Hybrid Bioinorganic Smart Membranes That Incorporate Protein

The biomolecular switches employed in this study are elastin-like polypeptides (ELPs), which were genetically engineered to allow for control of molec...
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Langmuir 2002, 18, 1819-1824

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Hybrid Bioinorganic Smart Membranes That Incorporate Protein-Based Molecular Switches G. V. Rama Rao,† S. Balamurugan,† Dan E. Meyer,‡ Ashutosh Chilkoti,*,‡ and Gabriel P. Lo´pez*,† Center for Micro-Engineered Materials, Department of Chemical and Nuclear Engineering, 209 Farris Engineering Building, The University of New Mexico, Albuquerque, New Mexico 87131, and Department of Biomedical Engineering, Box 90281, Duke University, Durham, North Carolina 27708 Received July 27, 2001. In Final Form: November 7, 2001 Polypeptide- and protein-based components have the potential to greatly enhance the functional character of hybrid organic/inorganic materials by imparting recognition, actuation, or transduction properties to these materials. In this study, we demonstrate a simple method of fabricating hybrid silica/polypeptide membranes that exhibit molecular-level control of permeability in response to an external stimulus, namely, heat. The biomolecular switches employed in this study are elastin-like polypeptides (ELPs), which were genetically engineered to allow for control of molecular size and thermal response. ELPs were chosen for incorporation into silica membranes because they exhibit a lower critical solution temperature (LCST) transition in aqueous solution. We demonstrate here that the LCST behavior of ELPs can be retained when they are entrapped in hydrated silica gels and that the LCST transition of these molecules can be used to toggle the permeability of hybrid membranes. Two different ELPs with different LCSTs and different molecular weights (60 and 13 kDa) were examined. They were incorporated into silica membranes using sol-gel synthesis conditions that resulted in the random encapsulation of the proteins in the silica matrixes such that significant porosity is present only upon hydrophobic collapse of the ELPs. Measurement of the permeation of solutions of poly(ethylene glycols) (PEGs) of various molecular weights through centrifugal and ultrafiltration membranes demonstrated that the ELPs in the hybrid membranes act as molecular switches. Below the LCSTs of the ELPs, the hybrid membranes are impermeable to all of the PEG solutions investigated, regardless of the molecular weight of the PEG; above the LCSTs, they are permeable only to those PEGs investigated with molecular weights less than 5000 Da. PEGs investigated of higher molecular weight did not permeate through the hybrid membranes. Thus, the hybrid ELP-containing membranes act as selective molecular weight cutoff filters whose permeability can be switched on and off.

Introduction The development of hybrid functional nanosystems that incorporate biological and inorganic components is a recent trend in materials synthesis.1,2 At the same time, stimuliresponsive polymers have found increased application in the field of biotechnology.3,4 There are several conventional polymers that undergo a transition from hydrophilic to hydrophobic conformations in response to small changes in environmental conditions.3-6 Poly(N-isopropyl acrylamide) (PNIPAAM), one of the most widely studied stimuliresponsive polymers, exhibits a lower critical solution temperature (LCST) transition at 32 °C in aqueous solutions.7 This interesting property has been exploited in various applications, including bioseparations,3-6 control of biofouling,8,9 and enzyme and cell immobilization.10,11 PNIPAAM and other smart polymers have also been used to control fluid and molecular transport in * Corresponding authors. E-mails: [email protected], chilkoti@ duke.edu. † University of New Mexico. ‡ Duke University. (1) Gomez-Romero, P. Adv. Mater. 2001, 13, 163. (2) Avnir, D.; Braun, S.; Lev, O.; Ottolenghi, M. Chem. Mater. 1994, 6, 1605. (3) Galaev, I. Y.; Mattiasson, B. Trends Biotechnol. 1999, 17, 335. (4) Hoffman, A. S. Clin. Chem. 2000, 46, 1478. (5) Galaev, I. Y. Russ. Chem. Rev. 1995, 64, 471. (6) Hoffman, A. S. Mater. Res. Bull. 1991, 42. (7) Schild, H. G. Prog. Polym. Sci. 1992, 17, 163. (8) Ista, L. K.; Lopez, G. P. J. Ind. Microbiol. Biotechnol. 1998, 20, 121. (9) Ista, L. K.; Perez-Luna, V. H.; Lopez, G. P. Appl. Environ. Microbiol. 1999, 65, 1603.

materials and devices in, for example, drug delivery,3,12-14 chromatography,y15,16 and micro-fluidic systems.17 Recently we reported that PNIPAAM can be dispersed in a dense, impermeable silica matrix through a sol-gel process and showed that the transition through the LCST results in the opening of a contiguous porous network of molecular dimensions.18 We carried out permeation experiments using monodisperse poly(ethylene glycol) solutions of various molecular weights and demonstrated the PNIPAAM in hybrid membranes can act as a molecular switch that can be used to control the selective permeability of the membranes.18 Elastin is a protein found in mammalian connective tissue. As the name suggests, it confers elasticity to structures such as skin and blood vessels. Contrary to the behavior of many globular proteins, elastin undergoes an inverse temperature transition, resulting in hydrophobic (10) Yamato, M.; Kwon, O. H.; Hirose, M.; Kikuchi, A.; Okano, T. J. Biomed. Mater. Res. 2001, 55, 137. (11) Kwon, O. H.; Kikuchi, A.; Yamato, M.; Sakurai, Y.; Okano, T. J. Biomed. Mater. Res. 2000, 50, 82. (12) Dong, L. C.; Yan, Q.; Hoffman, A. S. J. Controlled Release 1992, 19, 171. (13) Dong, L. C.; Yan, Q.; Hoffman, A. S. J. Controlled Release 1991, 15, 141. (14) Shin, Y. S.; Chang, J. H.; Liu, J.; Williford, R.; Shin, Y. K.; Exarhos, G. J. J. Controlled Release 2001, 73, 1. (15) Kanazawa, H.; Yamamoto, K.; Matsushima, Y.; Takai, N.; Kikuchi, A.; Sakurai, Y.; Okano, T. Anal. Chem. 1996, 68, 100. (16) Gewher, M.; Nakamura, K.; Ise, N. Makromol. Chem. 1992, 193, 249. (17) Beebe, D. J.; Moore, J. S.; Bauer, J. M.; Yu, A.; Liu, R. H.; Devadoss, C.; Jo, B. H. Nature 2000, 404, 588. (18) Rao, G. V. R.; Lopez G. P. Adv. Mater. 2000, 12, 1692.

10.1021/la011188i CCC: $22.00 © 2002 American Chemical Society Published on Web 01/23/2002

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dehydration; in its thermodynamics, this transition is analogous (and might be identical) to the LCST transition reported for PNIPAAM.19 Elastin-like polypetides (ELPs) are a class of synthetic polypeptides composed of ValPro-Gly-Xaa-Gly repeats (where Xaa is any amino acid with the exception of proline) that exhibit inverse solubility behavior in aqueous solutions as a function of temperature (i.e., LCSTs).19,20 Although the majority of the work on ELPs has been directed toward understanding the thermodynamics of the phase transition, recently, an increasing number of applications that exploit the environmentally responsive, chemomechanical properties of ELPs have been reported.20-26 ELPs are especially attractive as molecular actuators because they are genetically encodable, which enables important macromolecular properties such as sequence, chain length, and stereochemistry to be precisely specified to an extent that is impossible with synthetic polymer analogues.20,21 Control of the macromolecular properties of ELPs is critical because their transition temperatures are known to be sensitive to the primary amino acid sequence and chain length of the ELP.20,21 In applications, this allows one to specify independently the transition temperature and molecular weight as desired. For ELPs of similar compositions, an increase in molecular weight results in a decrease in transition temperature. Alternatively, to vary the LCST at a given molecular weight, one can substitute different amino acids (except proline) at the fourth position of the (Val-Pro-Gly-Xaa-Gly)n sequence. In general, substitution with a more hydrophobic amino acid results in a decrease in the LCST, whereas more hydrophilic residues increase the LCST. This paper describes the synthesis of hybrid materials composed of ELPs encapsulated in silica matrixes by the sol-gel process. In principle, we can choose other matrixes; we chose to use silica because its sol-gel chemistry is well-standardized and procedures for production of dense silica films, which are ideal for permeation studies of hybrid materials, are well-documented. We demonstrate that these hybrid materials can be formed to act as switchable membranes in which permeability can be controlled by the solubility behavior of the ELPs. By dispersing the encapsulated ELP molecules randomly in the silica matrix, it is possible to form “nanovalves” whose molecular permeability can be controlled by cycling through the LCST of the ELP component. The concentration of ELPs in the silica matrix (20 vol %) was chosen such that pores created in the silica matrix upon heating through the LCSTs of the ELPs would exhibit random percolation behavior.27 To understand the effect of the molecular weight on the pore size of the membranes, experiments were carried out on two different ELPs: an ELP with a molecular weight (Mw) of 60 kDa, termed ELP60, and an ELP with a Mw of 13 kDa, termed ELP-13. ELP-60 has a LCST of ∼42 °C, and ELP-13 has a LCST of ∼50 °C at a concentration of 25 µM in phosphate buffer saline (PBS). We note that the LCST of an ELP is not an (19) Urry, D. W. J. Protein Chem. 1988, 7, 1-34 and 81-114. (20) Urry, D. W. J. Phys. Chem. 1997, 101, 11007. (21) Meyer, D. E.; Chilkoti, A. Nature Biotechnol. 1999, 17, 1112. (22) Reiersen, H.; Rees, A. R. Biochemistry 1999, 38, 14897. (23) Luan, C. H.; Parker, T. M.; Prasad, K. U.; Urry, D. W. Biopolymers 1991, 31, 465. (24) Urry, D. W.; Haynes, B.; Zhang, H.; Harris, R. D.; Prasada, K. U. Proc. Nat. Acad. Sci. USA 1988, 85, 3407. (25) Meyer, D. E.; Kong, G. A.; Dewhirst, M. W.; Zalutsky, M. R.; Chilkoti, A. Cancer Res. 2001, 61, 1548. (26) Meyer, D. E.; Shin, B. C.; Kong, G. A.; Dewhirst, M. W.; Chilkoti, A. J. Controlled Release 2001, 74, 213. (27) Lu, Y.; Cao, G.; Kale, R. P.; Prabakar, S.; Lopez, G. P.; Brinker, C. J. Chem. Mater. 1999, 11, 1223.

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absolute parameter but is dependent on both the ELP concentration and the composition and concentration of other cosolutes in solution. The hybrid membranes were characterized by several techniques, and permeation experiments with monodisperse poly(ethylene glycol) solutions of various molecular weights showed that these membranes are capable of acting as switchable molecular weight cutoff filters. Experimental Section Materials. The chemicals used were tetraethyl orthosilicate (TEOS) (Aldrich), bovine serum albumin (BSA, from Sigma), ethyl alcohol (Aaper), crystal violet (Mw ) 408 Da, Fischer Scientific), poly(ethylene glycol) (PEG; polymer standard grade; Mw ) 1000, 2200, 5000, 7850, and 10 000 Da; Mw/Mn ) 1.1; Polymer Source), PEG 9000 (Mw/Mn ) 1.1, Polysciences), and PEG 400 (Aldrich). All chemicals were used as received. Deionized water with a resistivity greater than 18.2 MΩ cm was used for all of the experiments. ELPs were synthesized by the overexpression of a plasmidborne synthetic gene of the ELP in Escherichia coli.21,28 The primary acid sequence of ELP-60 is Ser-Lys-Gly-Pro-Gly-(ValGly-Val-Pro-Gly-Val-Gly-Val-Pro-Gly-Gly-Gly-Val-Pro-Gly-AlaGly-Val-Pro-Gly-Val-Gly-Val-Pro-Gly-Val-Gly-Val-Pro-Gly-ValGly-Val-Pro-Gly-Gly-Gly-Val-Pro-Gly-Ala-Gly-Val-Pro-Gly-GlyGly-Val-Pro-Gly)15-Trp-Pro, and that of ELP-13 is Ser-Lys-GlyPro-Gly-(Val-Gly-Val-Pro-Gly)30-Trp-Pro. Silica sol was prepared by a standard sol-gel process by mixing TEOS, ethanol, water, and HCl in a molar ratio of 1:3:1:0.0007 and allowing the mixture to react at 60 °C for 90 min.18,29 The resulting stock sol was stored at -20 °C until use. For each membrane prepared, 0.25 mL of stock sol was diluted with 0.043 mL of water and 0.6 mL of ethanol, and stirred well to yield a sol with a final ratio of TEOS:ethanol:water:HCl ) 1:20:5.02: 0.0007. One-milliliter samples (4.5 mg/mL) of either ELP-60, ELP-13, or BSA were prepared and added to the diluted stock sol, and the mixture was stirred to obtain a clear, transparent hybrid sols. The concentration of ELP in the silica membranes is 20 vol %. The hybrid sols were coated onto Millipore Microcon centrifugal filter units (diameter, 1.23 cm; active membrane area, 0.32 cm2) with 30 000-Da (YM-30), 50 000-Da (YM-50), and 100 000-Da (YM-100) molecular weight cutoff membranes at 1500-2000 rpm using a spin coater. Sols were also coated onto YM-30 disk membranes of 2.5-cm diameter that were used in pressurized ultrafiltration experiments. To obtain bulk gels, the sol was aged at room temperature until gelation. Characterization. Uncoated and silica/ELP-coated filters were sputter-coated with platinum and characterized by SEM (Hitachi S-800). Silica/ELP composite gels were washed repeatedly in water and subjected to thermal analytical studies to determine the LCST of the ELP in the silica gel matrix. Differential scanning calorimetric studies were carried out using a Universal V2.5H TA instrument. For the measurements, 4050 mg of sample was used. Experiments were carried out in a nitrogen atmosphere with a heating rate of 1 K/min. Silica gel prepared without the ELPs was used as a reference. Silica/ELP sols were also spin-coated onto gold-coated glass substrates. The films thus obtained were used for contact angle measurements using a Rame´-Hart model 100 contact angle goniometer. The sample was placed inside an environmental chamber saturated with water vapor and was heated by circulating water from the temperature-controlled water bath. The temperature inside the chamber was measured with a thermocouple. The sample was equilibrated at a specified temperature for 20 min, and then the contact angle values were measured using a sessile drop method. Permeation Experiments. Permeation experiments were carried out using an IEC Centra CL3R centrifuge at various temperatures ranging from 25 to 40 °C with the temperature controlled to within (1 °C. Figure 1a shows a schematic diagram (28) Meyer, D. E.; Chilkoti, A. Biomacromolecules, in press. (29) Brinker, C. J.; Scherrer, G. W. In Sol-Gel Science: The Physics and Chemistry of Sol- Gel Processing; Academic Press: New York, 1990.

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Figure 1. (A) Schematic representation of centrifugal filtration process indicating the direction of the flow through the membrane. (B) Experimental setup for permeation studies in an ultrafiltration cell. of the centrifugal filtration process. For permeation at higher temperatures (42 °C), an Eppendorf centrifuge (5415C) was equilibrated in an incubator, and centrifugation was carried out with the temperatures controlled to within (1 °C from the set temperatures. In these experiments, 100-300 µL of solute [crystal violet (0.1 wt % aqueous solution), PEGs (1-3 wt % aqueous solutions)] was centrifuged through uncoated, silica/BSA-coated and silica/ELP-coated YM-30, -50, and -100 centrifugal filters. Unless otherwise indicated, permeation experiments were performed at 1000 rpm, equivalent to an estimated centrifugal force of 200 × g (g ) gravities) for 3 min. Initially, the samples were equilibrated at the test temperature in an oven for 10-20 min before being transferred to the centrifuge, which was maintained at the set temperature, and the samples were again equilibrated at that temperature for 5 min before the centrifugation was carried out. The concentration of crystal violet in the filtrate was determined by UV spectrophotometric measurements at 591 nm. For PEG experiments, the concentration of the filtrate was determined from refractive index measurements using a Kernco refractometer by comparison to a calibration curve obtained by measuring the refractive index of known concentrations of PEG. The minimum concentration of PEG that can be measured using our refractometer was determined to be 0.3 wt %. Permeation experiments were also carried out using a standard Amicon stirred ultrafiltration 8003 cell with a capacity of 3 mL. Figure 1b shows the schematic of the ultrafiltration experimental setup. Silica/ELP-coated YM-30 membranes were mounted in the ultrafiltration cell, and nitrogen gas was used to pressurize the cell at various set pressures (20-60 psi). The amount of filtrate collected was weighed and used to estimate the flow rate of filtrate per minute. Refractive index measurements were carried out at room temperature to determine the concentration of PEG in the filtrate.

Results and Discussion To ensure that the spin-coating process produces defectfree overlayers of hybrid protein/silica coatings, the surface morphology of the silica/ELP membranes was observed by SEM (micrographs are shown in Figure 2). Uncoated YM-50 membranes have very large pores connected as channels. YM-50 is a molecular weight cutoff filter; globular proteins whose molecular weights are less than 50 kDa can pass through these membranes, but proteins

Figure 2. Scanning electron micrographs of membranes: (A) planar view of uncoated YM-50, (B) planar view of silica/ELP60-coated on YM-50, and (C) Cross-section of silica/ELP-60coated on YM-50.

with molecular weights greater than 50 kDa cannot.30 Random-coil polymers with molecular weights higher than 50 kDa can also snake through the pores of the membranes.30 After coating of the YM-50 filters, the silica/ ELP membranes were observed to be smooth and devoid of any defects. The thickness of the membranes was found to be ∼2.5 µm. Similar results were obtained for membranes coated onto YM-30 and YM-100 filters. Differential scanning calorimetry (DSC) has been widely employed to study thermally induced structural and phase transitions in biomolecular systems.31,32 Endotherms for (30) Encyclopedia of Separation Technology, Ruthven, D. M., Ed.; John Wiley and Sons: New York, 1997; Vol. 2, p 123. (31) Sturtevant, J. M. Annu. Rev. Phys. Chem. 1987, 38, 463. (32) Privalov, P. L. Adv. Protein Chem. 1979, 33, 167.

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Rao et al. Table 1. Permeation of Crystal Violet Solution through Silica/ELP Membranes Coated on YM-30 Centrifugal Filter Units temperature (°C)

silica/ELP-60a

silica/ELP-13a

25 27 29 31 33 35 37 39 42 45

no no no no no yes yes yes yes yes

no no no no no no no yes yes yes

a Yes indicates that permeation of crystal violet solution can be visually observed. No indicates that water permeation through the membranes is not observed.

Figure 3. Differential scanning thermograms of silica/ELP60 and silica/ELP-13 bulk gels.

Table 2. Permeation of PEG Solutions at 40 °C through Silica/ELP-60 Membranes Coated on YM-30 Centrifugal Filters PEG Mw (Da)

concentration (wt %)

RIa of feed ((0.0002)

RIa of filtrate ((0.0002)

% rejectionb

1000 2200 5000 7850 9000

3.5 2 2 2 3

1.3370 1.3348 1.3352 1.3355 1.3366

1.3370 1.3345 1.3328 1.3330 1.3330

0 0 100 100 100

a RI ) refractive index measured at 25 °C. The RI for pure water is 1.3330. b % rejection ) (concentration of feed - concentration of filtrate) × 100/(concentration of feed).

Table 3. Permeation of PEGs at 40 °C through for Silica/ELP-13 Membranes Coated on Centrifugal Filters PEG Mw (Da)

RIa of feed ((0.0002)

RIa of filtrate ((0.0002)

% rejectionb

1000 2200 5000 7850 9000

1.3352 1.3357 1.3352 1.3358 1.3355

1.3352 1.3355 1.3329 1.3327 1.3329

0 0 100 100 100

a RI ) refractive index. The RI for pure water is 1.3330. b % rejection ) (concentration of feed - concentration of filtrate) × 100/(concentration of feed).

Figure 4. Permeation of crystal violet solutions (200 µL) through (A) silica/BSA and (B) silica/ELP-60 membranes. “Permeation” indicates that the solution permeated through the membrane and the concentrations of the filtrate and the feed were measured to be the same. “No permeation” indicates that not even a trace of water was observed to permeate through the filter. Each data point was obtained after 3 min of centrifugation at the indicated centrifugal force.

silica/ELP-60 and silica/ELP-13 were observed at 33 and 44 °C, respectively (Figure 3), and are attributed to the hydrophilic-to-hydrophobic transition of the polypeptide.20,21,23 The inception temperature of the endotherm33 corresponds to the LCST of the ELP encapsulated in the silica matrix. In contrast to silica/PNIPAAM hybrids,18

we did not observe the corresponding exotherm upon cooling of the sample. However, when the samples were cooled to 5 °C and equilibrated at that temperature for 30 min, we were able to observe the endotherm again during heating. The LCST transition of the encapsulated ELPs was thus found to be essentially reversible, but these results suggest that the reverse process is slow.23 Although several reports of DSC studies on aqueous solutions of ELPs are available, none of these studies has revealed an exotherm corresponding to the LCST during cooling of the ELP samples.23,34-36 Permeation studies were carried out on silica/ELP membranes using solutions of crystal violet and PEG 400. At room temperature, all of the membranes were impermeable to crystal violet or PEG 400 when centrifuged at 200 × g for 3 min. In contrast, when the membranes were centrifuged under similar conditions at 40 °C, they were permeable to both molecules, and the concentrations of (33) Otake, K.; Inomata, H.; Konno, M.; Saito, S. Macromolecules 1990, 23, 283. (34) Luan, C. H.; Parker, T. M.; Gowda, D. C.; Urry, D. W. Biopolymers 1992, 32, 1251. (35) Luan, C. H.; Harris, R. D.; Urry, D. W. Biopolymers 1990, 29, 1699. (36) Rodriguez-Cabello, J. C.; Alonso, M.; Perez, T.; Herguedas, M. M. Biopolymers 2000, 54, 282.

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Table 4. Permeation of PEG Solutions in a Pressurized Ultrafiltration Cell (20 psi) through Silica/ELP-60 Membranes Coated on YM-30 Disk Membranes PEG Mw (Da)

RIa of feed

filtrate flow rate at 25 °C (mg/min)

filtrate flow rate at 40 °C (mg/min)

RIa of filtrate obtained at 40 °C

% rejectionb at 40 °C

1000 2200 5000 7850 10 000

1.3355 1.3355 1.3355 1.3356 1.3356

no flow no flow no flow no flow no flow

5 4.5 3.5 3.5 3

1.3354 1.3352 1.3330 1.3330 1.3330

0 0 100 100 100

a RI ) refractive index measured at 25 °C. The RI for pure water is 1.3330. b % rejection ) (concentration of feed - concentration of filtrate) × 100/(concentration of feed).

Table 5. Permeation of PEG Solutions in an Ultrafiltration Cell at 60 psi through Silica/ELP-60 Membranes Coated on YM-30 Disk Membranes T ) 25 °C

T ) 40 °C

PEG Mw (Da)

RIa of feed

filtrate flow rate (mg/min)

RIa of filtrate

% rejectionb

filtrate flow rate (mg/min)

RIa of filtrate

% rejectionb

1000 2200 5000 7850 10 000

1.3355 1.3352 1.3353 1.3358 1.3356

4 4 3.5 3.5 3

1.3330 1.3330 1.3330 1.3330 1.3330

100 100 100 100 100

17 10 8 8 7

1.3355 1.3350 1.3330 1.3330 1.3330

0 0 100 100 100

a RI ) refractive index measured at 25 °C. The RI for pure water is 1.3330. b % rejection ) (concentration of feed - concentration of filtrate) × 100/(concentration of feed).

crystal violet and PEG in the filtrate and feed solutions were similar. We hypothesize that, below the LCST, the ELPs are in an extended conformation, and permeation of the solution is blocked. Above their LCSTs, the ELPs are in a contracted conformation that creates pores to facilitate permeation. To test this hypothesis, the membranes were tested at various temperatures using crystal violet as the permeating solution to determine the transition temperature for permeation. The permeation transition was found to be 35 °C for silica/ELP-60 and 39 °C for silica/ELP-13 membranes (Table 1). The permeation transition temperature for silica/ELP-60 is thus similar to the LCST obtained from DSC measurements, whereas that for ELP-13 deviates slightly from the value obtained from DSC. The origins of this slight deviation might be related to the substantial differences in the processing conditions between the materials used for DSC and those used for permeation measurements; however, the precise reasons for the deviation are not clear at this time. The permeation measurements clearly indicate that silica/ELP membranes exhibit switchable behavior as a function of solution temperature. The effect of thermal cycling of the membranes between room temperature and 40-45 °C on their permeation characteristics was also studied to investigate the reversibility of the switches and the stability of the membranes. In control experiments, silica and silica/BSA membranes were also tested. Figure 4a shows the permeation results for silica/BSA membranes cycled between 25 and 40 °C. These membranes withstood more than 15 cycles, and no permeation was observed either at room temperature or at high temperatures. Similar behavior was also observed for silica membranes without any proteins. This confirms that the membranes are defect-free and do not exhibit temperature-dependent permeation. In contrast, Figure 4b shows that the silica/ ELP-60 membranes are thermally responsive and strongly suggests that they contain pores that close at room temperature and open at 40 °C. To retain the reversibility of the membranes, however, a gradual reduction in centrifugal force is required (from 200 to 100 × g) beginning with the second cycle, and at 100 × g, the membranes withstood at least 15 cycles. A similar trend was observed for the silica/ELP-13 membranes. Incomplete reversibility of the membranes at higher forces is attributed to either

fatigue of the membrane or incomplete closure of the pores as a result of slow relaxation of ELP chains.23 As discussed above, the reverse transition of ELP is a slow process, as suggested by the lack of an exotherm in DSC during cooling. Even though the silica/ELP membranes were cooled at 8 °C for 10-15 min between thermal cycles, the ELP chains might not have attained their original, extended conformation on this time scale. Similar behavior has also been observed for silica/PNIPAAM membranes.18 Neutron and X-ray scattering experiments are in progress to understand the contraction and relaxation of stimuliresponsive polymers within the silica matrix. Having established the LCST and stability of the silica/ ELP membranes, experiments were carried out to examine the filtration characteristics of the membranes. A series of monodisperse PEGs with Mw ranging from 1000 to 10 000 Da (Mw/Mn ≈ 1.0) was used to determine the sizedependent permeation behavior of these membranes as a function of temperature. As expected, permeation experiments with uncoated filters showed facile passage of each PEG solution. Prior to high-temperature permeation, the silica/ELP membranes were found to be impermeable to all PEG solutions when centrifuged at 800 × g for 1 h at 25 °C. Permeation data for silica/ELP-60 and silica/ELP-13 membranes on YM-30 filters are presented in Tables 2 and 3, respectively. Similar results were obtained for YM-50 and YM-100 filters. The data indicate that these membranes are permeable to PEGs with Mw’s less than 5000 Da and were impermeable to PEGs with molecular weights of 5000 Da and above. Thus, these results indicate that silica/ELP membranes can act as switchable, thermoresponsive molecular filters. Similar cutoff values (i.e.,