Modification of Gold Nanorods Using Phosphatidylcholine to Reduce

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Letters Modification of Gold Nanorods Using Phosphatidylcholine to Reduce Cytotoxicity Hironobu Takahashi,† Yasuro Niidome,*,†,‡ Takuro Niidome,†,‡,§ Kenji Kaneko,| Hideya Kawasaki,⊥ and Sunao Yamada*,†,‡ Department of Materials Physics and Chemistry, Graduate School of Engineering, Kyushu UniVersity, Hakozaki, Fukuoka 812-8581, Japan, Department of Applied Chemistry, Faculty of Engineering, Kyushu UniVersity, Hakozaki, Fukuoka 812-8581, Japan, Center of Future Chemistry, Kyushu UniVersity, Hakozaki, Fukuoka 812-8581, Japan, HVEM Laboratory, Kyushu UniVersity, Hakozaki, Fukuoka 812-8581, Japan, and Department of Chemistry, Faculty of Science, Kyushu UniVersity, Hakozaki, Fukuoka 812-8581, Japan ReceiVed July 23, 2005. In Final Form: October 27, 2005 Hexadecyltrimethylammonium bromide (CTAB), which is necessary for the preparation of gold nanorods (NRs), was extracted from a NR solution into a chloroform phase containing phosphatidylcholine (PC). After three extractions, the zeta potential of the NRs remained positive, but its magnitude decreased from +67 ( 1 to +15 ( 1 mV. Transmission electron microscopy and energy-dispersive X-ray analysis indicated that the NRs were passivated with PC. The PC layer on the NR surface contributed to the prevention of NR aggregation. The PC-passivated NRs showed low cytotoxicity in comparison with twice-centrifuged NRs. It was shown that a negligible amount of CTAB was dispersed in the NR solution after the extraction. The extraction using a chloroform phase containing PC was found to be a convenient way of replacing the CTAB with alternative capping agents such as PC. This is a key technique for preparing functional NRs that can have practical applications.

Introduction Gold nanoparticles show unique optical properties that are not observed in either molecules or bulk materials. A noteworthy characteristic of the nanoparticles is the presence of distinctive absorption bands in the visible region, due to surface plasmon (SP) oscillation of free electrons.1,2 This unique optical property has resulted in extensive research into possible applications of gold nanoparticles such as Raman sensors,3 photocatalysts,4 and photoelectrochemical applications.5,6 For these practical applications, especially for analytical or bio-scientific applications, surface modification of gold nanoparticles with functional molecules is essential. Because the surfaces of gold nanoparticles are easily modified, various kinds of surface modifications have been reported.7,8 For example, modification of gold nanoparticles with thiol-terminal DNA has been used for colorimetric sensing of a single base mismatch of DNA.9,10 Moreover, it has been reported that the cytotoxicity of nanoparticles to cultivated cells * Corresponding authors. (Y.N.) E-mail: [email protected]. Tel: 81-92-642-3581. Fax: 81-92-642-3611. (S.Y.) E-mail: sunaotcm @mbox.nc.kyushu-u.ac.jp. Tel: 81-92-642-3579. Fax: 81-92-642-3679. † Department of Materials Physics and Chemistry, Graduate School of Engineering. ‡ Department of Applied Chemistry, Faculty of Engineering. § Center of Future Chemistry. | HVEM Laboratory. ⊥ Department of Chemistry, Faculty of Science. (1) Hayat, M. A. Colloidal Gold; Academic Press: New York, 1989. (2) Schmidt, G. Chem. ReV. 1992, 92, 170. (3) Tian, Z.-Q.; Ren, B.; Wu, D.-Y. J. Phys. Chem. B 2002, 106, 9463. (4) Kamat, P. V. J. Phys. Chem. B 2002, 106, 7729. (5) Shipway, A. N.; Katz, E.; Willner, I. ChemPhysChem. 2000, 1, 18. (6) (a) Kuwahara, Y.; Akiyama, T.; Yamada, S. Thin Solid Films 2001, 393, 273. (b) Kuwahara, Y.; Akiyama, T.; Yamada, S. Langmuir 2001, 17, 5714. (7) Shenhar, R.; Rotello, V. M. Acc. Chem. Res. 2003, 36, 549. (8) Thomas, K. G.; Kamat, P. V. Acc. Chem. Res. 2003, 36, 888.

depends on characteristics of the molecules adsorbed on the nanoparticle surface.11,12 Connor et al. have clearly shown that the cytotoxicity of gold is negligible.12 Observation of nuclear localization of peptide-modified nanoparticles13 and gene delivery by cationic nanoparticles14-16 has also been realized. Recently, some anisotropic gold nanoparticles have been prepared.17-19 Gold nanorod (NR), a rod-shaped nanoparticle, is a typical example. The NRs have unique optical properties depending on their shapes.20,21 An absorption spectrum of NRs shows two peaks that are assignable to a transverse SP band and a longitudinal SP band (Figure 1).21 The transverse SP band is (9) (a) Mirkin, C. A.; Letsinger, R. L.; Mcic, R. C.; Storhoff, J. J. Nature 1995, 382, 607. (b) Elghanian, R.; Storhoff, J. J.; Mucic, R. C.; Letsinger, R. L.; Mirkin, C. A. Science 1997, 277, 1078. (c) Storhoff, J. J.; Elghanian, R.; Mucic, R. C.; Mirkin, C. A.; Letsinger, R. L. J. Am. Chem. Soc. 1998, 120, 1959. (10) Sato, K.; Hosokawa, K.; Maeda, M. J. Am. Chem. Soc. 2003, 125, 8102. (11) Goodman, C. M.; McCusker, C. D.; Yilmaz, T.; Rotello, V. M. Bioconjugate Chem. 2004, 15, 897. (12) Connor, E. E.; Mwamuka, J.; Gole, A.; Murphy, C. J.; Wyatt, M. D. Small 2005, 1, 325. (13) Tkachenko, A. G.; Xie, H.; Liu, Y.; Coleman, D.; Ryan, J.; Glomm, W. R.; Shipton, M. K.; Franzen, S.; Feldheim, D. L. Bioconjugate Chem. 2004, 15, 482. (14) Sandhu, K. K.; McIntosh, C. M.; Simard, J. M.; Smith, S. W.; Rotello, V. M. Bioconjugate Chem. 2002, 13, 3. (15) Niidome, T.; Nakashima, K.; Takahashi, H.; Niidome, Y. Chem. Commun. 2004, 1978. (16) Thomas, M.; Klibanov, A. M. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 9138. (17) Chen, S.; Wang, Z. L.; Ballato, J.; Foulger, S. H.; Carroll, D. L. J. Am. Chem. Soc. 2003, 125, 16186. (18) Sau, T. K.; Murphy, C. J. J. Am. Chem. Soc. 2004, 126, 8648. (19) Kuo, C.-H.; Huang, M. H. Langmuir 2005, 21, 2012. (20) Yu, Y.-Y.; Chang, S.-S.; Lee, C.-L.; Wang, C. R. C. J. Phys. Chem. B 1997, 101, 6661. (21) (a) Link, S.; Mohamed, M. B.; El-Sayed, M. A. J. Phys. Chem. B 1999, 103, 3073. (b) Brioude, A.; Jiang, X. C.; Pileni, M. P. J. Phys. Chem. B 2005, 109, 13138.

10.1021/la0520029 CCC: $33.50 © 2006 American Chemical Society Published on Web 11/24/2005

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Figure 1. A TEM image (A) and an absorption spectrum (B) of NRs in the initial solution.

located in the visible region at around 520 nm, whereas the longitudinal band is located in the near-infrared (near-IR) region. Thus, NRs are unusual materials with an intense absorption band in the near-IR region.22-24 Because the near-IR light is hardly absorbed by tissue, the optical properties of NRs are expected to offer novel applications in the bioscience field. Despite their unique characteristics, NRs have hardly ever been used in the bioscience field. This is due to hexadecyltrimethylammonium bromide (CTAB), which is a cationic detergent used as a stabilizing agent in the preparation of NRs.20,25,26 Nikoobackt and El-Sayed27 and Gao et al.28 have discussed characteristics of the CTAB layers on the surfaces of NRs. Their works indicated that the CTAB formed bilayers on NRs, and that the bilayers were essential for the anisotropic growth of NRs. On the other hand, the CTAB bilayer is obstructive for surface modification of NRs. Moreover, the large amount of CTAB dispersed in the solution interferes with biological processes and shows high cytotoxicity.12,29,30 The toxicity is not due to the CTAB layers that are statically bound to the nanoparticles, as Connor et al. have quantitatively discussed in their paper.12 They have shown that 1 µM of CTAB-passivated nanoparticles did not cause cytotoxicity to cultivated cells after three times of centrifugation. Their experiments proved that the unbound CTAB was toxic to cultivated cells. Thus, to obtain “biocompatible” NRs, excess CTAB other than that in the bilayers on NR surfaces should be removed. Repeated centrifugation to decrease the excess CTAB has already been reported.23,26,31 After the centrifugation, partial modification of the NRs is possible.32-34 In all cases, however, the CTAB bilayers remained on the NR surfaces. The CTAB bilayers on the NR surfaces are not static; in other words, (22) (a) Link, S.; Burda, C.; Nikoobakht, B.; El-Sayed, M. A. J. Phys. Chem. B 2000, 104, 6152. (b) Nikoobakht, B.; El-Sayed, M. A. J. Phys. Chem. A 2003, 107, 3372. (23) Niidome, Y.; Takahashi, H.; Urakawa, S.; Nishioka, K.; Yamada, S. Chem. Lett. 2004, 33, 454. (24) Gorelikov, I.; Field, L. M.; Kumacheva, E. J. Am. Chem. Soc. 2004, 126, 15938. (25) Kim, F.; Song, J. H.; Yang, P. J. Am. Chem. Soc. 2002, 124, 14316. (26) (a) Jana, N. R.; Gearheart, L.; Murphy, C. J. J. Phys. Chem. B 2001, 105, 4065. (b) Jana, N. R.; Gearheart, L.; Murphy, C. J. AdV. Mater. 2001, 13, 1389. (c) Jana, N. R. Chem. Commun. 2003, 1950. (d) Busbee, B. D.; Obare, S. O.; Murphy, C. J. AdV. Mater. 2003, 15, 414. (27) Nikoobakht, B.; El-Sayed, M. A. Langmuir 2001, 17, 6368. (28) Gao, J.; Bender, C. M.; Murphy, C. J. Langmuir 2003, 19, 9065. (29) Cortesi, R.; Esposito, E.; Menegatti, E.; Gambari, R.; Nastruzzi, C. Int. J. Pharm. 1996, 139, 69. (30) Mirska, D.; Schirmer, K.; Funari, S. S.; Langner, A.; Dobner, B.; Brezesinski, G. Colloids Surf. B 2005, 40, 51. (31) Hsieh, S.; Meltzer, S.; Wang, C. R. C.; Requicha, A. A. G.; Thompson, M. E.; Koel, B. E. J. Phys. Chem. B 2002, 106, 231. (32) Caswell, K. K.; Wilson, J. N.; Bunz, U. H. F.; Murphy, C. J. J. Am. Chem. Soc. 2003, 125, 13914. (33) (a) Thomas, K. G.; Barazzouk, S.; Ipe, B. I.; Joseph, S. T. S.; Kamat, P. V. J. Phys. Chem. B 2004, 108, 13066. (b) Sudeep, P. K.; Joseph, S. T. S.; Thomas, K. G. J. Am. Chem. Soc. 2005, 127, 6516. (34) Chang, J.-Y.; Wu, H.; Chen, H.; Ling, Y.-C.; Tan, W. Chem. Commun. 2005, 1092.

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the CTAB molecules can desorb from the NR surfaces. Thus, even if it were possible to remove all of the unbound CTAB from a NR solution, some CTAB would be provided to the solution from the NR surfaces. Further removal of the desorbed CTAB will result in aggregation of the NRs because of the lack of a repulsive interaction between the NRs. For these reasons, it is hard to eliminate all of the unbound CTAB without aggregation of NRs, if the NRs are passivated only with CTAB molecules. Thus, replacement of the CTAB with alternative stabilizing agents is essential for the realization of functional NR probes that can be used in cultivated cells as concentrated NR solutions. In this paper, we will discuss a method for replacing CTAB with phospholipids by extraction using a chloroform phase. This is a key technique for designing practically useful NRs for biochemical applications. Experimental Section NRs, prepared with a slight modification of our method,35 were supplied by Mitsubishi Materials Co. Ltd. The average length and width of the as-prepared NRs was +65 ( 5 nm and +11 ( 1 nm, respectively (aspect ratio: 5.9) (Figure 1A). The initial NR solution (∼1 mM (Au atoms)) contained 80 mM of CTAB. Some of the CTAB precipitated when the solution was kept in a refrigerator (∼4 °C). The precipitated CTAB was removed by using a membrane filter (pore size, 0.8 µm). The residual CTAB in the NR solution (20 mL) was extracted into chloroform (10 mL) or phosphatidylcholine-chloroform (PC-chloroform) solution (10 mg/mL, 10 mL; PC from egg yolk was purchased from Nacalai Tesque). After performing two more extraction procedures, the aqueous solutions containing NRs were centrifuged once and then dispersed again in pure water (2 mL). Absorption spectra were obtained after 20 times dilution of the NR solutions (JASCO V-570). The zeta potential of the NRs was evaluated by ELS-8000 (Otsuka Electronics, He-Ne laser). In this study, zeta potentials were calculated from the Smoluchowski equation, which is used as a theoretical model of spherical particles. Because the value indicates the sign and qualitative magnitude of the zeta potential of the NRs, this is sufficient for a relative evaluation of the surface charge of NRs. A combination of transmission electron microscopy (TEM) and energy-dispersive X-ray analysis (EDX) was carried out on a TECNAI-20 (Philips). Cytotoxicities of NRs were estimated from cell viabilities of HeLa cells using a slight modification of the MTT assay. Briefly, HeLa cells were plated at 5 × 103 cells/well in a 96-well plate. After 24 h of incubation (37 °C, 5% CO2), 10 µL of PC-NR solution (1-16 mM (Au atoms)) was added to the 100 µL of Dulbecco’s Modified Eagles Medium (DMEM) containing 10% (v/v) of heat-inactivated fetal bovine serum in each well. In this study, DMEM contained antibiotic-antimycotic solution (GIBCO). The cells were further incubated for 24 h. To evaluate cell viability, 10 µL of Cell Counting Kit-8 (Dojindo Laboratories) was added to the medium.36 After 2 h of incubation, the absorbance of lysates was measured at 450 nm by a 96-well microplate reader (model 680 Microplate Reader, BioRad Laboratories). For microscopic observations of cultivated cells treated with PC-NRs, the cells were cultivated in glass base dishes (35 mm, glass: 12φ). After 24 h of incubation, PC-NR solution was added to the medium. The cells were further incubated for 3 h, and were observed by a differential interference microscope (Nikon, TE2000).

Results and Discussion Absorption spectra of NR solutions after the extraction procedure in the absence of PC are shown in Figure 2. Curve a indicates the spectrum of the NR solution immediately after (35) Niidome, Y.; Nishioka, K.; Kawasaki, H.; Yamada, S. Chem. Commun. 2003, 2376. (36) The cell viabilities were evaluated by enzymatic reduction of the watersoluble tetrazolium salt, WST-8, to a water-soluble formazan. Ishiyama, M.; Miyazono, Y.; Sasamoto, K.; Ohkura, Y.; Ueno, K. Talanta 1997, 44, 1299.

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Figure 4. TEM image of PC-NR (a) and elemental mappings [gold (b, 2.122 keV), phosphorus (c, 2.013 keV)] estimated by EDX. Figure 2. Absorption spectra of NRs after three CTAB extractions using chloroform. (a) 0, (b) 15, and (c) 30 days after the three extractions.

Figure 3. TEM image (A) and absorption spectra (B) of NRs after three CTAB extractions using a chloroform solution containing PC (10 mg/mL). (a) 0, (b) 15, and (c) 30 days after the three extractions.

the three extractions. The spectrum shows quite similar characteristics to that of the initial NR solution (Figure 1B);20,21 two SP peaks corresponding to the longitudinal (∼900 nm) and the transverse (∼520 nm) oscillation modes can be seen. The zeta potential of NRs decreased from +67 ( 1 mV to +21 ( 1 mV after the extractions. When the initial NRs solution was centrifuged three times, some of the NRs aggregated, and the zeta potential of those was +27 ( 2 mV (Supporting Information). Thus, it is shown that the CTAB in the NRs solution was extracted to the chloroform phase, and then the surface density of CTAB on the NRs decreased. Fifteen days after the three extractions (b), however, the peak intensity of the longitudinal band decreased somewhat, and the half width of these peaks changed. Thirty days after the procedure (c), the spectrum showed two broad peaks, assignable to aggregation of NRs. Thus, extraction of CTAB is found to be an effective way of decreasing the CTAB in the NR solution; however, the NRs could not retain the colloidal dispersion in the long term, because of the lack of stabilizing agents. A TEM image of NRs after the extraction procedure with the PC-chloroform solution and absorption spectra of the NR solution are shown in Figure 3. The TEM image of the NRs indicates that the extraction procedure does not change the shape of the NRs. The absorption spectrum of the NR solution immediately after the three extractions followed by centrifugation (once) (a) is exactly the same as that obtained after 15 days (b) and shows no remarkable changes even after 30 days (c). This indicates that the NRs can disperse without forming aggregates. The zeta potential of the NRs after the three extractions with PC was +15 ( 1 mV. Thus, though the magnitude is smaller than that in the case of Figure 2, the colloidal dispersion of the NRs is retained in the long term. Considering that PC is a zwitterion-type molecule, the decrease of the zeta potential indicates the replacement of CTAB with PC, and the sign of the zeta potential

Figure 5. Viabilities of HeLa cells after contacting with the PCNR solutions (A-E) and twice-centrifuged NR solutions (a-e). A total of 10 µL of NR solution [1 (A, a), 2 (B, b), 4 (C, c), 8 (D, d), and 16 (E, e) mM (as Au atoms)] was added to 100 µL of DMEM. Final NR concentrations: 0.09 (A, a), 0.18 (B, b), 0.36 (C, c), 0.72 (D, d), and 1.45 (E, e) mM). The cells were incubated for 24 h in the presence of the NRs.

indicates that some CTAB molecules are retained on the NR surfaces. In Figure 4, a TEM image (a) of the NR and elemental mapping of gold (b) and phosphorus (c) are shown. Gold signals (b) (MR, 2.122 keV) overlapped with the position of the NR (a). Phosphorus signals (KR, 2.013 keV) were also observed in the same area. From these results, it is obvious that the surfaces of the NRs are modified with the PC. Thus, the colloidal dispersion of the NRs, which was shown in Figure 3B, would most probably be attributed to a so-called hydration repulsive force between the PC layers37 on the NR surfaces. As a control experiment, a PC-liposome solution prepared by the Bangham method38 was added to the initial NR solution. Centrifugation of the NR solution after addition of the PCliposome resulted in irretrievable aggregation (data not shown). Thus, replacement of CTAB with PC provided by a chloroform phase is an effective way of preparing PC-modified NRs (PCNRs) without forming irretrievable aggregates. We estimated the cytotoxicity of PC-NRs to HeLa cells after 24 h of incubation in the presence of PC-NRs. The cell viabilities are shown in Figure 5A-E. The same procedure as that for the PC-NRs was applied to twice-centrifuged NRs (a-e), to investigate the effects of residual CTAB (Supporting Information). In the case of the 0.09-0.72 mM PC-NR solutions (A-D), little cytotoxicity was observed. When the concentration of PCNR was increased to 1.45 mM, about 20% of the cells died. On the other hand, the twice-centrifuged NR solutions (a-e) showed significant cytotoxicity. Even when 0.09 mM of the centrifuged NR (a) was present in the medium, the viability was about 85% after 24 h of incubation. The viabilities decreased drastically with increasing concentrations of NRs (b-e). Hardly any cells survived when 1.45 mM of the NR (e) was present in the medium. Thus, the twice-centrifuged NRs are much more toxic than the (37) Dimitrova, M. N.; Matsumura, H.; Neitchev, V. Z. Langmuir 1997, 13, 6516. (38) Bangham, A. D.; Standish, M.; Watkins, J. C. J. Mol. Biol. 1965, 13, 238.

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words, despite the contact between PC-NR aggregates and cells, 80% of cells were hardly damaged. Thus, we can conclude that the PC-NRs themselves show low cytotoxicity.

Conclusion Figure 6. Microscopic images of HeLa cells in the medium with (a) and without (b) 1.45 mM of PC-NR. The cells were incubated for 3 h in the presence of the PC-NRs (a). The PC-NRs were observed as darker spots as indicated by arrows.

PC-NRs. Under our experimental conditions, the 50% inhibitory concentration (IC50) of CTAB was about 9.1 µM. This means that the concentration of unbound CTAB was much lower than 9.1 µM in our PC-NR solution even when 1.45 mM of PC-NR was present in the medium. The low cytotoxicity of the PCNRs probably originates from the PC layers which can suppress the desorption of CTAB retained on the surfaces of the PCNRs.39 Figure 6a shows a microscopic image of HeLa cells after 3 h of incubation in the medium containing 1.45 mM of PC-NR. Darker spots can be seen in the image as indicated by arrows. The spots can be assigned to aggregates of the PC-NRs that are taken up or in contact with the cells, because these spots are not observed when PC-NRs are not present in the medium (Figure 6b). Thus, it is obvious that PC-NRs form aggregates in or on the cells. It is probable that the NR aggregates affect the living cells; indeed, 20% cell death occurred after 24 h of incubation with 1.45 mM PC-NRs (Figure 5E). This cell death may be the result of a harmful influence by the NR aggregates. In other (39) Fan, H.; Leve, E. W.; Scullin, C.; Gabaldon, J.; Tallant, D.; Bunge, S.; Boyle, T.; Wilson, M. C.; Brinker, C. J. Nano Lett. 2005, 5, 645.

CTAB was successfully removed from the NR solution by a simple extraction using chloroform containing an additional stabilizing agent. It has been shown that PC is a possible candidate for suppressing the aggregation of NRs after the extraction of CTAB. Because the PC is not inherently toxic to living cells, the PC-NRs show reduced cytotoxicity. Under our experimental conditions, cytotoxicity of the PC-NRs was negligible when about 0.73 mM of PC-NRs was added to the medium. Thus, our method is a useful way of replacing CTAB with alternative stabilizing agents that can diminish the cytotoxicity of CTAB on NR surfaces. Based on this method, various kinds of surfacemodified NRs for practical applications, which can be applied to living cells as concentrated solutions, can be developed. Acknowledgment. This study was supported in part by Research Fellowships of the Japan Society for Promotion of Science (JSPS) for Young Scientists, and by a Grant-in-Aid for Scientific Research (KAKENHI) in the Priority Area “Molecular Nano Dynamics” from the Ministry of Education, Culture, Sports, Science and Technology. Supporting Information Available: Removal of CTAB from NR solution with and without polymer, using centrifugation. This material is available free of charge via the Internet at http://pubs.acs.org. LA0520029