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Optically Responsive Gold Nanorod-Polypeptide Assemblies Huang-Chiao Huang,† Piyush Koria,‡ Sarah M. Parker,† Luke Selby,‡ Zaki Megeed,‡ and Kaushal Rege*,† Department of Chemical Engineering, Arizona State UniVersity, Tempe, Arizona 85287-6006, The Center for Engineering in Medicine, Massachusetts General Hospital and HarVard Medical School, Boston, Massachusetts 02114 ReceiVed August 29, 2008. ReVised Manuscript ReceiVed September 30, 2008 Environmentally responsive nanoassemblies based on polypeptides and nanoparticles can have a number of promising biological/biomedical applications. We report the generation of gold nanorod (GNR)-elastin-like polypeptide (ELP) nanoassemblies whose optical response can be manipulated based on exposure to near-infrared (NIR) light. Cysteinecontaining ELPs were self-assembled on GNRs mediated by gold-thiol bonds, leading to the generation of GNR-ELP nanoassemblies. Exposure of GNR-ELP assemblies to NIR light resulted in the heating of GNRs due to surface plasmon resonance. Heat transfer from the GNRs resulted in an increase in temperature of the self-assembled ELP above its transition temperature (Tt), which led to a phase transition and aggregation of the GNR-ELP assemblies. This phase transition was detected using an optical readout (increase in optical density); no change in optical behavior was observed in the case of either ELP alone or GNR alone. The optical response was reproducibele and reversible across a number of cycles following exposure to or removal of the laser excitation. Our results indicate that polypeptides may be interfaced with GNRs resulting in optically responsive nanoasssemblies for sensing and drug delivery applications.
Introduction Novel optically responsive nanoassemblies can have promising applications in molecular-scale switching devices,1,2 sensors,3-5 drug delivery systems,6-10 and biomedical imaging modalities.11,12 In particular, the interfacing of proteins/polypeptides with nanoparticles can result in the generation of novel functional nanomaterials for biological/biomedical applications. Gold nanorods (GNRs) demonstrate a tunable photothermal response to near-infrared (NIR) light as a function of nanoparticle aspect ratio13,14 and have been investigated as potential diagnostics,15,16 therapeutic systems,11,17-19 imaging agents,20 and sensors.21-23 The ability to convert incident light energy to heat energy due to surface plasmon resonance activity makes GNRs attractive candidates for modulating polypeptide (or protein) structure/ * Corresponding author. Address: Department of Chemical Engineering, ECG 202, Arizona State University, Tempe, AZ 85287-6006. E-mail:
[email protected]. † Arizona State University. ‡ Massachusetts General Hospital and Harvard Medical School. (1) Ipe, B. I.; Mahima, S.; Thomas, K. G. J. Am. Chem. Soc. 2003, 125(24), 7174–7175. (2) Medintz, I. L.; Trammell, S. A.; Mattoussi, H.; Mauro, J. M. J. Am. Chem. Soc. 2004, 126(1), 30–31. (3) Elghanian, R.; Storhoff, J. J.; Mucic, R. C.; Letsinger, R. L.; Mirkin, C. A. Science 1997, 277(5329), 1078–1081. (4) Storhoff, J. J.; Marla, S. S.; Bao, P.; Hagenow, S.; Mehta, H.; Lucas, A.; Garimella, V.; Patno, T.; Buckingham, W.; Cork, W.; Muller, U. R. Biosens. Bioelectron. 2004, 19(8), 875–883. (5) Mitsuishi, M.; Koishikawa, Y.; Tanaka, H.; Sato, E.; Mikayama, T.; Matsui, J.; Miyashita, T. Langmuir 2007, 23(14), 7472–7474. (6) Hirsch, L. R.; Stafford, R. J.; Bankson, J. A.; Sershen, S. R.; Rivera, B.; Price, R. E.; Hazle, J. D.; Halas, N. J.; West, J. L. Proc. Natl. Acad. Sci. U.S.A. 2003, 100(23), 13549–13554. (7) Wu, C.; Chen, C.; Lai, J.; Chen, J.; Mu, X.; Zheng, J.; Zhao, Y. Chem. Commun. (Cambridge, U.K.) 2008, (23), 2662–2664. (8) Caruso, E. B.; Cicciarella, E.; Sortino, S. Chem. Commun. (Cambridge U.K.) 2007, (47), 5028–5030. (9) Norman, R. S.; Stone, J. W.; Gole, A.; Murphy, C. J.; Sabo-Attwood, T. L. Nano Lett. 2008, 8(1), 302–306. (10) Huang, W.-C.; Tsai, P.-J.; Chen, Y.-C. Nanomedicine 2007, 2(6), 777– 787. (11) Huang, X.; El-Sayed, I. H.; Qian, W.; El-Sayed, M. A. J. Am. Chem. Soc. 2006, 128(6), 2115–2120. (12) Lao, U. L.; Mulchandani, A.; Chen, W. J. Am. Chem. Soc. 2006, 128(46), 14756–14757.
phase transition using optical methods (i.e., NIR light). While the ability to induce irreversible structural change in proteins can play a role in therapeutic applications, the ability to reversibly control protein structure lends flexibility for a variety of applications including site-specific drug delivery, biosensors, and switching. Elastin-like polypeptides (ELPs) are derived from a portion of mammalian elastin characterized by the sequence, VPGXG, where V ) valine, P ) proline, G ) glycine, and X ) any amino acid except proline. ELPs exhibit a thermally induced phase transition at the inverse transition temperature, characterized by reversible intramolecular contraction and intermolecular coacervation.24 The thermal transition behavior of ELPs has been exploited in a number of applications, including bioseparations,25-27 drug delivery,28-32 sensors,33-35 and tissue engineering.36,37 These reports have traditionally employed thermal (13) Lee, K. S.; El-Sayed, M. A. J. Phys. Chem. B 2006, 110(39), 19220– 19225. (14) Lee, K. S.; El-Sayed, M. A. J. Phy.s Chem. B 2005, 109(43), 20331– 20338. (15) El-Sayed, I. H.; Huang, X.; El-Sayed, M. A. Nano Lett. 2005, 5(5), 829– 834. (16) Huang, X.; El-Sayed, I. H.; Qian, W.; El-Sayed, M. A. Nano Lett. 2007, 7(6), 1591–1597. (17) Huang, Y. F.; Chang, H. T.; Tan, W. Anal. Chem. 2008, 80(3), 567–572. (18) Skirtach, A. G.; Dejugnat, C.; Braun, D.; Susha, A. S.; Rogach, A. L.; Parak, W. J.; Mohwald, H.; Sukhorukov, G. B. Nano Lett. 2005, 5(7), 1371–1377. (19) Salem, A. K.; Searson, P. C.; Leong, K. W. Nat. Mater. 2003, 2(10), 668–671. (20) Durr, N. J.; Larson, T.; Smith, D. K.; Korgel, B. A.; Sokolov, K.; BenYakar, A. Nano Lett. 2007, 7(4), 941–945. (21) Sonnichsen, C.; Alivisatos, A. P. Nano Lett. 2005, 5(2), 301–304. (22) Yu, C.; Irudayaraj, J. Anal. Chem. 2007, 79(2), 572–579. (23) York, J.; Spetzler, D.; Xiong, F.; Frasch, W. D. Lab Chip 2008, 8(3), 415–419. (24) Urry, D. W. J. Phys. Chem. B 1997, 101(51), 11007–11028. (25) Meyer, D. E.; Chilkoti, A. Nat. Biotechnol. 1999, 17(11), 1112–1115. (26) Ge, X.; Yang, D. S.; Trabbic-Carlson, K.; Kim, B.; Chilkoti, A.; Filipe, C. D. J. Am. Chem. Soc. 2005, 127(32), 11228–11229. (27) Banki, M. R.; Feng, L.; Wood, D. W. Nat. Methods 2005, 2(9), 659–661. (28) Dreher, M. R.; Liu, W.; Michelich, C. R.; Dewhirst, M. W.; Chilkoti, A. Cancer Res. 2007, 67(9), 4418–4424. (29) Dreher, M. R.; Simnick, A. J.; Fischer, K.; Smith, R. J.; Patel, A.; Schmidt, M.; Chilkoti, A. J. Am. Chem. Soc. 2008, 130(2), 687–694. (30) Chen, T. H.; Bae, Y.; Furgeson, D. Y. Pharm. Res. 2008, 25(3), 683–691.
10.1021/la802842k CCC: $40.75 2008 American Chemical Society Published on Web 11/14/2008
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Scheme 1. Schematic of Optically Responsive GNR-ELP Nanoassemblies
activation as a means of inducing phase transitions in ELPs. However, the ability to remotely and reversibly control the thermal response may be critical in applications such as targeted hyperthermia to tumors that may require precise spatial control in deep-seated tissues. We report GNR-ELP nanoassemblies that demonstrate a reversible optical response following exposure to NIR light (Scheme 1). Energy from NIR light is converted to heat energy by GNRs, which, in turn, is transferred to ELPs, resulting in a reversible phase change in response to optical stimulation. Cysteine-containing ELPs were self-assembled on GNRs mediated by gold-thiol bonds, leading to the generation of GNR-ELP nanoassemblies. Exposure of GNR-ELP assemblies to NIR light resulted in the heating of GNRs due to surface plasmon resonance.38,39 Heat transfer from the laser-irradiated GNRs resulted in an increase in temperature above the transition temperature (Tt) of the self-assembled ELP,24,25,40 leading to aggregation of GNR-ELP assemblies. This was detected using an optical readout (increase in optical density); no change in optical behavior was observed in the case of either ELP alone or GNR alone. The increase in absorbance was reversible following removal of the laser excitation.
Experimental Section 1. Design, Expression, and Purification of C2ELP. C2ELP Gene Synthesis. Cysteine-containing ELPs (C2ELPs) were synthesized via the method of recursive directional ligation,40 yielding the following amino acid sequence, where cysteine residues are shown in bold and underlined font; the name C2ELP denotes the two cysteines that are part of the ELP chain: MVSACRGPG-[VG VPGVG VPGVG VPGVG VPGVG VPG]8-[ VG VPGVG VPGVG VPGCG VPGVG VPG]2-WP Briefly, oligonucleotides encoding the ELP were cloned into pUC19, followed by cloning into a modified version of the pET25b+ expression vector at the sfiI site.40 Escherichia coli BLR(DE3) (Novagen) was used as a bacterial host. C2ELP Expression and Purification. The pET25b+ vector containing the C2ELP cassette was transformed into BLR cells. A starter culture of 50 mL was then inoculated overnight in terrific (31) Bidwell, G. L., III; Fokt, I.; Priebe, W.; Raucher, D. Biochem. Pharmacol. 2007, 73(5), 620–631. (32) Megeed, Z.; Haider, M.; Li, D.; O’Malley, B. W., Jr.; Cappello, J.; Ghandehari, H. J. Controlled Release 2004, 94(2-3), 433–445. (33) Valiaev, A.; Abu-Lail, N. I.; Lim, D. W.; Chilkoti, A.; Zauscher, S. Langmuir 2007, 23(1), 339–344. (34) Fujita, Y.; Funabashi, H.; Mie, M.; Kobatake, E. Bioconjugate Chem. 2007, 18(5), 1619–1624. (35) Megeed, Z.; Winters, R. M.; Yarmush, M. L. Biomacromolecules 2006, 7(4), 999–1004. (36) Janorkar, A. V.; Rajagopalan, P.; Yarmush, M. L.; Megeed, Z. Biomaterials 2008, 29(6), 625–632. (37) Betre, H.; Setton, L. A.; Meyer, D. E.; Chilkoti, A. Biomacromolecules 2002, 3(5), 910–916. (38) Hu, M.; Chen, J.; Li, Z. Y.; Au, L.; Hartland, G. V.; Li, X.; Marquez, M.; Xia, Y. Chem. Soc. ReV. 2006, 35(11), 1084–1094. (39) El-Sayed, M. A. Acc. Chem. Res. 2001, 34(4), 257–264. (40) Meyer, D. E.; Chilkoti, A. Biomacromolecules 2002, 3(2), 357–367.
broth. The next day the 50 mL culture was added to a 1 L culture. The 1 L flasks were then inoculated overnight in an incubator shaker at 250 rpm and 37 °C. Bacterial cells were harvested by centrifugation at 4 °C the next day. The bacterial pellet was resuspended in 1X phosphate buffered saline (PBS), and the cells were disrupted by sonication on ice. The lysate was cleared by centrifugation followed by a polyethyleneimine treatment (final concentration: 0.5% w/v) in order to precipitate soluble nucleic acids. After another round of centrifugation to pellet nucleic acids, the cleared supernatant containing C2ELP was transferred to a clean centrifuge tube. The tube was heated to 40 °C in the presence of 1 M NaCl to precipitate the ELP. A warm centrifugation at 40 °C was carried out to pellet C2ELP. The supernatant was then discarded, and the pellet was resolubilized in PBS in the presence of 10 mM DTT. Another cold spin at 4 °C was performed to get rid of insoluble contaminants. This cycle was repeated two more times. For the final resuspension step, the ELP was resuspended in purified water and then lyophilized and stored at room temperature. C2ELP Characterization. The lyophilized C2ELP was resuspended in 1X PBS. The resuspended material was then subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under denaturing conditions in order to determine the purity of the polypeptide. The gel was then stained with simply blue safe stain (Invitrogen, Carlsbad, CA) to visualize the proteins. As seen in Figure 1a, C2ELP corresponds to a molecular weight of ∼22 kDa. 2. Determination of Tt of C2ELP. The transition temperature (Tt) of C2ELP was characterized by monitoring the absorbance at 610 nm as a function of temperature with a UV-visible spectrophotometer (Beckman DU530) in 0.5X PBS. Briefly, 1 mL of C2ELP was placed in a disposable cuvette. The temperature of C2ELP was tuned by placing the C2ELP-containing cuvette into a Precision 288 Digital Water Bath (Thermo Scientific), which was calibrated using a Digi-Sense type J Thermocouple. The absorbance of C2ELP was monitored at 610 nm with a UV-visible spectrophotometer (Beckman DU530) immediately after withdrawing the cuvette out of the water bath. The Tt is defined as the temperature at which the absorbance of C2ELP solution reaches 50% of the maximum value. The temperature response of the C2ELP indicated a Tt value of 33.4 °C (Figure 1b). Note that we chose absorbance at 610 nm for determining the Tt value since GNRs show the lowest absorbance at this wavelength (Figure S1, Supporting Information), and, as a result, the absorbance of the solution is indicative of turbidity increase due to C2ELP alone. 3. Generation of NIR-Absorbing GNRs. GNRs were synthesized using the seed-mediated method as described by El-Sayed et al.41 Briefly, the seed solution was prepared by adding iced watercooled sodium borohydride (0.01 M) to reduce a solution of 0.2 mL of cetyltrimethylammonium bromide (CTAB) in 0.0005 M auric acid (HAuCl4 · 3H2O). The growth solution was prepared by reducing 0.2 mL CTAB in 0.001 M auric acid (HAuCl4 · 3H2O) containing 0.004 M silver nitrate with 0.0788 M L-ascorbic acid solution. Seed solution (12 µL) was introduced to 10 mL of growth solution, which resulted in the generation of GNRs after 4 h of continuous stirring. This method was employed for generating two different GNRs that possessed absorbance maxima at (λmax) at 710 and 810 nm, (41) Nikoobakht, B.; El-Sayed, M. A. Chem. Mater. 2003, 15(10), 1957– 1962.
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Figure 1. (A) Denaturing SDS-PAGE analysis of C2ELP employed in the current study. The molecular weight of the polypeptide was 22 kDa. (B) Temperature-dependent phase transition of C2ELP as measured by the change in optical density at 610 nm indicates a transition temperature (Tt) of 33.4 °C. (C) Absorption spectra of GNRs generated using the seed-mediated method.41 Two separate populations of GNRs were generated as described in the Experimental Section, and their absorbance was determined as a function of wavelength; the nanorods demonstrated peak absorbances at 710 and 810 nm and are called GNR(λmax ) 710 nm) and GNR(λmax ) 810 nm), respectively. (D) Absorbance spectrum of GNR(λmax ) 710 nm) and GNR(λmax ) 710 nm)-C2ELP nanoassemblies at 25 °C (Tt) following the 5 min NIR exposure. This resulted in a sharp increase in the optical density, indicating that laser powers higher than 350 mW were required for inducing the optical response. The range of laser power (350-650 mW) and/or power densities (5-7 W/cm2) that induce the optical response described in the above experiments is either significantly lower47,49 or comparable50,51 to those used in other reports in the literature on NIR-mediated photothermal activation of gold nanoparticle-based systems. These results are consistent with expected behavior, in that the optical response due to ELP self-assembly was seen beyond the threshold laser (49) Reismann, M.; Bretschneider, J. C.; von Plessen, G.; Simon, U. Small 2008, 4(5), 607–610. (50) Shiotani, A.; Mori, T.; Niidome, T.; Niidome, Y.; Katayama, Y. Langmuir 2007, 23(7), 4012–4018. (51) Jones, C. D.; Lyon, L. A. J. Am. Chem. Soc. 2003, 125(2), 460–465.
power that was necessary to raise the ELP temperature beyond its corresponding Tt. The kinetics of the optical response were then investigated by measuring the absorbance of GNR-C2ELP (λmax ) 720 nm) as a function of time (Figure 2c); in addition, the kinetics of the optical response were correlated with the kinetics of temperature increase (Figure 2d) in order to explain the photothermal effect. Figure 2c shows the optical response of GNR-C2ELP (λmax ) 720 nm), GNR, and C2ELP as a function of laser exposure time. The laser power was fixed at the maximum power (460 mW) used in the previous experiments (Figure 2a) in order to reliably generate the maximal optical response. The optical density of the GNR-C2ELP (λmax ) 720 nm) nanoassemblies increased sharply after 2 min of laser exposure and reached a plateau after 5 min. Although the maximal optical response was observed 5 min after laser exposure, a detectable response was observed after only 2.5 min of laser exposure time. In contrast, no change in optical density was seen either in the case of nanorods alone or C2ELP alone, indicating that the optical response was specific to nanorod-polypeptide assemblies. Figure 3 shows digital snapshots of the time-dependent phase transition and optical response of GNR-C2ELP (λmax ) 720 nm) nanoassemblies as a function of time following laser exposure (laser power ) 460 mW); the respective laser exposure times are shown by the timer in the background. As seen in the figure, the GNR-C2ELP (λmax ) 720 nm) solution continued to be optically clear after 1 min of laser exposure time. However, the solution turbidity increased after 3 min of laser exposure and reached a maximum in 5 min. The optical density remained invariant following 5 min of laser exposure, consistent with the absorbance measurements in Figure 2c. The temperature response of the GNR-C2ELP (λmax ) 720 nm) was determined as a function of time and compared with the response of solutions containing C2ELP alone and nanorods alone. The observed temperatures of the GNR-C2ELP solution were 33.6 °C, 35.5 °C, and 37.4 °C following 3, 4, and 5 min of exposure with 720 nm laser, respectively (Figure 2d). The time required for the solution temperature to increase beyond the transition temperature of C2ELP following NIR irradiation was well correlated with the observed change in solution optical density (Figure 2c). In addition, the kinetics of temperature increase of GNR-C2ELP nanoassemblies closely followed the rise in temperature of GNR alone. Thus, although the temperature of GNRs alone rose faster than GNR-C2ELP nanoassemblies (as may be expected), no change in optical response was observed in the former case (Figures 2c and 2d). No change in solution
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Figure 5. Optical response (in absorbance units at 610 nm) of GNR(λmax ) 810 nm)-C2ELP assemblies. (a) Optical response and (b) temperature response as a function of laser power following 5 min exposure to 810 nm laser. Kinetics of (c) optical and (d) temperature response of GNR-C2ELP assemblies following excitation with an 810 nm laser. Lines connecting the data points are for visualization only.
temperature was observed with C2ELP alone upon exposure to the laser, which explains the lack of optical response in this case. In order to examine whether the observed optical response was reversible, GNR-C2ELP (λmax ) 720 nm) assemblies were subjected to five alternating cycles in which the laser exposure was turned on for 5 min and then turned off for 5 min. Following laser exposure (laser power ) 460 mW) for 5 min, the absorbance of the solution increased up to 1.2 absorbance units (AU), indicating formation of the aggregated nanoassemblies (Figure 4). The optical density of the solution returned to baseline values in approximately 2 min following removal of the laser excitation. This behavior was reproducible over the five cycles tested, indicating that the optical response of the GNR-C2ELP assemblies was indeed reversible and reproducible across multiple cycles. The optical response was also evaluated with another set of GNRs that possessed peak absorbance at 810 nm. In the case of these nanorods, i.e., GNR(λmax ) 810 nm), the laser wavelength was tuned to the maximal peak absorbance of the nanorods (i.e., 810 nm) and not to the peak absorbance of the nanoassemblies. ELP self-assembly was shown to result only in a minor red-shift of the peak absorbance peak (Figure 1d) of the nanorods. As a result, the laser was tuned to the peak absorbance of the nanorods in this case. As with the GNR-C2ELP (λmax ) 720 nm) assemblies, the temperature profile (Figure 5b) of these nanoassemblies closely followed the laser power employed (Figure 5a). The optical response was specific to the GNR-C2ELP nanoassemblies; no change in absorbance was seen with solutions containing C2ELP alone and nanorods alone (Figure 5c). Finally, the optical response of the GNR-C2ELP nanoassemblies closely followed the kinetics of temperature increase (Figure 5d). The increase in temperature in the case of GNR(λmax ) 810 nm)-C2ELP closely followed that of GNR(λmax ) 810 nm) alone.
However, the optical response was seen only in case of the former because of the presence of the self-assembled C2ELP. These results are consistent with those observed with the GNR-C2ELP (λmax ) 720 nm) assemblies, indicating that the optical response can be obtained with nanorods that absorb at different wavelengths of the NIR region of the absorption spectrum, which further expands the application range of these nanoassemblies.
Conclusions We have investigated novel optically responsive polypeptidebased nanoassemblies in which, heat transfer from NIR-absorbing GNRs resulted in a conformational change in the self-assembled ELP, leading to a detectable optical response. Such ability to control nanoscale assembly and nanomaterial properties by optical manipulation can be exploited in the development of novel sensors, drug delivery systems, functional molecular and nanoscale devices, and imaging agents. Acknowledgment. The authors thank Dr. Su Lin, Dr. Laimonas Kelbauskas, and Professor Neal Woodbury, Director, Center for Bio-Optical Nanotechnology at The Biodesign Institute, Arizona State University (ASU), for access to the laser facility. The authors also thank Fred Pena˜ at ASU for invaluable technical assistance. This work was supported by National Institutes of Health Grant 1R21CA133618-01, National Science Foundation Grant CBET0829128, and start-up funds from the state of Arizona to K.R. and a Department of Defense Grant OC060266 to Z.M. Supporting Information Available: Figures showing absorption spectra of GNR-C2ELP (λmax ) 720 nm) nanoassemblies below and above the C2ELP transition temperature (Tt), and the experimental set up. This information is available free of charge via the Internet at http://pubs.acs.org. LA802842K
