Elastin-Based Rubber-Like Hydrogels - Biomacromolecules (ACS

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Elastin-based rubber-like hydrogels Malav S Desai, Eddie Wang, Kyle Joyner, Tae Won Chung, Hyo-Eon Jin, and Seung-Wuk Lee Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b00515 • Publication Date (Web): 03 Jun 2016 Downloaded from http://pubs.acs.org on June 5, 2016

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Elastin-based rubber-like hydrogels Malav S. Desai, Eddie Wang, Kyle Joyner, Tae Won Chung, Hyo-Eon Jin† and Seung-Wuk Lee* Department of Bioengineering, University of California, Berkeley, Berkeley, California 94720, USA. Biological Systems and Engineering, Lawrence Berkeley National Laboratory, Berkeley, California 94720 USA.

KEYWORDS: Protein-based polymers, elastin-like polypeptides, ELP, PEG, elastic, resilient

ABSTRACT

We developed rubber-like elastomeric materials using a natural elastin derived sequence and genetic engineering to create precisely defined elastin-like polypeptides. The coiled elastin-like polypeptide chains, which behave like entropic springs, were crosslinked using an end-to-end tethering scheme to synthesize simple hydrogels with excellent extensibility and reversibility. Our hydrogels extend to strains as high as 1500% and remain highly resilient with elastic recovery as high as 94% even at 600% strain, significantly exceeding any other protein-based hydrogel. These materials are valuable as elastomeric hydrogels for designing extremely robust scaffolds useful for tissue engineering.

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1. INTRODUCTION: Hydrogels with diverse chemical, biological and mechanical properties are needed in many fields, including biomedicine.1,

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In particular, applications requiring large and repeated

deformations can benefit from rubber-like elastomeric hydrogels. Yet, typical hydrogels lack sufficient strength and elastic extensibility due to crosslink inhomogeneity in the networks. Short, uneven inter-crosslink distances and poor polymer chain extensibility limit the ability of the hydrogels to stretch. Various strategies have been used to improve hydrogel properties such as extensibility and toughness through physical bonds.2-6 Among these, hydrogels based on double networks

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, nanocomposites

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and slide-ring networks

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can exhibit good

toughness and high extensibility due to physical bonds that break and regenerate.2 However, time-dependent recovery of physical crosslinks in these hydrogels result in high hysteresis and stress softening that leads to a loss of performance under repeated deformations.15, 16 Therefore, there are continued efforts to develop robust, elastomeric hydrogels that have the durability to undergo reversible extensions.17-19 Creating a rubber-like elastomeric hydrogel requires control over the architecture of the crosslinked network and a judicious choice of polymer. The architecture of the hydrogel should be homogeneous. Specifically, the chain length between crosslinks should be constant to avoid local concentrations of stress that would lead to premature fracture. Furthermore, the crosslinking scheme should ensure proper chain incorporation to avoid dangling chains that do not participate in the elastic network. Meanwhile, the polymer should ideally behave like an entropic spring that recoils once unloaded. Unlike energy storage in bonds, the driving force restoring an entropic spring is an increase in entropy as the chain goes from a stretched state with


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limited movements to a coiled state. Using these principles, we can develop elastomeric hydrogels that can reversibly extend to large deformations. Protein-based polymers are genetically encoded and recombinantly expressed materials that provide unique opportunities for engineering hydrogels with the aforementioned network and polymer properties. We can finely control their sequence and length to the extent that the distance between the available crosslinking sites would be theoretically monodisperse. Moreover, we can access a library of peptide domain sequences with different mechanical behaviors to synthesize the desired polymers.20 For creating elastomers, elastin-like polypeptide (ELP) sequences are one natural choice because they have been characterized as entropic springs.21 ELPs are built on a repetition of the pentapeptide Val-Pro-Gly-X-Gly with a guest residue ‘X’ that cannot be proline. ELPs are also thermo-responsive and undergo a process known as the inverse temperature transition (ITT) at a characteristic transition temperature (Tt). The ELPs reversibly transition from a hydrophilic to a hydrophobic state and phase separate around their Tt.21-27 Specifically, during ITT, the hydrogen bonding between ELP backbone and water molecules breaks and the ELPs collapse. Thermodynamic factors including the hydrophobic interactions within the chains keep the polypeptides in an unstructured coil form after the transition and play a role in the elastomeric nature of the polypeptides.28 In general, crosslinked ELP networks maintain the properties of elasticity and thermo-responsiveness.29-31 Although chemically crosslinked ELP networks have excellent elastomeric properties, even the best hydrogels developed so far only stretch to a strain of 400% due to limited control over crosslinking that led to random chain tethering.18, 32 In this work, we created simple hydrogels that combine the inherent properties of ELPs with controlled crosslinking to create near ideal networks with end-to-end tethered chains using the two components shown in Figure 1a and 1b.


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The resulting rubber-like elastic hydrogels attain failure strains up to 1500%, significantly exceeding previous examples.18, 32 The properties of failure strain and high resilience even at large deformations were tuned by precisely varying the lengths of the monodispersed peptide chains. We attribute the robustness of these hydrogels to polymer architectures embedding entropic springs and their thermo-responsive deswelling at physiological temperature of 37°C (Figure 1c).

2. MATERIALS AND METHODS: Materials: Dimethyl sulfoxide (D5879) was obtained from Sigma Aldrich; N,Ndimethylformamide (D-131) was obtained from Fisher Scientific; Triethylamine (T0424) was obtained from TCI America; 4-arm peg succinimidyl NHS ester (PSB-485) was obtained from Creative PEGWorks. Nomenclature: The proteins were named using their guest residue (valine), number of elastinlike pentapeptide repeats and the presence of a single lysine near the C-terminal. Three proteins were synthesized and named V50CK1, V75CK1, and V100CK1, with the general sequence MSGVG[VPGVG]nVPGKG (n = 50, 75, 100) (Table 1, Figure 1a). Genetic engineering of ELP: Genetic engineering was performed in a similar manner to our previous publications.31, 33 ELP gene synthesis was performed using pJ54 vector with ampicillin resistance gene as the selection marker. Genes for V50, V75 and V100 sequences were generated though several rounds of restriction and ligation. The completed ELP genes were digested using BpiI and Eco31I to obtain the inserts for each ELP type. pET28b plasmid (EMD, Gibbstown, NJ) containing kanamycin resistance gene was used for synthesizing the final plasmids for ELP


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expression. pET28b plasmids were digested using NcoI and BamHI and oligos containing specific N-terminal and C-terminal modifications were inserted (supplementary table S3). The modified plasmids were then digested with Eco31I and the ELP gene insert was ligated to complete the expression plasmid. The completed plasmids were finally transformed into BLR(DE3) E. coli for expression once the sequences were confirmed (Table 1, Figure 2a). Recombinant expression of ELP: Recombinant expression of ELP is performed in a similar manner to previous works.33, 34 Briefly, on day 0, E. coli was cultured in 60 mL of LB media with kanamycin overnight. On day 1, LB culture was transferred to terrific broth with kanamycin for expansion and expression overnight. Since leaky expression of T7 RNA polymerase is enough to express the ELPs, we simply cultured the bacteria overnight without adding IPTG.34 On day 2, E. coli was centrifuged down at 6k RCF, supernatant was decanted and pellet was resuspended in chilled resuspension buffer (10 mM tris-HCL, 2 mM EDTA @ pH 8.0). Resuspended cells were lysed using a probe-tip sonicator in the presence of 1 mM phenylmethylsulfonyl fluoride to inhibit any proteases. Lysate was centrifuged at 30k RCF to remove cell debris. Polyethylene imine was added to a final concentration of 0.5% to precipitate DNA and the mixture was centrifuged at 20k RCF. Decanted solution was ready for inverse temperature cycling (ITC) to remove other debris and salts. During the first step of ITC, ammonium sulfate is added to the protein solution to a concentration of 0.5 M. The mixture is incubated at 40°C for 30 minutes to precipitate ELP. It is centrifuged at 30-35°C and 18k RCF for 20 minutes. Supernatant is discarded and the pellet is resuspended in chilled resuspension buffer. Pellet is broken up and dissolved as much as possible. Solution is centrifuged at 4°C and 20k RCF for 15 minutes. Supernatant is decanted into new tubes to mark the end of the cycle. This cycle is repeated 2 more times and the resuspension after the third high temperature


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centrifuge spin is performed in deionized water. Upon completion of ITC, the protein solution is dialyzed using a 6000-8000 Da dialysis tubing. Dialysis water is changed at least 6 times over 2 days. The solution is then filtered through a 0.45 µm syringe filter and frozen in a glass beaker. The frozen proteins are lyophilized over 2 days to obtain fibrous white sample that is stored in dry conditions until use. Mass spectrometry: Mass of each ELP was measured using AB SCIEX TF4800 MALDI TOFTOF mass spectrometer. Sinapinic acid (SA; sigma-aldrich D7927) was used as the matrix and prepared using crushed crystal method. Briefly, a 0.5 or 1 µL droplet of SA was dried on the MALDI plate. The dry crystal was crushed using a pencil eraser covered with a clean piece of aluminum soil. 1 µL of a mixture of SA and ELP was then dropped on top and allowed to dry. ELP was used at a concentration of 100 pg/µL. The samples were then analyzed in positive mode to determine mass (Figure 2b). Turbidity tests: Turbidity tests were carried out using Ocean Optics USB4000. ELP solutions were prepared in PBS at 4.7 x 10-5 M concentration such that V50CK1 was 1 mg/mL wt/vol, V75CK1 was 1.48 mg/mL and V100 was 1.96 mg/mL. Solutions were placed in cuvette with 2.5 mm path length and the bottom half of the cuvette was immersed in a water bath on a stirrer/hot plate. Absorbance was measured at 600 nm with the rise in temperature. ITT was determined to be the inflection point using a sigmoidal curve fit. While studying the effects of guanidinium hydrochloride (GuHCl), a series of ELP solutions were prepared using PBS with specified amounts of GuHCl. Tests were conducted in a similar manner unless stated otherwise. Hydrogel synthesis: Prior to hydrogel synthesis organic solvent was prepared by mixing DMSO and DMF at a ratio of 3:1. DMF lowers the freezing point of DMSO. This helps us keep the


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polymer/crosslinker reaction on ice to slow down the reaction before injecting into a mold. ELP gels were prepared at 14% wt/vol to ensure the viscosity was low enough to allow for homogeneous mixing. Dry ELP was dissolved in organic solvents at a concentration of 210 mg/mL. Next, triethylamine was added to the solution as a catalyst. Amount of triethylamine was calculated using the formula: [primary amines on ELP] x 1.2. 4-arm PEG/NHS, with average molecular weight of 2 kDa, was prepared separately in the same organic solvents using the necessary volume to adjust final gel protein concentration of 14%. Concentration of PEG/NHS was calculated at a stoichiometric ratio of 1:1 between primary amines on ELP and NHS esters. This was determined from the typical 90% to 94% NHS modification ratio on the commercially available 4-arm PEG product used in this work. Prior to combining ELP and the crosslinker, solutions were chilled on ice for 10 minutes. Once thoroughly mixed, any bubbles were removed by a quick centrifuge spin and the solutions were injected into a mold. The mold consists of 0.75 mm thick poly(dimethylsiloxane) spacers sandwiched between parafilm coated glass slides. Parafilm improves the flow of the uncrosslinked gel mixture compared to bare glass. Crosslinked materials were removed from the mold after 20 to 24 hour incubation at 37°C and hydrated in 10 mL of phosphate buffered saline containing glass scintillation vials. Prior to any tests, the hydrogels were washed at least 3 times by exchanging PBS every 6 hours to remove organic solvents. Furthermore, the hydrogels remain immersed in PBS throughout its use unless stated otherwise. Swelling tests: In order to characterize the hydrogel swelling properties, we measured the weight of each hydrogel at 26°C and 37°C. Specifically, each hydrogel was first placed on a preweighed clean glass slide and excess water was removed using a tissue paper. The slide with hydrogel was then weighted and the hydrogel was placed back in its container. Each hydrogel


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was weighed 2 more times with at least 30 minutes of incubation between each measurement in order to minimize measurement errors. Once the weights were recorded, the same hydrogels were incubated overnight at the next temperature and the measurements were repeated in the same manner. After recording hydrogel weights in wet conditions, they were incubated in 15 mL of deionized water at room temperature and the water was exchanged at least 3 times over 2 days to remove salt. After 2 days, most of the water was aspirated from the glass vial and the hydrogels were frozen at -80°C for 2 hours, followed by lyophilization over 24 hours. The dried samples were weighted 3 times to obtain the dry weight for the respective samples. Water content of the hydrogels was calculated using the formula: Water Content (%) = 100*(weightWET – weightDRY)/weightWET. Tensile tests: Tensile tests were carried out using Instron® 5544 and a custom setup to provide a temperature controlled liquid environment for the test. Liquid is cycled between a heater and the test chamber to maintain our desired temperature of 37°C during the tests. Before loading each gel, hydrogel profile pictures were taken to measure their width and thickness. After the gels were loaded, an additional picture was taken to measure the gauge length. Once loaded, the gels were allowed to equilibrate for 30 in warm PBS. During the test, the extension rate was kept constant at 10 mm/minute for the tensile tests. The elastic modulus for each gel was calculated using the stress data between strains of 15% and 30%. Failure strain and ultimate tensile stress were determined as the strain and stress values, respectively, at the failure point for each gel. Sample size was 5 for each gel type tested to perform statistical analysis. Resilience tests: Resilience tests were carried out using the same instrument and setup. After the gels were equilibrated for 30 minutes, the cyclic test was performed at a strain rate of 1 /minute. Each gel underwent 6 cycles of stretching and relaxation. Elastic recovery was calculated as the


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ratio between energy of relaxation and the energy of extension. Sample size was 5 for each gel type tested to perform statistical analysis. Sample size was 3 for V75CK1 hydrogels in the presence of GuHCl. Statistical analysis: One-way analysis of variance was used to determine differences between groups of 3 or more hydrogel types. Differences were further analyzed by post-hoc Tukey HSD test. 3. RESULTS AND DISCUSSION: ELP synthesis and characterization We used genetic engineering and recombinant expression to synthesize three different ELPs to fabricate the elastomeric hydrogels (Table 1). The target proteins were designed to contain a single primary amine on the C-terminal lysine and a second amine provided by the N-terminal for crosslinking and the genes were constructed for bacterial expression system (Figure 2a). Each synthesized protein was monodisperse with molecular weights of 21.2, 31.4 and 41.7 kDa for V50CK1, V75CK1, and V100CK1, respectively, based on mass spectrometry (Figure 2b). We also determined the ITT for solutions of each ELP by measuring solution turbidity with increasing temperature. With an increase in molecular weight, the transition temperature (Tt) of ELPs decreases from 40 °C for V50CK1 to 34 °C and 31 °C for V75CK1 and V100CK1, respectively (Figure 3a). Our monodisperse ELPs engineered with only terminal reactive sites serve as well-defined components for the synthesis of ideal networks. As is typical for ELP, Tt depends on many factors including the peptide molecular weight.20,

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previous studies, longer ELP likely has lower Tt due to increased and more stable hydrophobic interactions that disrupt peptide backbone hydrogen bonding with water.26 We also took


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advantage of the ITT process to synthesize the hydrogels at a low initial protein concentration at which the lower viscosity helps create homogeneous ELP mixtures. Hydrogel synthesis and temperature-dependent swelling We synthesized stimuli-responsive hydrogels with well-defined networks by end-linking our ELPs with four-arm crosslinkers (Figure 1b). Specifically, the ELPs’ N-terminal amine and Cterminal amine (lysine) groups reacted with the N-hydroxysuccinimide (NHS) esters on the end of each arm of a 4-armed polyethylene glycol (PEG) molecule (2 kDa). We performed crosslinking in a mixture of dimethyl sulfoxide (DMSO) and dimethylformamide (DMF) rather than in water to prevent any ITT-induced phase separation during crosslinking. This prevents regions of non-uniform ELP concentration and undesired hydrolysis of NHS-esters. In addition, the low viscosity of the 14% weight/volume (w/v) ELP solutions used for crosslinking permits easy mixing of reactants. These factors resulted in isotropic, homogeneous and optically transparent hydrogels. At room temperature, in physiological ionic strength solutions, the equilibrium water content of the hydrogels ranged from 74% to 84%. At 37 °C, the ITT caused the gels to contract and reduced the water content to 60% (Figure 3b). Hydrogel swelling behavior also depends on the molecular weight of ELP, for example at 26°C, equilibrium swelling increases from 74% to 84% as the corresponding ELP size decreases from 41.7 kDa to 21.2 kDa. However, the three gels approach similar water content at 37°C with only V50CK1 and V100CK1 being significantly different likely due to the limit to which the chains can contract. We hypothesized that the temperature-induced collapse of ELP chains into unstructured coils with only weak inter- and intra-chain interactions in combination with the uniform chain length between crosslinks as well as the low crosslink density would allow our hydrogels to be highly resilient and extensible (Figure 1c).


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Extensibility of ELP hydrogels The ELP hydrogels stretched to large magnitudes under uniaxial tensile testing. We conducted the tests using rectangular hydrogels of each ELP equilibrated at 37 °C in PBS (supplementary movie SM1). Failure strains increased significantly with increase in chain lengths. V50CK1, V75CK1, and V100CK1 achieved average strains of 1030%, 1210%, and 1440% (Figures 4a and 4b). Moreover, V100CK1s gel reached failure strain as high as 1530%, which is considerably more than the previous best ELP value of 400% and the largest observed for a protein-based hydrogel to our knowledge.18,

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Although we measured statistically significant differences

between failure strains among the three hydrogels, their elastic moduli were practically the same at 19 kPa (Figure 4c). Similarly, there was no difference between their ultimate tensile stresses (average of 100 kPa) (Figure 4d). We hypothesize that the hydrogel moduli are indistinguishable because at low strains the coiled chains are unraveling and the stress is mainly borne by entanglements and intra-/inter- chain interactions that should both be similar for all of the hydrogels. At higher strains, once the chains are uncoiled, the load starts to fall directly on the chains and crosslinks and there is a drastic increase in stress with plastic deformation. The onset of this behavior is reached by V50CK1 first, followed by V75CK1 and V100CK1 due to their greater chain lengths.

Rubber-like reversibility We further characterize the elastomeric properties of our ELP hydrogels by performing cyclic tensile tests and show high resilience and low stress softening. We hypothesized that the protein


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size should influence potential hysteresis among hydrogels due to differences in ELP interactions. The cyclic tensile tests were performed by stretching the hydrogel to a strain of 600% at a strain rate of 100%/minute for 6 cycles to clearly show their resilience (Figure 5a, supplementary movie SM2). We chose 600% strain in order to stay within the elastic limit of the shortest ELP, V50CK1, as determined from cyclic tests with increasing strains (supplementary information); V50CK1 hydrogels start to undergo plastic deformation beyond a strain of 700%, while the limit is 900% higher for V75CK1 and 1100% for V100CK1 (supplementary Fig. S1). Cyclic tests show that V50CK1 hydrogels have the lowest hysteresis with an energy recovery of 94%, while V75CK1 and V100CK1 recover 88% and 83%, respectively (Figure 5b). The values are averaged over the last five cycles and statistically significant differences among the three hydrogel groups clearly indicates the influence of polymer type on hydrogel performance. We attribute the rubber-like elastic behavior in our hydrogels to the random coiling based on weak hydrophobic interactions typical for ELP. As shown earlier, larger ELPs have lower Tt due to a greater amount of hydrophobic interactions that stabilize them in a coiled state at lower temperatures compared to small ELP. Thus, increased interactions can increase hysteresis in V100CK1 gels compared to V50CK1. The first cycle shows higher hysteresis (supplementary Fig. S2a) because of the unraveling of knotted and entangled polymer chains as well as permanent damage from rupturing of some chains. Observation of hydrogel stress softening during the first couple of cycles supports the argument of permanent structural changes that taper off upon repeated deformations (supplementary Fig. S2b). Majority of hysteresis observed beyond the first cycle is likely due to the weak hydrophobic interactions that ELP chains can undergo. Repeated coiling and uncoiling results in constant restructuring of chains and the consequential changes in inter-/intra- chain interactions would increase hysteresis (Figure 6).


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Chain interaction driven hysteresis We further studied the role of ELP interactions by altering the peptide chain interactions to affect hydrogel hysteresis. Earlier cyclic tests showed a clear trend for hysteresis in which shorter ELPs have higher energy recovery, however it is difficult to determine the mechanism through the presented data. We investigate this is by mitigating the interactions between ELPs to decrease hydrogel hysteresis and show a specific example by matching the cyclic characteristics of V75CK1 hydrogels to the highly resilient V50CK1 gels. We accomplish this using guanidinium hydrochloride (GuHCl), a well-known chaotropic protein denaturant to influence ELP ITT behavior. GuHCl increases ELP chain hydration by impeding chain interactions and thus preventing ELP hydrophobic collapse and increasing their Tt. Through our studies, we found a linear relationship between the concentration of GuHCl and the ELP Tt for both V75CK1 and V100CK1 (Figure 7a). We used this data to rationalize a concentration of GuHCl that would be enough to affect hydrogel properties assuming that if the ELP transition temperatures are matched, their interactions will be similar. We observed that Tt of V75CK1 (Figure 7a, green) matches that of V50CK1 (Figure 7a, red arrow) at close to 0.5 M GuHCl concentration. Using linear regression, we determined that 0.49 M GuHCl in PBS should be optimal for showing a significant change in hysteresis during the cyclic tests. We also confirmed that PBS maintains its pH after the addition of GuHCl to avoid potential effects of pH on ELP prior to testing. Similar to the previous cyclic tests, these hydrogels were equilibrated at 37°C in PBS with 0.49M GuHCl to ensure homogeneous swelling pre-test and stretched cyclically over 6 cycles using the same test parameters. Figure 7b shows the second loop for V75CK1 with and without GuHCl. We can observe a distinct change in the hydrogel hysteresis with a statistically significant increase in


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V75CK1 energy recovery in the presence of GuHCl. In fact, the energy recovery for V75CK1 with GuHCl closely matches V50CK1 with a 1% difference (Figure 7c). Thus, with the help of the chaotropic agent, we showed how the mitigation of inter-/intra- chain hydrophobic interactions allows V75CK1 chains to slide past each other with similar ease to V50CK1 and lower hysteresis in our rubber-like elastomeric hydrogels.

4. CONCLUSION: In conclusion, we used ELP to created protein-based mechanically robust hydrogels with rubberlike extensibility. End-to-end tethered coiled chains enabled our hydrogels extend to strains as high as 1500% and maintain high resilience. In order to design such mechanically robust hydrogels, we exploited ELP for its entropic spring nature as well as genetic engineering to conduct precise polymer design and synthesis. Control over ELP sizes and sequence along with crosslinking using 4-arm NHS/PEG helped regulate inter-crosslink distances. Our strategy led to observations of clear correlations between the molecular architectures of polymer networks and hydrogel extensibility as well as reversibility. Moreover, ELP’s unique ITT property and resulting chain interactions along with the capability of guanidinium to affect these interactions helped us examine how the polymer chain interactions influence hydrogel hysteresis. In addition to the exceptional properties described, the ELP hydrogels also have a biologically relevant elastic modulus matching soft tissues and numerous studies have already shown its excellent biocompatibility.35 Unlike many other hydrogels that lack the ability to deform reversibly, these ELP hydrogels can be used as tissue engineering materials to develop extraordinarily robust scaffolds. Through further incorporation of cell-stimulating motifs by genetic engineering, our rubber-like hydrogel scaffolds will be useful for regenerating diseased tissues such as skeletal


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and cardiac muscles, blood vessels, dermal tissues, etc. that undergo repetitive deformations.4, 6, 35-37

FIGURES

Figure 1. a. Chemical structure of ELP with pentapeptide ‘VPGVG’ repeated n = 50, 75 or 100 times. Amines on N-terminal and C-terminal lysine are designed to achieve end-to-end tethering. b. 4-arm PEG/NHS crosslinker reacts readily and specifically with primary amines on ELP to form a network. c. Once ELP is crosslinked, hydrogels are equilibrated in phosphate buffered saline. Due to ELP ITT, hydrogels remain thermo-responsive with high swelling at low


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temperatures and vice versa. At high swelling, ELP chains behave like loose springs with low extensibility, however, at the physiological temperature of 37°C, ELPs contract into more compact coils and allow hydrogels to undergo extreme deformations.


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Figure 2. ELP genetic engineering and recombinant expression a. Double digest of plasmids with XbaI and HindIII shows vectors at 5 kb and ELP genes at 0.8 kb, 1.2 kb and 1.6 kb for V50CK1, V75CK1 and V100CK1, respectively. b. Mass spectrometer data shows a single peak for each type of ELP. Measured masses: V50CK1 = 21.2 kDa, V75CK1 = 31.4 kDa, V100CK1 = 41.7 kDa).


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Figure 3. ELP thermo-responsiveness a. ITT of ELP measured by turbidity tests. V50CK1 has a high Tt of 40°C followed by V75CK1 with 34°C and V100CK1 with 31°C. b. Comparison of ELP hydrogel water content in different conditions. Water content decreases with higher temperatures and greater ELP size. (n = 5, except for V75CK1 with n = 6; statistically significant differences noted with ‘**’ for p < 0.01)


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Figure 4. Tensile tests to failure: a. Representative plots for tensile test to failure (red: V50CK1, green: V75CK1, blue: V100CK1). b. Extremely high failure strains were observed with strains as high as 1530% for ideal-network ELP hydrogels. Failure strains directly depend on ELP size. c. Elastic modulus of ELP hydrogels shows that between strains of 15% and 30%, all three types of proteins generate a similar modulus of about 19 kPa. d. Ultimate tensile stress measured prior to failure shows that regardless of ELP type, the hydrogels can handle about 105 kPa of stress before failure. (n = 5; statistically significant differences noted with ‘*’ for p < 0.05 and ‘**’ for p < 0.01)


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Figure 5. Cyclic tensile tests: a. Representative plots for the second cycle of a cyclic tensile test (V75CK1 is shifted up by 10 kPa and V50CK1 by 20 kPa for clarity) (red: V50CK1, green: V75CK1, blue: V100CK1). b. Cyclic tests show an increase in energy recovery with a decrease in ELP size. The increase in energy loss for longer ELPs is likely due to an increases in ELP interactions with an increase in size. (n = 5; statistically significant differences noted with ‘**’ for p < 0.01)


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Figure 6. Hysteresis during cyclic tensile tests is caused by chain interactions. a. Initially, ELP chains are coiled and entangled. b. Once the entanglements are unraveled in the first cycle, hydrophobic interactions dominate hysteresis seen during the subsequent cycles. c. Specifically, ELP-ELP chain interactions cause changes in conformations of relaxed ELP chains after each cycle leading to hysteresis.


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Figure 7. ELP chain interaction based hysteresis: a. Increasing concentration of GuHCl raises ELP Tt. V75CK1 (green) with 0.49M and V100CK1 (blue) with 0.81M GuHCl have Tt similar to V50CK1 at approximately 40°C. b. Representative plots for V75CK1 hydrogels with and without GuHCl showing a clear decrease in hysteresis in the presence of the chaotropic agent (green: V75CK1, violet: V75CK1 with GuHCl). c. In the presence of GuHCl, V75CK1 hydrogels show a statistically significant increase in elastic recovery to match that of V50CK1 in PBS. (n = 5 except for V75CK1 gels in GuHCl with n = 3; statistically significant differences noted with ‘**’ for p < 0.01)


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TABLES Molecular weight (kDa) Tt (°C)

ELP

Sequence

V50CK1

MSGVG[VPGVG]50VPGKG

21.2

40

V75CK1

MSGVG[VPGVG]75VPGKG

31.4

34

V100CK1 MSGVG[VPGVG]100VPGKG

41.7

31

Table 1. Summary table with ELP names and sequences along with characteristics such as molecular weight and transition temperature.


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ASSOCIATED CONTENT Supporting Information. Results and brief discussion regarding hydrogel behavior with increasing strain, data about the first cycle of the cyclic tests, table with oligo sequences used for ELP terminal medications and movies showing tensile test to failure and cyclic tensile test for V75CK1 hydrogel. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *Email: [email protected] Present Addresses † Present address: College of Pharmacy, Ajou University, Suwon 16499, Republic of Korea Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Funding Sources

ACKNOWLEDGMENT This work was supported by NIH ARRA supplement to an NIDCR R21 Grant (DE 018360-02) and Tsinghua-Berkeley Shenzhen Institute. It was also partially supported by the National Research Foundation of Korea Grant funded by the Korean Government (NRF2014S1A2A2027641) and Siebel Scholars Foundation. Work at the Molecular Foundry was


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supported by the Office of Science, Office of Basic Energy Sciences, Office of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231. The authors thank Paul Keselman for his help in fabricating the liquid testing chamber. ABBREVIATIONS ELP, Elastin-like polypeptides; ITT, Inverse temperature transition; PEG, Polyethylene glycol; NHS, N-hydroxysuccinimide; DMSO, Dimethyl sulfoxide; DMF, Dimethylformamide; PBS, Phosphate buffered saline; GuHCl, Guanidinium hydrochloride; SA, Sinapic Acid; MALDITOF, Matrix assisted laser desorption ionization – time of flight.


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