Protein-Based Aqueous−Multiphasic Systems - Langmuir (ACS

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Protein-Based Aqueous-Multiphasic Systems Xin Ge, Todd Hoare, and Carlos D. M. Filipe* Department of Chemical Engineering, McMaster University, Hamilton, ON L8S 4L7, Canada Received April 21, 2009. Revised Manuscript Received January 27, 2010 This paper reports the formation of aqueous-multiphasic systems (AMPS) exclusively made using elastin-like polypeptides (ELP) which have the ability to undergo reversible inverse phase transitions. Manipulating variables such as the salt concentration and the molecular weight and the composition of ELPs (using different amino acid sequences or by fusing the ELP with different functional proteins) permits modulation of the temperature at which phase transition takes place, the number of phases that are formed, and the composition of the multiple aqueous phases. Using these variables, isotropic hybrid colloids with tunable functionality (in this case, fluorescent intensity) and anisotropic colloids with variable morphologies could be generated. While formation of AMPS and anisotropic colloids has been reported in the literature using synthetic polymers, to our knowledge this is the first report of generating such systems using proteins.

Introduction Controlling material distributions in ultrasmall scales is a key challenge in nanotechnology. In particular, the formation of nanoparticles with multiple, well-defined compartments is a particular technical challenge which may have significant applications for separations, drug delivery, and other applications. There are currently few reports on synthesis of multicompartment nanoparticles, mostly consisting of biphasic and triphasic Janus droplets generated by simultaneous microfluidic jetting of parallel polymer solutions in an electric field1,2 or through sheath flowing.3-6 To date, fabricated anisotropic nanoparticles are mainly composed of polymers, and no report could be found on building these structures exclusively with proteins. Proteinbased materials have significant advantages over chemically synthesized polymers: (1) the polypeptide sequence and length can be precisely controlled at the genetic level;7 (2) protein-based materials can be produced with very specific and predefined sites for chemical conjugation to other functional molecules; (3) multiple functionality can be incorporated in a single molecule at the gene level by expression of chimeric proteins; (4) protein-based materials are generally biocompatible, which is an essential requirement for biomedical applications such as drug delivery,8 regenerative tissue engineering,9,10 and stem cell differentiation;11 (5) since proteins are expressed in living cells; these structures can *To whom correspondence should be addressed: Tel (905) 525-9140 ext 27278; Fax (905)-525-1350; e-mail [email protected].

(1) Roh, K. H.; Martin, D. C.; Lahann, J. Nat. Mater. 2005, 4(10), 759–763. (2) Roh, K. H.; Martin, D. C.; Lahann, J. J. Am. Chem. Soc. 2006, 128(21), 6796–6797. (3) Nisisako, T.; Torii, T. Adv. Mater. 2007, 19(11), 1489–þ. (4) Pannacci, N.; Bruus, H.; Bartolo, D.; Etchart, I.; Lockhart, T.; Hennequin, Y.; Willaime, H.; Tabeling, P. Phys. Rev. Lett. 2008, 101(16), 4. (5) Nie, Z. H.; Xu, S. Q.; Seo, M.; Lewis, P. C.; Kumacheva, E. J. Am. Chem. Soc. 2005, 127(22), 8058–8063. (6) Utada, A. S.; Lorenceau, E.; Link, D. R.; Kaplan, P. D.; Stone, H. A.; Weitz, D. A. Science 2005, 308(5721), 537–541. (7) Yu, S. J. M.; Conticello, V. P.; Zhang, G. H.; Kayser, C.; Fournier, M. J.; Mason, T. L.; Tirrell, D. A. Nature 1997, 389(6647), 167–170. (8) Meyer, D. E.; Kong, G. A.; Dewhirst, M. W.; Zalutsky, M. R.; Chilkoti, A. Cancer Res. 2001, 61(4), 1548–1554. (9) Ellis-Behnke, R. G.; Liang, Y. X.; You, S. W.; Tay, D. K. C.; Zhang, S.; Schneider, G. E.; So, K. F. Nanomedicine 2006, 2(4), 317. (10) Holmes, T. C.; de Lacalle, S.; Su, X.; Liu, G. S.; Rich, A.; Zhang, S. G. Proc. Natl. Acad. Sci. U.S.A. 2000, 97(12), 6728–6733. (11) Silva, G. A.; Czeisler, C.; Niece, K. L.; Beniash, E.; Harrington, D. A.; Kessler, J. A.; Stupp, S. I. Science 2004, 303(5662), 1352–1355.

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be used in vivo in applications such as the microcompartmentalization of living cells.12 Because of these advantages, protein-based materials are increasingly being used in nanotechnology.13,14 Elastin-like polypeptides (ELP) are protein biopolymers composed of repetitive sequences of the five-amino-acid motif VPGXG, where X, the guest residue, can be any amino acid except proline.15,16 Elastin-like polypeptides are particularly interesting in a nanotechnology context given that they undergo a reversible inverse phase transition in aqueous solutions.16-18 Below a certain temperature, called the phase transition temperature (Tt), aqueous solutions of ELPs are clear and homogeneous. Upon increasing the temperature of the solution above Tt, the solution becomes turbid17,18 due to coacervation of the ELP into droplets. Dynamic light scattering studies revealed that the size of these droplets ranges from submicrometer to a few micrometers.19,20 The size and size distribution of elastin droplets can be manipulated by changing the protein concentration and temperature.19,21 Nanoscale protein particles can also be constructed by fusing ELP with poly(aspartic acid)s.22 After coacervation, the ELP droplets spontaneously aggregate to form aqueous two-phase systems (ATPS),23 i.e., ELP-rich and ELPpoor phases. While these structures are typically only kinetically stable, the addition of surfactants such as SDS can stabilize the emulsion for several hours.23 The reversibility of the phase transition behavior of ELP has the potential to serve as a tool for fabrication of protein-based colloids that are responsive to external stimuli and that can be formed in a reversible manner. (12) Ge, X.; Conley, A. J.; Brandle, J. E.; Truant, R.; Filipe, C. D. M. J. Am. Chem. Soc. 2009, 131(25), 9094–9099. (13) Banta, S.; Megeed, Z.; Casali, M.; Rege, K.; Yarmush, M. L. J. Nanosci. Nanotechnol. 2007, 7(2), 387–401. (14) Sarikaya, M.; Tamerler, C.; Jen, A. K. Y.; Schulten, K.; Baneyx, F. Nat. Mater. 2003, 2(9), 577–585. (15) Urry, D. W.; Trapane, T. L.; Prasad, K. U. Biopolymers 1985, 24(12), 2345– 2356. (16) Urry, D. W. J. Protein Chem. 1988, 7(1), 1–34. (17) Urry, D. W. Prog. Biophys. Mol. Biol. 1992, 57(1), 23–57. (18) Urry, D. W. J. Phys. Chem. B 1997, 101(51), 11007–11028. (19) Meyer, D. E.; Chilkoti, A. Biomacromolecules 2002, 3(2), 357–367. (20) Meyer, D. E.; Trabbic-Carlson, K.; Chilkoti, A. Biotechnol. Prog. 2001, 17 (4), 720–728. (21) Kaibara, K.; Watanabe, T.; Miyakawa, K. Biopolymers 2000, 53(5), 369– 379. (22) Fujita, Y.; Mie, M.; Kobatake, E. Biomaterials 2009, 30(20), 3450–3457. (23) Zhang, Y. J.; Trabbic-Carlson, K.; Albertorio, F.; Chilkoti, A.; Cremer, P. S. Biomacromolecules 2006, 7(7), 2192–2199.

Published on Web 02/12/2010

DOI: 10.1021/la9045463

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Scheme 1. Generation of Aqueous-Multiphasic Systems Using Two Elastin-like Polypeptidesa

(denoted as ELP[V5A2G3-30]). Two initial plasmids, pTrx-ELP20 and pTrx-ELP90, encoding for the production of ELP[V-20] and ELP[V5A2G3-90] fused to the C-terminus of the E. coli protein thioredoxin (Trx), were assembled as previously described.19,20 Starting with these two plasmids and using standard molecular biology protocols, the following additional plasmids were generated: pELP90 encoding for the production of ELP[V5A2G3-90] alone; pELP20 encoding for the production of ELP[V-20] alone; pELP30 encoding for the production of ELP[V5A2G3-30] (a kind gift from Dr. Daniel Yang of Department of Biochemistry of McMaster University); pELP90-GFP encoding for the production of a variant of the green fluorescent protein (GFPuv4) with enhanced fluorescence excitation at 488 nm33 fused to the C terminus of ELP[V5A2G3-90]. The GFPuv4 gene was PCR amplified from the pCA24N34 template using 50 -gtacctgcaggggccgcagtaaaggagaagaa-30 and 50 -catggcatggatgaactatacaaataagcttgcgcc-30 primers.

Production and Purification of Elastin-like Polypeptides and Their Fusion Proteins. E. coli strain BLR (DE3) (Novagen) a ELP90 consists of 90 repeats of the pentapeptide Val-Pro-Gly-XaaGly, where Xaa is Val, Ala, and Gly in the ratio of 5:2:3; ELP20 consists of 20 repeats of the pentapeptide Val-Pro-Gly-Val-Gly.

Changing external conditions or the internal composition of ELP molecules can modify the phase transition temperature of ELPs. External conditions affecting Tt include salt type and concentration,17,24,25 pH,17 pressure,17 and ELP concentration.24,26 The Tt of ELP can be changed by fusing the gene for ELP to the gene for a protein partner,27 by changing the length of ELP,20,26 and/or by changing the amino acid sequence of ELP, especially at the guest residue site.19,28 Most of these parameters have been systematically studied in a quantitative manner17,18,24-29 and may thus be used as tools to generate ELPs with a large variety of Tt. It is also known that simultaneous phase transitions take place for mixtures of ELP and ELP fusion proteins as long as the molecular weight and chemistry of the ELPs are the same,30 a feature that has been used to recover recombinant proteins present at very low concentrations.31,32 On the basis of these facts, we hypothesized that aqueous multiphasic systems can be formed by mixtures of different ELPs; in other words, protein-based anisotropic colloids may be generated. In this paper, we report that isotropic hybrid colloids with varied functional density and anisotropic colloids with different morphologies could be generated in a controllable fashion (as illustrated in Scheme 1) using exclusively polypeptides.

Materials and Methods Plasmids. Three ELPs were used in this study: (1) 20 repeats of the pentapeptide Val-Pro-Gly-Val-Gly (denoted as ELP[V-20]); (2) 90 repeats of the pentapeptide Val-Pro-Gly-Xaa-Gly, where Xaa is Val, Ala, and Gly in the ratio of 5:2:3 (denoted as ELP[V5A2G3-90]); (3) 30 repeats of the pentapeptide Val-Pro-GlyXaa-Gly, where Xaa is Val, Ala, and Gly in the ratio of 5:2:3 (24) Yamaoka, T.; Tamura, T.; Seto, Y.; Tada, T.; Kunugi, S.; Tirrell, D. A. Biomacromolecules 2003, 4(6), 1680–1685. (25) Cho, Y. H.; Zhang, Y. J.; Christensen, T.; Sagle, L. B.; Chilkoti, A.; Cremer, P. S. J. Phys. Chem. B 2008, 112(44), 13765–13771. (26) Meyer, D. E.; Chilkoti, A. Biomacromolecules 2004, 5(3), 846–851. (27) Trabbic-Carlson, K.; Meyer, D. E.; Liu, L.; Piervincenzi, R.; Nath, N.; LaBean, T.; Chilkoti, A. Protein Eng., Des. Sel. 2004, 17(1), 57–66. (28) Urry, D. W.; Luan, C. H.; Parker, T. M.; Gowda, D. C.; Prasad, K. U.; Reid, M. C.; Safavy, A. J. Am. Chem. Soc. 1991, 113(11), 4346–4348. (29) Urry, D. W.; Gowda, D. C.; Parker, T. M.; Luan, C. H.; Reid, M. C.; Harris, C. M.; Pattanaik, A.; Harris, R. D. Biopolymers 1992, 32(9), 1243–1250. (30) Shimazu, M.; Mulchandani, A.; Chen, W. Biotechnol. Bioeng. 2003, 81(1), 74–79. (31) Ge, X.; Filipe, C. D. M. Biomacromolecules 2006, 7(9), 2475–2478. (32) Christensen, T.; Trabbic-Carlson, K.; Liu, W. G.; Chilkoti, A. Anal. Biochem. 2007, 360(1), 166–168.

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was used as the host cell to produce ELP and ELP fusion proteins since its recA deficiency helps stabilizing target plasmids containing repetitive sequences. The plasmids were transformed into the competent cells through the CaCl2 treatment method. The transformed cells were cultivated in rich media, Terrific Broth (12 g/L tryptone, 24 g/L yeast extract, 4 mL/L glycerol, 2.31 g/L KH2PO4, and 12.54 g/L K2HPO4). For protein expression, isopropyl β-thiogalactopyranoside (IPTG) was not used, since it has been shown that ELP and ELP fusion proteins are expressed to very high levels in the absence of IPTG and by extending the cultivation period to 24 h.35,36 The cells were harvested by centrifugation and subsequently resuspended in phosphate buffered saline (PBS) buffer in a 1:25 volumetric ratio. Ultrasonication was used for cell disruption, followed by centrifugation to obtain cell lysate. From cell lysate, ELP proteins were purified through three rounds of inverse transition cycling.20 The purity of the separated ELP proteins was verified by SDS-PAGE. The concentrations of the purified ELP proteins were measured with UV spectrophotometry at a wavelength of 280 nm, using extinction coefficients calculated using the amino acid composition of the expressed proteins. Typical yields for the ELP proteins (fused and nonfused) were more than 50 mg/L of culture media.

Characterization of Phase Transition by Absorbance Measurements as a Function of Temperature. The thermal response of the ELPs, ELP fused proteins, or their mixtures was determined using a Cary 100 UV-vis spectrophotometer equipped with a Peltier multicell temperature controller. The absorbance of the samples was monitored as a function of the solution temperature at a wavelength of 350 nm (OD350). A slow heating rate of 0.5 °C/min was used to minimize kinetic effects while avoiding precipitation of ELP aggregates to the bottom of the cuvette in the time scale of the temperature ramp. The phase transition temperature (Tt) was defined as the temperature at which the absorbance of the sample attained 50% of its maximum value. Preparation of Samples and Microscopic Imaging. A variety of samples consisting of mixtures of ELP/ELP fusion/ NaCl/PBS in different proportions and concentrations were prepared and kept on ice. Before imaging, these samples were exposed to room temperature for at least 5 min. An aliquot consisting of 5 μL of each sample was pipetted to a glass slide for imaging. All microscopic images were taken using a Leica TCS SP5 confocal microscope (Leica Microsystem) equipped with an Acousto-Optical beam splitter and a 63  1.3NA glycerol (33) Ito, Y.; Suzuki, M.; Husimi, Y. Biochem. Biophys. Res. Commun. 1999, 264 (2), 556–560. (34) Kitagawa, M.; Ara, T.; Arifuzzaman, M.; Ioka-Nakamichi, T.; Inamoto, E.; Toyonaga, H.; Mori, H. DNA Res. 2005, 12(5), 291–299. (35) Guda, C.; Zhang, X.; McPherson, D. T.; Xu, J.; Cherry, J. H.; Urry, D. W.; Daniell, H. Biotechnol. Lett. 1995, 17(7), 745–750. (36) Chow, D. C.; Dreher, M. R.; Trabbic-Carlson, K.; Chilkoti, A. Biotechnol. Prog. 2006, 22(3), 638–646.

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Article

Figure 1. Phase transition behavior of mixtures of Trx-ELP[V5A2G3-90] and ELP[V5A2G3-90] at different molar ratios: (A) phase transition profiles; (B) Tt as a function of the Trx-ELP[V5A2G3-90] molar ratio. For all samples, the total concentration of ELP (free and fused to Trx) was 20 μM, and all samples were prepared in PBS buffer. objective lens. The microscope was operated with the Leica Application Suite Advanced Fluorescence software. The laser source for excitation was argon ion (488 nm) for imaging the green fluorescence protein. Visible light microscopy was done using the differential interference contrast (DIC) channel. The fluorescence intensities of ELP[V5A2G3-90]-GFP-associated droplets were measured with the above-mentioned Leica software using a consistent laser power, gain, and focus at the plane of maximum intensity for the individual samples. Measurements were performed for five microphase droplets in each sample, reporting the average and standard deviation of the measured fluorescence intensity.

Results Modulating Tt Required for Colloid Formation Using Mixtures of ELP-Based Proteins. When two different tagged ELP-based polypeptides with different Tt values but the same ELP molecular weight and amino acid sequence are mixed, we hypothesized that the Tt required to induce a phase transition in the mixture should be a function of the proportion at which these two ELP polypeptides are present in that mixture. To test this hypothesis, mixtures of a free ELP[V5A2G3-90] and a thioredoxin-fused ELP with the same molecular weight and composition (Trx-ELP[V5A2G3-90]) were used. The Trx fusion was chosen since it has been previously demonstrated that fusing Trx to ELP results in an increase of Tt as compared to the nonfused ELP tag,37 given that thioredoxin is hydrophilic relative to the ELP tag.27 Samples containing different Trx-ELP[V5A2G3-90]/ ELP[V5A2G3-90] ratios were prepared in PBS, maintaining the total concentration of ELP[V5A2G3-90] (either free or fused to Trx) at 20 μM. The absorbance of these samples as a function of temperature is shown in Figure 1A. Each mixture appears to undergo a single phase transition event comparable to that observed for each individual component. However, mixtures containing higher Trx-ELP[V5A2G3-90] ratios exhibit slightly broader phase transitions (i.e., lower absorbance vs temperature gradients) than either the individual components or mixtures with higher ELP[V5A2G3-90] ratios. This broader phase transition is likely a kinetic effect indicative of slower droplet formation in samples with higher Trx-ELP[V5A2G3-90] ratios; indeed, when a faster heating rate was used (e.g., 1 °C/min or higher), an even broader phase transition starting at a higher temperature was observed. (37) Meyer, D. E.; Chilkoti, A. Nat. Biotechnol. 1999, 17(11), 1112–1115.

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Using the data from Figure 1A, the values for Tt (the temperature at which 50% of the maximum absorbance is obtained) are reported in Figure 1B as a function of the molar percentage of Trx-ELP[V5A2G3-90] in the samples. Tt increases progressively from the Tt associated with free ELP[V5A2G3-90] (49.3 °C) to that associated with Trx-ELP[V5A2G3-90] (56.6 °C). This result reflects the higher hydrophilicity of the Trx protein partner relative to the ELP tag, increasing the enthalpic penalty for losing the water of hydration around the Trx residues and thus increasing the Tt.27 However, for each ratio tested, a single coacervation event occurs in the sample. A single phase transition was also observed with samples of 50% Trx-ELP[V5A2G3-90] and 50% ELP[V5A2G3-90] at different total concentrations of ELP tags varying from 5 to 40 μM (Supporting Information Figure 1). Thus, hybrid coacervation between ELP and ELP fusion appears to occur over a broad range of mixing ratios and concentrations when the ELP tags have the same sequence and length. Separate Phase Transition in Mixtures of ELP with Different Sequences or Lengths. Given the single phase transitions observed in hybrid systems containing ELP tags with the same sequence and length, we were interested to investigate whether single phase transitions were also observed if mixtures of ELPs with different lengths and sequences were used. To this end, ELP[V-20] and ELP[V5A2G3-90] were selected for screening. These two ELP polypeptides differ both in length (20 vs 90 repeat units) and sequence (100% Val compared to 50% Val, 20% Ala, and 30% Gly in the fourth amino acid position in the repeat sequence). To investigate the independence of the ELP[V-20] and ELP[V5A2G3-90] phase transitions, we selected a unique set of experimental conditions (i.e., salt and ELP concentrations) for which the phase transitions resulted in a large increase in absorbance for both ELP[V-20] and ELP [V5A2G3-90] at widely different Tt values. To meet this criteria, we prepared three samples, all in 1.5 M NaCl, containing (1) 60 μM ELP[V-20], (2) 5 μM ELP[V5A2G3-90], and (3) a mixture of 60 μM ELP[V-20] and 5 μM ELP[V5A2G3-90]. The phase transition profiles for these three samples are shown in Figure 2A. For the ELP[V-20]-only and ELP[V5A2G3-90]-only samples, a single sharp increase in absorbance was observed at Tt = 34.7 and 41.8 °C, respectively. When the two proteins were mixed, the phase transition profile of the mixture exhibited two discrete increases of absorbance, indicating that two separate phase transition events took place. The first increase in absorbance started at ∼33 °C and ended at ∼37 °C (Tt = 34.3 °C); this Tt DOI: 10.1021/la9045463

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Figure 2. (A) Two phase transition events take place for a mixture of ELP[V-20] and ELP[V5A2G3-90], which differ both in amino acid sequences and molecular weight. All samples were prepared in 1.5 M NaCl. (B) Two phase transition events take place for a mixture of ELP[V5A2G3-90] and ELP[V5A2G3-30] have the same amino acid sequence but different molecular weights. All samples were prepared in 1 M NaCl.

value is very close to the Tt of a sample consisting of 5 μM ELP[V5A2G3-90] alone (34.7 °C). Moreover, the trajectory over which the absorbance changes with temperature and the absolute value of OD350 at the end of this first stage of coacervation closely matches that of the 5 μM ELP[V5A2G3-90] sample. Together, these results suggest that the first increase in absorbance is due to the single component phase transition of ELP[V5A2G3-90]. A second increase in absorbance occurs between ∼39 and ∼43 °C (Tt = 41.5 °C); in parallel, this Tt value is very close to that of the sample containing 60 μM ELP[V-20] alone (41.8 °C). These results suggest that two individual and sequential phase transition events took place in mixtures of ELP[V5A2G3-90] and ELP[V-20]. To decouple the effects of amino acid sequence (i.e., protein hydrophobicity) and protein length on the sequential phase transitions observed in ELP mixtures, a plasmid was constructed to encode the expression of ELP[V5A2G3-30], a polypeptide which has the same amino acid sequence as ELP[V5A2G3-90] but a lower number of repeat units. The absorbance of a sample comprised of 60 μM of ELP[V5A2G3-30] and 5 μM of ELP[V5A2G3-90] dissolved in 1.0 M NaCl was then monitored as a function of temperature, the results of which are shown in Figure 2B. Again, two independent phase transitions were observed in this system regardless of the fact that the amino acid sequences (and thus hydrophobicity) of both ELPs are identical. This result confirms that mixtures of ELPs with the same amino acid sequence but different molecular weights undergo independent phase transition events in solution. Thus, while simultaneous phase transitions occur between ELP and ELP-tagged molecules with the same ELP sequence and molecular weight, independent phase transition events occur when ELPs with different lengths and/or sequences are used. Analysis of Phase Morphology Using Green Fluorescent Protein-Tagged ELPs. For ELP mixtures resulting in independent phase transitions, multiple droplet populations or complex core-shell or “plum-pudding” morphologies may be formed. To assess how different ELP-based polypeptides are distributed within the droplets, a protein consisting of a fusion of green fluorescent protein (GFP) to the C-terminus of ELP[V5A2G3-90] was produced using E. coli cells. It must be emphasized that while the protein tags of GFP (27 kDa) and thioredoxin (12 kDa) differ in molecular weight, previous studies have demonstrated that GFP and Trx fused with same ELP tags have very similar Tt values under the same conditions (e.g., 52 and 54 °C for 25 μM of GFP- and Trx-fused ELP[V5A2G3-90], respectively, in PBS), a similarity likely attributable to their very similar hydrophobic 4090 DOI: 10.1021/la9045463

Figure 3. Isotropic hybrid colloid formation with ELP[V5A2G390] and ELP[V5A2G3-90]-GFP. (A) Homogeneous ATPS droplets using different ELP[V5A2G3-90]-GFP molar ratios (bar = 5 μm). (B) Average fluorescence intensity of the droplets as a function of the ELP[V5A2G3-90]-GFP molar ratio. For all samples, the total concentration of ELP[V5A2G3-90] (free and fused to GFP) was 40 μM and [NaCl] = 1.5 M. Fluorescent intensities (FI) were obtained by image analysis on five different droplets.

fraction of solvent-accessible surface.27 Therefore, GFP-ELP fusion provides a powerful and relevant imaging system permitting direct investigation of the formation of hybrid colloids. Formation of ELP-Based Isotropic Hybrid Phases with Varying Levels of Functionalization. To confirm that mixtures of tagged and untagged ELPs of the same sequence and length undergo a single phase transition event, confocal microscopy studies were conducted in which the total concentration of ELP[V5A2G3-90] (either free or fused to GFP) was kept constant at 40 μM and the molar percentage of ELP[V5A2G3-90]-GFP was varied between 25% and 100%. Sodium chloride was added to a final concentration of 1.5 M to lower Tt of all samples below room temperature (RT) and enable measurements in the microscope. Results from the imaging experiment are shown in Figure 3A. The images indicate that all the droplets have green fluorescence and that no colorless droplets could be found in these samples (fluorescent and DIC channels for the 50% sample are shown as Supporting Information Figure 3). These results demonstrate the formation of a single population of droplets composed of ELP[V5A2G3-90] and ELP[V5A2G3-90] fused with GFP. Measurement of the fluorescence intensity (FI) of a population of individual droplets indicates that FI is approximately identical for Langmuir 2010, 26(6), 4087–4094

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Article Table 1. Composition of Samples A-D and Associated Phase Transition Temperatures (°C) composition

Tt (°C) associated with

sample

NaCl (M)

ELP[V-20] (μM)

ELP[V5A2G3-90]-GFP (μM)

ELP[V-20]

ELP[V5A2G3-90]-GFP

A B C D

1.5 2.0 1.5 2.0

250 250 60 60

2 2 10 10

23.4 15.5 32.0 23.3

30.4 22.5 23.8 18.1

all droplets in the same sample. This apparent formation of a homogeneous droplet population and the uniformity of the FI inside the droplets provide direct evidence that simultaneous phase transition and formation of isotropic hybrid colloids composed of free ELP and ELP-tagged molecules. The average FIs of the five droplets from the image are plotted in Figure 3B as a function of molar ratio of ELP[V5A2G3-90]GFP in the mixture. It can be seen that the FI of the droplets progressively increases as the molar ratio of ELP[V5A2G3-90]GFP is increased. We would expect that FI would change in a linear fashion with molar ratio of the ELP[V5A2G3-90]-GFP, but that was not the case. The reason for this observation is currently unknown. One possible explanation could be that not all of the ELP90-GFP was incorporated into the micrometer scale droplets visible in the image; measurements of the FI on background regions had higher signals for the 25% and 50% ELP[V5A2G390]-GFP samples. This background signal may be comprised of nonaggregated GFP-tagged ELPs at a low concentration below the aggregation threshold or nanophases of ELP[V5A2G390]GFP sterically stabilized by the hydrophilic GFP tag formed concurrently with the visible microphases; neither population would be detectable by confocal imaging. However, based on the overall trend observed in Figure 3B, coaggregation of different concentrations of proteins or other molecules tagged to the same ELP provides a method to easily modulate the degree of functionalization of a droplet. Formation of ELP-Based Aqueous-Multiphasic Systems. To determine the morphologies of droplets formed via the phase transition(s) of ELPs with different amino acid sequences or molecular weights, emulsions of ELP[V5A2G3-90]GFP and ELP[V-20] mixtures were prepared and imaged using confocal microscopy. Two sets of experimental conditions (protein concentration and NaCl concentration) were targeted: one condition at which one ELP component would undergo a phase transition at the imaging temperature (25 °C) but the other ELP component would not and another condition at which both ELP components underwent phase transition at the imaging temperature. Table 1 shows the four combinations used and the Tt values associated with the sequential phase transitions occurring within these combinations. For example, in sample A, ELP[V-20] at a concentration of 250 μM in 1.5 M NaCl undergoes a phase transition at 23.4 °C (RT). The sequence of the phase transitions as well as the absolute temperatures at which the transitions take place can be modulated by varying the concentration of protein and/or the concentration of salt in the sample. For example, increasing the ELP concentration reduces the phase transition temperature of that ELP species, making it possible to reverse the sequence of phase transition events in a mixture of ELP components; this can be demonstrated by comparing the phase transition temperatures identified in sample A and sample C (Table 1). Alternately, by modulating the salt concentration, the Tt values of both proteins are lowered such that one or both of ELP[V5A2G3-90]-GFP and ELP[V-20] undergo phase transitions Langmuir 2010, 26(6), 4087–4094

Figure 4. Confocal microscopy images showing selective formation of multiple phases in emulsions containing mixtures of ELP[V5A2G3-90]-GFP and ELP[V-20] at different NaCl concentrations (see Table 1). Both the green channel and visible DIC channel are shown. The arrows indicate the droplets formed by ELP[V-20]. Bar = 5 μm.

at the imaging temperature; this can be demonstrated by comparing the phase transition temperatures identified in samples A and B or samples C and D. Thus, protein concentration can be used to control the sequence of transitions while salt concentration can be used to modulate whether a transition will occur or not under the imaging conditions. Figure 4 shows the imaging results in both the fluorescent channel (GFP) and optical channel for all samples A-D in Table 1. For the experimental conditions used for sample A (Figure 4A), all the droplets are colorless while the solution has uniform green florescence, revealing that ELP[V-20] forms droplets while the ELP[V5A2G3-90]-GFP remains soluble in solution. Reversing the order of the transitions by modulating the protein concentration (Figure 4C), all the droplets are fluorescent while the solution has no fluorescence, indicating that ELP[V5A2G390]-GFP forms droplets while ELP[V-20] remains in solution. Thus, it is possible to select any ELP component in the mixture to undergo phase transition to form a colloid while the other component remains in solution. When the NaCl concentration is increased to 2.0 M, the Tt values for both ELP[V5A2G3-90]GFP and ELP[V-20] lie below room temperature (samples B and D), inducing phase transitions in both ELP populations. The imaging results for these samples (Figure 4B,D) show that both ELP90-GFP droplets (green droplets) and ELP[V-20] droplets (colorless droplets, indicated by the arrows) were simultaneously formed. Thus, in this case, two populations of droplets coexist in the emulsion to form a protein-based aqueous triphasic system. It is expected that the finding demonstrated herein can be expanded to multiple phase systems by introducing other species of ELP. Morphology of ELP-Based Anisotropic Colloids. Given this observed potential to produce independent multiphasic colloidal systems in ELP mixtures, the generation of anisotropic colloids by intensive mixing of emulsions composed of both ELP[V5A2G3-90]-GFP and ELP[V-20] droplets is of significant interest. Various concentrations of ELP[V5A2G3-90]-GFP and ELP[V-20] were used to form the emulsions, and intensive vortexing was applied before imaging. A high NaCl concentration DOI: 10.1021/la9045463

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Figure 5. Different morphological shapes are formed after vortexing emulsions composed of ELP20 and ELP[V5A2G3-90]-GFP droplets. Concentration of ELP[V-20]/ELP[V5A2G3-90]-GFP in samples: (A) 0 μM/10 μM; (B, C) 350 μM/8 μM; (D) 60 μM/10 μM; (E) 400 μM/1.5 μM. For all samples, the NaCl concentration was 2.0 M. Bar = 2 μm.

(2.0 M) was chosen to ensure that both ELP species undergo a phase transition at the imaging temperature (25 °C). Figure 5 shows the range of morphologies that can be generated by applying shear to ELP droplet mixtures. While ELP[V5A2G390]-GFP droplets are completely miscible (Figure 5A), ELP[V5A2G3-90]-GFP and ELP[V-20] droplets are immiscible (Figure 5B,C). The coexistence of adjacent droplets (Figure 5B) and fused droplet in geometric parallel patterns (Figure 5C) in the same sample is likely indicative of different shear fields which may be experienced by different droplets during the vortexing step, imparting different kinetic energies to each droplet which result in different colloidal morphologies upon droplet collision. It is also possible that different ELP-GFP phases have different degrees of partitioning of the more hydrophilic GFP tag toward the droplet surface on the kinetic time scale of the collisions, resulting in droplets with slightly different surface energies and thus wetting characteristics upon collision with other droplets. By varying the relative concentrations of ELP[V-20] and ELP[V5A2G3-90]-GFP droplets in the original suspension, 4092 DOI: 10.1021/la9045463

Ge et al.

droplet-in-droplet morphologies can be generated. The order of the phases can be tuned by adjusting the ratios of the two ELPs used to form the droplets; a mixture of 60 μM ELP[V-20] and 10 μM ELP[V5A2G3-90]-GFP generated droplets of ELP[V-20] in a matrix of ELP[V5A2G3-90]-GFP (Figure 5D) while a mixture of 400 μM ELP[V-20] and 1.5 μM ELP[V5A2G3-90]-GFP generated droplets of ELP[V5A2G3-90]-GFP in a matrix of ELP[V-20] (Figure 5E). The formation of hybrid droplet-in-droplet morphologies comprised of the independent ELP[V5A2G3-90] and ELP[V-20] phases in a single anisotropic colloid was further confirmed by radiolabeling experiments in which radiolabeled Trx-ELP[V-20] was successfully trapped into droplets by the addition of ELP[V5A2G3-90] (>95% recovery) but not by the addition of an equal amount of the inert supplement bovine serum albumin (