Article pubs.acs.org/journal/abseba
Stimuli-Responsive Genetically Engineered Polymer Hydrogel Demonstrates Emergent Optical Responses Eva Rose M. Balog,†,‡ Koushik Ghosh,†,§ Young-Il Park,⊥,|| Vaughn Hartung,# Prakash Sista,†,○ Reginaldo C. Rocha,† Hsing-Lin Wang,⊥ and Jennifer S. Martinez*,†,△ †
Center for Integrated Nanotechnologies, Materials Physics and Applications Division, ⊥C-PCS, Chemistry Division, #MST-7, Materials Science and Technology Division, and △Institute for Materials Science, Los Alamos National Laboratory, Los Alamos, New Mexico 87545, United States S Supporting Information *
ABSTRACT: Biopolymer-based optical hydrogels represent an emerging class of materials with potential applications in biocompatible integrated optoelectronic devices, bioimaging applications, and stretchable/flexible photonics. We have synthesized stimuli-responsive three-dimensional hydrogels from genetically engineered elastin-like polymers (ELPs) and have loaded these hydrogels with an amine-containing pphenylenevinylene oligomer (OPPV) derivative featuring highly tunable, environmentally sensitive optical properties. The composite ELP/OPPV hydrogels exhibit both pH- and temperature-dependent fluorescence emission, from which we have characterized a unique optical behavior that emerged from OPPV within the hydrogel environment. By systematic comparison with free OPPV in solution, our results suggest that this distinct behavior is due to local electronic effects arising from interactions between the hydrophobic ELP microenvironment and the nonprotonated OPPV species at pH 7 or higher. KEYWORDS: conjugated oligomer, composite material, optically active material, photoluminescence, polymeric material, stimuli-responsive material, genetically encoded
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INTRODUCTION Protein-based polymers are an exciting option for future materials applications because they are sustainable, biodegradable, and can be designed at the DNA level, affording a degree of precision not attainable through conventional polymer synthesis. Additionally, through natural selection, proteins have evolved desirable materials properties that can be manipulated for specific applications. For example, elastin-like polymers (ELPs) are based on the vertebrate protein elastin, which is a specialized extracellular matrix protein that confers mechanical properties such as extensibility and resilience to our connective tissue. Unlike typical globular proteins, which have a biochemically heterogeneous composition leading to distinct 3D folds, elastin contains a series of pentameric (VPGXG) amino acid repeats that underlie its extraordinary elasticity.1 In aqueous environments, ELPs composed primarily of these VPGXG repeats exhibit reversible coacervation that depends upon temperature and ionic strength.1 This same stimuli-responsive behavior can be manifested within the 3D polymer network of a hydrogel.2 Upon heating, ELP gels condense in volume, expel water, and become opaque. Upon cooling, the gels swell, reabsorb water molecules, and become transparent. This volume shrinkage/expansion process is reversible and can be designed at the molecular level to occur in physiologically and experimentally practical temperature ranges at the macroscopic © XXXX American Chemical Society
level. ELPs have shown promise in applications such as drug delivery, coatings for implants, and hydrogels for tissue engineering.3−5 However, despite their appealing properties, there has been limited research into the integration of ELPs with optical or electronic materials, and none of this past work has taken advantage of ELP hydrogel elasticity and stimuliresponsiveness for optically active “smart” materials applications.6−11 Oligomers of p-phenylenevinylene (OPPV) are a promising type of conjugated system with applications in light-emitting diodes, organic solar cells, and semiconducting devices.12−14 Recently, we have synthesized an OPPV that displays pHdependent optical properties over a wide range of pH (2− 12).15 Herein, we incorporate this OPPV into elastin-based hydrogel materials to generate emergent optical properties and explore their potential applications as highly stimuli-responsive biomaterials. Specifically, we chose to create hydrogels made of ELPs to impart temperature-sensitivity to our OPPV system, with the potential to engineer additional sensitivities through genetic engineering of polymer composition. Because the optical properties of OPPV are affected by the formation of Received: March 8, 2016 Accepted: June 14, 2016
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DOI: 10.1021/acsbiomaterials.6b00137 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
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weight of the hydrogel in its swollen state after removal of the soluble fraction and Wd is the weight of that same hydrogel following lyophilization). The reported ratio is the average ± standard deviation of three gels. Scanning Electron Microscopy (SEM). SEM micrographs (n = 11) were obtained on an FEI Quanta 400 FEG-E-SEM environmental microscope (resolution 3−4 nm, high voltage range from 500 V-30 kV) after sputter coating lyophilized hydrogel samples with 1 nm gold. SEM images were collected from a range of voltage spanning from 12.5 to 20 kV. Rheology. Rheological data were obtained on a TA Instruments Advanced Rheometric Expansion System (ARES) rheometer equipped with a forced air convection environmental chamber and parallel plate geometry (8 mm diameter). Though applied shear strain is known to vary under parallel plate configuration, plate radius was small enough to assume that the applied shear strain gradient was insignificant. Hydrogel discs were tested under 1 mm (4 and 37 °C) and 2 mm (25 °C) gaps. Gap height was adjusted for hydrogel shrinkage due to rapid water loss in nonambient environmental chamber conditions. A dynamic oscillatory strain sweep was performed at 25 °C across a range of 0.1−10% strain at a frequency of 1 rad/s. Dynamic oscillatory frequency sweeps (n = 4) from 0.1 to 100 rad/s were performed at 5% percent strain at both 4 and 37 °C. Incorporation and Leaching of OPPV. After casting and removal of hydrogels from molds, hydrogels were equilibrated in water and stored at 4 °C. To load gels with OPPV, we mixed 1 mL of 200 μM OPPV in water with 1 mL of 0.1 M buffers (pH 3 = citric acid/sodium citrate; pH 5 = acetic acid/sodium acetate; pH 7 = sodium phosphate monobasic/sodium phosphate dibasic; and pH 9 = sodium carbonate/sodium bicarbonate). Gels were soaked in buffered OPPV mixtures for >48 h at 4 °C. To remove excess free OPPV, 2 mL of 50 mM buffer was replaced every 24 h until the amount of remaining OPPV plateaued. The initial amount of adsorbed OPPV in the gel was determined using absorbance spectroscopy to measure the amount of OPPV remaining in the buffered loading solution after the gel was removed. This quantity, which varied with pH, was then considered to be 100%. Experimental leaching data were plotted and the first 60% were fit to the Peppas−Sahlin equation Mt/M∞ = k1tm + k2t2m, where Mt/M∞ is the fraction of OPPV released, k1 and k2 are kinetic constants, t is the time in days, and m is the Fickian diffusion exponent determined using the aspect ratio of the gels (8 mm diameter/3 mm thickness, m = 0.46). The k1 term represents the Fickian contribution, whereas the k2 term represents the relaxational contribution. Optical Spectroscopy. Absorbance measurements were made on a Varian Cary 300 Bio UV−visible spectrophotometer (at 1.0 nm resolution) and a small-volume sample cells (150 μL) with a 1.0 cm path length. Fluorescence measurements were obtained on a Horiba Jobin Yvon Fluoromax-4 spectrofluorometer and on a Varian Cary Eclipse fluorescence spectrometer. Hydrogels were placed directly in cuvettes against a piece of quartz for vertical support. Temperature control was achieved using temperature-controlled cuvette holders (Quantum Northwest). Spectra in Figures 5−8 and Figures S4 and S5 are representative of five independent gelation experiments with at least two technical replicates (scans) performed per sample. Prior to fluorescence experiments, gels were placed in fresh buffer at the appropriate pH. Following these experiments, gels were removed and the buffer was assayed to ensure that any contributions from the buffer itself to observed spectra were negligible and that no leaching of OPPV occurred during the course of the experiment. For lifetime measurements, the spectrofluorometer was coupled with a timecorrelated single photon counting (TCSPC) system from Horiba Jobin Yvon. The apparatus was equipped with a pulsed laser diode source (NanoLED) operating at 1 MHz and with excitation centered at 390 nm. Each measurement was terminated when a maximum peak preset of 20 000 photon counts was reached for the monitored fluorescence. Analysis of fluorescence decay profiles was performed with the Horiba DAS6 software.
aggregates, as well as by the nature of these aggregates and the local environment, we hypothesized that temperature-dependent changes to a hydrogel consisting of OPPV should generate tunable optical outputs in combination with pH-dependent responses. Toward the development of ELP biomaterials with improved integration of optical and biological components, we systematically explored these temperature- and pH-dependent optical responses and discovered emergent optical properties specific to the nature of the hydrogel system.
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MATERIALS AND METHODS
Biosynthesis and Characterization of ELPs. The K-sEL gene was designed based on the ELP-1 construct described previously.16,17 To add an amine group to facilitate cross-linking, the N-terminal sequence AGKGS was introduced using a PCR primer to amplify ELP1 (Table S1). Purified PCR product was subcloned into the BsshII and NheI sites of the POE expression vector, which contributes C-terminal SV5 and 6XHis tags and an N-terminal pelB leader sequence (MKKIWLALAGLVLAFSAHA) that is removed upon protein secretion to the periplasm. The sEL-W expression construct was created by ligation of sEL into a modified version of a tagless POE vector called POE-W, where the SV5 and 6XHis tags have been removed and a single Trp residue has been introduced downstream of the NheI 3′ restriction site using site-directed mutagenesis. Successful clones were verified by sequencing (MWG Operon). Sequences of oligos and plasmid descriptions are provided in Tables S1 and S2. ELPs were expressed from BL21(DE3) E. coli cells without induction using the leaky T7 promoter. Typically, 1 L SuperBroth (MP Biomedicals) supplemented with 100 μg/mL carbenicillin was inoculated with 15 mL of overnight culture grown from freshly transformed colonies. Following cell harvesting by centrifugation, ELP was released from the periplasm via cold osmotic shock (20% sucrose/ 1XPBS) and purified as described elsewhere.16 ELP purity was verified by SDS-PAGE. Dynamic light scattering (DLS) experiments to study temperature-dependent coacervation were performed on a Zetasizer NanoZS (Malvern). Three volume measurements of 10 mg/mL K-sEL were acquired at 2 °C intervals from 4 to 40 °C with 2 min equilibration times at each temperature. The average hydrodynamic diameter was plotted ± standard deviation (error bars). Synthesis and characterization of OPPV is described in detail elsewhere.15 Preparation and Characterization of ELP Hydrogels. Hydrogels were generated by dissolving lyophilized K-sEL at a concentration of 106.7 mg/mL in 85% DMSO:15% DMF (10.67% w/v K-sEL). The trifunctional cross-linker tris-succinimidyl aminotriacetate (TSAT) was added dry to a final concentration of 3.7 mg/mL and the solution was vortexed immediately and quickly pipetted (∼100 μL per gel) into caps of 1.5 mL Eppendorf tubes that served as disc-shaped molds. Dry TSAT stored at 4 °C was found to be more stable than resuspended aliquots of TSAT in DMSO/DMF stored at −80 °C. Although the solution became too viscous to pipet within a few minutes, gelation was allowed to continue undisturbed overnight before gels were removed by shrinking with 1 mL of 5 M NaCl for several hours. Gels were then swelled and washed for at least 48 h at 4 °C in sterile water or buffer containing 0.01% NaN3 as an antimicrobial preservative. Gels were stable for months of storage at 4 °C without apparent loss of integrity. To determine the insoluble (gel) fraction of the hydrogels, three 100 μL hydrogels (10.67 mg of polymer) were weighed after lyophilization following extraction of the soluble polymer fraction by immersion in 10 mL of water for 48 h. The insoluble fraction was determined by using the formula: gel fraction (hydrogel%) = (Wd/ Wi)100( [where Wi is the initial weight of the polymer in the sample (10.67 mg) and Wd is the weight of the insoluble fraction after extraction and drying).18,19 The reported solubility percentage is the average ± standard deviation of three measurements; the weight contribution of the cross-linker was ignored. The degree of swelling was calculated as follows: swelling = (Ws − Wd)/Wd (where Ws is the B
DOI: 10.1021/acsbiomaterials.6b00137 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
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Figure 1. (A) Amino acid sequence of K-sEL. Amine sites available for cross-linking are shown in bold. (B) Schematic of cross-linking strategy and OPPV. (C) ELP hydrogels exhibit visible temperature-dependent swelling and condensing behavior.
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RESULTS AND DISCUSSION Preparation of Elastin Hydrogels. We first designed, expressed, and purified a recombinant ELP for hydrogel synthesis via chemical cross-linking. The ELP construct was designed to permit cross-linking chemistry using amines in Lys3 and Lys137 and the N-terminus of the protein chain itself. This polymer is called “K-sEL” here to indicate the addition of Lys (K) to our previously designed “short elastin” (sEL) construct called ELP-1.16,17 The amino acid sequence of K-sEL is shown in Figure 1A. The temperature-dependent coacervation of K-sEL was characterized by dynamic light scattering across the pH and temperature ranges used in subsequent experiments (Figure 2). The slight increase in transition
Table 1. Summary of the Physical and Rheological Properties of K-sEL Hydrogels characterization
value
solubility (gel fraction) mass swelling ratio elastic (storage) modulus (G′) (1 rad/s, 5% strain) dynamic shear modulus (G*) (1 rad/s, 5% strain)
79.3 ± 4.6% 33.5 ± 2.3 11.1 kPa (4 °C); 11.9 kPa (37 °C) 11.4 kPa (4 °C); 13.9 kPa (37 °C)
hydrogel refers to the ratio of hydrated mass to its dry mass. Mass swelling ratio was determined after gels were swollen at 4 °C for at least 48 h and was calculated to be 33.5 ± 2.3, indicating that the K-sEL component accounts for approximately 3% of the swollen gel mass.22 This degree of swelling is on par with responses achieved in synthetic tropoelastin, αelastin, and other ELP hydrogels.2,5,23−26 The porosity and overall surface topography of these hydrogels were observed using scanning electron microscopy after water-swelling, snapfreezing and lyophilizing the hydrogels (Figure 3A). Although SEM analysis cannot be taken to fully represent the hydrated state of the gels because of structural artifacts introduced by the lyophilization process, our gels show a porous microstructure and the presence of channels similar to other freeze-dried ELP hydrogels.23,26 After equilibration in water, hydrogels were loaded with OPPVs at four different pHs (pH 3, 5, 7, or 9). Excess OPPV was removed by passive leaching (Figure S2). When the amount of OPPV remaining in the gel plateaued (typically after 1 week), gels were transferred to freshly prepared buffer for use in subsequent experiments. To gain quantitative insight into the leaching mechanism of OPPV from the gels, experimental data (n = 1) were fit to the Peppas−Sahlin coupled diffusion/ relaxation release model.27 Interestingly, this analysis indicated that OPPV leaching was due primarily to a Fickian diffusion mechanism at low pH, whereas the diffusional contribution was less dominant at higher pH. Kinetic and model fit parameters of OPPV leaching are reported in Table S3. Stimuli-responsive, optoelectronic hydrogels are promising materials for applications such as artificial skins and tissue engineering scaffolds because they offer an expanded suite of tools for controlled communication with biological systems. The mechanical properties of 3D tissue mimetics are very important because they must resemble those of the native
Figure 2. Dynamic light scattering experiments measure the stimuliresponsive coacervation of K-sEL as a function of temperature and pH.
temperature observed with lower pH can be attributed, in part, to the increased mean polarity of the polymer upon protonation of His residues (pKa ≈ 6).20 Recombinantly expressed K-sEL was purified to homogeneity by inverse temperature cycling (Figure S1).21 A gelation strategy was developed based on the report by Trabbic-Carlson et al.2 Gelation was performed in DMSO/DMF solution to avoid the heterogeneity associated with ELP behavior in aqueous environments. An equimolar amount of TSAT was added to provide a 1:1 ratio of the three amines on K-sEL to the three succinimidyl ester groups on TSAT. As observed with other ELP hydrogels, K-sEL gels also retained temperatureresponsive swelling and condensing behavior (Figure 1C). Morphological and Rheological Characterization. A summary of the physical properties characterized for K-sEL gels is given in Table 1. The percentage of polymer incorporated into hydrogel was 79.3 ± 4.6%.19 The mass swelling ratio of a C
DOI: 10.1021/acsbiomaterials.6b00137 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
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Figure 4. Rheology experiments show that K-sEL hydrogels are viscoelastic. (A) Storage (G′) and loss (G″) moduli and (B) complex viscosity as a function of frequency (ω) for K-sEL hydrogels measured at 4 and 37 °C.
ELP hydrogels as well as for soft biological materials such as muscle.2,26,31 pH- and Temperature-Dependent Optical Characterization. The same polymeric features that provide ELP/OPPV hydrogels with temperature-dependent viscoelasticity also supply a unique, stimuli-responsive environment in which to investigate the photophysical properties of OPPV. We compared the fluorescence emission properties of gelimmobilized OPPV to those of OPPV in buffered aqueous solutions across a pH range of 3−9. The pKa of this OPPV has been previously estimated to be 3.3−4.0.15 We found that, at each pH, the emission spectrum of the gel-immobilized OPPV was distinct from that of free OPPV (Figure 5). The fluorescence spectra were collected at 4 °C upon excitation at 390 nm, the wavelength where both free and gel-adsorbed OPPV displays maximum absorption across the temperatures and pHs used in this study. At pH 3, there is a slight red shift in the emission of the gel compared to free OPPV (with a λmax of 479 nm for the gel and 473 nm for free OPPV). For free OPPV at pH 5, two bands were observed: one at 478 nm and another less intense at 609 nm, while for gel-immobilized OPPV at pH 5, a single broader band at 500 nm was observed rather than two separate bands. At pHs 7 and 9, again the spectrum of free OPPV exhibits two distinct emission bands (with the dominant component at 600 nm in this pH range), while the gels display a single band at 558 nm. Clearly, the optical response of OPPV to pH changes is altered in gel-immobilized versus free OPPV. This important observation is likely due to a combination of factors: the interaction between the OPPV and ELP components of the gel, and the intrinsic physical microenvironment of the gel itself. To determine the optical responses as a function of temperature-related effects on the gel structure, we next measured the fluorescence spectra as a function of temperature for each pH. Gels were equilibrated for at least 10 min at 37 °C, allowing sufficient time for a gel to complete its temperature
Figure 3. Representative SEM micrographs show the structure of lyophilized K-sEL hydrogels at different magnifications.
extracellular matrix to achieve proper structural and biological functions. Therefore, we performed dynamic oscillatory rheology to investigate the mechanical properties of our ELP/OPPV hydrogels. Similar to most hydrogels, ELP/OPPV hydrogels are viscoelastic. At 25 °C and 1 rad/s, a dynamic oscillatory strain sweep showed that the shear moduli were independent of strain from 0.1−10% strain (Figure S3), thereby providing an estimate of the linear viscoelastic region (LVR). Subsequent dynamic frequency tests at temperatures of 4 and 37 °C were performed at 5% strain and assumed to be within the LVR. Figure 4A shows the elastic (storage) modulus (G′) and viscous (loss) modulus (G″) plotted versus oscillation frequency (ω) below (4 °C) and above (37 °C) the transition temperature. We observed that G′ is greater than G″ and that both moduli exhibit a plateau across the frequency range from 1 × 10−1 to 1 × 102 rad/s, indicating a solid-like, elastic gel and the formation of a cross-linked network.28−30 Complex viscosity η* decreased with increasing frequency, which is also typical for viscoelastic solids (Figure 4B). Increasing the temperature to 37 °C resulted in large frictional energy losses observed as an increase in both G″ and η*. The high temperature strain induced loss response suggests that intermolecular contact between reoriented K-sEL chains increases as hydrogels condense and contract with water loss. For applied strain at low frequencies, an apparent increase in G′ and η* was also detected when increasing the temperature to 37 °C. Higher storage moduli at low frequencies indicate that cross-linked chain network relaxations are reduced due to hydrogel network confinement and extensive entanglement dampening. Our values for elastic and shear moduli are comparable to those reported for other D
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Figure 6. ELP/OPPV hydrogels show temperature-dependent changes in emission. Normalized fluorescence spectra of ELP/OPPV hydrogel at 4 and 37 °C at (A) pH 3, (B) pH 5, (C) pH 7, and (D) pH 9. (E) The spectra of ELP/OPPV hydrogels at 37 °C across the pH range 3− 9 are shown at the bottom.
9, the opposite trend in response to temperature is observed: the emission maxima are blue-shifted by 13 and 10 nm, respectively. All of the temperature-dependent changes in emission were completely reversible upon return to 4 °C. Nonnormalized emission spectra of gels are provided in Figure S5. In control experiments, the fluorescence of free OPPV was observed to be temperature-dependent, specifically at pHs 5 and 7. Using free OPPV, we observed a change in relative intensities of 478 and 600 nm bands due to decreased intensity of the 478 nm band with increasing temperature (Figure S4). However, the temperature sensitivity of the gel-immobilized OPPV showed a unique behavior not observed for free OPPV as seen by the appearance of new temperature-dependent emission maxima. Taken together, these results suggest a number of interesting phenomena, for which different plausible interpretations are possible. First, the band broadening observed for the gel emission at pH 3, in response to increased temperature, indicates that the gel-embedded OPPV experiences a different
Figure 5. ELP/OPPV hydrogel emission spectra are distinct from those of free OPPV. Normalized fluorescence spectra of ELP/OPPV hydrogel (labeled “gel”) compared with free OPPV at (A) pH 3, (B) pH 5, (C) pH 7, and (D) pH 9. (E) The normalized fluorescence spectra of ELP/OPPV hydrogels at 4 °C across the pH range 3−9 are shown at the bottom.
transition before the measurement. At each pH, temperaturedependent fluorescence emission was observed that was distinct from the effect of temperature on free OPPV (Figure 6 and Figure S4). At pH 3, the emission spectrum of the gel at 37 °C shows a broadening and a slight red-shift relative to the spectrum of the gel at 4 °C. This broadening only occurs for OPPV in the gel environment. At pH 5, the gel emission is redshifted by 13 nm after transition to 37 °C. At pH values 7 and E
DOI: 10.1021/acsbiomaterials.6b00137 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
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ACS Biomaterials Science & Engineering environment as compared to free OPPV in solution. One hypothesis is that, as the temperature is increased, the gel microenvironment becomes more rigid and the local inhomogeneity in this confined environment is reflected in the optical properties of OPPV. According to computational studies, two forms of free OPPV exist in solution: monomer and dimer.15 At pH 5, both monomer and dimer forms of OPPV are present in solution before loading into the gel. For the OPPV-loaded gel at pH 5, we observed a fluorescence redshift in response to temperature. This intriguing response appears to be unique to the particular equilibrium between monomer and dimer species. At pH 7 and above, deprotonation of the amine group of the OPPV increasingly favors formation of OPPV dimers because of a molecular dipole in the deprotonated OPPV. Therefore, the dimer is the main species entering the hydrophobic gel environment. One possible explanation for the blue-shift upon OPPV encapsulation within the gel environment is that the nonpolar, hydrophobic environment of the gel destabilizes the transition dipole of the dimer. Consequently, the excited state is destabilized and the LUMO is raised, all leading to the increased transition energy and resulting blue shift. Recently, we observed an analogous behavior in the solvatochromism of an NH2−OPPV.32 This derivative has a permanent dipole and when placed in different solvent environments with varying polarity, it exhibited incremental blue-shifts with decreasing solvent polarity.32 The further blue-shift upon raising the temperature of the gel at pH 7 and above may be attributed to an additional decrease in polarity of the environment as water is excluded from the gel when the ELP component condenses and desolvates. Alternatively, the increase in temperature may affect the nature of OPPV adsorption, for example, by causing planarization changes resulting in slightly higher energy conformations within the ELP hydrogel. In seeking further evidence for whether the 558 nm peak observed in the gels originated from dimeric OPPV, we performed fluorescence lifetime measurements comparing gels and free OPPV at 4 and 37 °C. The fact that only small differences are observed between the fluorescence lifetimes of free and gel-encapsulated OPPV (Table S4) indicates that the same OPPV species is present within the gel as it is in solution. These comparative results thus provide another indication that the discussed spectral changes arise simply from the exposure of OPPV to different interactions with the microenvironment in the gel context. Control experiments mixing OPPV with non-cross-linked KsEL demonstrate that association between K-sEL and OPPV in solution is sufficient to create the environment responsible for the 558 nm emission of gels at pH 7 or higher (Figure 7). This indicates that the gel-specific emission properties can be attributed to intermolecular interactions between K-sEL and OPPV. In an additional control experiment using an ELP that lacks the His6 tag and all charged residues (“sEL-W”, so named because the tags that are C-terminal relative to the VPGIG repeats in K-sEL have been replaced with a single Trp residue), once again the band around 550 nm was observed, suggesting that the ELP content (VPGIG repeats) of our polymers is responsible for OPPV association. We asked whether OPPV retains any pH-responsive behavior after encapsulation in the gels. Two gels prepared and loaded with OPPV at either pH 3 or pH 9 were equilibrated to the other pH (to pH 3 for the gel loaded with OPPV at pH 9, and vice versa) and their emission spectra were collected (Figure 8).
Figure 7. Mixture of non-cross-linked ELP and OPPV in solution results in the same emission band observed in ELP/OPPV hydrogels. Normalized fluorescence spectra of OPPV at pH 9 with varying concentrations of K-sEL or sEL-W polymer.
Figure 8. ELP/OPPV gels retain pH-responsive emission behavior. Normalized fluorescence spectra of gels that were loaded with OPPV at either pH 3 or pH 9 for a starting measurement and then allowed to equilibrate at the other pH and observed again.
Emission spectra of both gels were observed to change upon shifting to a new pH. The emission band corresponding to the gel that was loaded with OPPV at pH 3 and moved to pH 9 is red-shifted and significantly broadened, and displays a shoulder around 550 nm. The band corresponding to the gel loaded with OPPV at pH 9 and moved to pH 3 is significantly blue-shifted and broadened as well, although no shoulder is observed. Spectra of both gels are fairly similar, which suggest that they have same end point. These data also suggest heterogeneity of OPPV resulting from association and dissociation with other OPPV molecules within the scaffold of the gel. We speculate that some proportion of the OPPV is immobilized in the gel in a way that prevents protonation and deprotonation of the OPPV amine as pH is changed, such as hydrogen bonding between the OPPV and the ELP backbone.
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CONCLUSIONS We have introduced and characterized a novel optically active hydrogel in which a genetically engineered ELP induces unique optical behaviors in a pH-sensitive OPPV, underscoring the tunability and versatility of such composite materials. A dramatic advantage of genetically engineered polymers is the ability to program the composition and length of polymers with high precision, which in turn allows engineering of tailored intermolecular interactions to, for example, alter the photophysical properties of OPPVs. Our results offer compelling motivation for the adoption of genetically engineered polymers for integrated optoelectronic hydrogel materials. F
DOI: 10.1021/acsbiomaterials.6b00137 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
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(7) Del Mercato, L. L.; Pompa, P. P.; Maruccio, G.; Della Torre, A.; Sabella, S.; Tamburro, A. M.; Cingolani, R.; Rinaldi, R. Charge transport and intrinsic fluorescence in amyloid-like fibrils. Proc. Natl. Acad. Sci. U. S. A. 2007, 104 (46), 18019−18024. (8) Alvarez-Rodriguez, R.; Arias, F. J.; Santos, M.; Testera, A. M.; Rodriguez-Cabello, J. C. Gold Tailored Photosensitive Elastin-like Polymer: Synthesis of Temperature, pH and UV-vis Sensitive Probes. Macromol. Rapid Commun. 2010, 31 (6), 568−573. (9) Huang, H. C.; Nanda, A.; Rege, K. Investigation of phase separation behavior and formation of plasmonic nanocomposites from polypeptide-gold nanorod nanoassemblies. Langmuir 2012, 28 (16), 6645−6655. (10) Sun, Z.; Qin, G.; Xia, X.; Cronin-Golomb, M.; Omenetto, F. G.; Kaplan, D. L. Photoresponsive Retinal-Modified Silk-Elastin Copolymer. J. Am. Chem. Soc. 2013, 135 (9), 3675−3679. (11) Wang, E.; Desai, M. S.; Lee, S. W. Light-Controlled GrapheneElastin Composite Hydrogel Actuators. Nano Lett. 2013, 13, 2826. (12) Brouwer, H. J.; Hilberer, A.; Krasnikov, V. V.; Werts, M.; Wildeman, J.; Hadziioannou, G. LEDs based on conjugated PPV block copolymers. Synth. Met. 1997, 84 (1−3), 881−882. (13) Friend, R. H.; Gymer, R. W.; Holmes, A. B.; Burroughes, J. H.; Marks, R. N.; Taliani, C.; Bradley, D. D. C.; Dos Santos, D. A.; Brdas, J. L.; Lgdlund, M.; Salaneck, W. R. Electroluminescence in conjugated polymers. Nature 1999, 397 (6715), 121−128. (14) Jørgensen, M.; Krebs, F. C. Stepwise and Directional Synthesis of End-Functionalized Single-Oligomer OPVs and Their Application in Organic Solar Cells. J. Org. Chem. 2004, 69 (20), 6688−6696. (15) Park, Y.; Postupna, O.; Zhugayevych, A.; Shin, H.; Park, Y.-S.; Kim, B.; Yen, H.-J.; Cheruku, P.; Martinez, J. S.; Park, J.; Tretiak, S.; Wang, H.-L. A New pH Sensitive Fluorescent and White Light Emissive Material through Controlled Intermolecular Charge Transfer. Chem. Sci. 2015, 6, 789. (16) Ghosh, K.; Balog, E. R. M.; Sista, P.; Williams, D. J.; Kelly, D.; Martinez, J. S.; Rocha, R. C. Temperature-dependent morphology of hybrid nanoflowers from elastin-like polypeptides. APL Mater. 2014, 2 (2), 021101. (17) Ghosh, K.; Balog, E. R. M.; Kahn, J. L.; Shepherd, D. P.; Martinez, J. S.; Rocha, R. C. Multicolor Luminescence from Conjugates of Genetically Encoded Elastin-like Polymers and Terpyridine-Lanthanides. Macromol. Chem. Phys. 2015, 216 (18), 1856−1861. (18) Gulrez, S. K. H.; Al-Assaf, S.; Phillips, G. O.; Hydrogels: Methods of Preparation, Characterisation and Applications. Progress in Molecular and Environmental Bioengineering: From Analysis and Modeling to Technology Applications; Carpi, A., Ed.; Intech Open: Rijeka, Croatia, 2003; Chapter 5, pp 117−150; DOI: 10.5772/24553. (19) Katayama, T.; Nakauma, M.; Todoriki, S.; Phillips, G. O.; Tada, M. Radiation-induced polymerization of gum arabic (Acacia senegal) in aqueous solution. Food Hydrocolloids 2006, 20 (7), 983−989. (20) Callahan, D. J.; Liu, W.; Li, X.; Dreher, M. R.; Hassouneh, W.; Kim, M.; Marszalek, P.; Chilkoti, A. Triple stimulus-responsive polypeptide nanoparticles that enhance intratumoral spatial distribution. Nano Lett. 2012, 12 (4), 2165−2170. (21) Hassouneh, W.; Christensen, T.; Chilkoti, A. Elastin-like polypeptides as a purification tag for recombinant proteins. Current Protocols in Protein Science; Wiley: New York, 2010; Chapter 6, Unit 6.11; DOI: 10.1002/0471140864.ps0611s61. (22) Nagasawa, N.; Yagi, T.; Kume, T.; Yoshii, F. Radiation crosslinking of carboxymethyl starch. Carbohydr. Polym. 2004, 58 (2), 109−113. (23) Mithieux, S. M.; Rasko, J. E. J.; Weiss, A. S. Synthetic elastin hydrogels derived from massive elastic assemblies of self-organized human protein monomers. Biomaterials 2004, 25 (20), 4921−4927. (24) Leach, J.; Wolinsky, J.; Stone, P.; Wong, J. Crosslinked α-elastin biomaterials: towards a processable elastin mimetic scaffold. Acta Biomater. 2005, 1 (2), 155−164. (25) Lim, D. W.; Nettles, D. L.; Setton, L. A.; Chilkoti, A. In Situ Cross-Linking of Elastin-like Polypeptide Block Copolymers for Tissue Repair. Biomacromolecules 2008, 9 (1), 222−230.
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomaterials.6b00137. Primers, DNA fragments, and plasmids used in this study, SDS-PAGE of K-sEL purification, OPPV leaching kinetics analysis, rheological strain sweep data, emission spectra of OPPV and ELP/OPPV controls, and fluorescence decay lifetimes (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Present Addresses ‡
E. R. M. B. is currently at Department of Chemistry and Physics, University of New England, Biddeford, ME 04005 § K. G. is currently at Eastman Chemical, Kingsport, TN 37660 || Y.-I. P. is currently at Research Center for Green Fine Chemicals, Korean Research Institute of Chemical Technology, Ulsan 681−802, Korea ○ P. S. is currently at SABIC Innovative Plastics, Mount Vernon, IN 47620. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors acknowledge financial support by the Laboratory Directed Research and Development (LDRD) program for synthesis of conjugated oligomers and hydrogels (E. R. M. B., K. G., P. S., R. C. R.). Photophysical and rheological characterization of ELP/OPPV hydrogels was supported by the Basic Energy Science, Biomolecular Materials Program, Division of Materials Science & Engineering (H.-L. W., J. S. M., and Y.-I. P.). This work was performed, in part, at the Center for Integrated Nanotechnologies, an Office of Science User Facility operated for the U.S. Department of Energy (DOE) Office of Science. Los Alamos National Laboratory, an affirmative action equal opportunity employer, is operated by Los Alamos National Security, LLC, for the National Nuclear Security Administration of the U.S. Department of Energy under contract DE-AC52-06NA25396.
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REFERENCES
(1) Urry, D. W. Physical Chemistry of Biological Free Energy Transduction As Demonstrated by Elastic Protein-Based Polymers. J. Phys. Chem. B 1997, 101 (51), 11007−11028. (2) Trabbic-Carlson, K.; Setton, L. A.; Chilkoti, A. Swelling and Mechanical Behaviors of Chemically Cross-Linked Hydrogels of Elastin-like Polypeptides. Biomacromolecules 2003, 4 (3), 572−580. (3) Raphel, J.; Parisi-Amon, A.; Heilshorn, S. C. Photoreactive elastin-like proteins for use as versatile bioactive materials and surface coatings. J. Mater. Chem. 2012, 22 (37), 19429−19437. (4) McDaniel, J. R.; Callahan, D. J.; Chilkoti, A. Drug delivery to solid tumors by elastin-like polypeptides. Adv. Drug Delivery Rev. 2010, 62 (15), 1456−1467. (5) Annabi, N.; Mithieux, S. M.; Weiss, A. S.; Dehghani, F. The fabrication of elastin-based hydrogels using high pressure CO2. Biomaterials 2009, 30 (1), 1−7. (6) Alonso, M.; Reboto, V.; Guiscardo, L.; San Martín, A.; RodríguezCabello, J. C. Spiropyran Derivative of an Elastin-like Bioelastic Polymer: Photoresponsive Molecular Machine to Convert Sunlight into Mechanical Work. Macromolecules 2000, 33 (26), 9480−9482. G
DOI: 10.1021/acsbiomaterials.6b00137 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
Article
ACS Biomaterials Science & Engineering (26) Martín, L.; Alonso, M.; Girotti, A.; Arias, F. J.; RodríguezCabello, J. C. Synthesis and Characterization of Macroporous Thermosensitive Hydrogels from Recombinant Elastin-Like Polymers. Biomacromolecules 2009, 10 (11), 3015−3022. (27) Peppas, N. A.; Sahlin, J. J. A simple equation for the description of solute release. III. Coupling of diffusion and relaxation. Int. J. Pharm. 1989, 57 (2), 169−172. (28) Almdal, K.; Dyre, J.; Hvidt, S.; Kramer, O. Towards a phenomenological definition of the term “gel. Polym. Gels Networks 1993, 1 (1), 5−17. (29) Nishinari, K. Some Thoughts on The Definition of a Gel. In Gels: Structures, Properties, and Functions; Springer: Berlin, 2009; Vol. 136, pp 87−94. (30) Asai, D.; Xu, D.; Liu, W.; Garcia Quiroz, F.; Callahan, D. J.; Zalutsky, M. R.; Craig, S. L.; Chilkoti, A. Protein polymer hydrogels by in situ, rapid and reversible self-gelation. Biomaterials 2012, 33 (21), 5451−5458. (31) Levental, I.; Georges, P. C.; Janmey, P. A. Soft biological materials and their impact on cell function. Soft Matter 2007, 3 (3), 299−306. (32) Park, Y. Il; Kuo, C.-Y.; Martinez, J. S.; Park, Y.-S.; Postupna, O.; Zhugayevych, A.; Kim, S.; Park, J.; Tretiak, S.; Wang, H.-L. Tailored Electronic Structure and Optical Properties of Conjugated Systems through Aggregates and Dipole-Dipole Interactions. ACS Appl. Mater. Interfaces 2013, 5 (11), 4685−4695.
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DOI: 10.1021/acsbiomaterials.6b00137 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
