pubs.acs.org/Langmuir © 2010 American Chemical Society
Mechanically Robust, Rapidly Actuating, and Biologically Functionalized Macroporous Poly(N-isopropylacrylamide)/Silk Hybrid Hydrogels Eun Seok Gil,† Sang-Hyug Park,† Lee W. Tien,† Barry Trimmer,†,‡ Samuel M. Hudson,§ and David L. Kaplan*,† † Department of Biomedical Engineering, and ‡Department of Biology, Tufts University, Medford, Massachusetts 02155, and §Fiber and Polymer Science Program, Box 8301, North Carolina State University, Raleigh, North Carolina 27695
Received June 21, 2010. Revised Manuscript Received July 30, 2010 A route toward mechanically robust, rapidly actuating, and biologically functionalized polymeric actuators using macroporous soft materials is described. The materials were prepared by combining silk protein and a synthetic polymer (poly(N-isopropylacrylamide) (PNIAPPm)) to form interpenetrating network materials and macroporous structures by freeze-drying, with hundreds of micrometer diameter pores and exploiting the features of both polymers related to dynamic materials and structures. The chemically cross-linked PNIPAAm networks provided stimuli-responsive features, while the silk interpenetrating network formed by inducing protein β-sheet crystallinity in situ for physical cross-links provided material robustness, improved expansion force, and enzymatic degradability. The macroporous hybrid hydrogels showed enhanced thermal-responsive properties in comparison to pure PNIPAAm hydrogels, nonporous silk/PNIPAAm hybrid hydrogels, and previously reported macroporous PNIPAAm hydrogels. These new systems reach near equilibrium sizes in shrunken/swollen states in less than 1 min, with the structural features providing improved actuation rates and stable oscillatory properties due to the macroporous transport and the mechanically robust silk network. Confocal images of the hydrated hydrogels around the lower critical solution temperature (LCST) revealed macropores that could be used to track changes in the real time morphology upon thermal stimulus. The material system transformed from a macroporous to a nonporous structure upon enzymatic degradation. To extend the utility of the system, an affinity platform for a switchable or tunable system was developed by immobilizing biotin and avidin on the macropore surfaces.
Introduction When considering lessons from nature, biomimetic actuators/ muscles are intriguing. Current challenges in forming devices similar to muscles include achieving fast response with mechanical robustness.1 Synthetic materials, genetically engineered recombinant proteins, or muscle tissue thin films have been fabricated toward devices with biomimetic movement.2-5 Unfortunately, natural or synthetic materials have not yet satisfied the demands of such actuator systems, failing either in speed or in mechanics.2,6-8 In general, polyacrylamide-based stimuli-responsive hydrogels require relatively long response times for dimensional change due to slow diffusion of water.2 Protein-based gels, such as engineered elastin-like proteins, as hydrogels are weak mechanically compared to chemically cross-linked hydrogels.9-12 The *To whom correspondence should be addressed. E-mail: david.kaplan@ tufts.edu.
(1) Spatz, J. P. Nat. Mater. 2005, 4, 115–116. (2) Gil, E. S.; Hudson, S. A. Prog. Polym. Sci. 2004, 29, 1173–1222. (3) Lao, U. L.; Sun, M. W.; Matsumoto, M.; Mulchandani, A.; Chen, W. Biomacromolecules 2007, 8, 3736–3739. (4) Petka, W. A.; Harden, J. L.; McGrath, K. P.; Wirtz, D.; Tirrell, D. A. Science 1998, 281, 389–392. (5) Feinberg, A. W.; Feigel, A.; Shevkoplyas, S. S.; Sheehy, S.; Whitesides, G. M.; Parker, K. K. Science 2007, 317, 1366–1370. (6) Galaev, I. Y.; Mattiasson, B. Trends Biotechnol. 1999, 17, 335–340. (7) Liang, L.; Liu, J.; Gong, X. Y. Langmuir 2000, 16, 9895–9899. (8) Sidorenko, A.; Krupenkin, T.; Taylor, A.; Fratzl, P.; Aizenberg, J. Science 2007, 315, 487–490. (9) McHale, M. K.; Setton, L. A.; Chilkoti, A. Tissue Eng. 2005, 11, 1768–1779. (10) Gil, E. S.; Hudson, S. M. Biomacromolecules 2007, 8, 258–264. (11) Vernon, B.; Martinez, A. J. Biomater. Sci., Polym. Ed. 2005, 16, 1153–1166. (12) Lee, B. H.; West, B.; McLemore, R.; Pauken, C.; Vernon, B. L. Biomacromolecules 2006, 7, 2059–2064.
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fusion of natural and synthetic polymeric systems may offer to bridge this gap and combine the most useful features of both systems. Silk derived from spiders or silkworms is known to be the strongest and toughest natural fibrous material.13-15 Regenerated silk offers new opportunities in many applications such as tissue engineering scaffolding,16 drug delivery,17 and optical platforms18 due to the novel material features; providing a mechanically robust and biocompatible material base. Moreover, the ability to control the formation of β-sheet crystalline physical networks in regenerated silk systems enables the fabrication of freestanding biomaterials including films, hydrogels, and macroporous sponges.19 Poly(N-isopropylacrylamide) (PNIPAAm) has been extensively studied as an actuating material due to its sharp phase transition at 32 C, between room and body temperatures.2 Cross-linked PNIPAAm hydrogels provide reversible expansion/contraction in aqueous environments upon thermal stimulus; however, these systems suffer from slow swelling/deswelling response, weak mechanical properties, and limited ability to be functionalized with biological components. The study of PNIPAAm hydrogels (13) Jin, H. J.; Kaplan, D. L. Nature 2003, 424, 1057–1061. (14) Becker, N.; Oroudjev, E.; Mutz, S.; Cleveland, J. P.; Hansma, P. K.; Hayashi, C. Y.; Makarov, D. E.; Hansma, H. G. Nat. Mater. 2003, 2, 278–283. (15) Vollrath, F.; Knight, D. P. Nature 2001, 410, 541–548. (16) Wang, Y. Z.; Blasioli, D. J.; Kim, H. J.; Kim, H. S.; Kaplan, D. L. Biomaterials 2006, 27, 4434–4442. (17) Wang, X.; Wenk, E.; Hu, X.; Castro, G. R.; Meinel, L.; Wang, X.; Li, C.; Merkle, H.; Kaplan, D. L. Biomaterials 2007, 28, 4161–4169. (18) Omenetto, F. G.; KapLan, D. L. Nat. Photonics 2008, 2, 641–643. (19) Vepari, C.; Kaplan, D. L. Prog. Polym. Sci. 2007, 32, 991–1007.
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has progressed via chemical and physical modifications.20 Chemical modification of PNIPAAm networks, such as copolymerization with acrylic acid (AAc)21 and incorporation of graft chains with PNIPAAm22 or poly(ethylene oxide) (PEO),23 has been reported to accelerate swelling kinetics by suppressing the formation of a skin layer. Physical modification of PNIPAAm hydrogels with heterogeneous or porous structures can provide faster responses due to convection flow through macropores rather than diffusion through the gel network. A heterogeneous structure was formed with PNIPAAm hydrogels by polymerizing NIPAAm with a cross-linker above the lower critical solution temperature (LCST)24 or with γ-ray irradiation.25 To enhance the response time of PNIPAAm hydrogels, freeze-drying26,27 and radical polymerization under freezing temperatures28,29 have been utilized, resulting in mesoscopic porous networks or macroporous structures, respectively. A homogeneous porous structure was also synthesized in PNIPAAm hydrogels by using pore-forming agents such as silica particles,30,31 poly(ethylene glycol),32,33 dimethyl sulfoxide,34 and nonionic surfactants35 during polymerization of NIPAAm (N-isopropylacrylamide) with a cross-linking monomer. Emulsion templating has also been utilized as another strategy to prepare porous hydrogels.36,37 Most recently, Tokuyama and Kanehara reported that porous PNIPAAm hydrogels synthesized through an oil-in-water emulsion templating methodology had a pore diameter distribution in the range of 1-40 μm and showed rapid swelling/shrinking in accordance with temperature oscillations.38 We have previously reported the formation of interpenetrating polymer networks (IPNs) of PNIPAAm and silk. These hydrogels demonstrated higher mechanical strength and accelerated deswelling kinetics compared to pure PNIPAAm gels by suppressing skin layer formation.10 However, the rates of swelling and deswelling were still low, with the IPN hydrogels taking over 1 h to reach the equilibrium when the temperature was raised from 20 to 45 C. Moreover, the silk/PNIPAAm IPN hydrogels showed slow swelling kinetics, similar to pure PNIPAAm hydrogels: with 1524 h required for the IPN hydrogels to reach equilibrium in the swollen state when the temperature was dropped from 45 to 20 C. Therefore, in the present work, we examined the feasibility of synthesizing hybrid macroporous hydrogels by using silk fibroin and PNIPAAm, with the main goal to accelerate the swelling/ (20) Zhang, X. Z.; Xu, X. D.; Cheng, S. X.; Zhuo, R. X. Soft Matter 2008, 4, 385–391. (21) Diez-Pena, E.; Quijada-Garrido, I.; Barrales-Rienda, J. M. Polymer 2002, 43, 4341–4348. (22) Yoshida, R.; Uchida, K.; Kaneko, Y.; Sakai, K.; Kikuchi, A.; Sakurai, Y.; Okano, T. Nature 1995, 374, 240–242. (23) Kaneko, Y.; Nakamura, S.; Sakai, K.; Aoyagi, T.; Kikuchi, A.; Sakurai, Y.; Okano, T. Macromolecules 1998, 31, 6099–6105. (24) Gotoh, T.; Nakatani, Y.; Sakohara, S. J. Appl. Polym. Sci. 1998, 69, 895– 906. (25) Kishi, R.; Kihara, H.; Miura, T. Colloid Polym. Sci. 2004, 283, 133–138. (26) Kato, N.; Takahashi, F. Bull. Chem. Soc. Jpn. 1997, 70, 1289–1295. (27) Kato, N.; Hasegawa, H.; Takahashi, F. Bull. Chem. Soc. Jpn. 2000, 73, 1089–1095. (28) Xue, W.; Hamley, I. W.; Huglin, M. B. Polymer 2002, 43, 5181–5186. (29) Srivastava, A.; Jain, E.; Kumar, A. Mater. Sci. Eng., A 2007, 464, 93–100. (30) Kaneko, T.; Asoh, T. A.; Akashi, M. Macromol. Chem. Phys. 2005, 206, 566–574. (31) Takeoka, Y.; Watanabe, M. Langmuir 2002, 18, 5977–5980. (32) Dogu, Y.; Okay, O. J. Appl. Polym. Sci. 2006, 99, 37–44. (33) Zhang, X. Z.; Yang, Y. Y.; Chung, T. S.; Ma, K. X. Langmuir 2001, 17, 6094–6099. (34) Zhang, X. Z.; Chu, C. C. Chem. Commun. 2003, 1446–1447. (35) Antonietti, M.; Caruso, R. A.; Goltner, C. G.; Weissenberger, M. C. Macromolecules 1999, 32, 1383–1389. (36) Bennett, D. J.; Burford, R. P.; Davis, T. P.; Tilley, H. J. Polym. Int. 1995, 36, 219–226. (37) Okay, O. Prog. Polym. Sci. 2000, 25, 711–779. (38) Tokuyama, H.; Kanehara, A. Langmuir 2007, 23, 11246–11251.
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deswelling rates in response to temperature. Figure 1 illustrates the fabrication and function of macroporous hybrid hydrogels with PNIPAAm as the functional synthetic component and silk as the biopolymer component. The ability to fabricate controlled porous structures is of interest in many applications such as tissue engineering scaffolds,16 microreactors,39 energy storage devices,40 insulators,41 and sensors.42 Macroporous structures in hydrogels can provide elastic properties, biocompatible environments due to high water content, and improved transport.43 These structures have increased surface area which can result in faster dimensional changes with a stimulus when compared to the corresponding nonporous systems.38 The challenge is to achieve this type of macroporous morphology with retention of robust material features. Toward this end, silk/PNIPAAm hybrid macroporous hydrogels were prepared and investigated for the structure of the materials, actuating properties, mechanics, degradation, and as an affinity platform for a switchable or tunable systems.
Experimental Section Preparation of Macroporous Hybrid Hydrogels. Silk fibroin (SF) and N-isopropylacrylamide (NIPAAm) (SigmaAldrich) (recrystallized twice from n-hexane) blend solutions were prepared to a final total concentration of 10% w/v. The SF solution was prepared by following previous reports.44 Cocoons of B. mori silkworm silk were supplied by Tajima Shoji Co (Yokohama, Japan). Briefly, the cocoons were degummed in a boiled 0.02 M Na2CO3 (Sigma-Aldrich, St. Louis, MO) solution for 30 min. The fibroin extract was then rinsed three times in MilliQ water, dissolved in a 9.3 M LiBr solution yielding a 20% (w/v) solution, and subsequently dialyzed (MWCO 3500) against distilled water for 2 days to obtain silk fibroin aqueous solution (ca. 8 wt/vol %). In 10 mL of NIPAAm/SF blend solution, N,N0 -methylenebis(acrylamide) (BIS; Sigma-Aldrich; 2.7 wt % of NIPAAm) and N,N,N0 ,N0 -tetramethylethylenediamine (TEMED; SigmaAldrich; 10 μL) were added. After nitrogen gas was bubbled for 10 min, ammonium persulfate (APS, 10 mg) as an initiator was added to the solution. The mixture was immediately poured into a mold. The solution mixture was polymerized at room temperature for 1 day. The prepared hydrogels were frozen at -20 C and lyophilized. The gel membrane which formed was immersed in 80% aqueous methanol solution for 1 day and dried in air for 2 days. The dried samples were immersed in deionized water for 7 days by changing deionized water daily to remove unreacted chemicals. The macroporous PNIPAAm/SF hydrogels were cut into disks (6 mm in diameter and 3 mm in height) with a cork borer. The gel disks were dried in air for 2 days and additionally dried in vacuum for 1 day. A hybrid macroporous hydrogel ball (9.2 cm diameter) was fabricated from a poly(dimethylsiloxane) (PDMS; GE Plastics, Pittsfield, MA) replica mold. The PDMS mold was prepared by casting a toy rubber ball. Air in the toy rubber ball was replaced with water when casting the PDMS mold to prevent the ball from buoying in PDMS liquid. The mixture described above was added to the prepared PDMS mold. The obtained hybrid hydrogel was frozen at -20 C, and lyophilized.
Secondary Structure of Silk Component in the Hybrid System. Fourier transform infrared (FTIR) spectra were obtained (39) Basheer, C.; Swaminathan, S.; Lee, H. K.; Valiyaveettil, S. Chem. Commun. 2005, 409–410. (40) Wang, D. W.; Li, F.; Liu, M.; Lu, G. Q.; Cheng, H. M. Angew. Chem., Int. Ed. 2008, 47, 373–376. (41) Lee, Y. J.; Huang, J. M.; Kuo, S. W.; Chang, F. C. Polymer 2005, 46, 10056– 10065. (42) Ju, Y. M.; Yu, B. Z.; Koob, T. J.; Moussy, Y.; Moussy, F. J. Biomed. Mater. Res., Part A 2008, 87A, 136–146. (43) Dainiak, M. B.; Kumar, A.; Galaev, I. Y.; Mattiasson, B. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 849–854. (44) Kim, U. J.; Park, J.; Kim, H. J.; Wada, M.; Kaplan, D. L. Biomaterials 2005, 26, 2775–2785.
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Figure 1. Schematic of the fabrication of macroporous hybrid hydrogels with PNIPAAm as the functional synthetic component and silk as the biopolymer component. (A) Nonporous silk/PNIPAAm hybrid hydrogels: N-isopropylacrylamide monomer mixed with silk fibroin to form homogeneous semi-interpenetrating polymer networks after radical polymerization with cross-linker. Exposure to aqueous MeOH promotes crystallization of the silk fibroin into β-sheets, thereby producing silk/synthetic interpenetrating polymer networks with two coexisting nanostructures and insolubility in water. (B) Freeze-drying temporarily generates the porous structure. PNIPAAm hydrogel networks return to the original nonporous topology after reswelling (Figure 3A-C). (C) In this scenario, silk forms β-sheet crystals in the temporarily induced porous topology of the hybrid networks by freeze-drying, thereby promoting a stable macroporous structure in the silk/ synthetic hybrid hydrogels (Figure 3A-C). (D) Scheme of the temperature-responsive macroporous structure of the hybrid hydrogels. The enzymatic environment selectively degrades the silk networks in the macroporous hybrid hydrogels, resulting in transformation of macroporous structure to a nonporous material (Figure 7). using a JASCO FT/IR-6200 instrument (Easton, MD). Attenuated total reflectance (ATR) was used for the dried hydrogels. All scans were performed with an average of 32 repeated scans and 4 cm-1 scan resolution. To identify the secondary structure of the silk component in the hybrid structures, Fourier transform selfdeconvolution of the FTIR absorbance spectra in the amide I region (1585-1720 cm-1) was performed using Opus 5.0 software as described previously.45 Swelling. For equilibrium water content and swelling ratio, the dried hydrogels were swollen in deionized water at 20 and 45 C for at least 1 day, removed, wiped with moistened tissue paper, and weighed. The equilibrium swelling ratio was defined as the weight of absorbed water (Ww) per weight of dried gel (Wdry gel). The equilibrium water content is defined as 100 Ww/Wwet gel, where Wwet gel is the weight of wet gel. For the temperature dependence study of deswelling/swelling kinetics, the weight of the hydrogels was monitored by changing the temperature stepwise from 20 to 45 C and then back to 20 C. The hydrogels were taken out of the water at regular intervals and weighed after removing excess water from the hydrogel surface. Temperature dependent weight change of the hydrogels was monitored and then normalized between equilibrium swollen (100%) state at (45) Hu, X.; Kaplan, D.; Cebe, P. Macromolecules 2006, 39, 6161–6170.
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20 C and equilibrium shrunken (0%) state at 45 C. The desired temperature was controlled via a thermostatted water bath (NESLAB RTE-100, Newington, NH) with a temperature stability of (0.01 C. Mechanical Assessments. Mechanical characterization of compression properties of the hydrated hydrogels was determined on an Instron (Norwood, MA) 3366 testing frame equipped with a 0.1 kN load cell. The tests were carried out at room temperature with a conventional open-sided (nonconfined) configuration. The load cell moved at a rate of 5 mm/min. Four samples were tested that were 6 mm in diameter and 3 mm in height. The slopes of the stress-strain curves from initial loading until just beyond initial failure of the samples were used for the calculation of the compressive modulus. The compressive strength was determined using an offset-yield approach. A line was drawn parallel to the modulus line, but offset by 0.5% of the sample gauge length. The corresponding stress value at which the offset line crossed the stress-strain curve was defined as the compressive strength of the hydrogels. Expansion properties of the hydrogels responding to a temperature change were also monitored on an Instron 3366 testing frame equipped with a 0.1 kN load cell. The hydrogel samples (6 mm in diameter and 3 mm in height) were kept in a water bath at 45 C for at least 24 h before test. The shrunken hydrogels were subsequently removed from the water bath and Langmuir 2010, 26(19), 15614–15624
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Figure 2. (A) FTIR absorbance spectra in the amide I and II regions for the macroporous hybrid hydrogels. MeOH (80% in water) treated silk film as a control (i), macroporous hybrid hydrogels with different silk weight fractions: (ii) 0.8, (iii) 0.6, (iv) 0.4, (v) 0.2, and (vi) 0. (B) FTIR absorbance spectra in the amide I region, deduced by deconvolution. then wiped with moistened tissue paper. After measuring the diameter and height of the samples, they were loaded on the equipment. The load cell was approached to the samples and held when it recorded 0.02 N. Subsequently, deionized water at 20 C filled the space between the two load cell plates which were lightly holding the samples. The curves of expansion stress verse time were obtained and normalized to the curves of expansion stress per the loaded sample height verse time. Degradation. The hydrogels were incubated in protease XIV solution (Sigma-Aldrich, 10 unit/mL in 7.4 pH, PBS) for the desired time periods at 37 C. After incubation, the samples were kept at 20 C to be in equilibrium swollen state before image acquisition. Biotin and Avidin Conjugation. Succinimidyl-6-(biotinamido)hexanoate (NHS-LC-Biotin; Pierce, Rockford, IL) was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 20 mM for a stock solution. The stock solution was diluted to 2 mM in phosphatebuffered saline (PBS, pH 8). The hydrogels were suspended in PBS (pH 8.0) and moved to the prepared NHS-LC-Biotin solution (1 mL of 2 mM in PBS, pH 8.0). After 30 min incubation at room temperature, the hydrogels were immersed in PBS (pH 8) for 3 days by changing PBS daily to remove unreacted NHS-LC-Biotin. Subsequently, the hydrogels were transferred to FITC-avidin (Pierce, Rockford, IL) solution (in PBS, pH 8) and incubated for 2 h at room temperature. The reacted hydrogels were immersed in PBS (pH 8) for 3 days by changing PBS daily to remove unreacted FITC-avidin. Langmuir 2010, 26(19), 15614–15624
Morphology. Scanning electron microscopy (SEM) of the dried macroporous hybrid hydrogels was performed by fracturing the samples in liquid nitrogen. Secondary-electron images of fractures were acquired on a Hitachi S-3200N microscope (Tokyo, Japan) at an accelerating voltage of 5 kV. The prepared hydrogels were sufficiently swollen in deionized water and then air-dried before SEM measurement to demonstrate the permanently formed porous features. The hydrogel samples were Au-coated to increase conductivity and reduce charging. In the fabrication process, all samples were lyophilized and treated with MeOH. Then the samples were swollen in deionized water at room temperature before being air-dried. The macroporous hydrogels were visualized by confocal laser scanning microscopy (CLSM) using a Leica (Wetzlar, Germany) DMIRE2 confocal laser scanning microscope with a TCS SP2 scanner equipped with 488 nm argon and 543 nm He/Ne lasers. The CLSM images of the hydrogel matrixes were acquired with 488 nm excitation and 500-700 nm or 460-490 nm emission. The CLSM images of the conjugated FITC-avidin were taken with 488 nm excitation and 520-530 nm emission. Analysis was performed with the Leica Confocal Software (Wetzlar, Germany) and ImagePRO Plus 6.0.
Results and Discussion In the current study, macroporous soft materials were synthesized by chemically cross-linking PNIPAAm networks followed DOI: 10.1021/la102509a
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by the formation of silk protein β-sheet crystalline networks through physical cross-linking (Figure 1). The property of silk proteins, to self-organize and physically cross-link, was exploited to construct these hybrid structures in PNIPAAm hydrogels. We reasoned that two polymers in interpenetrating synthetic/silk networks should govern hybrid material functions. A freezeTable 1. Secondary Structure of Silk in the Macroporous Hybrid Hydrogels with Different Mixing Ratios Determined by Deconvolution silk weight fraction
β-sheet
random coil and R-helix
turns
side chains
27.8 51.2 11.1 10.0 0a 0.1 38.1 35.5 15.4 11.0 0.2 38.8 34.8 15.7 10.6 0.4 40.7 30.0 15.6 13.8 0.6 40.1 30.8 15.3 13.8 0.8 39.4 29.5 18.1 12.9 b 43.7 23.0 20.7 12.6 1 a PNIPAAm and PNIPAAm/SF hybrids determined by Fourier transform self-deconvolution do not represent an accurate measure of secondary structure but were analyzed in order to understand the secondary structure of silk for comparison. b MeOH treated silk film as a control.
drying step was added to the process for morphological control of the system in order to generate macroporous features, followed by solvent treatment to lock in the β-sheet crystalline network via the silk physical cross-linking (Figure 1). The total concentration of silk and NIPAAm monomers in water for the synthesis was 10 w/v%, with the silk weight fraction varied from 0 to 0.8 and PNIPAAm weight fraction from 1 to 0.2, where the sum of both silk and PNIPAAm weight fractions was 1. The hybrid hydrogels are denoted by the silk weight fraction in the PNIPAAm/silk blends, ranging from 0 to 0.8. Secondary Structure of the Silk Component in the Hybrid Structures. To identify the secondary structure of the silk component in the hybrid structures, FTIR absorbance spectra of the hybrid samples and the Fourier transform self-deconvolution in the amide I region (1585-1720 cm-1) were studied (Figure 2). The peaks for β-sheet at 1620 and 1515 cm-1 became more predominant in the spectra of the hybrid hydrogels with higher silk weight fraction (Figure 2A). Deconvolution of the amide I bands to determine the fraction of secondary structural elements, including MeOH treated silk films (as a control), PNIPAAm hydrogel, and the hybrid hydrogels (0.2 and 0.8 silk weight fraction), are shown in Figure 2B. The outer solid pink curve is the deconvoluted amide I band, and the inner small individual
Figure 3. Macroporous structures of the hybrid hydrogels in the dry (by SEM) (A) and wet (by CLSM) states at 20 C (B) or 45 C (C). Scale
bar represents 200 μm. Each CLSM image represents a z-stack average image of 25 sectional images with 3.2 μm interval in a depth of 80 μm. Autofluorescence of silk enables visualization of the CLSM images of the macroporous hybrid hydrogels. (D) Pore size in dry (red square) and hydrated states at 20 C (purple down triangle) and 45 C (blue up triangle) (N = 40). (E) Wall thickness in dry (red square) and hydrated states at 20 C (purple down triangle) and 45 C (blue up triangle) (N = 15). Error bars denote the standard deviation. The pore size and wall thickness were measured by using the ImagePro Plus 6.0 program.
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Figure 4. (A) Equilibrium swelling ratio with different silk fibroin weight fractions at 20 C (red square) and 45 C (blue up triangle) (N = 4). (B) Equilibrium water content with different silk fibroin weight fractions at 20 C (red square) and 45 C (blue up triangle) (N = 4). Error bars denote the standard deviation.
Figure 5. (A) Weight change of the reversibly actuating macroporous hydrogels at below/above the transition temperature, with different compositions (silk fibroin weight fractions: 0 (red square), 0.1 (gold up triangle), 0.2 (green down triangle), 0.3 (blue tilted square), 0.6 (purple circle)) (N = 3). The pure PNIPAAm hydrogels show a slow deswelling rate so the dewelling and swelling kinetics were separately tested for comparison. Error bars denote the standard deviation. (B) Oscillating swelling/deswelling properties of a hybrid macroporous hydrogel (silk weight fraction: 0.3) over 40 s cycles between 20 and 45 C.
curves are Gaussian curve-fitted peaks. The fractional small peaks were assigned to each secondary component, such as β-sheets and random coils, by ref 45, and the amount (%) of the secondary structural element was determined by quantifying the assigned peak area (Table 1). The hybrid hydrogels (even with 0.1 silk fraction) showed significant β-sheet fraction (approximately 40%) similar to that of MeOH treated silk film (43.7%). This implies that the amide bands of the hybrid hydrogels are governed mainly by the silk component, and also that the silk component in all the hybrid hydrogels has as high of a β-sheet content as the MeOH treated silk film control.19,46 Therefore, after methanol treatment, the hybrid PNIPAAm/silk macroporous hydrogels have postinduced silk crystalline networks in the presence of chemically crosslinked PNIPAAm networks. In contrast, a major fraction in the PNIPAAm amide I band is ascribed to random coils (16381646 cm-1) and R-helix (1656-1662 cm-1) (51.2%), although this polymer is not a protein and the assignments are not reflective of true secondary structures. For this reason, we observe somewhat higher random coil and R-helix fractions in the hybrid hydrogels (35.5-29.5%) than in the MeOH treated film (23.0%). Morphology of the Temperature Responsive Macroporous Structures. The morphology of the macroporous structures was investigated in both dry and wet states using SEM and CLSM, respectively (Figure 3A-C). The fracture surfaces via SEM of the hydrated and then air-dried macroporous hybrid (46) Jin, H. J.; Park, J.; Karageorgiou, V.; Kim, U. J.; Valluzzi, R.; Kaplan, D. L. Adv. Funct. Mater. 2005, 15, 1241–1247.
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hydrogels are shown in Figure 3A. After reswelling, PNIPAAm hydrogels were transparent and had no macropores (Figure 3A-C), thus the freeze-drying process apparently did not change the original topology of the PNIPAAm network. Kato and co-workers reported that freeze-dried PNIPAAm hydrogels exhibited accelerated deswelling kinetics and proposed that mesoscopic (2-50 nm) porous networks formed by the freeze-drying treatment may provide channels for fast release of water molecules; however, no porous structures were observed by SEM of the PNIPAAm hydrogels when not swelled.26,27 Given that the collapsed PNIPAAm hydrogels above the LCST contain a low water content comparable to that of the air-dried PNIPAAm hydrogels, the nonporous morphology of the freeze-dried PNIPAAm hydrogels under deswelling conditions supports our results. However, macroporous structures were observed in the hybrid hydrogels (silk plus PNIPAAm) after hydration and then air-drying. Therefore, the macroporous structures in the hybrid hydrogels were preserved and governed by the presence of the physically cross-linked silk networks. The pore structures were controllable with different mixing ratios of silk to PNIPAAm. Pore size (Figure 3D) increased ranging from 110 ( 22 to 272 ( 45 μm and wall thickness (Figure 3E) decreased ranging from 20 ( 9 to 7 ( 2 μm with higher silk weight fractions. All SEM fracture images showed smooth surfaces, demonstrating good miscibility between the two polymers. In order to understand the internal features and the actuator responsiveness of the macroporous soft materials, their real time CLSM morphology in an aqueous environment was studied at 20 DOI: 10.1021/la102509a
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Gil et al. Table 2. Comparison of Various Types of Macroporous PNIPAAm Hydrogels hydrogel size for swelling test
ref
methods
pore size
modulus
this study, (38)
pure PNIPAAm hydrogel
nonporous
30.0 KPaa (27.3 KPab) 17-319 KPac 81-169 KPaa 33-65 KPaa
10 this study 29
time to reach time to reach near equilibrium near equilibrium deswollen size swollen size
6 mm dia/3 mm height (6 mm dia/6 mm height) 12 mm dia/3 mm height 6 mm dia/3 mm height 10 mm dia/20 mm height
∼¥
silk/PNIPAAm IPNs nonporous 80 min 40 s macroporous silk IPNs 129-432 μm 2 min radical polymerization under 33-99 μm freezing temperatures (Cryogel) 6 mm dia./6 mm height 1 min 38 oil-in-water emulsion 1-40 μm 0.8-2.7 KPab 34 DMSO as pore-forming agent ∼5 μm 3 min 30 silica nanoparticles as 10 nm 1 mm dia/10 mm height 10 min pore-forming agent 33 PEO as pore-forming agent ∼20 μm 10 mm dia/3 mm height 20 min 35 surfactant as pore-forming agent 0.5-10 μm 0.8-2.1 KPac a Compressive modulus at RT. b Shear modulus measured by compression at RT. c Oscillatory shear modulus at RT.
and 45 C, respectively (Figure 3B,C). The autofluorescent property of silk enabled the material to be visualized using CLSM.47 The pore size and wall thickness in the wet state were dependent on the silk weight fraction and temperature. Below the LCST (e.g., at 20 C; note that the LCST of all hybrid hydrogels was around 32 C10), the pore sizes varied from 221 ( 49 to 432 ( 127 μm (Figure 3D), while the wall thickness decreased from 57 ( 22 to 15 ( 6 μm with 0.1-0.8 silk weight fraction (Figure 3E). When the temperature was shifted above the LCST (45 C), the pore sizes and wall thickness decreased (Figure 3D,E). However, the extent of decrease was reduced as the weight fraction of silk was increased. The pore sizes ranged from 129 ( 23 to 432 ( 127 μm at 20 and 45 C, respectively. Real time CLSM studies revealed that temperature stimuli dramatically changed the features and dimensions of the internal macropores. Swelling Behaviors of the Temperature Responsive Macroporous Hydrogels. The equilibrium swelling ratio and equilibrium water content of the hydrogels were studied (Figure 4). The hydrogels were swollen for at least 24 h at 20 and 45 C before measuring weights. At 20 C, all hydrogels showed around 90% water content regardless of silk/PNIPAAm composition. We note that the ratio of water to NIPAAm monomer/silk mixture was 9 to 1 (i.e., the water content was 90%) before polymerization and postprocessing. At 20 C, the hydrogels maintained the ratio of water to material (monomer plus silk) prepared during synthesis. However, at 45 C, the equilibrium swelling ratio and water content of the hydrogels varied according to the silk weight fraction. At 45 C, hydrogels with lower silk content exhibited a lower equilibrium swelling ratio and water content. Dynamic deswelling/swelling curves of the macroporous hybrid hydrogels are shown in Figure 5A. The hydrogels swollen to their equilibrium state at 20 C were quickly transferred to a water bath at 45 C, and the weight change was determined for 60 min. The samples were then placed back in a 20 C water bath and monitored until returning to the original equilibrium weight at 20 C. Individual deswelling/swelling curves of pure PNIPAAm hydrogels from 20 and 45 C were studied as controls. Pure silk hydrogels are not stimuli-responsive. PNIPAAm hydrogels showed slow deswelling kinetics when the temperature was increased above the LCST, due to skin layer formation effects as previously described.10,22 Slow swelling kinetics were also observed with these systems, with around 15 h required to recover equilibrium weight at 20 C. In contrast, the macroporous hybrid hydrogels showed accelerated (47) Rice, W. L.; Firdous, S.; Gupta, S.; Hunter, M.; Foo, C. W. P.; Wang, Y.; Kim, H. J.; Kaplan, D. L.; Georgakoudi, I. Biomaterials 2008, 29, 2015–2024.
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15-24 h 15-24 h 40 s 20 min 5 min 3 min 60 min >300 min
Figure 6. Mechanical properties of the hydrated hybrid macroporous hydrogels. (A) Compressive modulus (red square) and yield modulus (blue up triangle) at 20 C (N = 4). Error bars denote the standard deviation. The photo shows the elastic properties of the hybrid macroporous hydrogels (silk weight fraction: 0.3). (B) Dynamic expansion stress during swelling upon temperature change from 45 to 20 C, differing in composition (silk fibroin weight fraction in the hydrogels: (i) 0 (pure PNIPAAm), (ii) 0.2, (iii) 0.3, (iv) 0.4). All samples were prepared in discs (6 mm diameter and 3 mm thickness) in deionized water at room temperature. Error bars correspond to the standard deviation. (C) Schematic of a hybrid macroporous actuating hydrogel system; silk networks act in a positive role as elastic nanosprings to foster the swollen state. Also, in the deswelling process above the LCST, silk networks govern the extent of shrinkage of the hybrid hydrogels. (D) Hybrid macroporous hydrogel ball (9.2 cm diameter below the LCST and 6.8 cm diameter above the LCST) with a 0.3 silk weight fraction. The hydrogel ball shows reversible dimensional change by almost reaching equilibrium shrunken/expanded state in 2 min.
rates of actuation when compared to the pure PNIPAAm hydrogel controls and previously reported nonporous silk/PNIPAAm Langmuir 2010, 26(19), 15614–15624
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Figure 7. CLSM images of hybrid macroporous hydrogels after enzymatic degradation (protease XIV, at 37 C, 10 units/mL) with different silk fibroin weight fractions: (A) 0.1, (B) 0.2, (C) 0.4, and (D) 0.6 at 0, 1, 5, and 14 days. Scale bars represent 300 μm.
IPN hydrogels, with the speed dependent on the mixing ratio with silk. The deswelling rate was faster in the systems containing 0.1 and 0.2 weight fraction of silk, with the enhancement effect of silk reduced with higher silk weight fractions (0.6 and 0.8). In contrast, the swelling rate consistently increased as the content of silk in the system was increased. The complex factors governing the actuating speed of the macroporous hybrid hydrogels include skin layer formation in the deswelling process, increased surface area for mass transfer from the marcroporous structure, the presence of elastic polymer networks, and the actuating force of the PNIPAAm chains. In the deswelling process, mixtures with silk reduce skin layer formation,10 and the porous structure promotes higher surface area. In the deswelling process above the LCST, silk networks govern the extent of shrinkage of the macroporous hybrid hydrogels by reducing the equilibrium weight change with higher silk content (Figure 5A). Although the restriction against shrinkage by the silk network on PNIPAAm movement has an impact, the reduced skin layer formation and the induced porous structure with the silk component provided an overall enhanced swelling speed. The deswelling rate also becomes faster, reaching the equilibrium shrunken state in less than 1 min, independent of the level of silk Langmuir 2010, 26(19), 15614–15624
content. Thus, the macroporous hybrid hydrogels take advantage of the positive effect of the silk while avoiding the negative effect of a high silk weight fraction. In contrast, the compressed silk networks function positively in the swelling process, because these networks want to return to the swollen state and therefore accelerate PNIPAAm network expansion. Therefore, the deswelling rate is maximum at lower silk content, where the macroporous hybrid hydrogels take advantage of the positive effect of the silk and avoid the negative effect of a high silk weight fraction. Unlike in the deswelling process, silk networks act in a positive role as nanosprings to foster the swollen state. This mechanism results in faster expansion of the material system with higher silk weight fractions. Thus, the macroporous hybrid hydrogels with 0.3 silk weight fraction shrinks and swells repetitively at a constant rate by switching the temperature at 40 s intervals between 20 and 45 C (Figure 5B). This is a significant improvement in performance when compared to other macroporous PNIPAAm hydrogels24,25,30-36,38 as well as to our previous nonporous silk/ PNIPAAm hydrogels10 (Table 2). Another important feature with the new systems is that the expansion and contraction speeds were almost symmetrical with each cycle. The hydrogels gained back 100% of their original equilibrium weight at 20 C after DOI: 10.1021/la102509a
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Figure 8. CLSM images of FITC-avidin conjugated hybrid macroporous hydrogels. (A) Schematic depicting sequential conjugation of NHS-LC-biotin and FITC-avidin on the actuating macroporous soft materials. Red color represents the hydrogel matrices (488 nm excitation, 460-490 nm emission), and green color corresponds to the conjugated FITC-avidin (488 nm excitation, 520-530 nm emission). The overlay image of the FITC-avidin reacted PNIPAAm hydrogels revealed no conjugation on the hydrogels (B). CLSM images of macroporous hybrid hydrogel matrices, differing in silk fibroin weight fraction: (C) 0.1, (F) 0.4, and (I) 0.6. CLSM images of the hydrogels conjugated with FITC-avidin (silk fibroin weight fractions: (D) 0.1, (G) 0.4, and (J) 0.6). Overlay images: silk fibroin weight fractions (E) 0.1, (H) 0.4, and (K) 0.6. Scale bar represents 200 μm.
reswelling, and thus, the silk networks were reduced within the linear elastic region without permanent deformation. Mechanical Assessments of the Temperature Responsive Macroporous Hydrogels. The compressive modulus of the macroporous hybrid hydrogels was assessed (Figure 6A). Enhanced mechanical properties were found with a higher weight fraction of silk, with about a 3-fold increase in compressive modulus and compressive stress with a 0.1 silk weight fraction (80.8 KPa) compared to pure PNIPAAm hydrogels (30.0 KPa). These 15622 DOI: 10.1021/la102509a
values plateau up to 0.4 silk weight fraction and then increase up to 168.7 KPa above 0.4. These results indicate that the silk networks in the macroporous hybrid hydrogels enhanced mechanical robustness and macroporous structure. Other fabrication methods to generate porous structures in PNIPAAm hydrogels severely reduced the original PNIPAAm hydrogel modulus. The reported moduli of the macroporous PNIPAAm hydrogels, using either porogen extraction35 or emulsion templating,38 were 0.82.7 KPa. These values are approximately 10-30 times lower than Langmuir 2010, 26(19), 15614–15624
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those of pure PNIPAAm hydrogels and around 30-100 times lower than those of the silk/PNIPAAm macroporous hydrogels with 0.1-0.4 silk weight fraction generated in the present study when measured at room temperature (Table 2). The incorporation of the silk component in the PNIPAAm hydrogels also enhanced the actuating force. The expansion force of the macroporous hybrid and pure PNIPAAm hydrogels during the temperature transition from 45 to 20 C was determined (Figure 6B). The macroporous hybrid hydrogels with 0.2 and 0.3 silk weight fractions exhibited significantly higher expansion stress (10.9 ( 3.2 and 7.9 ( 3.0 KPa over 1 h, respectively) than the PNIPAAm hydrogels (4.9 ( 0.8 KPa). When we consider that the intermolecular interaction of the PNIPAAm networks is the major factor in the hydrogels, with 0.2 (or 0.3) silk fraction or 20% (or 30%) less PNIPAAm than pure PNIPAAm hydrogels, a significantly higher expansion force was found. This can be explained by the more rapid swelling features of the macroporous hybrid gels versus the pure PNIPAAm system. In a macroporous hybrid actuating hydrogel system, silk networks act in a positive role as elastic nanosprings to foster the swollen state as illustrated in Figure 6C. However, the macroporous hybrid hydrogels with 0.4 silk weight fraction displayed reduced expansion stress over 1 h (1.6 ( 0.2 KPa), likely due to the lower content of PNIPAAm. In order to demonstrate a mechanically robust and rapidly actuating larger device, a macroporous hybrid hydrogel ball (9.2 cm in diameter) with 0.3 silk weight fraction was synthesized (Figure 6D). A PDMS replica mold was fabricated from a toy rubber ball (ca. 9.2 cm in diameter), and the macroporous hybrid hydrogels were obtained using this PDMS replica mold. The hydrogel ball obtained in this process showed reversible swelling/ deswelling behavior by reaching equilibrium expansion/contraction (9.2 cm dia/6.8 cm dia.) in 2 min, which is comparable to the results (less than 1 min) obtained from the small sized hydrogel discs above (6 mm diameter and 3 mm height). Considering the time for heat transfer from the hydrogel ball surface to inside in a water bath, bound water molecules rapidly permeate in and out through the interconnected macropores of this larger object. The percent change in diameter of the shrunken size compared to the expanded size of the hydrogel ball was 74%, which is similar to the result (70%) with the small size discs with 0.3 silk weight fraction. The calculated volumes of the macroporous hybrid hydrogel ball in the equilibrium expanded and contracted states were 407.5 and 164.6 cm3, respectively. Biodegradability of the Macroprous Hybrid Hydrogels. Aside from improved actuator function, these new natural/ synthetic blends offer biological function, particularly as the silk component exhibits biodegradability.46,48 Therefore, the morphology of the macroporous hybrid hydrogels was assessed during protease XIV enzymatic degradation (Figure 7). Changes in morphology were evident depending on the initial ratio of the two polymers. With low silk weight fractions (0.1 and 0.2), the macroporous structure disappeared by the first and 14th days, respectively (Figure 7A,B). At higher silk fractions, slower degradation of the macroporous structure occurred. Broadened wall thickness with fewer pores was seen in the case of 0.4 silk weight fraction (i.e., PNIPAAm-rich composition) during degradation (Figure 7C), while diminished pore size was seen during degradation in the case of 0.6 silk fraction (i.e., silk-rich composition) (Figure 7D). These changes in morphology may be related to whether the PNIPAAm network expands to replace the void volume of pores in PNIPAAm-rich composition or the (48) Wang, Y.; Rudym, D. D.; Walsh, A.; Abrahamsen, L.; Kim, H. J.; Kim, H. S.; Kirker-Head, C.; Kaplan, D. L. Biomaterials 2008, 29, 3415–3428.
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pores shrink to occupy the void space due to weak PNIPAAm networks in the silk-rich composition, after the silk degradation. Immobilization of Biotin and Avidin on the Hybrid Hydrogel Macropore Surface. Macroporous structures in soft materials should be useful for biological applications because of the increased interface for interactions with biologically active molecules such as affinity ligands43 and enzymes49 when immobilized on the surface. The macroporous hydrogels were further modified for biomolecule conjugation by using immobilized biotin and avidin, in order to demonstrate the utility of the silk chemistry for further functionalization of these stimuli responsive hydrogel systems (Figure 8). The free primary amines on silk in the hybrid materials allow for facile chemical conjugation of NHS-biotin on the hybrid hydrogel macropore surface.50 After biotin was immobilized, FITC-avidin was conjugated and visualized by CLSM. Fluorescence was not observed in nonporous PNIPAAm hydrogels, due to the absence of the NHS reactive groups, as there are no free primary amine groups in PNIPAAm (Figure 8B). In contrast, FITC-avidin was imaged on the macropore surfaces of all hybrid hydrogels (Figure 8C-K). This affinity platform on the actuating macroporous hydrogels can be further decorated with other biotin-biomolecules, such as enzymes, growth factors, drugs, cell-adhesive proteins, and other polymers, leading to potential switchable or tunable biofunctional interfaces with these biomimetic macroporous soft materials. The engineered macroporous hydrogels also provide useful features for tissue engineering and drug delivery if the PNIPAAm component is found biologically acceptable.51,52 Pore size is an important factor for cell penetration and tissue ingrowth.53 Similarly, the ability to actuate the material could provide a control point for drug release.54
Conclusions Robust, actuating, and biologically functional macroporous soft materials were prepared by exploiting a combination of the unique material features of a synthetic polymer and a biological protein. In preparation of the materials, the unique properties of silk fibroin protein played an important role by forming β-sheet crystals in the porous topology of the hybrid silk/PNIPAAm networks temporarily induced by freeze-drying, thereby promoting stable macroporous structures in the silk/synthetic hybrid hydrogels. Real time CLSM studies revealed that temperature stimuli dramatically changed the features and dimensions of the internal macropores. The equilibrium swelling ratio and equilibrium water content of the macroporous hybrid hydrogels at 20 C were constant regardless of silk/PNIPAAm composition, while the values at 45 C were affected by the silk content. The macroporous hybrid hydrogels showed enhanced thermal-responsive swelling/deswelling kinetics. The silk networks enhanced the macroporous hybrid hydrogel swelling/deswelling behavior mainly due to the convection flow of water through the macropores and the reduced skin layer formation, while the silk networks act as nanosprings to foster the swollen state. The macroporous hybrid hydrogels with 0.3 silk weight fraction showed rapid and stable shrinking and swelling, repetitively, at (49) Yang, X. Y.; Li, Z. Q.; Liu, B.; Klein-Hofmann, A.; Tian, G.; Feng, Y. F.; Ding, Y.; Su, D. S.; Xiao, F. S. Adv. Mater. 2006, 18, 410. (50) Pandori, M. W.; Sano, T. Gene Ther. 2005, 12, 521–533. (51) Huang, X.; Zhang, Y.; Donahue, H. J.; Lowe, T. L. Tissue Eng. 2007, 13, 2645–2652. (52) Levesque, S. G.; Lim, R. M.; Shoichet, M. S. Biomaterials 2005, 26, 7436– 7446. (53) Itala, A. I.; Ylanen, H. O.; Ekholm, C.; Karlsson, K. H.; Aro, H. T. J. Biomed. Mater. Res. 2001, 58, 679–683. (54) Sershen, S.; West, J. Adv. Drug Delivery Rev. 2002, 54, 1225–1235.
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a constant rate by switching the temperature at 40 s intervals between 20 and 45 C. The incorporation of the silk component in the PNIPAAm hydrogels also enhanced the mechanical properties and actuating force. A 10 cm scale actuating device was fabricated and shown to demonstrate swelling/deswelling behavior comparable to millimeter scale macroporous hybrid hydrogels. An enzymatic environment selectively degrades the silk networks in the macroporous hybrid hydrogels, resulting in the transformation of the macroporous structure to a nonporous material morphology. Biotin and avidin were successfully immobilized on the
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macropore surfaces to demonstrate an affinity platform for a switchable or tunable system. These systems are versatile due to the control of composition and biofunctionallity, as well as more dynamically responsive than the single polymer counterparts. These types of materials could offer new options for switchable surfaces and dynamic soft materials for a variety of sensing and related functions. Acknowledgment. We thank the AFOSR and DARPA for support of this work.
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