J. Phys. Chem. C 2007, 111, 16161-16166
16161
Covalently Immobilizing a Biological Molecule onto a Carbon Nanotube via a Stimuli-Sensitive Bond Ye-Zi You,*,† Chun-Yan Hong,‡ and Cai-Yuan Pan‡ Department of Polymer Science and Engineering, Hefei UniVersity of Technology, Hefei, 230009, Anhui, People’s Republic of China, and Department of Polymer Science and Engineering, UniVersity of Science and Technology of China, Hefei, 230026, Anhui, People’s Republic of China ReceiVed: April 30, 2007; In Final Form: July 23, 2007
Carbon nanotubes can act as new kinds of materials for the delivery of drugs, DNA, protein, RNA, and other biological molecules. However, immobilizing these biological molecules onto carbon nanotubes via stimulisensitive covalent bonds and employing controlled cleaving of these covalent bonds to release biological molecules from carbon nanotube in the presence of stimuli have been seldom explored until now. Here, we reported that simple chemically decorated carbon nanotubes can easily conjugate with biological molecules such as protein. We also demonstrated the ability to release these biological molecules at a late stage in response to biological or chemical stimuli. On the basis of the results of atomic force microscopy and highresolution transmission electron microscopy, this is very important to realize the application of carbon nanotubes to nanomedicine, biotechnology, or drug delivery.
Introduction Recently, it was reported that carbon nanotubes are potential carriers for the delivery of drugs, DNA, protein, RNA, and other biological molecules into cells;1-11 for example, Pantarotto et al. found that carbon nanotubes can transfer protein into a cell without damage of the cell;1 Liu et al. reported that polyethylenimine-grafted multiwalled carbon nanotubes can be used for secure noncovalent immobilization and efficient delivery of DNA;2 Kam et al. prepared biotin-decorated carbon nanotubes (carbon nanotube-biotin), and the streptavidin could go inside the cell when complexed with carbon nanotube-biotin.10 Serving as a delivery material, carbon nanotube is required to be modified for easily conjugating and releasing biological molecules without spoiling their structures.2,12 Two major methods have been used to conjugate protein or DNA onto carbon nanotubes. One is conjugating biological molecules onto carbon nanotubes via noncovalent interactions with functionalized carbon nanotube.2,8,11,13-17 For example, Zheng et al. reported that wrapping carbon nanotube with single-stranded DNA was found to be sequence-dependent;13 Pantarotto et al. found that the ammonium-functionalized nanotube was able to associate with plasmid DNA through electrostatic interaction;11 Kam et al. prepared a phospholipid molecule with a poly(ethylene glycol) (PL-PEG) chain and terminal amine or maleimide group. PL-PEG can be strongly bound to short carbon nanotubes via van der Waals and hydrophobic interactions between the PL alkyl chains and carbon nanotube sidewall, with the PEG chain extending into the aqueous phase to impart solubility of carbon nanotubes in water and the amine or maleimide terminal being used to conjugate with a wide range of biological molecules.3 The other one is covalently bonding biological molecules onto carbon nanotubes.18-20 For example, Huang et al. obtained a carbon nanotube-protein conjugate via * Corresponding author. E-mail:
[email protected]. † Hefei University of Technology. ‡ University of Science and Technology of China.
diimide-activated amidation reaction of the carboxylic acids on the surface of carbon nanotubes with the amino units in protein;21 Williams et al. linked peptide onto carbon nanotube through the displacement of N-hydroxysuccinimide in the presence of EDC;22 Moghaddam et al. functionalized carbon nanotube using azide photochemistry, and DNA oligonucleotides can be synthesized in situ from the reactive group on each photoadduct to produce water-soluble DNA-coated nanotubes.23 Although these methods have provided beautiful tools to conjugate biological molecules onto carbon nanotubes, owing to the broad applications of carbon nanotube-biological molecules in biotechnology and nanomedicine, finding a new and easy method to conjugate biological molecule onto carbon nanotubes via a stimuli-sensitive covalent bond will be advantageous. Recently, Bontempo et al. reported the synthesis of pyridyldithio-ended polymer using an activated disulfide-functionalized ATRP initiator, and this pyridyldithio terminal has high efficiency to conjugate with protein under mild condition.24 Zugates et al. synthesized poly(amido ester) with thiol-reactive sides via Michael-type addition reaction; the thiol-reactive sides in poly(amido ester) were highly reactive toward thiol.25 We obtained thiol-ended poly(N-isopropylacrylamide) (PNIPAMSH) and poly(N-(2-hydroxypropyl) methacrylamide) (PHPMASH) via reversible addition-fragmentation chain transfer (RAFT) polymerization and aminolysis; the pyridyldithio functionalities on the surface of carbon nanotube are highly efficient in directly coupling the thiol end of PNIPAM or PHPMA at room temperature without any catalyst.26 Bulmus et al. incorporated a pyridyldithio unit into different copolymers, and it is convenient to conjugate therapeutic molecules or targeting moieties to these copolymers via disulfide-exchange reaction at mild conditions.27 On the basis of this research, it is obvious that the pyridyldithio functionalities have high reactivity toward thiol without the requirement of elevated temperatures and catalysts; thus, this disulfide-exchange reaction should be very valuable for the conjugation of heat-sensitive sugar, protein,
10.1021/jp073324j CCC: $37.00 © 2007 American Chemical Society Published on Web 10/11/2007
16162 J. Phys. Chem. C, Vol. 111, No. 44, 2007
You et al.
SCHEME 1: Preparation of Pyridyldithio Functionalities Modified MWNT (MWNT-S-S-Py)
targeting peptides, DNA, and antibody ligands. Therefore, the pyridyldithio functionalities decorated carbon nanotubes will be very valuable for conjugating the thiolated biological molecules onto carbon nanotubes via disulfide-exchange at room temperature; moreover, the disulfide linkages between carbon nanotubes and biological molecules are sensitive to biological stimuli (such as glutathione) and chemical stimuli such as dithiothreitol, which can be employed to realize smart release of biological molecules from carbon nanotubes.25,28-35 As far as we know, there are very few reports on conjugating biological molecules or organic molecules onto carbon nanotubes via responsive covalent linkages by far although numerous biological molecules or organic molecules conjugated onto carbon nanotubes via covalent linkages have been reported. Here, we report that the simple chemically decorated carbon nanotubes not only have the ability of effectively conjugating biological molecules (such as bovine serum albumin) onto carbon nanotubes via responsive covalent linkages but also have the ability of smartly releasing conjugated biological molecules in the presence of stimuli. Experimental Section Materials. Multiwalled carbon nanotubes (MWNTs) were purchased from Tsinghua-Nafine NanoPowder Commercialization Engineering Center in Beijing. Aldrithiol-2 (Aldrich, 98%), methanol (99.8%, Aldrich), diethyl ether (g99.0%, SigmaAldrich), 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (crystalline, Sigma-Aldrich), 2-aminoethylthiol hydrochloride (98%, Sigma-Aldrich), acetic acid (g99.7%, SigmaAldrich), reduced glutathione (g99%, Sigma-Aldrich), and bovine serum albumin (BSA, Sigma-Aldrich) were used without further purification. Synthesis of MWNT-S-S-Py. MWNT-COOH (300.00 mg) was dispersed in PBS buffer solution (30.0 mL, pH 7.4) and sonicated for 30 min. 1-[3-(Dimethylamino)propyl]-3ethylcarbodiimide hydrochloride (EDC) (1725.0 mg, 9.0 mmol) and S-(2-aminoethylthio)-2-thiopyridine hydrochloride (2000.0 mg, 9.0 mmol) were added to this buffer solution. The reaction mixture was allowed to stir for 36 h, followed by centrifuging the suspension at 14 500 rpm for 10 min and washing/sonication/centrifugation cycles for five times to remove unreacted S-(2-aminoethylthio)-2-thiopyridine hydrochloride and EDC. The resulting pyridyldithio functionalities modified multiwalled carbon nanotube (MWNT-S-S-Py) was isolated and dried under vacuum at 40 °C for 30 h. Conjugate Protein BSA onto MWNT (MWNT-S-SBSA). MWNT-S-S-Py (10. 0 mg) was dispersed in PBS buffer solution (10.0 mL, pH 7.0) and sonicated for 30 min. BSA (800.0 mg) was added to this buffer solution. The reaction
mixture was allowed to stir for 36 h at room temperature, followed by centrifuging the suspension at 14 500 rpm for 10 min and washing/sonication/centrifugation cycles for five times to remove unreacted BSA. The resulting MWNT conjugated with BSA (MWNT-S-S-BSA) was isolated and dried under vacuum for 30 h. Controlled Release of BSA from MWNT in the Presence of Glutathione. MWNT-S-S-BSA conjugates (2.0 mg) were added into the vials with 20 mM glutathione PBS buffer and 2 µM glutathione PBS buffer, respectively. Then the mixture was sonicated for 5 min. After being stirred for 36 h, MWNTs were isolated by centrifuging the mixture at 14 500 rpm for 20 min and washing/sonication/centrifugation cycles for five times to remove physically absorbed BSA. Measurements. Fourier transform infrared (FT-IR) spectra were recorded on a Bruker VECTOR-22 IR spectrometer. Thermal gravimetric analyses (TGA) were carried out on a PE TGA-7 instrument with a heating rate of 20 °C/min under N2. X-ray photoelectron spectroscopy (XPS) was carried out on an EscalabII instrument. 1H and 13C nuclear magnetic resonance (NMR) spectra were analyzed on a Bruker DMX-500 instrument. Transmission electron microscopy (TEM) images were obtained from a JEOL 2010 electron microscope. Atomic force microscopy (AFM) observations were performed using a NanoScope III MultiMode AFM. Results and Discussion S-(2-Aminoethylthio)-2-thiopyridine hydrochloride reacting with MWNT-COOH under the catalysis of EDC produced the pyridyldithio functionalities decorated MWNT (MWNT-SS-Py) as shown in Scheme 1. In the FT-IR spectrum of MWNT-S-S-Py (see the Supporting Information), the characteristic peak of acylamide at 1650 cm-1 is obvious, but the peak at 1710 cm-1 for the carboxyl unit is absent, indicating that the amidation reaction of S-(2-aminoethylthio)-2-thiopyridine hydrochloride with carboxylic acid on the surface of MWNT occurred. The XPS results of MWNT-S-S-Py are shown Figure 1A-D. The major peak component at the binding energy (BE) of about 285.6 eV is assigned to C1s, the minor peak component at the BE of 532.8 eV is attributable to O1s of acylamide groups on the surface of MWNT, and the small peak at the BE of 399.9 eV corresponds to N1s. In the C1s area, the peak for CdO was found at a BE of 288 eV. In the N1s area, two different N atoms are found: one coming from the pyridyldithio groups and the other coming from acylamides. S2p for the S atom (two kinds of S atoms) in pyridyldithio groups on the surface of MWNTs appeared as the peak at the BE of 163.8 eV. The mole content of the pyridyldithio
Immobilizing a Biomolecule onto a Carbon Nanotube
J. Phys. Chem. C, Vol. 111, No. 44, 2007 16163
Figure 1. XPS spectra of pyridyldithio functionalities decorated MWNT (MWNT-S-S-Py (A), C1s of MWNT-S-S-Py (B), N1s of MWNTS-S-Py (C), S2p of MWNT-S-S-Py (D), MWNT loaded with BSA (MWNT-BSA) (E), C1s of MWNT-BSA (F), N1s of MWNT-BSA (G), and S2p of MWNT-BSA (H).
16164 J. Phys. Chem. C, Vol. 111, No. 44, 2007
You et al.
SCHEME 2: Procedure of Conjugating BSA onto and Controlled Releasing of BSA from the Surface of MWNTs
functionalities on the surface of MWNTs is about 1.7% with respect to carbon. The pyridyldithio functionalities are highly reactive toward thiol through a disulfide-exchange reaction at room temperature. It is well-known that many heat-sensitive biological molecules, such as protein, sugar, antibody ligands, DNA, and targeting peptides, have a thiol unit; therefore, MWNT-S-S-Py will be very valuable to conjugate these biological molecules onto carbon nanotubes. In the experiment, we employed BSA as a model biological molecule. BSA was conjugated onto carbon nanotube simply via stirring MWNT-S-S-Py and BSA in PBS buffer at room temperature. The pyridyldithio functionalities on the surface of the carbon nanotube underwent directly coupling free thiol in BSA, forming BSA and carbon nanotube conjugate with a stimuli-responsive disulfide linkage between BSA and the carbon nanotube. From the released amount of pyridinethione (absorption at 343 nm), ∼66% of the pyridyldithio functionalities on the surface of the carbon nanotube have reacted with BSA (Scheme 2). In the XPS spectra of MWNT-BSA (Figure 1E-H), the peaks at the BE of 400.5 eV corresponding to N1s and BE of 532.8 eV for O1s are much stronger than those of MWNT-S-S-Py due to BSA having many N and O atoms. The strength of C1s for CdO
also increased because BSA has many CdO species. The N1s peak for pyridine is not clear in the N1s area of MWNT-SS-BSA, which resulted from that most pyridine units left from the carbon nanotube after the disulfide-exchange reaction; the changes in the S2p area before and after disulfide-exchange reaction are not obvious. In the FT-IR spectrum of MWNTS-S-BSA (see the Supporting Information), the peaks for C-H at 2820 to ∼2990 cm-1 and the peaks at the range of 1590 to ∼1710 cm-1 for acylamide become obvious. All these facts demonstrated that BSA has been conjugated onto the carbon nanotube. Figure 2, parts A2 and A3, shows representative highresolution TEM (HRTEM) and AFM images of the BSAfunctionalized MWNT via stimuli-responsive linkages. It is clear that there was BSA on the surface of the carbon nanotube in the HRTEM and AFM images. MWNT-S-S-BSA has good stability in PBS buffer as shown in Figure 2A1, and there was no sediment of carbon nanotube found even after 18 days. In the control experiment, some carbon nanotubes settled down to the bottom of vials in several hours if stirring was stopped. Also, we separated carbon nanotube, and no BSA was found on the surface of the separated MWNT based on HRTEM and FT-IR results, indicating that BSA on the surface of MWNT was bonded via a stimuli-responsive disulfide linkage, not
Immobilizing a Biomolecule onto a Carbon Nanotube
J. Phys. Chem. C, Vol. 111, No. 44, 2007 16165
Figure 2. Solubility photos of MWNT-S-S-BSA in PBS buffer after 18 days (A1), MWNT-S-S-BSA in 2 µM glutathione PBS buffer after 36 h (B1), and MWNT loaded with BSA in 20 mM glutathione PBS buffer after 36 h (C1); HRTEM images of MWNT-S-S-BSA (A2), MWNTS-S-BSA treated with 2 µM glutathione PBS buffer for 36 h (B2), and MWNT-S-S-BSA treated with 20 mM glutathione PBS buffer for 36 h (C2); AFM images of MWNT-S-S-BSA (A3), MWNT-S-S-BSA treated with 2 µM glutathione PBS buffer for 36 h (B3), and MWNTS-S-BSA treated with 20 mM glutathione PBS buffer for 36 h (C3). The scale bars are 10 nm.
physically absorbed. All these facts show that the pyridyldithio functionalities on the surface of the carbon nanotube are effective to conjugate protein onto carbon nanotube via the disulfide-exchange reaction. Intracellular glutathione concentrations are sufficient to break the disulfide bond thereby releasing the biological molecules from the surface of the carbon nanotube; however, lower glutathione concentration outside the cell has a minimal effect, which provides a valuable mechanism for smartly releasing the loaded molecules from the carbon nanotube.25 To identify the smart release property, we prepared 20 mM glutathione and 2 µM glutathione solution. MWNT-BSA was treated with 20 mM glutathione and 2 µM glutathione solution for 36 h, respectively. In 20 mM glutathione solution, carbon nanotube sediments were found at the bottom of vial as shown in Figure 2C1, whereas almost no MWNT sediment was found in 2 µM glutathione solution as shown in Figure 2B1, indicating that the covalent linkages between MWNT and BSA are glutathioneresponsive; 20 mM glutathione cleaved the disulfide linkages between BSA and carbon nanotube, whereas 2 µM glutathione has very little effect on the disulfide linkage. Therefore, BSA can be smartly released from the carbon nanotube by glutathione. MWNT-BSA after being treated in 20 mM or 2 µM glutathione solution for 36 h were separated by centrifuge (14 500 rpm) for HRTEM and AFM measurements. It is clear that there is no BSA found on the surface of MWNT after being treated in 20 mM glutathione solution as shown in Figure 2, parts C2 and C3; however, it is obvious that BSA remains on the surface of MWNT after being treated with 2 µM glutathione solution as shown in Figure 2, parts B2 and B3, which further indicates
that the release of BSA from the surface of MWNT can be controlled by the concentration of glutathione. In Conclusion. Pyridyldithio functionalities decorated MWNTs were obtained, which provides an easy method to covalently conjugate protein such as BSA onto MWNT via stimuliresponsive covalent linkages. The protein conjugated on the surface of MWNT can be controlled to release in the presence of a suitable concentration of glutathione. The pyridyldithio functionalities decorated MWNT provides a platform for further conjugating many biological molecules onto carbon nanotubes, which potentially includes the attachment of thiolated targeting ligands such as sugar, peptides, and proteins to provide tissueand cell-specific delivery, thiolated functional polymers to improve serum stability, and other antibody, peptide transduction domains, nuclear localization sequences, and other functionalities to overcome cellular transfection barriers, and the corresponding experiments are in progress. Acknowledgment. This work was supported by Natural Science Foundation of China Grants 50403015 and 20404003. Supporting Information Available: 1H NMR, 13C NMR, FT-IR, and TGA results on products. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Pantarotto, D.; Briand, J. P.; Prato, M.; Bianco, A. Chem. Commun. 2004, 16. (2) Liu, Y.; Wu, D. C.; Zhang, W. D.; Jiang, X.; He, C. B.; Chung, T. S.; Goh, S. H.; Leong, K. W. Angew. Chem., Int. Ed. 2005, 44, 4782.
16166 J. Phys. Chem. C, Vol. 111, No. 44, 2007 (3) Kam, N. W. S.; Liu, Z.; Dai, H. J. J. Am. Chem. Soc. 2005, 127, 12492. (4) Zhang, Z. H.; Yang, X. Y.; Zhang, Y.; Zeng, B.; Wang, Z. J.; Zhu, T. H.; Roden, R. B. S.; Chen, Y. S.; Yang, R. C. Clin. Cancer Res. 2006, 12, 4933. (5) Gao, L. Z.; Nie, L.; Wang, T. H.; Qin, Y. J.; Guo, Z. X.; Yang, D. L.; Yan, X. Y. ChemBioChem 2006, 7, 239. (6) Cai, D.; Mataraza, J. M.; Qin, Z. H.; Huang, Z. P.; Huang, J. Y.; Chiles, T. C.; Carnahan, D.; Kempa, K.; Ren, Z. F. Nat. Methods 2005, 2, 449. (7) Kostarelos, K.; Lacerda, L.; Partidos, C. D.; Prato, M.; Blanco, A. J. Drug DeliVery Sci. Technol. 2005, 15, 41. (8) Singh, R.; Pantarotto, D.; McCarthy, D.; Chaloin, O.; Hoebeke, J.; Partidos, C. D.; Briand, J. P.; Prato, M.; Bianco, A.; Kostarelos, K. J. Am. Chem. Soc. 2005, 127, 4388. (9) Lu, Q.; Moore, J. M.; Huang, G.; Mount, A. S.; Rao, A. M.; Larcom, L. L.; Ke, P. C. Nano Lett. 2004, 4, 2473. (10) Kam, N. W. S.; Jessop, T. C.; Wender, P. A.; Dai, H. J. J. Am. Chem. Soc. 2004, 126, 6850. (11) Pantarotto, D.; Singh, R.; McCarthy, D.; Erhardt, M.; Briand, J. P.; Prato, M.; Kostarelos, K.; Bianco, A. Angew. Chem., Int. Ed. 2004, 43, 5242. (12) Tasis, D.; Tagmatarchis, N.; Bianco, A.; Prato, M. Chem. ReV. 2006, 106, 1105. (13) Zheng, M.; Jagota, A.; Strano, M. S.; Santos, A. P.; Barone, P.; Chou, S. G.; Diner, B. A.; Dresselhaus, M. S.; McLean, R. S.; Onoa, G. B.; Samsonidze, G. G.; Semke, E. D.; Usrey, M.; Walls, D. J. Science 2003, 302, 1545. (14) Tsang, S. C.; Guo, Z. J.; Chen, Y. K.; Green, M. L. H.; Hill, H. A. O.; Hambley, T. W.; Sadler, P. J. Angew. Chem., Int. Ed. 1997, 36, 2198. (15) Guo, Z. J.; Sadler, P. J.; Tsang, S. C. AdV. Mater. 1998, 10, 701. (16) Taft, B. J.; Lazareck, A. D.; Withey, G. D.; Yin, A. J.; Xu, J. M.; Kelley, S. O. J. Am. Chem. Soc. 2004, 126, 12750.
You et al. (17) McKnight, T. E.; Melechko, A. V.; Griffin, G. D.; Guillorn, M. A.; Merkulov, V. I.; Serna, F.; Hensley, D. K.; Doktycz, M. J.; Lowndes, D. H.; Simpson, M. L. Nanotechnology 2003, 14, 551. (18) Baker, S. E.; Cai, W.; Lasseter, T. L.; Weidkamp, K. P.; Hamers, R. J. Nano Lett. 2002, 2, 1413. (19) Dwyer, C.; Guthold, M.; Falvo, M.; Washburn, S.; Superfine, R.; Erie, D. Nanotechnology 2002, 13, 601. (20) McKnight, T. E.; Melechko, A. V.; Hensley, D. K.; Mann, D. G. J.; Griffin, G. D.; Simpson, M. L. Nano Lett. 2004, 4, 1213. (21) Huang, W. J.; Taylor, S.; Fu, K. F.; Lin, Y.; Zhang, D. H.; Hanks, T. W.; Rao, A. M.; Sun, Y. P. Nano Lett. 2002, 2, 311. (22) Williams, K. A.; Veenhuizen, P. T. M.; de la Torre, B. G.; Eritja, R.; Dekker, C. Nature 2002, 420, 761. (23) Moghaddam, M. J.; Taylor, S.; Gao, M.; Huang, S. M.; Dai, L. M.; McCall, M. J. Nano Lett. 2004, 4, 89. (24) Bontempo, D.; Heredia, K. L.; Fish, B. A.; Maynard, H. D. J. Am. Chem. Soc. 2004, 126, 15372. (25) Zugates, G. T.; Anderson, D. G.; Little, S. R.; Lawhorn, I. E. B.; Langer, R. J. Am. Chem. Soc. 2006, 128, 12726. (26) You, Y-Z.; Hong, C-Y.; Pan, C-Y. Macromol. Rapid Commun. 2006, 27, 2001 and AdV. Funct. Mater. 2007, 17, 2470. (27) Bulmus, V.; Woodward, M.; Lin, L.; Murthy, N.; Stayton, P.; Hoffman, A. J. Controlled Release 2003, 93 (2), 105-120. (28) Li, C. M.; Madsen, J.; Armes, S. P.; Lewis, A. L. Angew. Chem., Int. Ed. 2006, 45, 3510. (29) Tsarevsky, N. V.; Matyjaszewski, K. Macromolecules 2002, 35, 9009. (30) Tsarevsky, N. V.; Matyjaszewski, K. Macromolecules 2005, 38, 3087. (31) Li, Y. T.; Lokitz, B. S.; Armes, S. P.; McCormick, C. L. Macromolecules 2006, 39, 2726. (32) Oh, J. K.; Tang, C. B.; Gao, H. F.; Tsarevsky, N. V.; Matyjaszewski, K. J. Am. Chem. Soc. 2006, 128, 5578. (33) Hong, C.-Y.; You, Y.-Z.; Wu, D.-C., Liu, Y.; Pan, C.-Y. J. Am. Chem. Soc. 2007, 129, 5354. (34) Lin, C.; Zhong, Z. Y.; Lok, M. C; Jiang, X. L.; Hennink, W. E.; Feijen, J.; Engbersen, J. F. J. Bioconjugate Chem. 2007, 18, 138. (35) You, Y.-Z.; Manickam, D. S.; Zhou, Q.-H.; Oupicky´, D. Biomacromolecules 2007, 8, 2038.
