Pyrene-Based Macrocrosslinkers with Supramolecular Mechanochromism for Elastic Deformation Sensing in Hydrogel Networks

Excimer-containing polymers with supramolecular mecha- nochromism are an attractive and well-investigated class of mechanor-esponsive materials. However, only recently steps toward mechano- phore-like mechanochromic systems that are anchored within the par-ent polymer structure and that show defined optical transitions on the molecular scale have been reported. However, the multi-step syntheses of these constructs are tedious. Here we report the development of a series of pyrene-based macrocrosslinkers that display supramolecular mechanochromism and are readily synthesized from mostly commercial reagents. We incorporate the water-soluble macrocrosslinkers in hydrogel networks and demonstrate their reversible mechanochromic behav- ior in the elastic deformation regime.


Introduction
Pyrene is a well-established fluorophore 1 that is commonly used because of its ability to form excimers, long excitedstate lifetime, 2 high fluorescence quantum yield, and good photostability. 3 Excimer formation is a diffusion-controlled process that leads to a bathochromically shifted emission when a pyrene in the ground state binds to a pyrene in the excited state. 4 Hence, excimer emission strongly depends on the distance between the excimer-forming molecules rendering pyrene a popular distance-based probe in applications ranging from organic electronics 5 to sensors for temperature, 6 pH, 7 pressure, 8 small molecules, 9 protein conformation, 10 lipid structure, 11 or nucleic acid recognition. 12 The strong distance sensitivity of excimer systems is particularly attractive for the visualization of stress and strain in bulk polymer materials. [13][14][15] Weder and coworkers have pioneered early research in this field focusing on blending excimer-forming dyes in polymer films. 16 Yet, this has led to only a weak correlation between mechanical polymer deformation and the measured optical signal. While these systems hence have allowed the qualitative observation of force-induced effects, polymer-anchored mechanophorelike 17,18 systems have only recently been developed that enable the visualization of discrete force-induced molecular events despite the absence of a clear bond dissociation reaction. For example, emitter-quencher pairs have been bridged and anchored within rotaxanes [19][20][21][22] and cyclophanes, 23 and bond torsion with subsequent restructuring of conjugated systems has been demonstrated. [24][25][26][27] However, the multi-step syntheses of these constructs are tedious and occasionally low yielding. Solving this, Weder, Schrettl, and coworkers have developed a readily obtainable folded perylene diimide loop, 28 which shows strain-correlated mechanochromism in commodity polymers. 29 This has inspired us to investigate whether we could reduce the synthetic complexity of mechanochromic force sensors even further while simultaneously opening up new application fields. Therefore, we here report the development of a series of pyrene-based macrocrosslinkers (PyMCs) with supramolecular mechanochromism, which we incorporate into hydrogels formed with star-shaped poly(ethylene glycol) (sPEG; Figure 1). We achieve this by copolymerization of tetraethylene glycol methyl ether methacrylamide (TEGMEMAAm) and pyrene methacrylamide (PyMAAm) yielding watersoluble PyMC chains. We deliberately choose to incorporate PyMC into sPEG hydrogels due to their regular network structure leading to outstanding mechanical properties. Unlike randomly crosslinked networks, sPEG hydrogels evenly distribute applied mechanical loads on all bonds and dissipate mechanical energy by controlled uncoiling 30 of the network segments. [31][32][33][34][35] This renders any detection of force-in-duced events using optical force probes 36 based on covalent bond scission difficult and hence serves as an ideal proof-ofconcept application for our mechanochromic macrocrosslinkers. We show that PyMC endows the sPEG hydrogels with reversible mechanochromic behavior in the elastic deformation regime. While pyrene has been investigated as mechanochromic unit in polymers before, 37-39 the observed emission changes have been fairly small and the conception of distinct macrocrosslinkers and their application in sPEG hydrogels is unknown.

Synthesis and Optical Properties of Macrocrosslinkers and Hydrogels
The hydrophobic pyrene units were incorporated into water-soluble PyMC chains by reversible addition-fragmentation chain-transfer (RAFT) polymerization starting from a bifunctional chain transfer agent (CTA), the one-step synthesis of which we reported before. 40 The water-soluble polymers of several widely employed monomers, such as Nisopropylacrylamide (NIPAAm) or N-vinylcaprolactam (VCL), exhibit lower critical solution temperatures (LCSTs) at 32.0 and 33.5°C, respectively. Phase separation occurs above the LCST. 41 Since the copolymerization with hydrophobic monomers decreases the LCST (occasionally even below ambient temperature), 42 the desirable incorporation of high pyrene concentrations for strong fluorescence signals would have been unattainable with most common watersoluble polymers. Therefore, we synthesized copolymers of TEGMEMAAm and PyMAAm by statistical RAFT copolymerization (Scheme 1). We aimed at the incorporation of ca. 70 monomer units resulting in PyMCs with a contour length shorter than the commercial 10 kDa HS-PEG-SH employed as a co-crosslinker for hydrogel production. This was to ensure that PyMC would be extended first upon mechanically loading the hydrogel. The co-crosslinker was necessary to control the overall pyrene concentration in the hydrogels. The intrinsically high water-solubility of TEGMEMAAm was exploited to maintain a high water-solubility of the PyMCs even at high pyrene fractions, which ranged from 1 to 40 mol%. In the following text, the pyrene content of the PyMCs in mol% is denoted as subscript, e.g., PyMC 40 for 40 mol% pyrene. Since the LCST behavior is also concentration-dependent, 43 the macrocrosslinkers started to exhibit turbidity at high PyMC concentrations from PyMC 40 upwards. Therefore, PyMC 32 was identified as an ideal compromise between solubility and a strong fluorescence signal and hence subsequently used for further investigation and the synthesis of the hydrogels. Expectedly, the ratio of the excimer emission band at 480 nm and the third vibronic monomer emission band at 396 nm (I E /I M3 ) increased with increasing PyMC 32 concentration ( Figure 2). In earlier work we found that this was caused by either increasing interchain excimer formation dominating over intrachain excimer formation or by increasing interchain hydrodynamic compression due to passing the overlap concentration. 40 While PyMC 40 showed high excimer emission in a broad concentration regime, control PyMC 01 bearing exactly one pyrene unit per chain showed no excimer emission at all (Scheme S1 and Figure S1). Hydrogels were then synthesized using four-arm vinyl sulfone-terminated sPEG (M n = 10 kDa), co-crosslinker HS-PEG-SH (M n = 10 kDa), and PyMC 32 in H 2 O with Et 3 N ( Figure 1). 40 The PyMC 32 concentration was varied while the overall reactant composition was maintained constant by compensation with HS-PEG-SH to maintain identical mechanical properties of the hydrogels. The hydrogels were purified by swelling in a DMSO/THF mixture and subsequent replacement of the solvent with H 2 O. To obtain mechanoresponsive hydrogels that use the indicative I E /I M3 ratio as output signal, a suitable PyMC 32 concentration within the hydrogels must be identified ( Figure 3 and Table S1). At low PyMC 32 concentrations, I E /I M3 converged to a minimum and fluorescence measurements were dominated by noise due to the low pyrene content. With increasing PyMC 32 concentration, I E /I M3 also increased until the fluorescence spectra were dominated by the excimer emission band and I M3 was hardly measurable. For further characterization, we hence chose PyMC 32 concentrations c PyMC between 2.7 · 10 −5 and 1.4 · 10 −4 mol · L −1 to achieve a reasonable compromise between measurable I E /I M3 ratios and a high overall fluorescence intensity.

Photostability of Macrocrosslinkers and Hydrogels
Subsequently, we investigated the photostability of PyMC 32 and PyMC 32 -crosslinked hydrogels. Pristine pyrene is photostable and not prone to photobleaching. 44 However, others and we showed that pyrene esters undergo photoinduced solvolysis 40,45 leading to a rapidly decreasing I E and increasing I M featuring an isostilbic point. 4 We hypothesized that pyrene amides, as in the employed PyMAAm, would not show such behavior. Emission spectra taken over the course of the irradiation of aqueous PyMC 32 solutions revealed a decrease in I E (Figure 4a). However, this was significantly slower compared to the photoinduced solvolysis of pyrene esters and showed neither an increase in I M3 nor an isostilbic point ( Figure 4b). O 2 did not contribute to this process since similar experiments under a N 2 atmosphere showed comparable trends ( Figure S2). While the origin of this effect remains unclear, we speculate that it may result from the  Table S1.  complex interactions of pyrene in hydrophilic environments. Excimer formation is usually the result of diffusioncontrolled interaction of a pyrene in the ground state with a pyrene in the excited state. 46 Pyrene was shown to form preassociated excimers in aqueous environments. 44 The excitation of such preassociates may lead to a separation of the ground and excited state pyrenes, which may cause the loss of excimer emission. However, we found that this effect was very slow in hydrogels and hence negligible in the investigated timeframes for mechanical analysis ( Figure 5).

Influence of the Degree of Swelling on Hydrogel Emission
In addition to the PyMC concentration, we investigated the influence of the degree of swelling (d s ) on the I E /I M3 ratio, which was calculated from the masses of the dry polymer (m d ) and of added H 2 O (m w ) using Equation 1. Equilibrium swelling was reached when the sample mass was constant for 3 d upon immersion in excess H 2 O. We used a hydrogel with a PyMC 32 mass concentration of ρ PyMC = 2.08 · 10 −4 g · L −1 ( Figure 6). Only little variation of I E /I M3 upon varying d s occurred in the investigated swelling regime from 1000 to 8000 %. However, we found in earlier work that considerably dried hydrogels with a very low d s reached a threshold value where I E /I M3 decreased steeply to almost no excimer emission at all caused by the dissolution of preassociated pyrene within the polymer backbone. 40 This implied that it was critical to prevent the hydrogels from drying out over the course of the performed measurements. Since we observed that mechanical testing in uniaxial extension mode led to rapid drying effects, we hence performed subsequent mechanical testing in uniaxial compression mode.
Formula used to calculate the degree of swelling of the hydrogels in %.

Mechanochromic Hydrogel Response
To investigate the mechanochromic properties of the PyMC 32 hydrogels, we used three different variants with ρ PyMC ranging from 0.025 to 0.129 g · L −1 . Since the network segments of the hydrogels at equilibrium d s were already subject to maximum uncoiling and hence became rather brittle, we decided to investigate each of the three hydrogel samples at three distinct d s below equilibrium d s . The resulting nine investigated samples are denoted H1.1 to H3.3 and their associated ρ PyMC , c pyrene , and d s can be found in Table 1.   To circumvent the drying effect observed under uniaxial extension, we placed the hydrogel samples between two glass plates and subjected these to maximum compression using a micrometer screw stopping shortly before the glass plates broke (Figure 7). For example, upon compression of H3.2, I E /I M3 decreased from 7.2 to 4.3 corresponding to a relative change of ca. 40 %. Notably, I E /I M3 recovered almost fully after de-loading the hydrogel. Renewed compression then proved the reversibility of this effect measured up to 13 times ( Figure S3). Importantly, we observed the mechanochromic changes in the elastic deformation regime highlighting the advantage of the excimer-based supramolecular force sensors over traditional bond scission-based optical force probes for application in soft but elastically very extendible materials.
The results were qualitatively similar for all investigated hydrogels ( Figure 8) where deviations were observed mainly in the relative change of I E /I M3 upon compression ranging from 20 to 50 %. Notably, these relative changes are considerably higher than those reported to the literature before. [37][38][39] We believe that the molecular origin of the me-chanochromic behavior is the uncoiling and gliding of PyMC during hydrogel compression (and thereby lateral extension). We hypothesize that the preassociated intra-and intermolecular pyrene aggregates are broken and an increase in pyrene monomer emission is consequently observed.
Since H2 showed the largest relative changes, we subsequently used this hydrogel batch for further mechanical testing. H2.2 was loaded 3× with different defined weights. The engineering stress σ was calculated from the ratio of the force F exerted by the weight and the initial surface area of the hydrogel A 0 . It became clear that I E /I M3 decreased with increasing σ (Figure 9). Although σ correlated to I E /I M3 , the relation was non-linear. The largest changes were observed in the initial stages of the elastic deformation. At higher stresses, the relative change in I E /I M3 appeared to reach a limiting value. Mechanistically, this may be explained by the maximum possible displacement of the preassociated pyrenes from their equilibrium where residual excimer emission was caused by interchain pyrene interactions.

Conclusions
We have shown the straightforward synthesis of PyMC macrocrosslinkers that display supramolecular mechanochromism. PyMCs were obtained in a two-step reaction from monomers that are commercial or can likewise be prepared in a one-step reaction. From these, we prepared sPEG-based hydrogels. We fully characterized the fluorescence properties and photostability of PyMCs in solution and in hydrogels depending on concentration effects and degree of swelling. We then presented the reversible mechanochromic behavior of the sPEG hydrogels in the elastic deformation regime. Thereby, we provide a protocol for the facile synthesis of mechanochromic hydrogels and anticipate that this will incentivize easier entry into the field for non-chemical researchers.

Procedures
Optical experiments. UV-vis absorption spectroscopy was performed on a Thermo Scientific Evolution 300 and fluorescence spectroscopy on a Horiba Fluoromax-4P at room temperature in H 2 O (VWR, HPLC grade) and THF (VWR, Reag. Ph Eur). Excitation and emission spectra of the hydrogels were recorded with a compact spectrometer (CCS200, Thorlabs). A fiber-coupled LED (M340F3, Thorlabs) was used as a light source. The emission was detected simultaneously with the excitation with a fiber Y-bundle reflection probe (RP24, Thorlabs), which also transmitted the excitation light. The spectra were analyzed with the Thorlabs OSA software (version 2.90). Compression experiments. A rectangular hydrogel sample (ca. 5 × 5 mm) was cut out by a scalpel. This was placed on the surface of a microscopy-grade glass slide and the surface area was determined with a caliper. Afterwards, the sample was covered with a second glass slide and placed on a pedestal. Force was applied by placing defined metal weights on top of the cover slide. The deformed hydrogel increased in surface but did not exit the glass slide edges. The compressed surface area was determined again with a caliper through the bottom glass slide. Synthesis of TEGMEMAAm. 2,5,8,11-Tetraoxatridecan-13amine (0.91 g, 4.40 mmol) was dissolved in dry THF (20 mL) with DMAP (0.108 g, 0.88 mmol). The flask was flushed with Ar for 7 min and then dry Et 3 N (0.39 mL, 5.29 mmol) was added. Then, methacryloyl chloride (0.33 mL, 4.85 mmol) was added dropwise at 0°C under Ar. The reaction was stirred and slowly warmed to r. t. overnight. The reaction was quenched with aq. Na 2 CO 3 solution and then extracted with CH 2 Cl 2 . The organic layer was washed with 0.1 M aq. HCl, brine, and then dried over MgSO 4 . The solvent was evaporated in vacuo and the product was purified by silica column chromatography with EtOAc and MeOH (95 : 5, R f = 0.35). A yellow oil was obtained (87 %). 1 Figure S9. Synthesis of PyMAAm. Pyren-1-ylmethanaminium chloride (2.46 g, 10.20 mmol) was added to a reaction flask, dried in vacuo, and then suspended in dry THF (100 mL). By adding DMAP (0.25 g, 2.04 mmol) and Et 3 N (3.11 mL, 22.40 mmol), the suspension slowly dissolved. The reaction mixture was stirred for 30 min. Then, methacryloyl chloride was added dropwise at 0°C and the reaction was stirred at r. t. overnight. The reaction was then diluted with EtOAc and extracted 3× with 0.1 M aq. HCl and 3× with brine. The organic layer was dried over MgSO 4 , collected, and the solvent was evaporated. The product was purified by silica column chromatography with CH 2 Cl 2 and EtOH (97 : 3, R f = 0.5) yielding a white-yellow solid (91 %). 1 Figure S12. General procedure for PyMC synthesis. The bifunctional CTA (1 equiv.) was used in statistical copolymerizations of PyMAAm (2 · o equiv.) and TEGMEMAAm (2 · n = 70 -2 · o equiv.) in dry DMSO (66 wt%), cf. Scheme 1. To this, AIBN (0.4 equiv.) was added, and the solution was kept at 67°C for 18 h achieving conversions > 90 % as determined by 1 H NMR. The obtained polymers were dialyzed against THF and then against H 2 O. Yellow polymers were obtained with typical yields of~90 %. The polymers were analyzed by 1 H NMR and GPC (Figures S17 -S19). The feed fraction of PyMAAm during polymerization was slightly higher than the incorporated fraction that was found in the polymer. Afterwards, the terminal trithiocarbonates were aminolyzed by the addition of n-butylamine (30 equiv.) under Schlenk conditions in THF (0.012 mol · L −1 ) in the presence of TCEP (ca. 1 mg). Hereafter, the polymer was dialyzed against THF and then H 2 O, dried in vacuo, and colorless PyMCs were received. Đ M was typically~1.3 ( Figure S18). General procedure for hydrogel preparation. Polymerization was performed by step-growth polymerization of PyMC macrocrosslinkers (z equiv.) alongside HS-PEG-SH (2-z equiv., 10 kDa) and 4-armed sPEG terminally functionalized with 4× vinyl sulfone (1 equiv.) via a thiol-ene reaction at pH = 8 -10. Exemplarily, sPEG (1.3 µmol, 6.60 mg), HS-PEG-SH (2.66 µmol, 26.66 mg), and PyMC (0.068 mg) were dissolved in H 2 O (200 µL) and cast into a PTFE-coated metal mold. After the addition of Et 3 N (30 µL), the mold was covered and left to react overnight. The hydrogels were immersed first in DMSO/THF then in H 2 O to remove residual reactants yielding transparent hydrogels with visible blue or green fluorescence based on the identity and fraction of the incorporated PyMC.

Funding Information
D. R. and R. G. are grateful for support by a Freigeist-Fellowship of the Volkswagen Foundation (92 888). Parts of the analytical investigations were performed at the Center for Chemical Polymer Technology CPT, which was supported by the European Commission and the federal state of North Rhine-Westphalia (300 088 302).

Supporting Information
Supporting Information for this article is available online at https://doi.org/10.1055/s-0042-1757972.

Primary Data
Primary Data for this article are available online at https:// zenodo.org/record/6984827.