Reversible Hydrogel Photopatterning: Spatial and Temporal Control

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Cite This: ACS Appl. Mater. Interfaces 2019, 11, 24627−24638

Reversible Hydrogel Photopatterning: Spatial and Temporal Control over Gel Mechanical Properties Using Visible Light Photoredox Catalysis Faheem Amir,†,§ Kevin P. Liles,†,§ Abigail O. Delawder,† Nathan D. Colley,† Mark S. Palmquist,† Houston R. Linder,‡ Scott A. Sell,‡ and Jonathan C. Barnes*,† †

Department of Chemistry, Washington University, St. Louis, Missouri 63130, United States Department of Biomedical Engineering, Saint Louis University, St. Louis, Missouri 63103, United States

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‡

S Supporting Information *

ABSTRACT: There is a growing interest in being able to control the mechanical properties of hydrogels for applications in materials, medicine, and biology. Primarily, changes in the hydrogel’s physical properties, i.e., stiffness, toughness, etc., are achieved by modulating the network cross-linking chemistry. Common cross-linking strategies rely on (i) irreversible network bond degradation and reformation in response to an external stimulus, (ii) using dynamic covalent chemistry, or (iii) isomerization of integrated functional groups (e.g., azobenzene or spiropyran). Many of these strategies are executed using ultraviolet or visible light since the incident photons serve as an external stimulus that affords spatial and temporal control over the mechanical adaptation process. Here, we describe a different type of hydrogel cross-linking strategy that uses a redox-responsive cross-linker, incorporated in poly(hydroxyethyl acrylate)based hydrogels at three different weight percent loadings, which consists of two viologen subunits tethered by hexaethylene glycol and capped with styrene groups at each terminus. These dicationic viologen subunits (V2+) can be reduced to their monoradical cations (V•+) through a photoinduced electron transfer (PET) process using a visible light-absorbing photocatalyst (tris(bipyridine)ruthenium(II) dichloride) embedded in the hydrogel, resulting in the intramolecular stacking of viologen radical cations, through radical−radical pairing interactions, while losing two positive charges and the corresponding counteranions from the hydrogel. It is shown how this concerted process ultimately leads to collapse of the hydrogel network and significantly (p < 0.05) increases (by nearly a factor of 2) the soft material’s stiffness, tensile strength, and percent elongation at break, all of which is easily reversed via oxidation of the viologen subunits and swelling in water. Application of this reversible PET process was demonstrated by photopatterning the same hydrogel multiple times, where the pattern was “erased” each time by turning off the blue light (∼450 nm) source and allowing for oxidation and reswelling in between patterning steps. The areas of the hydrogel that were masked exhibited lower (by 1−2 kPa) shear storage moduli (G′) than the areas that were irradiated for 1.5 h. Moreover, because the viologen subunits in the functional cross-linker are electrochromic, it is possible to visualize the regions of the hydrogel that undergo changes in mechanical properties. This visualization process was illustrated by photopatterning a larger hydrogel (∼9.5 cm on its longest side) with a photomask in the design of an array of stars. KEYWORDS: hydrogel, redox-responsive materials, viologen, photomechanical actuator, photoredox catalysis, photopatterning, artificial molecular muscles

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to hydrolytic cleavage3,4 or more rapidly in response to (i) changes in pH,5 (ii) the introduction of degradative enzymes,6,7 or (iii) using ultraviolet (UV) light.8−10 Usually, the unidirectional breakdown in network structure is desirable in an effort to release therapeutics or cells from a hydrogel for applications in drug delivery11 and tissue engineering,12−14 respectively. Conversely, degradation followed by irreversible chemical reactions or polymerizations within existing hydrogel

INTRODUCTION The mechanical properties of hydrogel polymer networks are largely dictated by the cross-linking density, namely, the number (usually given in moles) of physical or chemical junctions between macromolecular chains in a given volume (m3 or cm3).1,2 Depending on the desired application, there are several strategies that chemists and material scientists have employed to control the cross-linking density. One approach involves the irreversible degradation of covalent bonds in a network to decrease the cross-linking density, thus allowing for larger mesh sizes, greater degrees of swelling, and softer materials. The degradation process can occur either slowly due © 2019 American Chemical Society

Received: May 21, 2019 Accepted: June 17, 2019 Published: June 17, 2019 24627

DOI: 10.1021/acsami.9b08853 ACS Appl. Mater. Interfaces 2019, 11, 24627−24638

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ACS Applied Materials & Interfaces

Figure 1. Modified hydrogel actuator design and synthesis. (a) Previously, azide-terminated polyviologens (poly(V2+)-N3) were mixed with azideterminated poly(ethylene glycol) chains (PEG2000-N3) (5/95 mol %, respectively) and cross-linked with a stoichiometric amount of tetraalkyne cross-linker (TAXL) to afford PEG-based hydrogel actuators using copper-mediated azide−alkyne cycloaddition. The photoreduction/contraction of the soft hydrogels was demonstrated for the first time using a photoinduced electron transfer (PET) mechanism. (b) In this work, a novel 2V4+St cross-linker was used in a free radical polymerization to synthesize poly(HEA) hydrogels possessing lower cross-linking densities, which, upon doping with a ruthenium photocatalyst (Ru(bpy)3Cl2) and irradiating with blue light (∼450 nm), resulted in the formation of intramolecular viologen radical dimers. The photoreduction of 2V4+-St to 2V2(•+)-St in situ resulted in the contraction of the hydrogel while changing its color and mechanical properties. (c) The photomechanical process was employed along with photomasks to pattern hydrogels reversibly over several cycles.

the amines, the macrocycle and side chain form host−guest complexes that hold the hydrogel together. They demonstrated that these supramolecular-based hydrogels could encapsulate cells and be used for tissue engineering applications. For networks that rely on reversible covalent chemistry, i.e., covalent-adaptable networks,21 many different types of chemical bonding have been investigated. Diels−Alder cycloaddition/reversion,22,23 imine bond formation,24,25 boronic esters,26 and photoresponsive cycloadducts like coumarin27,28 are some examples that have been explored to modulate network cross-linking and hydrogel properties. Even more recently, covalent-adaptable networks consisting of photolabile disulfide bonds have been investigated29 in the context of phase-changing media, such as water. Specifically, Zhao and co-workers demonstrated the ability to use ice formed inside a dynamic cross-linked network to template well-defined pores reversibly into the microstructure of a gel using the photoinduced exchange of disulfide bonds to accommodate the newly formed ice, the latter of which could be removed simply by melting. Alternatively, the cross-linking chemistry can employ molecules that isomerize in response to an external stimulus instead of undergoing bond breakage and reformation. Because light affords spatial and temporal control over chemical and

networks has been employed to modulate cross-linking density and hydrogel stiffness. For example, Anseth and co-workers demonstrated15 UV-induced degradation of o-nitrobenzyl groups that cross-linked hyaluronic acid (HA) polymers, followed by establishing new cross-links by adding a watersoluble photoinitiator and irradiating with visible light (400− 500 nm), a process that resulted in the in situ polymerization of methacrylate groups preattached to the HA polymers. This method of using orthogonal wavelengths of light makes it possible to have spatiotemporal control over hydrogel stiffness, which they showed could mimic the dynamic nature of native extracellular matrix and impart mechanical cues to human mesenchymal stem cells positioned directly on the mechanically dynamic substrate. Another strategy to control the cross-linking density in hydrogels involves the use of dynamic bonding,16 which relies on supramolecular interactions17,18 or reversible covalent chemistry19 that does not permanently degrade the hydrogel network. In the case where reversible noncovalent bonding is used, the interactions are dynamic and exchange quickly, often through host−guest pairing. For example, Kim and co-workers demonstrated20 that HA polymers functionalized with cucurbit[6]uril could be mixed with HA polymers bearing alkylamino side chains to induce gelation. Upon protonation of 24628


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regions. Moreover, the exact location of the patterned regions of the hydrogels was readily identified because the in situ dimerization of viologen radical cations exhibits a dark purple, almost black, color, thus effectively behaving as a mechanophore sensor for changes in mechanical properties in the irradiated portions of the hydrogel. To demonstrate the scalability of our approach, a larger hydrogel (∼9.5 cm on its longest side) was photopatterned in the design of an array of stars and monitored after the light source was turned off and the integrated viologens oxidized.

physical changes in a material, it is frequently used to activate materials.30 Azobenzene31 is the most commonly studied lightresponsive molecule that has been integrated into polymers,32−34 liquid-crystal networks,35,36 and proteins37 on account of its ability to undergo a reversible trans-to-cis isomerization in response to UV light. This photoinduced isomerization results in a shorter length scale and can lead to macroscopic changes in the corresponding hydrogel, such as contraction followed by expansion (i.e., actuation),38 or changes in the gel’s mechanical properties,39 as well as decreasing the average network mesh size. Similarly, spiropyran40 has been investigated as a photoresponsive linker because it also isomerizes in response to UV and visible light.41 Moreover, spiropyran can also serve as a mechanophore, which isomerizes and changes color in response to applied mechanical forces.42 In this article, we describe a novel redox-responsive crosslinker, 2V4+-St, which consists of two 4,4′-bipyridinium units (a.k.a. viologens43,44) tethered by a flexible and water-soluble hexaethylene glycol (HEG) spacer and capped at each end with styrene groups. Because viologens are electron-deficient molecules, they are easily reduced by one electron, either chemically or photochemically, from their dicationic oxidation state (V2+) to the corresponding monoradical cation (V•+).45 The resultant viologen radical cations can undergo selfassembly through radical−radical pairing interactions,46−48 which when stacked generate dark-purple-colored solutions and solid-state materials that appear almost black and are opaque. Recently, we demonstrated (Figure 1a) for the first time a novel mechanism for actuating soft materials, whereby oligomers and polymers containing main-chain viologen subunits could be incorporated into hydrogel networks using copper-mediated “click” chemistry and then reduced either chemically49 or photochemically,50 resulting in contraction of the poly(ethylene glycol) (PEG)-based hydrogels down to 9 or 45% of their starting volumes, respectively. However, these previously reported hydrogel actuator materials were synthesized using copper-mediated click chemistry with a theoretical 100% cross-linking stoichiometry, which made their rates of contraction somewhat sluggish. Additionally, the previous PEG-based gels did not possess high tensile strengths and therefore could not sustain heavy loads. In the current investigation, a different network design based on acrylates and a more efficient gelation protocol were employed (Figure 1b), whereby a water-soluble photoinitiator (lithium phenyl2,4,6-trimethylbenzoylphosphinate, LAP)51 was used to synthesize more elastic hydrogels by polymerizing 2-hydroxyethyl acrylate (HEA) in the presence of different weight percent loadings of 2V4+-St (0.875, 1.75, and 3.50%). The ability of this second-generation hydrogel to perform as a more mechanically robust reversible photomechanical actuator in the presence of a visible light-absorbing photoredox catalyst (tris(bipyridine)ruthenium(II) dichloride) (Ru(bpy)3) and a sacrificial reductant (triethanolamine, TEOA) was assessed in terms of its contraction kinetics, stiffnessas measured by shear oscillatory rheology and tensile experimentsand improved tensile strength. A correlation between viologen cross-linked poly(HEA) hydrogel stiffness and the length of time it is irradiated with blue light-emitting diode (LED) lights (∼450 nm) at a fixed distance was also determined. This correlation was used to reversibly photopattern the same set of hydrogels over three cycles, where each irradiated region of the hydrogel became stiffer (by 1−2 kPa) relative to the masked

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MATERIALS AND EXPERIMENTAL METHODS

Synthesis of Viologen-Based Cross-Linker (2V4+-St). All reagents were purchased from commercial suppliers (i.e., SigmaAldrich, Oakwood Inc.) and used without further purification. Precursor 2V2+ (1.6 g, 1.8 mmol, 1 equiv) and 4-vinylbenzyl chloride (11.0 g, 72 mmol, 40 equiv) were dissolved in MeCN (dry, 40 mL, 40 mg/mL 2V2+) and heated to 55 °C for 72 h. After 72 h, MeOH (10 mL) was added to the solution to dissolve the precipitate, and the solution was transferred to four 50 mL centrifuge tubes and diluted with 40 mL toluene (PhMe) to precipitate the product. The tubes were centrifuged at 4500 rpm at −10 °C for 30 min. The resulting supernatant was decanted, and the viscous, brown oil was washed by adding 50 mL MeCN to each tube, followed by sonication and centrifugation. The supernatant was decanted, and this process was repeated three times. The oil was then dissolved in 5 mL MeOH and precipitated a final time by adding 50 mL of 1:1 PhMe/Et2O to yield the desired product, 2V4+-St, as a dark brown, viscous solid (0.8 g, 39% yield). 1H NMR (500 MHz, (CD3)2SO): δH 9.65 (d, J = 6.4 Hz, 4H); 9.40 (t, J = 8.5 Hz, 4H); 8.93−8.76 (m, 8H); 7.66 (d, J = 8.1 Hz, 4H); 7.55 (d, J = 8.1 Hz, 4H); 7.47 (d, J = 7.9 Hz, 4H); 7.08 (d, J = 7.8 Hz, 4H); 6.74 (dd, J = 17.6, 11.0 Hz, 2H); 6.02 (s, 4H); 5.91 (t, J = 16.4 Hz, 2H); 5.32 (d, J = 11.0 Hz, 2H); 4.97−4.88 (m, 4H); 4.02−3.90 (m, 4H); 3.61−3.52 (m, 4H); 3.49−3.31 (m, 5H); 2.26 (s, 1H). 13C NMR (125 MHz, (CD3)2SO): δC 148.96, 148.76, 146.19, 145.72, 145.63, 138.22, 137.60, 135.77, 133.62, 129.41, 128.03, 127.12, 126.82, 126.33, 125.44, 115.76, 69.64, 69.57, 69.45, 68.61, 62.87, 60.25, 20.73. Counteranion exchange occurred during the reaction, thus decreasing the values of the tosylate anions. Highresolution mass spectrometry (electrospray ionization): m/z calcd for C57H65ClN4O8S [M − Cl − OTs]2+ 500.2100, found 500.2078. Representative Hydrogel (3.5 wt % Cross-Linker) Synthetic Protocol. All reagents were purchased from commercial suppliers (i.e., Sigma-Aldrich, Oakwood Inc.) and used without further purification. 2-Hydroxyethyl acrylate (HEA) (426.8 mg), 2V4+-St (15.5 mg), and lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) (1.2 mg) were dissolved in 1.11 mL degassed H2O inside a N2filled glovebox and vortexed for 10−15 s to ensure proper mixing. The entire solution was then plated into a 2.54 × 2.54 cm2 clear, square mold and irradiated with 365 nm light for 15 min (Analytik Jena UVP UVGL-15 lamp/4 W/0.16 A). The cured hydrogels were then carefully removed from the gel mold using a spatula and placed in a Petri dish. The gel(s) were then soaked in degassed Milli-Q water for a minimum of 48 h to allow for full swelling inside an inert atmosphere glovebag. Fourier transform infrared (FTIR) spectroscopy (Bruker α II Platinum ATR) was performed on a separate, assynthesized gel that was lyophilized for 18 h to confirm complete conversion of the cross-linker. Calculations for Cross-Linking Density, Volumetric Swelling Ratio, and Molecular Weight between Junctions. The volumetric swelling ratio, Q, was calculated as follows:

Q=1+

ρHEA ρH

2O

(

Ms Md

)

− 1 , where ρHEA is the density of poly(2-

hydroxyethyl acrylate) at 298 K (1.30 g/mL, average calculated data), ρH2O is the density of water at 298 K (0.997 g/mL), Ms is the mass of the swollen gel, and Md is the mass of the dried gel. The crosslinking density was calculated as follows: crosslinking density = 24629

G( 3 Q ) RT

, where G is the equilibrium shear DOI: 10.1021/acsami.9b08853 ACS Appl. Mater. Interfaces 2019, 11, 24627−24638

Research Article


same procedure as described in the hydrogel synthesis section for 3.50 wt % cross-linker, except that 3× the amount of material was used in a 4.8 × 3.1 cm2 rectangular mold. Hydrogels containing 3.50 wt % cross-linker, both with and without [Ru(bpy)3]Cl2 and TEOA, were brought into a N2-filled glovebox and irradiated in a transparent, closed Petri dish for 5 h by two 450 nm light sources that were 6.5 cm from the vessel. After irradiation, the gel was removed from the vessel, wrapped with wax paper on each end, and clamped with two 50 g clamps. The gels (experimental vs control) were then suspended, and weights were added in 10 g increments until the gel broke (Videos 1 and 2, respectively). Procedure for Hydrogels Lifting Weights While Being Photoirradiated. All hydrogels used for the dynamic weight-lifting experiments were prepared using the same procedure as described in the hydrogel synthesis section for 3.50 wt % cross-linker, except that 3× the amount of material was used in a 4.8 × 3.1 cm2 rectangular mold. Hydrogels containing 3.50 wt % cross-linker, both with and without [Ru(bpy)3]Cl2 and TEOA, were brought into a N2-filled glovebox. The end of each gel was wrapped with wax paper, clamped with two 50 g clamps, and 40 g of additional weight was suspended from the bottom clamp. The suspended gel was then irradiated with a single 450 nm light source from one side for 11.4 h. The irradiation was monitored via video to determine the change in length over time. The length of each gel (experiment vs control) was measured by determining the distance between clamps, and the length was measured every 30 min (Videos 3 and 4, respectively). Procedure for Quantitative Tensile Testing of Oxidized and Photoreduced Hydrogels. All hydrogels used for the tensile experiments were prepared using the same procedure as described in the hydrogel synthesis section for 3.50 wt % cross-linker. Hydrogels containing 0.875, 1.75, and 3.50 wt % cross-linkers were brought into a N2-filled glovebox and soaked for 24 h in degassed aqueous solutions with or without [Ru(bpy)3]Cl2 (0.15 mM) and triethanolamine (TEOA) (3.0 mM) to serve as the photoredox catalyst and sacrificial electron donor, respectively. The gels were then removed from solution and placed in a 9 cm diameter glass Petri dish. A watersoaked Kimwipe was also placed inside the Petri dish, which was covered by a lid and sealed with electrical tape to mitigate gel dehydration. The gels (n = 3 or more) were irradiated with ∼450 nm light from the top and bottom for 5 h, maintaining a 5 cm distance between the gel and the light source (Hampton Bay desk lamp with an ABI LED aquarium light bulb (450 nm/12 W/740 lm)). After irradiation, the gels were removed from the light source, and samples shaped like “dog bones” (2.75 mm wide at their narrowest point and a 7.5 mm gage length) were punched out of the material. Each sample was loaded onto the mechanical testing system (MTS) tensile instrument and extended at a rate of 5.0 mm/min until break. The stress (kPa) was measured as a function of strain (%), and Young’s modulus was determined using Origin Pro 8 by finding a linear line of best fit for the elastic region of the stress versus strain curve. The ultimate tensile strength was determined by taking the stress value at the point just before breaking. Procedure for Large-Scale Photopatterning. Larger hydrogels for photopatterning contained 1.75 wt % cross-linker fabricated using 35× the amount of material listed in Table S1. The mold used was a rectangular 15.4 × 11.3 cm2 plastic mold. The gel was produced on the benchtop (Great Value GVS14BK blacklight (14 W/0.23 A)) upon irradiating the pregel solution for 25 min. The gel was then removed from the mold and placed directly into a solution containing [Ru(bpy)3]Cl2 (0.15 mM) and TEOA (3.0 mM). After 72 h, the gel was carefully transferred onto the star array photomask. The photomask consisted of a sheet of glass covered by electrical tape and star stickers (Figure S12a). The gel was then irradiated from beneath by two 450 nm light sources on the benchtop, with the lamps repositioned every 15 min to ensure even irradiation of the gel (Figure S12b). After 2 h of irradiation, the star-patterned hydrogel was transferred to a different sheet of glass and the rate of oxidation was monitored using both video and pictures taken every 2 min (Video 5).

modulus ( (G′)2 + (G″)2 from oscillatory shear rheology at 10 rad/ s and 1% strain), R is the gas constant 8.314459848 m3 Pa/(mol K), and T = 298 K in units of mol/m3. The average molecular weight (g/ ρgel RT

mol) between cross-links was calculated as follows: Mc = G , where ρgel is the density of the gel: ((Md/(Ms − Md)) × ρH2O) in g/cm3, R is the gas constant 8.314459848 × 106 cm3 Pa/(mol K), and T = 298 K. Photoredox-Activated Hydrogel Contraction Kinetics. All kinetic experiments were performed in triplicate on gels containing 0.875, 1.75, and 3.50 wt % of 2V4+-St viologen cross-linker. These gels were fabricated and then swollen for 48 h in Milli-Q H2O. The gels were then brought into a N2-filled glovebox and soaked for 24 h in a degassed aqueous solution containing [Ru(bpy)3]Cl2 (0.15 mM) and triethanolamine (TEOA) (3.0 mM) to serve as the photoredox catalyst and sacrificial electron donor, respectively. The average (n = 3) gel volumes at t = 0 were 6.60, 6.72, and 6.97 cm3 for the 0.875, 1.75, and 3.50 wt % gels, respectively. The gels were then removed from solution and placed in a 9 cm diameter glass Petri dish. A watersoaked Kimwipe was also placed inside the Petri dish, which was covered by a lid and sealed with electrical tape to mitigate gel dehydration. The gels were irradiated with ∼450 nm light from the top and bottom for 5 h, maintaining a 5 cm distance between the gel and the light source (Hampton Bay desk lamp with an ABI LED aquarium light bulb (450 nm/12 W/740 lm)) with volume measurements taken at regular intervals. After irradiation, the gels were removed from the light source and a 20 mm diameter disc was punched out of the material. The gel discs were placed into an airtight container, parafilmed, and taped and rheological experiments (frequency sweep (1.0% strain, 0.1−30 rad/s) and strain sweep (1 rad/s, 0.1−50% strain)) were performed using a TA AR-G2 oscillatory shear rheometer to obtain the reduced/contracted storage and loss moduli data for each hydrogel. The resulting discs were oxidized and swollen in Milli-Q H2O. A new 20 mm diameter disc was punched out from the resulting reswollen gels, and the oxidized rheological data were recorded. For the control hydrogels, the same protocol was repeated on a swollen triplicate set of 3.50 wt % 2V4+-St gels without soaking in the [Ru(bpy)3]Cl2/TEOA solution. General Procedure for Photopatterning and Rheology of Hydrogels. All hydrogels used for the photopatterning experiments were prepared using the same procedure as described in the hydrogel synthesis section for 3.50 wt % cross-linker, except that 3× the amount of material was used in a 5.3 cm diameter circular mold. The hydrogels were then taken into a N2-filled glovebox and soaked in a Petri dish with a degassed solution containing [Ru(bpy)3]Cl2 (0.15 mM) and TEOA (3.0 mM) for at least 12 h. The gels were then removed from the solution and placed in a Petri dish with a photomask comprising strips of electrical tape adhered to the bottom. These masks covered a variety of patterns, such as a “plus” sign, stripes, and a “half-moon”. The hydrogel was then irradiated from below by a 450 nm blue light for 1.5 h to complete the photopatterning process. Oxidation was achieved by exposing the hydrogel to atmospheric oxygen. Images were taken of the gel every 2 min to monitor the rate of oxidation over time. The fully oxidized gel was then placed back into the degassed [Ru(bpy)3]Cl2 and TEOA solution, thus allowing the same gel to be cycled multiple times with different patterns. Oscillatory shear rheology was used to determine the difference in physical properties between areas that were exposed to light and those that were not. Rheology was done on hydrogels that were on their first, second, or third photoirradiation cycle, with the second and third hydrogels cycled through different patterns for each irradiation. Rheology was also done on a control hydrogel composed of 3.50 wt % cross-linker that was not soaked in [Ru(bpy)3]Cl2 and TEOA but was irradiated in the half-moon pattern. The lack of [Ru(bpy)3]Cl2 and TEOA prevents the viologen cross-linkers from reducing, which does not produce a pattern; thus, only a small change in mechanical properties is attributed to dehydration. Procedure for Gradual Weight Increase Tensile Tests. All hydrogels used for the tensile experiments were prepared using the 24630


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ACS Applied Materials & Interfaces Table 1. Summary of Hydrogel Parameters as a Function of Cross-Linker Loading cross-linker wt %

Q (swelling ratio)a

G (Pa)b

cross-linking density (mol/m3)c

Mc (kDa)d

0.875 1.750 3.500

14.21 17.64 24.92

4451 4544 4847

4.35 4.77 5.71

54.8 42.6 27.8

a

Determined by weighing hydrogels swollen for 48 h and after 48 h lyophilization (see Materials and Experimental Methods for calculation). Obtained by measuring the storage (G′) and loss (G″) moduli and taking the square root of each modulus squared. cCalculated using the theory of elastic rubbers for dilute polymer solutions (see Materials and Experimental Methods for calculation). dThe average molecular weight between cross-linking junctions was determined using the density of polymer in the hydrogel (see Materials and Experimental Methods for calculation). b

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RESULTS AND DISCUSSION Although viologens have been investigated extensively in polymers over the years,52−54 either as redox-active pendant groups or linear chains that are useful for tuning network properties through noncovalent host−guest interactions,55−57 viologen-based cross-linkers that establish networks purely through chemical bonding are far less common. One example of viologen-based covalent cross-linking was demonstrated recently by Scherman and co-workers who reported a bis(styrene)-functionalized mono(viologen) cross-linker that was used to synthesize highly branched cucurbit[8]uril polyrotaxanes.58 Through donor−acceptor interactions, they demonstrated control over a hydrogel’s mechanical properties by mixing a naphthalene-functionalized cellulose polymer with the viologen-containing polyrotaxanes, effectively employing both physical and chemical cross-links to modulate network properties. Here, we demonstrate the use of a bis(viologen)-based cross-linker, 2V4+-St, to synthesize poly(HEA) hydrogels and tune their mechanical properties by inducing intramolecular dimerization (Figure 1b) of viologen radicals that are generated by a catalytic photoinduced electron transfer (PET) from an embedded ruthenium-based photocatalyst (Ru(bpy)3Cl2). In addition to the intramolecular dimerization that effectively decreases the average mesh size of the hydrogel network, the loss of two positive charges and corresponding counteranions per cross-linker collectively results in the collapse of the hydrogel upon photoreduction. The photoinduced contraction of the hydrogel also causes H2O to leach out and therefore generates stiffer materials with significantly (p < 0.05) greater tensile strengths and elasticity (vide infra). The reduced/contracted hydrogels are easily converted back to the oxidized, swollen hydrogels by soaking them in O2saturated H2O. To demonstrate the spatiotemporal control over the photomechanical process, photomasks were used to block certain portions of the hydrogels, which led to a type of photopatterning of the unmasked regions of the hydrogel (Figure 1c). This process was investigated to determine whether it was possible to change the mechanical properties in only the irradiated portions of the hydrogel relative to the masked areas. In this study, the same set of hydrogels was patterned multiple times, while using oxidation and reswelling in H2O to “erase” the pattern. The synthesis of 2V4+-St was carried out on a gram scale using precursors previously reported by us, see the Supporting Information (SI) for more details about the synthesis (Schemes S1−S4), characterization (Figures S1−S4), and dimerization (Figure S5) of the cross-linker. A series of hydrogels were synthesized in triplicate using 0.25 wt % of the photoinitiator LAP and 0.875, 1.75, or 3.50 wt % 2V4+-St in the presence of a HEA monomer. The total mass of all pregel materials was 443.5 mg dissolved in a 1.11 mL degassed H2O

in each case (Table S1). The gelation time was 15 min, while the pregel solution was contained in 2.54 × 2.54 cm2 molds and irradiated at long-wavelength UV (365 nm). Complete conversion of the cross-linker and monomer vinyl groups was determined (Figure S6) using Fourier transform infrared (FTIR) spectroscopy. The resulting three-dimensional polymer network was swelled in H2O for at least 48 h to yield swollen hydrogels with volumes ranging between 6.60 and 7.75 cm3. The volumetric swelling ratio (Q)59 was determined (Table 1) for each cross-linker wt % by weighing the hydrogels swollen and dry (see Materials and Experimental Methods and SI for equations). The shear modulus (G) was obtained by measuring the storage (G′) and loss (G″) moduli (Figure S7) for each hydrogel, and the cross-linking density was determined using the theory for elastic rubbers.2 Although it is expected that G would increase as the cross-linking density increases, it is interesting to point out that the swelling ratio also increases in this case. We speculate that this is because increasing the amount of tetracationic cross-linker makes the corresponding hydrogels more hydrophilic and capable of greater degrees of swelling. When the average molecular weight of the polymer chains in between cross-linking junctions (Mc) was calculated using the density of polymer in the gel and G (Table 1),60 lower values were obtained as the amount of the cross-linker was increased. This trend is an indication that the addition of more cross-linker results in a greater cross-linking density and expected lower Mc values. With each hydrogel synthesized in triplicate, the ability of each to perform as a photomechanical actuator was assessed by soaking them in an aqueous solution containing 0.15 mM Ru(bpy)3Cl2 and 3.0 mM TEOA. Each hydrogel was irradiated with blue light (from top and bottom; Figure S8) in a clear, sealed Petri dish inside a N2-filled glovebox. The volume was measured (Figures 2a and S9) as the hydrogels contracted over the course of 5 h. As a control experiment, a set of hydrogels containing 3.50 wt % of cross-linker were soaked only in MilliQ H2O and irradiated with blue light for 5 h. After 2 h, the volumes measured for the 0.875 and 1.75 wt % hydrogels (∼70−75% of the starting size) are statistically significant (p value = 0.001) compared to that of the hydrogel containing 3.50 wt % cross-linker (∼45%). Beyond 2 h, there appears to be a limit in the amount of contraction that was observed for each of the hydrogels, where the 0.875, 1.75, and 3.50 wt % hydrogels settle at approx. the same final volumes. The control hydrogel on the other hand only exhibited 5% loss in size within the first 2 h, and even after 5 h, it only reduced in size by a total of 15% (i.e., the final volume was 85% of the starting size). In comparison to our previously reported click-based PEG hydrogels, the contraction kinetics of the poly(HEA) hydrogels are more than twice as fast, even though the crosslinker that was used to make these second-generation hydrogels consisted of only 2 viologen subunits versus the 24631


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Figure 2. Photoactivated contraction kinetics and rheological measurements for contracted and expanded hydrogels. (a) Volume for the 0.875, 1.75, 3.50, and 3.50 (control) wt % hydrogels was measured at different time points to quantify the photoinduced contraction kinetics. (b) Pictures of an as-synthesized, swollen hydrogel, the photoreduced/contracted hydrogel, and the oxidized/expanded hydrogel. The orange color in the oxidized hydrogel comes from the embedded Ru(bpy)3 photocatalyst. (c) Rheological data plot of the storage modulus (G′) as a function of angular frequency for each set of photoreduced hydrogels. (d) Rheological data plot of the storage modulus (G′) vs angular frequency for the corresponding sets of oxidized hydrogels. Each photoreduced-to-oxidized storage modulus was statistically significant (p values between 0.0005 and 0.0087).

Figure 3. Tensile data plots of the stress (kPa) as a function of strain (%) for photoreduced and oxidized hydrogels with 0.875 (a), 1.750 (b), and 3.50 (c) wt % cross-linkers. The stress vs strain plot for the 3.50 wt % hydrogel (c) also includes a control sample that was irradiated for 5 h but did not contain any photocatalyst. The photoreduced (contracted) hydrogels exhibit a noticeable increase in both the tensile strength and elongation at break point (see Table 2 for the exact numerical values). (d) Pictures of the 1.75 wt % hydrogels before (upper left) and after (bottom left) photoreduction, as well as during tensile testing.

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ACS Applied Materials & Interfaces Table 2. Summary of Hydrogel Mechanical Properties as a Function of Viologen Oxidation State 0.875 wt % mechanical properties a

Young’s modulus, E (kPa) tensile strength (kPa)b elongation at break, εB (%)c

1.75 wt %

3.50 wt %

ox

red

ox

red

ox

red

control

8.5 ± 0.9 20.4 ± 1.2 138.4 ± 2.5

12.3 ± 1.5 33.4 ± 5.5 222.6 ± 19.0

10.5 ± 0.2 18.2 ± 1.6 133.5 ± 12.9

18.6 ± 3.4 58.7 ± 12.4 243.3 ± 5.5

12.9 ± 1.2 21.6 ± 0.4 111.6 ± 2.6

20.6 ± 2.1 60.7 ± 3.1 255.8 ± 7.6

11.2 ± 0.4 24.1 ± 3.2 114.6 ± 10.7

a

Determined from the slope of the linear elastic region of each stress vs strain curve. bThe tensile strength of each sample was determined from the maximum stress value just before break. cElongation at break was determined from the maximum percent strain when the sample broke.

Figure 4. Dynamic tensile testing of hydrogels containing 3.50 wt % cross-linker after they were photoirradiated for 5 h. (a) Photoreduction/ contraction of a hydrogel with a photocatalyst while sustaining a 90 g load (highlighted in a red box). (b) Photoirradiation of a hydrogel with no photocatalyst present (control) while bearing a 90 g load. (c) Difference in the overall amount of contraction, as well as the rate of contraction, is shown over the course of irradiating for 11.4 h (bottom plot) and in the first 5 h (top, zoomed-in plot). The Z coordinate length was measured by monitoring the movement of the bottom clamp affixed to the bottom of each hydrogel and a ruler adjacent to the irradiated hydrogels.

10 viologen subunits used in our previous work49 on polyviologen-based actuation of hydrogels. The photoinduced contraction of the poly(HEA) hydrogels is completely reversible, as shown in Figure 2b. The in situ reduction of the cross-linker viologen subunits results in shrunken hydrogels that appear black in color. Once placed in a Petri dish containing O2-saturated H2O, the viologens oxidize within 20−60 min and the hydrogels reswell in a few hours. We measured the change in stiffness of the hydrogels before and after a 5 h photoreduction, using shear oscillatory rheology (Figure 2c,d). The hydrogels containing 1.75 and 3.50 wt % cross-linkers possess similar storage moduli (near 6.0 kPa) after photoreduction and contraction (Figure 2c), whereas the 0.875 and 3.50 (control) wt % hydrogels produced storage moduli at 4.8 and 5.2 kPa. After oxidation and reswelling, the hydrogels become softer, as evidenced by the 1.2−3.5 kPa decrease in their respective storage moduli. The 3.50 (control) wt % hydrogel exhibited the smallest change in stiffness from the photoreduction/contraction (5.2 kPa) to oxidation/ expansion (4.8 kPa), which is largely due to some dehydration during the photoreduction step but no viologen radical-based actuation. Next, the tensile strengths of photoreduced and control (i.e., no Ru(bpy)3) hydrogels containing 3.5 wt % cross-linker were

assessed (Figure S10) by photoirradiating each from top and bottom for 5 h with two blue LED bulbs distanced 6.5 cm from the Petri dish containing each hydrogel. After irradiating for 5 h, the ends of the hydrogels were covered with small pieces of weighing paper and secured at each end with 50 g clamps. One clamp was stabilized to a ring stand, while the other dangled off the other end of the hydrogel. Then, weights were added sequentially such that 100, 120, 130, 140, and 150 g were hung from a paper clip attached to the bottom clamp. The photoreduced and contracted hydrogel managed the 140 g load without tearing (Figure S10a) but could only sustain the 150 g weight for a few seconds before tearing. The control hydrogel (bearing no photocatalyst) started to show signs of tearing near the top clamp at 130 g and tore immediately once 140 g of weight had been added (Figure S10b). These experiments demonstrate the increased tensile strength of the hydrogels after photoreduction and contraction. See Videos 1 and 2 for videos of the experimental and control tensile tests. To quantify Young’s modulus (E), tensile strength, and percent elongation at break (εB) for each hydrogel possessing 0.875, 1.75, or 3.50 wt % cross-linker, tensile experiments (Figure 3) were carried out using an MTS Systems Corp. mechanical testing system (MTS) criterion 42 with a 100 N load cell. Cubic hydrogels at each wt % were prepared (Figure 24633


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ACS Applied Materials & Interfaces 3d, see Materials and Experimental Methods for more details) and a “dog-bone”-shaped hydrogel was punched out and loaded onto the MTS. Each hydrogel was extended at a rate of 5.0 mm/min until it broke. Similarly, cubic hydrogels were prepared, except each was soaked in the photoredox solution for 24 h before being irradiated (at 450 nm) for 5 h in a N2 atmosphere, followed by punching out hydrogels in the shape of a dog bone. These photoreduced hydrogels were loaded onto the MTS, and tensile testing was carried out for comparison to the oxidized hydrogels. The stress versus strain data plots (Figure 3a−c) for the oxidized and photoreduced hydrogels illustrate increases in E, tensile strength, and εB in all three cases. The exact values (as the average of four runs) for each mechanical parameter are provided in Table 2. Comparison of the mechanical parameters for only the oxidized hydrogels reveals the expected trends of larger values of E and decreasing values of εB as the amount of the cross-linker is increased, whereas the tensile strengths remained approximately the same throughout. After the hydrogels were photoreduced, however, a 45−60% increase in E, a 64−180% increase in tensile strength, and a 60−129% increase in εB were observed relative to the oxidized hydrogels at the same amounts of the cross-linker. Additionally, a control experiment was carried out on a hydrogel (with 3.50 wt % cross-linker) that was not soaked in the photoredox solution (i.e., no photocatalyst present) but was irradiated with blue light for 5 h in a N2 atmosphere. Mechanical testing of this control hydrogel (Figure 3c, green trace) showed similar behavior as the oxidized 3.50 wt % hydrogel (Figure 3c, black trace), and each mechanical parameter (E, tensile strength, and εB) was also within the margin of error of the oxidized 3.50 wt % hydrogel (Table 2). These results indicate that the photoreduction of hydrogels containing viologen-based crosslinkers enhances the mechanical properties of the material. The tensile performance of the hydrogels consisting of 3.50 wt % cross-linker was also tested during the photoreduction/ contraction process (Figure 4) by adopting a similar setup as that shown in Figure S10, except with the photoirradiation of the hydrogel carried out after the hydrogel was placed under a 90 g load (highlighted by a red box, Figure 4). The photoreduction/contraction of the hydrogel with a photocatalyst (Figure 4a) caused the hydrogel to change to a dark purple color and contract more rapidly than the control hydrogel with no photocatalyst present (Figure 4b). Even with each of the hydrogels dangling freely in an arid glovebox, and therefore susceptible to dehydration, the one capable of PET and viologen-based actuation due to the presence of the photocatalyst exhibited a much larger degree of contraction in the Z direction (Figure 4c) after irradiation. This difference is readily seen within the first 5 h of irradiation (see zoom in of the lower plot in Figure 4c), where the hydrogel with a photocatalyst contracted by 22% of the original size and the control hydrogel only contracted 9%. See Videos 3 and 4 for videos of the experimental and control dynamic tensile tests. Having demonstrated contraction kinetics and the enhanced mechanical properties of the acrylate-based hydrogels, which are superior to our previously reported PEG hydrogels, we then sought to correlate the length of time a photoactive hydrogel is irradiated to changes in its stiffness. To carry out this experiment, three hydrogels containing 3.50 wt % crosslinker were irradiated with blue light for 30, 60, 120, 180, and 300 min. After each time point, G′ was measured (Figure 5a) for the photoreduced/contracted hydrogels. Plotting the

Figure 5. Correlating changes in hydrogel stiffness to irradiation time. (a) Hydrogels containing 3.5 wt % cross-linker were irradiated for different lengths of time, and their stiffness was determined by measuring each hydrogel’s storage modulus (G′). (b) Plot of storage modulus (G′) versus irradiation time (min) reveals a plateau at 2 h, after which no change in the modulus was observed.

storage modulus versus irradiation time (Figure 5b) reveals a plateau after 2 h of hydrogel irradiation. Beyond this point, little change in G′ was observed, even after irradiating for up to 5 h. This correlation was applied to the photopatterning of hydrogels containing 3.5 wt % cross-linker, where the hydrogel was rested on a plate of glass with a photomask on the other side made from strips of black electrical tape. Additionally, the photoirradiation was carried out from underneath the gel− photomask setup instead of being irradiated from top and bottom and no moist Kimwipe was included. This experimental setup is different from how the hydrogels were irradiated for the contraction kinetics and tensile testing, which means that more friction between the hydrogel and the glass photomask is possible and could potentially mitigate contraction of the hydrogel during photoirradiation. To test this approach, a hydrogel embedded with a photocatalyst and sacrificial reductant was irradiated for 1.5 h while sitting on top of a photomask in the design of a plus sign (Figure 6a, cycle 1). The regions of the hydrogel that were irradiated immediately showed changes in color within the first few minutes. After the full irradiation period, the exposed regions were dark purple in color and the masked areas retained the same color as the starting hydrogel embedded with the photocatalyst (orange in color). After turning off the light source and allowing the hydrogel to oxidize on the benchtop for approx. 60 min, the pattern was “erased”. After the hydrogel was reswollen in a solution containing the photocatalyst and sacrificial reductant, it was photopatterned a second time in the design of vertical stripes (Figure 6a, cycle 2). This pattern could also be erased and the hydrogel reswollen in the photoredox solution. The 24634


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Figure 6. Continuous reversible photopatterning of a hydrogel containing 3.50 wt % cross-linker. (a) Different patterns were imparted on a hydrogel, followed by “erasing” the pattern by way of oxidation and reswelling of the hydrogel in H2O. (b) Storage modulus (G′) for irradiated and masked photopatterned hydrogels was assessed using oscillatory shear rheology. This was performed on hydrogels that were photopatterned once, twice, or thrice, and in each case, an increase in stiffness (1−2 kPa) was observed between the irradiated and masked areas of the hydrogel.

same hydrogel was photopatterned again, in the design of a half-moon, followed by oxidation and reswelling. This process could be repeated well beyond just three cycles without noticing any major degradation in the material. To confirm that changes in the hydrogel’s stiffness were possible, even while experiencing minimal degrees of contraction, the storage modulus was measured for three hydrogels that were photopatterned over one, two, or three cycles (thus, nine hydrogels in total). The patterns used to measure the changes in stiffness were somewhat different from the images of the representative photopatterning cycle shown in Figure 6a. Instead, whenever rheological measurements were needed, the final pattern in the sequence was the half-moon design, and disc-shaped hydrogels were punched out (20 mm in diameter) and their storage moduli measured (see Figure S11 for the exact sequence of photopatterning that was used to obtain rheological data). The storage modulus for the irradiated portions of the hydrogel from the first photopatterning cycle (Figure 6b, first cycle) was nearly 1.2 kPa higher than the values obtained for the masked areas of the same hydrogel. Likewise, the photopatterned hydrogel from the second cycle displayed an even larger difference (2.3 kPa) between the irradiated and masked areas of the hydrogel (Figure 6b, second cycle). This trend continued in the third photopatterning cycle (Figure 6b, third cycle), where the difference in storage moduli for the irradiated and masked areas of the hydrogel was still approx. 2 kPa. In each case, the photopatterned hydrogel could be erased and a new pattern “written” onto the same hydrogel. Additionally, it is important to note that as a consequence of the setup for the photopatterning experiments, little to no contraction in the X and Y directions was observed. However, small changes (∼5−10%) in the height (Z direction) of the hydrogels was observed (Figure S12). To demonstrate that patterns with more intricate photomask designs can be readily achieved, a larger plate of glass was fashioned with opaque stickers (Figure 7a) to afford a

Figure 7. Reversibly photopatterning a hydrogel (1.75 wt % crosslinker) with an array of stars. (a) Photomask was prepared by applying star-shaped stickers to a glass plate, and the hydrogel was transferred to this plate and irradiated from underneath by two blue LED light bulbs. The irradiated area of the hydrogel was 9.5 cm on its longest side, and after irradiation, a well-resolved pattern of stars (b) was obtained. The patterned hydrogel was erased (left to slowly oxidize) over the course of 2 h and can be repatterned multiple times.

photomask in the design of an array of stars. A larger hydrogel with a photocatalyst and 1.75 wt % cross-linker was synthesized (Figure 7a) and placed on top of the photomask. 24635


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ACS Applied Materials & Interfaces This larger hydrogel was irradiated from underneath by two blue LED light sources for 1.5 h. It is important to point out that this photopatterning was performed on the benchtop, which is an indication that the PET process can outcompete any potential O2 that may diffuse into the hydrogel during the photopatterning step. Once the photopatterning had been completed, the “stars” hydrogel was removed from the photomask and its oxidation over the course of 2 h was monitored (Figure 7b; Video 5). This benchtop photopatterning experiment confirms that it is feasible to utilize this patterning strategy on larger hydrogels, potentially ones that contain cells for mechanobiological studies, for example.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Abigail O. Delawder: 0000-0002-2297-9817 Jonathan C. Barnes: 0000-0003-2945-8691

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Author Contributions

CONCLUSIONS In this report, we describe a new functional cross-linker comprising two viologen subunits tethered by HEG and endcapped with styrene groups. The viologen subunits in the cross-linker can dimerize intramolecularly upon a one-electron reduction of each subunit, which we demonstrated could be achieved using a PET mechanism involving Ru(bpy)3Cl2 and TEOA as a sacrificial reductant. Once incorporated into an acrylate-based hydrogel, the photoreduction of the cross-linker resulted in hydrogel contraction down to approx. 45% of the original starting volume as part of a concerted mechanism involving radical−radical pairing and loss of positive charges and the corresponding counterions. These physical changes in the hydrogel network expelled H2O and led to faster rates of contraction, increased stiffness, and greater values for Young’s modulus, tensile strength, and percent elongation at break in the contracted hydrogels. The use of visible light as a spatiotemporal trigger to control the hydrogel’s mechanical properties was exploited to photopattern reversibly only certain areas of hydrogels that were unmasked. Each irradiated portion not only exhibited an approx. 1.0−2.3 increase in their storage modulus relative to the masked areas of the hydrogel, but the written photopattern could also be easily erased by turning off the blue light source and allowing the viologens to oxidize and the hydrogel to reswell in H2O. We envision that this scalable photopatterning strategy will find application in several areas in material science and biology on account of the demonstrated reversibility, as well as the versatility that can be had when pairing the functional bis(viologen) cross-linker with any type of a monomer, thus allowing for an even greater level of tuning, depending on the desired application.

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Photoirradiation of a control hydrogel (not reduced) while lifting a load of 90 g (1052× speed) (Video 4) (MP4) Monitoring oxidation of stars photopatterned hydrogel (512× speed) (Video 5) (MP4)

§

F.A. and K.P.L. contributed equally to this work.

Author Contributions

J.C.B. conceived the idea for the project, and J.C.B., F.A., and K.P.L. designed the experiments. F.A. and K.P.L. carried out the synthesis, rheological measurements, gel contraction kinetics, and photopatterning experiments. A.O.D. obtained FTIR data and calculated the cross-linking densities and molecular weight of chains between junctions. N.D.C. and M.S.P. helped synthesize precursors and the viologen-based cross-linker. F.A., K.P.L., and H.R.L. performed the tensile experiments, which were supervised by J.C.B. and S.A.S. J.C.B., F.A., K.P.L., and A.O.D. co-wrote the manuscript and Supporting Information documents, and all authors contributed to the refinement of each document. Funding

The research reported here was supported by the David and Lucille Packard Foundation in the form of J.C.B.’s Packard Fellowship for Science and Engineering. A.O.D. also acknowledges support from the National Science Foundation Graduate Research Fellowship Program (NSF GRFP). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The mass spectrometry data was obtained using the NIH/ NIGMS Biomedical Mass Spectrometry Resource at WUSTL, which is supported by a grant from the National Institutes of Health/National Institute of General Medical Sciences (#8P41GM103422). The rheological data was obtained through the Department of Mechanical Engineering and Materials Science at WUSTL.

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ASSOCIATED CONTENT

S Supporting Information *

REFERENCES

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