Plasmonic Gold Nanorods Can Carry Sulfonated Aluminum

Dec 7, 2010 - Plasmonic Gold Nanorods Can Carry Sulfonated Aluminum Phthalocyanine To Improve. Photodynamic Detection and Therapy of Cancers...
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Plasmonic Gold Nanorods Can Carry Sulfonated Aluminum Phthalocyanine To Improve Photodynamic Detection and Therapy of Cancers Lei Li,† Ji-Yao Chen,*,†,‡ Xi Wu,† Pei-Nan Wang,§ and Qian Peng‡,| State Key Laboratory of Surface Physics and Department of Physics, The Key Laboratory of Micro/Nano Photonics Structure (Ministry of Education), and Department of Optical Science and Engineering, Fudan UniVersity, Shanghai 200433, China; and Department of Pathology, Oslo UniVersity Hospital, Montebello, 0310 Oslo, Norway ReceiVed: September 30, 2010; ReVised Manuscript ReceiVed: NoVember 14, 2010

Hexadecyltrimethylammonium bromide-coated gold nanorods (AuNRs) with positive charges were effectively bound to negatively charged sulfonated aluminum phthalocyanine (AlPcS), a photosensitizer for photodynamic detection and therapy, due to the electrostatic force, with a loading content of 104 AlPcS molecules per rod. A 5-fold increase in the AlPcS fluorescence of the AlPcS-AuNRs complex was seen. The excitation fluorescence spectrum of the AlPcS-AuNRs with a typical 520 nm band fits well with the resonance band of AuNR surface plasmons, suggesting that such increased AlPcS fluorescence is produced from the strong surface plasmons of AuNRs. The intracellular distribution of AlPcS-AuNRs was studied in the QGY liver cancer cells by respectively imaging the AlPcS fluorescence and AuNRs reflectance with a confocal microscope. Furthermore, the AlPcS-AuNRs-loaded cells were photodynamically damaged after being exposed to red light in a light-dose-dependent manner. In contrast, no phototoxicity of the cells was seen after incubation with the same amount of free AlPcS, indicating that the AlPcS-AuNRs can enhance the AlPcS-mediated photodynamic effect. In addition, the loaded AlPcS can be photothermally released from AuNRs in the cells by the irradiation with an 800 nm femtosecond laser, demonstrating the potential for controlled drug release. 1. Introduction Photodynamic therapy (PDT) has been established as a new treatment modality for a number of malignant and nonmalignant diseases during the past two decades. The principle of this modality is that a photosensitizing drug can preferably accumulate in a lesion with abnormally proliferative cells and produce active oxygen species when excited with the light of appropriate wavelengths to destroy the lesion.1 Several photosensitizers (PSs) have officially been approved for clinical use including Photofrin and metal phthalocyanines.2-4 Although silicon phthalocyanine 4 (Pc4) and sulfonated aluminum phthalocyanine (AlPcS) are often used in clinical PDT,5-8 the selective and effective delivery of the two drugs to a target lesion still faces a great challenge. With the rapid development of nanotechnology, the use of nanoparticles as vehicles for the drug delivery has been conducted.9-13 The delivery of the hydrophobic PS, mTHPC with liposomal vesicles, has been reported.14 Quantum dots (QDs) as PS carriers are also attractive because they have excellent optical properties such as the large extinction coefficients allowing them to effectively absorb the incident photons at a broad wavelength region and transfer the energy to PSs for the indirect excitation of PSs, thus improving the PDT effect.15 PSs conjugated to water-soluble QDs result in a fluorescence * Corresponding author. Address: Department of Physics, Fudan University, No. 220 Handan Road, Shanghai 200433, PR China. Tel.: +862165643084. Fax: +8621-65104949. E-mail: [email protected]. † State Key Laboratory of Surface Physics and Department of Physics, Fudan University. ‡ The Key Laboratory of Micro/Nano Photonics Structure (Ministry of Education), Fudan University. § Department of Optical Science and Engineering, Fudan University. | Oslo University Hospital.

resonance energy transfer (FRET) from QDs to PSs,16,17 suggesting a promising potential for PDT. Recently, gold nanoparticles have been explored in various biological applications because of their chemical inertness, excellent optical properties, and minimum biological toxicity;18-23 particularly, gold nanoparticles were found to be good carriers for genes and drugs including PSs for PDT.24-26 Cheng et al. have found that the gold nanoparticles with a diameter of about 5 nm can adsorb Pc4 on their surfaces with a loading rate of about 30 Pc4 molecules per particle and such loaded Pc4 can be detected in the tumor.27 However, such Pc4 loading was not so encouraging for photodynamic detection because the fluorescence of these surface-attached Pc4 is partially quenched as compared to that of free Pc4.27 This is probably due to a relatively large distance between Pc4 and gold surface and/or to relatively weak surface plasmons on these gold spheres. Generally, strong surface plasmons on metals can enhance Raman and fluorescence of the adsorbed molecules if they are close to the surface within a range of a few nanometers. Therefore, the use of differently structured gold nanoparticles with stronger surface plasmons and a shorter distance between PSs and gold surfaces may enhance the PSs fluorescence. Among gold nanoparticles with different structures, AuNR has proved to be the most flexible structure owing to the synthetic control of its size and aspect ratio.28 AuNRs have been reported to have much strong surface plasmons29,30 and may be suitable gold carriers for PSs. Furthermore, AuNRs coated with cetyltrimethylammonium bromide (CTAB) have positive charges on their surfaces, while AlPcS has negative charges of the sulfonated groups (SO3-) on its benzene rings, so that the force of the electric attraction between gold surface and AlPcS may be strong enough to hold AuNRs and AlPcS closely to enhance the AlPcS fluorescence. In this work, we show that the AlPcS

10.1021/jp109363n  2010 American Chemical Society Published on Web 12/07/2010

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Figure 1. (A) TEM image of AuNRs with length of about 85 nm and aspect ratio of 3.6. (B) The extinction spectrum of AuNRs in aqueous solution with transverse mode band of 520 nm and longitudinal mode band of 770 nm.

Figure 2. (A) Molecular structure of tetrasulfonated aluminum phthalocyanine. (B) Normalized absorbance and emission spectra of AlPcSs.

attached to the surface of AuNRs has 5-fold stronger fluorescence signals than that of free AlPcS, thus facilitating its photodetection. Further, the effective PDT result is achieved on QGY (human liver cancer cell) cells with a very low concentration of AlPcS-AuNRs, while the same amount of free AlPcS has neglectable PDT effect. 2. Materials and Methods Preparation of AuNRs. AuNRs were prepared using a seeding growth method as described previously.31,32 The prepared nanorods were stabilized by the CTAB bilayer to form CTAB-coated gold nanoparticles. The positive charges on the outside terminals of CTAB make AuNRs repel each other to disperse in the aqueous solution. The average length of the nanorods used in the present work was 85 nm with the aspect ratio of about 3.6, a similar size to 50 nm gold nanoparticle that has been reported to have the highest uptake by cells.33 Transmission electron microscopy (TEM) was carried out with a FEI CM 120 microscope (120 kV) to measure the size of AuNRs (Figure 1A). The extinction spectrum of these AuNRs shows two typical resonance bands of the surface plasmons with the transverse one at 520 nm and the longitudinal band at around 770 nm (Figure 1B). Preparation of AlPcS-AuNRs. The photosensitizer AlPcS (Frontier Scientific, Inc.) with the maximum four negative charges of the sulfonations on its four benzene rings (Figure 2A) can bind to the surfaces of AuNRs by electrostatic force. The absorption and fluorescence spectra of AlPcS, measured

by a spectrophotometer (Hitachi, F-2500), are shown in Figure 2B. Different amounts of AlPcS were added into the AuNRs solution of 0.01 nM to find a suitable molar ratio for the AlPcS-AuNRs complex. The conjugates were then stirred overnight in the dark at room temperature (25 °C) and the solutions were filtrated by a filtration film (Microcon YM-50, Millipore Acquires Serologicals Corp.) with apertures of 10 nm to separate the free AlPcS from the AlPcS-AuNRs complex. The harvested complex of AlPcS-AuNRs was resuspended in aqueous solution for biological experiments. Zeta-Potential Measurements. The ζ-potentials of AuNR and AlPcS-AuNR particles were measured in water solution by a Malvern Zetasizer (ZEN 3600, Worcestershire, U.K.). Cell Culture. The QGY cells (human hepatocellular carcinoma cell line 7701) were obtained from the cell bank of Shanghai Science Academy. The cells were seeded in culture dishes containing DMEM medium with 10% calf serum, 100 units mL-1 penicillin, 100 µg mL-1 streptomycin, and 100 µg mL-1 neomycin, and incubated in a fully humidified incubator at 37 °C with 5% of CO2. When the cells reached 80% confluence with normal morphology, AlPcS-AuNRs was added and incubated in an incubator for 30 min. After incubation these cells were washed with PBS for three times to remove unbound AlPcS-AuNRs for further experiments. Imaging of AlPcS-AuNRs in Cells. The fluorescence images of AlPcS in cells were made with a laser scanning confocal microscope (LSCM) (Olympus. FV300, IX71) equipped with a matched pinhole and a long-pass filter of 590 nm in a

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Figure 3. (A) Fluorescence intensities of the AlPcS solutions before and after filtration. (B) Fluorescence spectra of the AlPcS-AuNRs solution before filtration (1), resuspended AlPcS-AuNRs solution after filtration (2), filtrated solution separated from the AlPcS-AuNRs (4), and free AlPcS solution at the concentration of 0.5 µM (3). The excitation is 380 nm. (C) ζ-potentials of AuNRs and AlPcS-AuNRs.

detection channel. A 405 nm laser was used to carry out the excitation for the fluorescence images of cellular AlPcS, as AlPcS still has partial absorption around 405 nm (Figure 2B). Differential interference contrast (DIC) images were recorded simultaneously in a transmission channel to exhibit the cell morphology. A water immersion objective (60×) was used in the experiments. The cellular AuNRs were measured in the same microscope with a confocal reflectance mode. In this mode, the reflecting light from cellular AuNRs was also recorded in the detection channel by removing the 590 nm long-pass filter to allow the reflecting light to be collected. Cytotoxicity Assay. The 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) tetrazolium reduction assay was used to measure the killing effect of PDT with AlPcSAuNRs on cells. The QGY cells with the concentration of 3 × 104 cells/mL were seeded in each well of a 96 well flat bottom tissue culture plate and allowed to attach to the plate overnight. The AlPcS (0.5 µM), AuNRs (0.01 nM), and AlPcS-AuNRs (0.01 nM) were then added into different wells, respectively, and incubated for 30 min. After incubation, cells were washed with PBS for three times and added with the fresh medium. The cell samples were then irradiated with a 630-680 nm bandpass filtered lamp. After illumination, cells were incubated for 24 h and 10 µL of MTT solution (5 mg/mL) was then added into each well for another incubation of 1.5 h. Finally, the 96well plates were put into an iEMS Analyzer (Lab-system) to measure optical densities (OD) at 450 nm. All results were presented as the mean ( SD from three independent experiments with four wells in each. 3. Results and Discussion Fluorescence Enhancement of AlPcS-AuNRs. The harvested AlPcS-AuNRs solution may contain unconjugated free AlPcSs, so that a filtration film with an aperture of 10 nm was used to purify the AlPcS-AuNR solution. As shown in Figure

3A, the fluorescence intensity of free AlPcS solution after filtration has the same value as compared to that of AlPcS solution before the filtration, confirming that such filtration can completely get rid of those unconjugated free AlPcS in the AlPcS-AuNR solution. The different molar ratios of AlPcS to AuNRs were examined and the ratio of 0.5 µM AlPcS to 0.01 nM AuNRs was optimal. Figure 3B demonstrates the fluorescence intensity of the AlPcS-AuNRs solution with such ratio. After filtration the harvested AlPcS-AuNRs complex was resuspended in the same volume of water and its fluorescence intensity was slightly lower but not significantly than that of the solution before the filtration (Figure 3B). Further, the filtrated solution shows no fluorescence signals of AlPcSs. These results indicate that almost all AlPcSs were bound to the AuNRs with 5 × 104 AlPcSs per rod. The Zeta-potential of AuNRS was about +38 mV demonstrating the positive charges on the AuNRs surfaces, and that of AlPcS-AuNRs decreased to +1.5 mV (Figure 3C) reflecting that the negative charges of AlPcS neutralized positive charges on the AuNRs surfaces and confirming the surface binding of AlPcS on AuNRs. Cheng et al have found that the loading rate of gold spheres with a diameter of 5 nm to carry Pc4 photosensitizer was about 30 Pc4s per gold sphere,27 a similar finding to the results of the present study, as the surface area of the rod used in the present study is at least 103-fold larger than that of the 5 nm gold sphere. Moreover, a 5-fold enhancement of the AlPcS-AuNRs conjugates for AlPcS fluorescence was shown as compared to the fluorescence of same amount free AlPcSs (Figure 3B). The phenomenon of metal enhanced fluorescence (MEF) has recently been reported, as a fluorophore in close proximity to metal surface can emit more fluorescence via the surface plasmon coupling.34 Based on the radiation plasmon model of MEF, however, only big metal colloids with the size larger than 0.05 λ can enhance the fluorescence of fluorophore and such enhancement depends largely on the distance between the fluorophore and matal surface.35 The conjugate of AlPcS-AuNRs

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Figure 4. (A) Excitation fluorescence spectra of free AlPcS and AlPcS-AuNRs. (B) The differential spectrum made by the AlPcS-AuNR spectrum divided by the free AlPcS spectrum. (C) Fluorescence emission spectra of free AlPcS and AlPcS-AuNRs at the excitation of 520 nm.

Figure 5. Confocal images of uptake of AlPcS and AlPcS-AuNRs by QGY cells: (A) AlPcS fluorescence images, (B) AlPcS-AuNRs fluorescence images, and (C) AlPcS-AuNRs reflectance images in the same cells as those in B. Left, fluorescence images; center, DIC images; and right, merged images.

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Figure 6. Viability of QGY cells incubated with free AlPcS or AlPcS-AuNRs for 30 min, followed by irradiation with different light doses, measured by MTT assay.

used in the present study appears to meet both requirements, so that an increased fluorescence of AlPcS can be obtained. To find out the effect of AuNRs on enhanced AlPcS fluorescence, the excitation fluorescence spectra of free AlPcS and AlPcS-AuNRs were compared at the emission peak of 680 nm. As shown in Figure 4A, the excitation fluorescence spectrum of free AlPcS is almost the same as its absorption spectrum, but that of AlPcS-AuNRs has an additional band around 520 nm. This difference can be seen more clearly in a differential spectrum (Figure 4B) by the AlPcS-AuNRs spectrum divided by free AlPcS spectrum. The excitation band in the differential spectrum is at about 520 nm, which is just the transverse resonance band of surface plasmons in AuNRs (Figure 1B), implying that the AlPcS emission of AlPcS-AuNRs

Li et al. under 520 nm excitation results from the surface plasmon coupling. Moreover, with the excitation of 520 nm where the AlPcS has no absorption, the fluorescence emission of AlPcS-AuNRs can be clearly seen, while no emission of free AlPcS can be found (Figure 4C), supporting the mechanism of surface plasmon coupling involved. Cellular Uptake of AlPcS-AuNRs. AlPcS, the photosensitizer for PDT, is water soluble with a high singlet oxygen yield. However, it is not easily taken up by cells36 probably due to the negative charges of sulfonation groups of the AlPcS molecule, as there are also net negative charges on the surface of cell membrane. Figure 5A shows little AlPcS fluorescence in the QGY cells after incubation with AlPcSs (0.5 µM) for 30 min. In contrast, AlPcS-AuNRs, conjugated from 0.5 µM AlPcSs and 0.1 nM AuNRs, are distributed in the whole cytoplasm of the QGY cells after 30 min incubation (Figure 5B), since the surfaces of AuNRs in the AlPcS-AuNRs conjugates still contain uncovered positive charges with the positive ζ-potential of +1.5 mV (Figure 3C), thus promoting the AuNRs to bind to cell membranes and facilitate the cellular endocytosis. In order to examine if AlPcS is still bound to AuNRs when they are taken up by cells, confocal reflectance images of cellular AuNRs were made by scanning the same regions of cellular AlPcS fluorescence excited with a 405 nm laser but recorded with the confocal reflectance model of the imaging system (Figure 5C). The excellent overlapping of the AlPcS fluorescence (Figure 5B) and AuNRs reflectance (Figure 5C) images demonstrates the still binding of AlPcS to the AuNRs in the cells after the complex of AlPcS-AuNRs has passed through the cell membranes. This indicates that such complex can effectively deliver AlPcS into the cells, and perhaps

Figure 7. Fluorescence images of intracellular AlPcS-AuNRs distribution before (A) and after (B) irradiation of the 800 nm fs laser. (C) The AlPcS-AuNRs fluorescence intensity pattern in the circle area of the cell before the femtosecond laser irradiation as indicated in A. (D) The AlPcS-AuNRs fluorescence intensity pattern in the circled area of the cell after the femtosecond laser irradiation as indicated in (B).

Plasmonic Gold Nanorods also that AuNRs have the potential for delivering photosensitizers with negative charges. PDT Effect of AlPcS-AuNRs on QGY Cells. No photodamage to the QGY cells was seen after incubation with 0.5 µM AlPcSs for 30 min followed by irradiation with the red light of 11 J/cm2 (Figure 6) because of little fluorescence of AlPcSs seen in the cells after such a short incubation time of AlPcSs (Figure 5A). However, the cells incubated with AlPcS-AuNRs were destroyed after light irradiations (Figure 6), although the AlPcS-AuNRs complex shows a slight dark toxicity on cells probably due to the toxic effect of CTAB. Such photodestruction of cells was enhanced with increasing light doses (Figure 6), exhibiting a typical light-dose-dependent PDT damaging mode. This indicates that AuNRs enable to deliver a therapeutic dose of AlPcSs to the cells, and AuNRs thus are the promising vehicles for intracellular delivery of AlPcSs. Photothermal Release of AlPcS from AuNRs in Cells. The irradiation with a near-infrared femtosecond laser can melt AuNRs, since they have a high absorption coefficient at this wavelength region (Figure 1B).37 The high temperature in melted AuNRs makes surface-bound molecules have high kinetic energy that can break the binding to release AlPcS from the AuNRs. In the present study, an 800 nm fs laser was used to examine if such irradiation can induce a photothermal effect on the release of AlPcS from AlPcS-AuNRs conjugates in the QGY cells. Once released from AuNRs, AlPcS fluorescence decreases, as the effect of metal surface enhancement is lost. Figure 7, A and B, demonstrates the fluorescence of AlPcSAuNRs in a cell before and after irradiation on a small area (circled part) with the 800 nm fs laser at a power density of 0.7 mW/(µm)2 for 10 s. Figure 7, C and D, shows the detailed fluorescence intensity of AlPcS-AuNRs at a selected area of the cell (circle part) before and after the irradiation. The decreased fluorescence intensity of AlPcS-AuNRs after the femtosecond laser irradiation indicates the release of AlPcS from the gold surfaces. This finding suggests that it is possible for the AlPcS-AuNRs complex to release the surface-bound AlPcS with the 800 nm fs laser irradiation in the cells. To further convince that the decreased fluorescence of AlPcS was produced by the AlPcS release from AlPcS-AuNRs but not caused by the AlPcS photobleaching under femtosecond laser irradiation, a control experiment for AlPcS alone was carried out. We used a higher incubation concentration of AlPcS (10 µM) and longer incubation time (3 h) to make the AlPcS enter the cells as demonstrated in the fluorescence image of cellular AlPcS (Supporting Information, Figure S-A). Then we selected a small area (circled part) in the cell to do the irradiation with this fs laser for the same time (10 s) and remeasured the fluorescence of cellular AlPcS after the irradiation (Figure S-B). As shown in Figure S-C,S-D, the fluorescence intensities of cellular AlPcS at the irradiated area (circle part) before and after the irradiation are almost the same, demonstrating that the femtosecond laser irradiation did not result in a fluorescence photobleaching of cellular AlPcS. 4. Conclusion Positively charged AuNRs are capable of carrying negatively charged AlPcSs with a high loading rate of 5 × 104 AlPcSs molecules per rod, resulting in a 5-fold increase in the fluorescence intensity of AlPcS via its surface plasmons. AlPcS-AuNRs can effectively deliver AlPcSs into QGY cancer cells and perform the PDT to destroy the cancers, while free AlPcSs with the same amount are difficult to associate with cells and have negligible PDT effects. This finding may indicate

J. Phys. Chem. B, Vol. 114, No. 51, 2010 17199 that AuNRs are promising carriers of AlPcS-like photosensitizers with negative charges for PDT and photodetection. In addition, the result that AlPcSs are photothermally released from AuNRs by the irradiation of an 800 nm fs laser in the QGY cells demonstrates the potential of the AlPcS-AuNRs conjugates for a controllable drug release. Acknowledgment. Financial support from the National Natural Science Foundation of China (10774027 and 11074053) and Visiting Scholar Foundation of Key Lab in Fudan University is gratefully acknowledged. Supporting Information Available: Fluorescence images of intracellular AlPcS distribution before (A) and after (B) irradiation of the 800 nm fs laser. (C) The AlPcS fluorescence intensity pattern in the circle area of the cell before the femtosecond laser irradiation as indicated in A). (D) The AlPcS fluorescence intensity pattern in the circle area of the cell after the femtosecond laser irradiation as indicated in (B). This material is available free of charge via the Internet at http:// pubs.acs.org. References and Notes (1) Dougherty, T. J.; Gomer, C. J.; Henderson, B. W.; Jori, G.; Kessel, D.; Peng, Q. J. Natl. Cancer Inst. 1998, 90, 889–905. (2) Bonnett, R. Chem. Soc. ReV. 1995, 24, 19–33. (3) Whitacre, C. M.; Feyes, D. K.; Satoh, T.; Grossmann, J.; Mulvihill, J. W.; Oleinick, N. L. Clin. Cancer Res. 2000, 6, 2021–2027. (4) Peng, Q.; Moan, J.; Kongshaug, M.; Evensen, J. F.; Anholt, H.; Rimington, C. Int. J. Cancer 1991, 48, 258–264. (5) Dolmans, D. E. J. G. J.; Fukumura, D.; Jain, R. K. Nature ReV. Cancer 2003, 3, 380–387. (6) Macdonald, I. J.; Dougherty, T. J. J. Porphyrins Phthalocyanines 2001, 5, 105–129. (7) Rosenthal, I. Photochem. Photobiol. 1991, 53, 859–870. (8) Spikes, J. D. J. Photochem. Photobiol. B 1990, 6, 259–274. (9) Farokhzad, O. C.; Langer, R. ACS Nano 2009, 3, 16–20. (10) Schwiertz, J.; Wiehe, A.; Grafe, S.; Gitter, B.; Epple, M. Biomaterials 2009, 30, 3324–3331. (11) Thurn, K. T.; Paunesku, T.; Wu, A.; Brown, E. B.; Lai, B.; Voget, S.; Maser, J.; Aslam, M.; Dravid, V.; Bergan, R.; Woloschak, G. E. Small 2009, 5, 1318–1324. (12) Lewinski, N.; Colvin, V.; Drezek, R. Small 2008, 4, 26–29. (13) Wang, L.; Luo, J.; Fan, Q.; Suzuki, M.; Suzuki, I. S.; Engelhard, M. H.; Lin, Y.; Kim, N.; Wang, J. Q.; Zhong, C. J. J. Phys. Chem. B 2005, 109, 21593–21601. (14) Bombelli, C.; Stringaro, A.; Borocci, S.; Bozzuto, G.; Colone, M.; Molinari, A. Mol. Pharmaceutics 2010, 7, 130–137. (15) Samia, A. C. S.; Chen, X. B.; Burda, C. J. Am. Chem. Soc. 2003, 125, 15736–15737. (16) Shi, L. X.; Hernandez, B.; Selke, M. J. Am. Chem. Soc. 2006, 128, 6278–6279. (17) Tsay, J. M.; Trzoss, M.; Shi, L. X.; Kong, X. X.; Selke, M.; Weiss, S. J. Am. Chem. Soc. 2007, 129, 6865–6871. (18) Chen, C. L.; Kuo, L. R.; Chang, C. L.; Hwua, Y. K.; Huang, C. K.; Chen, Y. Y. Biomaterials 2010, 31, 4104–4112. (19) Reismann, M.; Bretschneider, J. C.; Plessen, G. V.; Simon, U. Small 2008, 4, 607–611. (20) Jain, P. K.; Lee, K. S.; El-Sayed, I. H.; El-Sayed, M. A. J. Phys. Chem. B 2006, 110, 7238–7248. (21) Mishra, A.; Ram, S.; Ghosh, G. J. Phys. Chem. C 2009, 113, 6976– 6982. (22) Louit, G.; Asahi, T.; Tanaka, G.; Uwada, T.; Masuhara, H. J. Phys. Chem. C 2009, 113, 11766–11772. (23) Schwartzberg, A. M.; Olson, T. Y.; Talley, C. E.; Zhang, J. Z. J. Phys. Chem. B 2006, 110, 19935–19944. (24) Lee, O. S.; Schatz, G. C. J. Phys. Chem. C 2009, 113, 2316–2321. (25) Peled, D.; Naaman, R.; Daudbe, S. S. J. Phys. Chem. B 2010, 114, 8581–8584. (26) Housni, A.; Ahmed, M.; Liu, S.; Narain, R. J. Phys. Chem. C 2008, 112, 12282–12290. (27) Cheng, Y.; Samia, A. C.; Meyers, J. D.; Panagopoulos, I.; Fei, B.; Burda, C. J. Am. Chem. Soc. 2008, 130, 10643–10647. (28) Chou, C. H.; Chen, C. D.; Wang, C. R. C. J. Phys. Chem. B 2005, 109, 11135–11138.

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