shape in mechanotransduction. In an effort to investigate the effect that cell shape might have on 3D mechanotransduction of muscle myoblasts, photostiffening was performed at a delayed time point (day 7), which led to a similar magnitude of stiffening (Figure S6). By day 7, myoblasts in soft SPAAC hydrogels with peptide crosslinks have already spread throughout the gel via proteasemediated degradation of the surrounding matrix. Interestingly, stiffening the matrix around these cells through the DBCO photocrosslinking causes an increase in nuclear localization of YAP, notably even higher than those maintained in stiff networks from day 1 (Figure 5d). This result implies that, as opposed to rounded cells, spread myoblasts are able to sense and respond to the increased matrix stiffness, and maintain a spread morphology and nuclear YAP reminiscent of myoblasts in soft networks. In previous findings with MSCs, decreased matrix degradability promoted by photostiffening led to decreased mechanical activation.24 Therefore, different modes of environmental sensing may exist and be dominant depending on the cell type.

Figure 4. Viability of myoblasts encapsulated in PEG-peptide hydrogels containing 1 mM RGD at 2:1 (alkyne:azide) stoichiometry. a) Structure of the enzyme-degradable azide crosslinker. b-c) Calcein-AM/ethidium homodimer staining for living (green) and dead (red) cells in SPAAC only and photocrosslinked (SPAAC+PC) hydrogels. d) Quantification of myoblast viability 24 h post encapsulation, p=0.10. Scale bars 100 µm.

The DBCO photocrosslinking and resulting hydrogel stiffening caused a marked change in the morphology and nuclear localization of YAP of encapsulated cells (Figure 5). In the baseline (710 Pa) hydrogels without light exposure, myoblasts were able to spread in 3D, decreasing cell circularity (Figure 5a), and the YAP nuclear-to-cytosolic ratio increased from 1.47±0.05 on day 3 to 1.70±0.18 on day 15 as cells remodeled their local microenvironment (Figure 5b). In contrast, myoblasts in hydrogels that were stiffened to 5 kPa after 24 h displayed a rounded morphology and a decreasing YAP nuclear to cytosolic ratio of 1.48±0.07 on day 3 and 1.17±0.03 on day 15. These results indicate that myoblasts in softer gels are more mechanically activated than their counterparts in stiff gels, likely due to the ability of cells to degrade and spread within the softer hydrogel.24 We also hypothesized that the cell-mediated degradability would be hindered by introducing non-degradable crosslinks into the network. Indeed, upon treatment with collagenase (2 mg ml-1) for 24 h, the peptide crosslinked SPAAC hydrogels (i.e., non-stiffened through photocrosslinking of DBCO) were completely dissolved, whereas the stiffened hydrogels swelled, but remained struc-

Figure 5. Spreading and mechanotransduction of myoblasts in dynamic 3D environments. Hydrogels were stiffened by photocrosslinking excess cyclooctynes on day 1 or day 7 of the experiment. a) Shape of cells encapsulated in PEG-peptide SPAAC hydrogels after 15 days, visualized with CellMask Green. b) Cell shape quantified through circularity, with values closer to unity corresponding to rounder cells. c) Immunofluorescence of encapsulated myoblasts on day 15 reveals trends in subcellular YAP localization for cells in soft gels, or matrices stiffened on day 1 or day 7. d) Quantification of YAP nuclear:cytoplasmic intensity ratio. Scale bars 10 µm.

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We have shown photocrosslinking of DBCO can be used in a biological setting, providing spatiotemporal control over mechanics in SPAAC networks. Notably, photo-activatable cyclooctynes26 can be used in a conceptually similar manner; however, there is added synthetic complexity to these molecules. Moreover, along with the radical-mediated27 and nucleophilic28 reactions of thiols with cyclooctynes, this reaction adds to the list of azide-independent reactions of cyclooctynes. Therefore, care must be taken when performing radical reactions in the presence of these functionalities, but selected conditions can exploit secondary reactions to impart unique material properties. While the exact mechanisms that permit rapid, radical mediated crosslinking of cyclooctynes are not known and currently under investigation, the reaction offers a cytocompatible strategy to provide dynamic control over click hydrogels. We have employed this reaction as a means to alter the 3D microenvironment of myoblasts to mimic the physical stiffening associated with muscle disease and aging. Upon stiffening, cells embedded in 3D fail to adopt a spread morphology and display decreased nuclear localization of YAP relative to their counterparts in SPAAC control gels. However, by delaying the secondary photocrosslinking of DBCO for 7 days, spread myoblasts are able to sense the increase in matrix elasticity and respond through nuclear localization of YAP.

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ASSOCIATED CONTENT Supporting Information (13)

Experimental details and Figures S1-S6.

AUTHOR INFORMATION Corresponding Author

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* Email: [email protected]

Author Contributions All authors have given approval to the final version of the manuscript. ‡T.E.B. and J.S.S. contributed equally to this work.

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Funding Sources

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Funding for this work was provided by the NSF DMR (1408955). The authors also gratefully acknowledge fellowship support from the NSF GFRP (T.E.B and I.A.M), the NIH/CU Biophysics training program (T32 GM-065103) (T.E.B.), the CU MSTP (NIH T32 GM008497-26) (J.S.S) and the Arnold and Mabel Beckman Postdoctoral Fellowship (B.T.W). Image analysis was performed at the BioFrontiers Advanced Light Microscopy Core (NIH 1S10RR026680-01A).

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ACKNOWLEDGMENTS The authors would like to thank Dr. Sudheendran Mavila for assistance with GPC and Dr. Alex Kislukhin for helpful discussions.

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