Mechanochemical Reaction Cascade for Sensitive Detection of

Nov 13, 2014 - ... of poly(olefin sulfone). Xuewen Guo , Xin Liu , Hongying Su , Shaoyun Shan , Qingming Jia. Polymer International 2017 66 (11), 1530...
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Mechanochemical Reaction Cascade for Sensitive Detection of Covalent Bond Breakage in Hydrogels Kirsten R. Fitch and Andrew P. Goodwin* Department of Chemical and Biological Engineering, University of Colorado Boulder, 3415 Colorado Avenue, 596 UCB, Boulder, Colorado 80309, United States S Supporting Information *

ABSTRACT: A novel strategy is reported for sensing chemical bond breakage in hydrogels at low levels of mechanical stress using a mechanochemical reaction cascade to generate fluorescence. Hydrogels are promising substrates and frameworks for cell growth and tissue engineering, particularly for cardiovascular repair and cartilage replacement. For these applications, it is important to maintain careful control over gel mechanical properties so that these hydrogels not only match the properties of the desired tissue for replacement but also retain their integrity for extended periods. Since the failure of hydrogels begins with the breakage of cross-links within the structure, methods are needed to sense these initial events for monitoring the performance of implants. In this work, it was hypothesized that nonspecific covalent bond breakage would produce radicals that would react with water to produce reactive oxygen species, which in turn could activate fluorophores sensitive to these. A series of multiarm poly(ethylene glycol) hydrogels were synthesized with a variety of cross-links of different bond dissociation energies. It was found that gels loaded with the masked fluorophore 3′-(p-aminophenyl) fluorescein became fluorescent during compression, even with as little as 5 kPa of pressure. The effect of compression on fluorescence activation was found to depend primarily on the strength of the cross-linking functional group. Future studies include utilizing this system to image mechanical variability in heterogeneous gel structures.

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under tension,20,26 ultrasound-induced shearing of macromolecules in solution,27−29 and bulk mechanical activation of polymer materials -- have shown that macromolecular shearing tends to occur at the bond with the smallest bond dissociation energy (BDE).9,10 Examples include S−S (∼60 kcal mol−1) and C−S (∼70 kcal mol−1) bonds through disulfide and sulfide groups, respectively, rather than C−C (83 kcal mol−1) or C−O (85 kcal mol−1) bonds.19,30 The breakage of covalent bonds generally results in the formation of radicals,17,29 which is included in the definition of BDE, although pericyclic reactions are a notable exception.9,11,14,22,24,28,31 Once the radicals are formed, they tend to react with their surroundings to seek a lower energy state by forming new covalent bonds.29 For example, protein microspheres have been formed by ultrasound-induced scrambling of the disulfide bonds between albumin chains,32 and sonication of poly(ethylene glycol) dimers containing a diazo bridging group led to alcohol adducts from ambient water.29 In polymer materials, this process was studied closely by Baytekin and Grzybowski, who discovered that if poly(dimethylsiloxane) (PDMS) was contacted with water and compressed, enough hydrogen peroxide could be

ydrogels have shown promise as substrates and frameworks for cell growth and tissue engineering.1,2 In particular, cell matrices for cardiovascular repair and cartilage replacement require careful tuning of gel structure to both mimic the mechanical properties of the target tissue and remain viable for the duration of the desired repair or replacement.1−4 For cross-linked gels, like other elastic polymers, failure mechanisms generally begin with small amounts of covalent bond breakage followed by irreversible deformation.5,6 In recent years, significant effort has been devoted to both theoretical and empirical study of how covalent bonds in polymers and polymer materials react or break under sufficient mechanical force.6−20 In some cases, mechanochemically sensitive functional groups have been designed to produce an optical signal in a polymer material to signify strain or failure.16,21−25 However, despite the groundbreaking work in this area, these latter methods not only require the implantation of specialized functional groups that may affect the mechanical properties of the polymer but also are only able to sense activation of those specific groups, as opposed to any bond in the polymer network. Moreover, these chemical structures have, to our knowledge, only been tested in more mechanically rigid polymers, such as polyacrylates or epoxies, and thus would require greater mechanical force to induce activation than what would be experienced by a typical hydrogel implant. Previous studies of mechanically induced polymer fracture -including single-molecule studies of macromolecular dynamics © XXXX American Chemical Society

Received: September 4, 2014 Revised: November 11, 2014

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APF and HPF Activation Tests. For most studies, gels were compressed to and held at 10, 20, and 30% strain for 10 min. For the repetition studies, the gels were compressed at increasing strain from 0 to 50% at 1% min−1. A drop of additional dye solution was added to the gel each run to prevent the gel from drying out during compression. Gels were imaged before and after each compression point with the VersaDoc in both fluorescence and nonfluorescence mode at 80× magnification. Image Analysis. ImageJ was used to analyze the fluorescent images of each gel in order to determine the fluorescence intensity. The mean intensity was divided by exposure time to obtain a normalized fluorescence emission value. The linear relationship between intensity and exposure was verified with a calibration curve of uranine-loaded gels imaged at 1−10 s exposure. In each case, background fluorescence was negligible. Analysis of Relationship between Fluorescence and Stress. To normalize APF fluorescence to expected changes as a result of compression, APF fluorescence emission was divided by fluorescence emission from uranine-loaded gels for each gel composition at each compression level. Next, a linear regression for data points representing 10−30% strain was applied using Microsoft Excel, and standard errors were obtained from these. Fenton Reaction and Analysis of Effect of FeCl2 on Activation. Five 1 mL solutions of 10 μM APF were made in 50 mM Tris buffer, pH 7.4, with 1 mM H2O2 and 0, 10 μM, 100 μM, 1 mM, or 10 mM FeCl2. The fluorescence intensity was then measured on the fluorimeter using a 480 nm excitation wavelength and an emission wavelength range of 500−560 nm, with a 2 nm slit width. The voltage was averaged over three scans. As a control, this was repeated with five 1 mL solutions of 0.06 μM uranine with the same concentrations of H2O2 and FeCl2. The pH of each sample was checked with litmus paper immediately after measuring the fluorescence. To test for hydrogen peroxide formation from bond breakage, gels were loaded with 10 μM APF in 50 mM Tris Base, pH 7.4, and compressed to 10, 20, and 30% strain for 10 min. Before each compression 100 μL of the dye solution was added to the gel to prevent drying. After the 30% strain test, 80 μL of 1 mM FeCl2 was added to the dye solution (a final approximate concentration of 100 μM FeCl2). The gels were imaged via VersaDoc in fluorescence and nonfluorescence mode at 80× magnification after soaking in the dye/ iron solution for the time indicated.

produced to react with a number of different substrates, including masked fluorophores.33 Here we report a novel mechanochemical technique for the detection of generalized bond breakage in hydrogels using simple fluorescence measurements. This technique can detect bond breakage with less than 5 kPa of compression, even when the mechanical properties appear unchanged by simple mechanical testing. Finally, as this technique appears to be general for poly(ethylene glycol)-based hydrogels, we detail the relationship between the material and chemical structure of the gels and their propensity toward bond degradation.

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EXPERIMENTAL SECTION

Materials and Instrumentation. Fluorescein disodium salt (uranine) and iron(II) chloride (FeCl2) were purchased from Acros Organics. All functionalized 4-arm poly(ethylene glycol) (PEG) reagents were purchased from Laysan Bio, Inc. 5 mM solutions of 3′-(p-aminophenyl) fluorescein (APF) and 3′-(p-hydroxyphenyl) fluorescein (HPF) in N,N-dimethylformamide (DMF) were purchased from Life Technologies and further diluted to 100 μM in DMF (Sigma-Aldrich). Sodium hydroxide (NaOH) pellets and a 30% aqueous solution of hydrogen peroxide (H2O2) were both purchased from Macron Fine Chemicals. Hydrochloric acid (HCl), Tris Base, and 10X phosphate buffered saline (PBS) were purchased from Fisher Scientific. The 10X PBS was diluted to 1X using Millipore water prior to use. A 6 mg mL−1 aqueous solution of Irgacure 2959 (I2959) was donated by the Bryant lab at the University of Colorado, Boulder. Compressive testing was performed on an MTS Synergie 100, model LCCA-122-96 with load cell SMT1-10N-166 and a tolerance of 1 mN. All images were taken with a VersaDoc MP 4000 imaging system from Bio-Rad. Fenton experiments were performed with a Photon Technology International fluorimeter with lamp power supply model LPS-220B; motor driver model MD-5020; and shutter control model SC-500. Gel Synthesis. The PEG precursors for all gels were either 4armPEG(10 kDa)-tetrathiol (PEGSH), 4arm-PEG(10 kDa)-tetraacrylate (PEGTA), or both. In a typical procedure, the gelation components (Tables S1−S3) were mixed 1:1 at the appropriate polymer wt % briefly in a centrifuge tube and transferred in 70 μL aliquots into 1 mL syringes. Thiol−acrylate and disulfide gels were wrapped in Parafilm and allowed to react overnight. Acrylate gels were polymerized through exposure to 20 W of 350 nm ultraviolet light (Sankyo Denki, model F20T10BLB) for 10 min. Aspect ratios for all gels were near unity to ensure accurate mechanical testing. After polymerization, the gels were washed by soaking 3 times in 10 mL of water for at least 8 h per soak. They were then transferred to either 1 mL of 10 μM APF, 10 μM HPF, or 0.06 μM uranine in 1X PBS and stored overnight in the dark. All DMF was removed from the APF and HPF by rotary evaporation followed by storage under high vacuum (or by high vacuum alone) before addition to PBS. Compression Testing. First, the compressive modulus, E, was determined by ramping the strain from 0 to 10% at a rate of 1% min−1 on an MTS machine. The modulus could be calculated using the compressive stress at 10% strain, τ, and the ratio of the gel height before and after compression, α:18,34

τ = E(α − α −2)

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RESULTS We hypothesized that any radicals formed through covalent bond breakage within the hydrogel itself would first react with the surrounding water to produce radical adducts.29 Reaction with water should produce some fraction of reactive oxygen species (ROS), such as hydrogen peroxide, hydroxyl radical, and hypochlorite anion. These latter two species can activate masked fluorophores such as 3′-(p-aminophenyl) fluorescein (APF, Figure 1).36 Poly(ethylene glycol)-derived hydrogels were chosen as a test substrate because their synthesis is wellestablished, their overall mechanical properties can be easily tuned, and they can be cross-linked with a variety of functional groups of varying bond strength.18 The weakest cross-linkers in this study were disulfides (BDE ∼ 60 kcal mol−1), formed by oxidation of 4arm-PEG-thiols with hydrogen peroxide. The next weakest were adducts of 4arm-PEG-thiol and 4arm-PEGacrylate (hereby thiol−acrylate, C−S BDE ∼ 70 kcal mol−1), which polymerize through base-catalyzed Michael addition. Finally, the strongest gels were formed through photopolymerization of 4arm-PEG-acrylate, a common formulation for hydrogels. The C−C and C−O bonds in this polymer are of similar strength, with BDE ∼ 84 kcal mol−1 (specific formulations are listed in Table S1−3). In a typical hydrogelation procedure, a mixture of hydrogel precursor was allowed to cure in a cylindrical mold either overnight or

(1)

Calipers were used to measure gel dimensions. For higher strains, the stress−strain relationship became nonlinear and instead followed Jcurve behavior. This was approximately modeled by the following equation, where n represents an exponent reflecting non-Hookean behavior:35

τ = E H(α − α −2)n

(2) −1

The strain was increased at 1% min to 80% or until the MTS reached its maximum load and fit the resulting stress−strain data to the linearized form of this equation in order to find the modulus of the corresponding Hookean material. B

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Figure 2. (a) Compressive moduli of hydrogels vs weight % loading for disulfide (blue diamonds), thiol−acrylate (red squares), and acrylate (green triangles) cross-linkers. Error bars represent standard error and are smaller than the size of the points in the chart. (b) Stress−strain curves for 10 wt % thiol−acrylate (blue) and 20 wt % thiol−acrylate (red) gels for 0−50% strain. The log−log plot shows linear dependence for most strains, allowing use of eq 2. Linear fits are shown in Figure S1.

applied to 10% and 20% thiol−acrylate hydrogels (Figure 2b). As these data showed a good linear fit up through 50% strain, the amount of degradation in the gel was small. Next, the effect of compression on APF activation was measured. A series of APF-loaded hydrogels was prepared, and each was compressed at 10, 20, and 30% strain for 10 min, with the fluorescence of each gel recorded after each compression test and plotted as a function of compressive strain (Figure 3). Figure 1. (a) Proposed mechanism of fluorescence activation by mechanical stress. Mechanical force on a generic covalent bond (R-XX-R) creates two radicals of the form R-X•. These then react with the surrounding water to produce ROS such as hydroxyl radical, among other adducts. Hydroxyl radical can then react with the APF, producing the green-emissive fluorescein (a similar reaction occurs with hypochlorite anion). (b) Structure of 4arm-PEG-SH. (c) Structure of 4arm-PEG-acrylate. (d-f) Specific cross-linking units of (d) disulfide (S−S), (e) thiol−acrylate (C−S), and (f) acrylate (C−C) formed from 4arm-PEG derivatives.

through exposure to UV light. The gels were washed and swelled several times in plain water, followed by transfer to 1X PBS to shrink the gels and control for pH. During this step, 10 μM APF was loaded into some gels for detecting bond breakage, while in other gels 0.06 μM uranine was added to control for any fluorescence affects resulting from compression itself. Initial mechanical testing showed that disulfide and thiol− acrylate gels loaded with 10−25 wt % PEG had compressive moduli of 5−40 kPa (Figure 2a, Table S4). To produce photopolymerized acrylate gels with similar moduli, polymer loadings were decreased to 5.0−12.5 wt % to compensate for the additional stiffness caused by the reduced distance between PEG branches.6,15 As expected, the dependence of the compressive modulus on weight % changed significantly as the cross-linking method changed from step-growth (disulfide, thiol−acrylate) to chain-growth (acrylate only), but the relationship between polymer loading and the compressive modulus remained consistent within each formulation (Figure 2a).37 The stress−strain curves of the gels exhibited a “J-shape” (concave upward), which is characteristic of hydrogels and other elastic polymer materials, and thus they do not exhibit Hookean mechanics.35 This dependence may be corrected by applying an exponential factor n (n = 1 for Hookean materials), for which a value EH may be calculated after linearization (eq 2). A linear fit of the log−log plot reveals an approximate Hookean modulus for the gels. As examples, this analysis was

Figure 3. (a-c) Fluorescence emission of APF for different 4arm-PEG loadings as a function of compressive strain. (a) Disulfide cross-linker. Red square: 10 wt %; blue diamond: 15 wt %; green triangle: 20 wt %; purple circle: 25 wt %. (b) Thiol−acrylate cross-linker. Red square: 10 wt %; blue diamond: 15 wt %; green triangle: 20 wt %; purple circle: 25 wt %. (c) Acrylate. Red square: 5 wt %; blue diamond: 7.5 wt %; green triangle: 10 wt %; purple circle: 12.5 wt %. Each data point represents at least two averaged data points; error bars represent standard error. (d-f) Fluorescence micrographs of (d) 10 wt % disulfide, (e) 10 wt % thiol−acrylate, and 5 wt % acrylate gels compressed from 0 to 30% strain. (g) APF fluorescence emission for a 20 wt % thiol−acrylate gel compressed from 0 to 50% strain three separate times, after background subtraction of APF from an uncompressed gel imaged under same conditions.

As the gel was compressed, the total fluorescence from the gel increased with a nonlinear dependence. Compression of the gels did not result in a significant change in pH from initial pH of 7.4 (data not shown). While hydroxyl radicals are known to activate APF, other ROS may be present as well. Our initial hypothesis was that hydroxyl radical would be formed as a C

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of similar strength, so U can be approximated at this value. This effect has been observed by others in 4arm PEG hydrogels with strong (C−O or C−C) cross-linkers.38 Thus, the fracture energy of a hydrogel based on strong bonds, or the amount of energy required to fracture a covalent bond in the chain, should scale with the density of the polymer ρ. To analyze acrylate gels via a Lake-Thomas model, it was necessary to take into account any changes in fluorescence emission as a result of the compression process itself. For this, the measured emission from each APF-loaded gel was divided by the measured emission from gels loaded with 0.06 μM fluorescein for each composition at each compression level (Figure S5). This normalized fluorescence (ϕ) was then plotted against stress (σ) instead of strain (Figure 4a) as measured by

result of the bond breaking process (Figure 1a), but as stated in the Introduction, Baytekin and Grzybowski showed that hydrogen peroxide formed when polymer materials were compressed.33 Hydrogen peroxide can be converted to hydroxyl radical using iron(II) salts via the Fenton reaction. Having determined that 100 μM FeCl2 could activate APF in the presence of hydrogen peroxide without sacrificing significant fluorescein emission (Figure S2), 10 wt % disulfide gels loaded with either APF or fluorescein were compressed to 30% strain and then incubated with 100 μM FeCl2. As the FeCl2 diffused into the gel the fluorescence of the APF increased while the fluorescein-loaded gel decreased, indicating the presence of hydrogen peroxide in the compressed gel (Figure S3). Since APF can also react with hypochlorite, an additional experiment was conducted comparing the activation of APF against HPF, a similar masked fluorophore with little reactivity to hypochlorite, in a compressed 10 wt % disulfide gel.36 The HPF exhibited far less activation than the APF, indicating that hypochlorite was a significant contributor to the produced ROS (Figure S4). The plots of APF fluorescence vs strain show an initial jump in fluorescence from 0 to 10% strain, followed by a shallower increase from 10 to 30% strain. This initial increase is most likely due to selective activation of bonds at areas with less polymer reinforcement, such as at the edge of the gel or at heterogeneities within the gel environment, where there is less bulk polymer to reinforce the chemical bonds. However, owing to the diffusion of free APF through the gel, correlating APF activation to specific heterogeneities within the gel proved to be difficult; this will be the focus of future studies. For each of the formulations, the gel with the smallest wt % of polymer loading (10 wt % for disulfide and thiol−acrylate, 5 wt % for acrylate) gave the largest change in fluorescence as a function of compressive strain. For the other polymer loadings, the type of cross-linker had a more significant effect on the relationship between compressive strain and APF activation, with the disulfide cross-links showing the greatest level of APF activation, followed by thiol−acrylate, then acrylate (Figure 3ac). To further confirm the effect of mechanical stress on APF activation, a 20 wt % thiol−acrylate gel was compressed multiple times to 50% strain. The APF fluorescence increased with each compression cycle relative to an uncompressed gel (Figure 3g). This result shows that the APF does convert to fluorescein as a result of the gel compression process. Since the concentration of APF was only 10 μM, and the fluorescence emission is similar to that of fluorescein at 0.06 μM (Figure S5), a small fraction of the approximately 10−100 mM crosslinks present may be broken without apparent plastic deformation.

Figure 4. (a) Fluorescence emission of APF normalized to fluorescein (Φ) vs compressive stress (σ) for 4arm-PEG acrylate gels. Red square: 5 wt %; blue diamond: 7.5 wt %; green triangle: 10 wt %; purple circle: 12.5 wt %. Each data point represents at least two averaged data points; error bars represent standard error. (b) Slope of stress vs normalized fluorescence (kPa per arbitrary unit) vs polymer loading. Slope is expressed as Δσ/Δϕ. Error bars indicate standard error derived from linear regression; error bars for all but 12.5 wt % are smaller than the data point in the graph.

the MTS. In this treatment, the differences between the smallest (10 wt %) and the other polymer loadings are much more pronounced, as is the initial increase in APF fluorescence as a result of stress on the wall of the hydrogel. However, beyond this point the emission appears to increase with stress with linear dependence. As a way to describe the amount of stress required for breaking a bond in the gel, a linear fit was obtained at these latter data points (Figure S6) to obtain the change in compressive stress (σ) as a function of normalized fluorescence emission (ϕ), which is expressed as the slope Δσ/ Δϕ. The stress per bond breakage for 10−30% appears to scale linearly with polymer density, which is consistent with the Lake-Thomas model (Figure 4b); however, the small changes in fluorescence for the 12.5 wt % polymer lead to a large error so this cannot be confirmed based on the data obtained. However, the introduction of weaker bonds in the gel complicates this description. If the Lake-Thomas equation were expanded to consider each bond, the tearing energy would simply be the average of each bond dissociation energy applied over each of the bonds within a given fracture area. Instead, the fracture may be considered via mechanochemical analysis of each bond. A description of the behavior of a covalent bond under stress is provided by the Bell-Evans model, which follows the Arrhenius form

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DISCUSSION The establishment of this new tool for observing covalent bond breakage in polymers allows evaluation of two different approaches toward theoretical approach to this question. One of the first descriptions of mechanically induced covalent bond breakage was described by Lake and Thomas,15 who derived the following equation T0 = ρNU

k(F ) = k 0e

(3)

−(

Ea − F Δx ) kBT

(4)

where k(F) is the rate constant of dissociation as a function of applied force F, k0 is the pre-exponential factor (>1013 s),39 Ea is the activation energy (i.e., bond dissociation energy), Δx is the distance required to achieve dissociation, kB is Boltzmann’s constant, and T is temperature. Application of mechanical force

where fracture energy T0 is the product of the polymer density (loading) ρ, the number of monomer units between cross-links N, and the bond dissociation energy U. In the case of the photopolymerized acrylate gels, the C−O and C−C bonds are D

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across the polymer chain shrinks the activation barrier necessary for reaction and reduces the bond lifetime to that which can be measurably obtained within the 10 min of the experiment. When the same analysis as in Figure 4 is applied to the disulfide and thiol−acrylate gels, rather than the slope increasing with polymer loading it instead appears to reach a constant value that is far less than those of the acrylate gels (Figure 5). To provide a semiquantitative comparison, the

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ASSOCIATED CONTENT

S Supporting Information *

Specific hydrogel formulations, compressive moduli of gels, fluorescence emission from gels loaded with uranine, results of FeCl2 incubation with APF and uranine, results of HPF-loaded gels, and example of calculation of slope. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions

The manuscript was written through contributions of all authors. Funding

This research was supported by University of Colorado startup funds. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to thank Prof. Kristi Anseth for use of her Mechanical Testing System Synergy 100 and Prof. Stephanie Bryant for use of her Bio-Rad VersaDoc MP 4000 Imaging System and the photoinitiator Irgacure 2959. The authors would also like to thank Prof. Chris Bowman, Dr. Chelsea Kirschner, Mr. Kyle Kyburz, Mr. Chris Fenoli, Dr. Stacey Skaalure, and Prof. Jennifer Cha for helpful discussions and suggestions. This work was supported by start-up funds from the University of Colorado Boulder.

Figure 5. (a, b) Fluorescence emission of APF normalized to fluorescein (Φ) vs compressive stress (σ) for 4arm-PEG with different cross-linkers. Each data point represents at least two averaged data points; error bars represent standard error. (a) Disulfide cross-linker. Red square: 10 wt %; blue diamond: 15 wt %; green triangle: 20 wt %; purple circle: 25 wt %. (b) Thiol−acrylate cross-linker. Red square: 10 wt %; blue diamond: 15 wt %; green triangle: 20 wt %; purple circle: 25 wt %. (c) Slope of stress vs normalized fluorescence (kPa per arbitrary unit) vs polymer loading. Slope is expressed as Δσ/Δϕ. Error bars indicate standard error derived from linear regression. (d) Natural logarithm of normalized emission (ln(Δσ/Δϕ)) for 15 wt % disulfide, 15 wt % thiol−acrylate, and 7.5 wt % acrylate hydrogels.

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ABBREVIATIONS PEG, poly(ethylene glycol); APF, 3′-(p-aminophenyl) fluorescein; HPF, 3′-(p-hydroxyphenyl) fluorescein; PBS, phosphate buffered saline; BDE, bond dissociation energy; MTS, mechanical testing system

natural logarithm of the Bell-Evans equation was taken, and the natural log of the slope was plotted against the estimated bond dissociation energy (i.e., activation energy) of the weakest bond in the gel (Figure 5d). This analysis showed that the amount of APF fluorescence produced as a function of compression showed a clear dependence on cross-linker bond dissociation energy (Figure 5d).19,30 These findings, and the analysis presented above, imply that the mechanochemical breakage of bonds in the gel can be considered both from the standpoint of the polymer material (Lake-Thomas) and the individual bonds in the chain (Bell-Evans), depending on the applied force and bond strength. In conclusion, a novel technique was designed to sense for mechanically induced fracture of covalent bonds by taking advantage of the formation of ROS caused by homolytic cleavage of polymer chains to activate fluorescent molecules in a hydrogel. As test systems, hydrogels were synthesized using branched 4arm-PEG cross-linked with either disulfide, thiol− acrylate, or acrylate cross-linkers. For hydrogels with smaller moduli and polymer loading, APF was found to activate quite readily with compression less than 5 kPa. At higher molecular weight loading, the disulfide-cross-linked hydrogels were found to activate most readily, followed by thiol−acrylate, then acrylate. These findings were attributed to a dependence of mechanical degradation on bond dissociation energy within the gel through analysis via the Lake-Thomas and Bell-Evans model for covalent bond breakage in polymers. In future research, we plan to utilize this technique to perform 3-D imaging of mechanical variability in heterogeneous gel structures.

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