Peptide-Conjugated Gold Nanorods for Nuclear Targeting - American

Parker H. Petit Institute for Bioengineering and Biosciences, Laser Dynamics Laboratory ... Surgery, Comprehensive Cancer Center, University of Califo...
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Bioconjugate Chem. 2007, 18, 1490−1497

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Peptide-Conjugated Gold Nanorods for Nuclear Targeting Adegboyega K. Oyelere,*,† Po C. Chen,† Xiaohua Huang,‡ Ivan H. El-Sayed,§ and Mostafa A. El-Sayed*,‡ Parker H. Petit Institute for Bioengineering and Biosciences, Laser Dynamics Laboratory, School of Chemistry and Biochemistry, Georgia Institute of Technology, 901 Atlantic Drive, Atlanta, Georgia 30332, Otolaryngology-Head and Neck Surgery, Comprehensive Cancer Center, University of California, San Francisco, 400 Parnassus Avenue, California 94143-0342. Received April 18, 2007; Revised Manuscript Received June 6, 2007

Resonant electron oscillations on the surface of noble metal nanoparticles (Au, Ag, Cu) create the surface plasmon resonance (SPR) that greatly enhances the absorption and Rayleigh (Mie) scattering of light by these particles. By adjusting the size and shape of the particles from spheres to rods, the SPR absorption and scattering can be tuned from the visible to the near-infrared region (NIR) where biologic tissues are relatively transparent. Further, gold nanorods greatly enhance surface Raman scattering of adsorbed molecules. These unique properties make gold nanorods especially attractive as optical sensors for biological and medical applications. In the present work, gold nanorods are covalently conjugated with a nuclear localization signal peptide through a thioalkyl-triazole linker and incubated with an immortalized benign epithelial cell line and an oral cancer cell line. Dark field light SPR scattering images demonstrate that nanorods are located in both the cytoplasm and nucleus of both cell lines. Single cell micro-Raman spectra reveal enhanced Raman bands of the peptide as well as molecules in the cytoplasm and the nucleus. Further, the Raman spectra reveal a difference between benign and cancer cell lines. This work represents an important step toward both imaging and Raman-based intracellular biosensing with covalently linked ligand-nanorod probes.

INTRODUCTION Nanobiotechnology and nanomedicine are emerging fields engendered by the convergence of multiple scientific fields for biologic applications and the recently discovered novel properties of materials at the nanoscale. Nanoparticles, because of their small sizes and unique properties, have been widely used in drug delivery (1-4), cellular imaging (5-9), and biomedical diagnostics and therapeutics (10-13). Plasmonic nanoparticles are especially useful for these applications because of their enhanced resonant absorption and scattering properties as well as strong Raman scattering, which are essential properties for their applications in photothermal therapy (14-25), optical imaging (26-31), and Raman probe design (32-38). Nuclear targeting of nanoparticles in live cells is generating widespread interest because of the prospect of developing novel diagnostic and therapeutic strategies such as gene therapy. However, nuclear delivery of the nanoparticles requires bypassing the formidable barriers of the cellular membrane and the nuclear membrane. The cellular membrane endocytoses nonspecific particles on the cell surface, enveloping them with a phopholipid membrane and restricting interaction with the general cell cytoplasm. The nuclear membrane is a double-layer membrane marked with nuclear pores that allow diffusion of molecules and selective uptake into the nucleus. One common approach to targeted nuclear delivery is the conjugation of drug molecules and nanoparticles to nuclear membrane-penetrating peptides. Gold nanoparticles are examples of extremely attractive candidates for attachment to such carrier peptides due to their * To whom correspondence may be addressed. Adegboyega K. Oyelere. Phone: 404-894-4047. Fax: 404-894-7452. E-mail: [email protected], Mostafa A. El-Sayed. Phone: 404-894-0292. Fax: 404-894-0294. E-mail: [email protected]. † Parker H. Petit Institute for Bioengineering and Biosciences, Georgia Institute of Technology. ‡ Laser Dynamics Laboratory, Georgia Institute of Technology. § University of California, San Francisco.

small sizes, ease of preparation and bioconjugation (39, 40), strong absorbing and scattering properties (41-44), as well as their well-known biocompatibility (45). Tkachenko et al. (46, 47), using video-enhanced color differential interference contrast microscopy, have demonstrated nuclear entry of 20 nm gold nanospheres indirectly conjugated to various nuclear localization signal (NLS) peptides through a shell of bovine serum albumin (BSA) protein. However, these gold nanospheres only absorb in the visible region, thus limiting their use in in ViVo applications such as in photothermal therapy, which requires near-infrared region where the light penetration is optimal. Unlike gold nanospheres, gold nanorods possess unique optical properties ideal for in ViVo applications. They have two surface plasmon absorption bands, a strong long-wavelength band due to longitudinal oscillation of their electrons and a weak short wavelength band around 520 nm due to the transverse electronic oscillation (42, 43, 48-51). The longitudinal absorption band is very sensitive to the size of the nanorods. By increasing the aspect ratio (length divided by width), the longitudinal absorption maximum red-shifts with an increase in the absorption intensity. This provides the opportunity for their applications as near-infrared photoabsorbers and scatterers (23). Gold nanorods also provide a scaffold for observing surfaceenhanced Raman scattering (SERS) (52-59). The welldeveloped synthesis of nanorods with different aspect ratios provides the opportunity to tune the surface plasmon band to the excitation laser to obtain the largest enhancement. It has been shown that the enhancement factors on the order of 104105 could be observed for the adsorbed molecules on gold nanorods, while no such enhancement was observed on nanospheres (52). Biological species are known to have small Raman scattering cross section and sensitivity toward photochemical damage and interference from fluorescence when UV or visible laser excitation is used. In order to overcome these difficulties, the size of gold nanorods can be adjusted to have its enhancement in the near-infrared region away from the biomolecular

10.1021/bc070132i CCC: $37.00 © 2007 American Chemical Society Published on Web 07/13/2007

Peptide-Au Nanorods for Nuclear Targeting

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Scheme 1. Synthesis of Thiolalkyl-Triazole Linked NLS Peptidea

a Conditions: (a) NaN3, DMF, 110 °C; (b) MsCl, Et3N, CH2Cl2, rt; (c) potassium thioacetate, THF, reflux; (d) conc. HCl, methanol, reflux; (e) alkyne-modified NLS (PC-37044-PI), TBTA, CuI, DMF, rt. Abbreviations: MsCl, methanesulfonyl chloride; TBTA, Tris-(benzyltriazolylmethyl)amine.

excitation transitions that could lead to fluorescence or photochemical decomposition. Moreover, the surface chemistry of gold nanorods allows for multiple functionalization so that the capping molecules, such as cetyltrimethylammonium bromide (CTAB), could be replaced or conjugated with many functional groups (60-63). In the present work, we describe the preparation of gold nanorods directly conjugated to a simian virus (SV40) NLS peptide through a thioalkyl-triazole linker. Using a simple conventional microscope in the dark field, we are able to image the intracellular localization of these nanorods. Furthermore, cellular components are observed by surface-enhanced Raman scattering of these gold nanorod-peptide conjugates. Our results demonstrate that these nanorods are useful for cellular delivery applications, especially nuclear targeting and cellular components sensing.

EXPERIMENTAL PROCEDURES Materials. 11-Bromoundecan-11-ol and all chemicals used for the synthesis of gold nanorods are purchased from Sigma Aldrich. Anhydrous solvents and other reagents are purchased and used without purification. Analtech silical gel plate (60 F254) is used for analytical TLC, and UV light is used to examine the spots. Tris-(benzyltriazolylmethyl)amine (TBTA) is prepared by using the procedure described by Chan et al. (64). Alkynemodified NLS (PCS-37044-PI) is synthesized by the solid-phase method at Peptide International, Louisville, Kentucky. NMR spectra are recorded on a Varian-Gemini 400 magnetic reso-

nance spectrometer. 1H NMR spectra are recorded in parts per million (ppm) relative to the peak of CDCl3, (7.24 ppm). Mass spectra are recorded at the Georgia Institute of Technology mass spectrometry facility in Atlanta. Synthesis of Thiolalkyl-Triazole Linked NLS Peptide (Scheme 1). 1. Synthesis of 1-Azidoundecan-11-ol (1). This compound is synthesized by adapting the method of Shon et al. (65). Briefly, sodium azide (1.2 g, 18.5 mmol) is added to a solution of 1-bromoundecan-11-ol (2.0 g, 7.96 mmol) anhydrous DMF (20 mL). The reaction mixture is heated to 110 °C in a pressure tube and stirred continuously for 24 h. The reaction mixture is then cooled to room temperature and quenched with distilled water (30 mL). The cooled reaction mixture is extracted with anhydrous diethyl ether (3 × 20 mL), and the combined organic layers are washed with distilled water (3 × 20 mL) and dried over Na2SO4. The solvent is removed and concentrated in Vacuo to give 1.275 g (75%) of 1 as pale yellowish oil. 1H NMR (CDCl3, 400 MHz) δ 1.25-1.38 (14H, m), δ 1.50-1.60 (4H, m), δ 3.22 (2H, t, J ) 7.2 Hz), δ 3.61 (2H, t, J ) 7.2 Hz). 2. Synthesis of 1-Azidoundecan-11-methylsulfonate (2). Triethylamine (4.35 mL, 31.2 mmol) is added to a solution of 1-azidoundecan-11-ol 1 (2.42 g, 11.3 mmol) in anhydrous CH2Cl2 (100 mL). The reaction mixture is stirred for 15 min at 0 °C, and then methanesulfonyl chloride (2.41 mL, 31.2 mmol) is added. The reaction mixture is allowed to warm up to room temperature and stirring continued for 2 h. The reaction mixture is quenched with ice-cold distilled water (35 mL) and extracted

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with anhydrous diethyl ether (2 × 35 mL). The combined organic layer is washed in succession with 1 N HCl, distilled water, saturated NaHCO3, and again with distilled water. The organic layer is dried over Na2SO4 and concentrated in Vacuo to give 2.934 g (89%) of 2 as pale yellowish oil. 1H NMR (CDCl3, 400 MHz) δ 1.25-1.40 (14H, m), δ 1.53-1.60 (2H, m), δ 1.68-1.75 (2H, m), δ 2.97 (3H, s), δ 3.22 (2H, t, J ) 6.8 Hz), δ 4.19 (2H, t, J ) 6.8 Hz). 3. Synthesis of 1-Azidoundecan-11-thioacetate (3). Potassium thioacetate (2.02 g, 6.79 mmol) is added to a solution of 1-azidoundecan-11-sulfonate 2 (1.00 g, 3.43 mmol) in anhydrous THF (10 mL). The reaction mixture is degassed, refilled with argon, and heated under reflux for 3 h. The reaction mixture is quenched with ice-cold distilled water (30 mL) and extracted with anhydrous diethyl ether (3 × 30 mL). The combined organic layer is dried over Na2SO4 and concentrated in Vacuo to give 856 mg (87%) of 3 as yellowish oil. 1H NMR (CDCl3, 400 MHz) δ 1.23-1.38 (14H, m), δ 1.50-1.60 (4H, m), δ 2.29 (3H, s), δ 2.83 (2H, t, J ) 7.6 Hz), δ 3.22 (3H, t, J ) 7.6 Hz). 4. Synthesis of 1-Azidoundecane-11-thiol (4). A solution of 1-azidoundecane-11-thiolacetate 3 (0.1 g. 0.369 mmol) in anhydrous methanol (7.0 mL) is first degassed and refilled with argon. Concentrated HCl (0.4 mL) is added to the solution, and the resulting mixture is refluxed for 3 h. The reaction mixture is quenched with distilled water (10 mL) and extracted with anhydrous diethyl ether (2 × 10 mL). The organic layer is dried over Na2SO4 and concentrated in Vacuo to give 85 mg (100%) of 4 as yellowish oil. 1H NMR (CDCl3, 400 MHz) δ 1.251.38 (14H, m), δ 1.53-1.62 (4H, m), δ 2.50 (2H, q, J ) 15.2 Hz, 7.2 Hz), δ 3.23 (2H, t, J ) 5.6 Hz). 5. Synthesis of Thiolalkyl-Triazole Linked NLS Peptitde (5). Anhydrous CuI (0.003 g, 0.011 mmol) and tris-(benzyltriazolylmethyl)amine (0.007 g, 0.011 mmol) is added to a solution of 1-azidoundecane-11-thiol 4 (0.02 g, 0.079 mmol) and alkynemodified NLS peptides PC-37044-PI (0.05 g, 0.039 mmol) in anhydrous DMF (2 mL). The solution is stirred at room temperature under argon for 48 h. Excess DMF is removed in Vacuo, and the residue is quenched with distilled water (5 mL). The resulting mixture is extracted with dichloromethane (2 × 10 mL) to remove unreacted 1-azidoundecane-11-thiol 4. The remaining aqueous layer is frozen in an acetone-dry ice bath and concentrated in Vacuo to give 50 mg (43%) of 5 as a brown solid. 1H NMR (DMSO-d6, 400 MHz) δ 0.83 (2H, t, J ) 6.0 Hz), δ 1.23-1.36 (6H, m), δ 1.42-1.54 (3H, m), δ 1.56-1.68 (1H, m), δ 2.13-2.24 (2H, m), δ 2.40-2.50 (5H, m), δ 2.662.80 (2H, m), δ 2.87 (1H, s), δ 3.29 (1H, t, J ) 8.0 Hz), δ 3.34-3.54 (1H, m), δ 3.62-3.78 (1H, m), δ 4.10-4.30 (1H, m), δ 7.26-7.36 (1H, m), δ 7.64-7.88 (3H, m), δ 7.94 (1H, s), δ 8.10-8.32 (2H, m). MS (MALDI, CHCA) calcs for [C67H123N23O13S + H]+ 1490.9, found 1491.0. Synthesis of Gold Nanorods and Peptide Conjugation. The gold nanorods are synthesized according to the seed-mediated growth method (66). Briefly, 600 uL 0.01 M ice-cold sodium borohydride is quickly added into a 10 mL 0.5 mM stirring auric acid, which is dissolved in 0.2 M CTAB solution. 2-5 nm gold nanoparticles are formed after 2 min as a seed solution. In a separate flask, 1 mL of 4 mM silver citrate is added to 50 mL growth solution, which contains 0.2 M CTAB, 0.15 M benzyldimethylammonioum chloride hydrate (BDAC), and 1 mM auric acid. 0.07 mL of 0.0788 M ascorbic acid is added to the growth solution to reduce the auric acid to form HAuCl2 solution. 0.08 mL seed solution is injected into the growth solution to initiate the rod formation and growth. In this procedure, gold nanorods with absorption maximum at 650 nm (aspect ratio of 2.4) are obtained overnight. For the conjugation of peptide 5 to gold nanorods, 100 µL 1 mM peptide 5 is added to 10 mL gold nanorod solution (OD650 ) 1.0), and the mixture

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is left to react for 72 h. The unbound peptide is separated by centrifugation at 5000 rpm for 10 min. Cell Culture and Cellular Delivery of Peptide-Conjugated Gold Nanorod. One nonmalignant epithelial cell line, HaCaT (human keratinocytes), and one malignant epithelial cell lines, HSC 3 (human oral squamous cell carcinoma), are cultured on coverslips in DMEM (Dulbecco’s Modification of Eagle’s Medium, Cellgro) plus 10% FBS (fetal bovine serum, Gem Cell) at 37 °C under 5% CO2 for 24 h. The DMEM medium is then taken out and rinsed with PBS buffer. Fresh DMEM medium containing 1 nM thioalkyl-triazole peptide/Au nanorod conjugates is added, and the cells are put back in the incubator for 2 h. After the incubation of the nanorods, the cells are rinsed with PBS buffer, fixed with paraformaldehyde, coated with glycerol, and sealed with another coverslip. Dark Field Imaging. The dark field images are taken under an inverted Olympus IX70 microscope. A dark field condenser (U-DCW) with a numerical aperture between 0.9 and 1.2 is used to deliver a very narrow beam of white light from a tungsten lamp to the sample. A 100×/1.35 oil Iris objective (UPLANAPO) is used to collect only the scattered light from samples. In this mode, the samples with highly scattering properties are shown as a bright object in a dark background. Raman Measurements. The Raman spectra are obtained with a Holoprobe series 5000 Micro-spectrometer (Kaiser Optical Systems, Inc., Ann Arbor, MI) in a 180° reflective configuration with a 50× objective. The excitation wavelength is 785 nm from a diode laser. The spectral resolution is 5 cm-1, and the laser power is 25 mW. Each spectrum is obtained in 10 s collection time with six accumulations.

RESULTS AND DISCUSSION Peptide Conjugation to Gold Nanorods. The use of NLS for targeting of proteins, peptides, nucleic acids, and small organic molecules to the cell nucleus has been extensively investigated (46, 47, 67-72). One of the most studied is the NLS of SV40 large T antigen, which has been shown to act as a “Trojan horse”, efficiently distributing appended payloads into the cell nucleus (68). However, the chemical syntheses of NLSconjugated payloads are very difficult, often requiring technically challenging compound purification and characterization protocols. In the case of nanoparticle-peptide conjugates, an alternative synthetic approach has been to indirectly couple these nanoparticles to carrier peptides through a secondary protein, such as bovine serum albumin (BSA) protein (46, 47). The resulting conjugates are large and possess highly branched polypeptides that are very difficult to characterize. We observe in this study that thiol-terminated NLS peptides incorporating appropriate spacer groups could be easily accessed by Cu(I)-catalyzed cycloaddition reaction (click chemistry) (71) between an alkyne-terminated NLS, such as PC-37044-PI, and an appropriate azidothiols (Scheme 1). Subsequent reaction of the resulting thioalkyl-triazole linked NLS 5 with gold nanorods which are stabilized with CTAB molecules yields peptideconjugated gold nanorods by forming Au-S bonds between the peptide and the gold surface. There are some other previous reports of bioconjugation to gold nanoparticles through Au-S bonds. For example, HS-mPEG has been reported to rapidly assemble onto gold nanoshells through Au-S bonds within 1 h (14). In this case, there are no capping molecules on the surface of the gold layer. In comparison to a bare gold surface, it takes a longer time to assemble thiolated molecules to the surface of gold nanorods. The adsorption of HS-mPEG onto gold nanorods requires stirring for 24 h (61), while thiolated DNA is bound to gold nanorods after 72 h of incubation (73). Gold nanorods are capped with CTAB surfactants, which form a bilayer structure around the gold surface (74). The inner layer

Peptide-Au Nanorods for Nuclear Targeting

Figure 1. A. Absorption spectra of gold nanorods (solid line) and peptide conjugated gold nanorods (dashed line). B. TEM image of gold nanorods. Scale bar: 50 nm.

of the surfactants is bound to the gold surface via the surfactant headgroups. In our work, the thiolated peptide and gold nanorod solution are left for up to 72 h. The absorption spectra in Figure 1 show that there is no change in the peak position after CTAB replacement, but the peak intensity decreases slightly. This intensity damping is due to the increase of the positive charge on the capping molecules when positive CTAB is replaced by more positively charged peptide molecules. SERS of the Peptide-Conjugated Gold Nanorods. Figure 2 shows a comparison of the Raman spectrum of the peptideconjugated gold nanorods with that of the pure peptide and CTAB-capped gold nanorods. It can be seen that the Raman spectrum of the peptide-conjugated gold nanorods show some signals from CTAB molecules, which indicates that a small proportion of the CTAB molecules remained on the rod surface. It is very interesting to note that the amide I CdO stretching vibration of the peptide at 1665 cm-1 is not enhanced when it is bound to the nanorod surface. The CH bending at 1432 cm-1 for the peptide is shifted to 1440 cm-1 on the rod surface. The amide III C-N stretching at 1240 cm-1 is the same for peptide and peptide bound to gold nanorods. The amide II N-H vibrations (1500-1570 cm-1 region) are not observed in both the peptide powder and the peptide bound onto gold nanorods. The thioalkyl-triazole linker shows a strong CH2 rocking

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Figure 2. SERS of CTAB capping molecules on gold nanorods, thioalkyl-triazole-peptide on gold nanorods and Raman spectrum of peptide. A. 300-1400 cm-1 region. B. 1400-3000 cm-1 region.

vibration at 760 cm-1 (75) and C-C stretch at 1092 cm-1 (75, 76). The CH bending is found to overlap with the CH bending in the peptide at 1440 cm-1 (52). The C-N stretch in CTAB molecules overlaps with C-N stretch in the thioalkyl-triazole to give a strong peak at 1002 cm-1 (52). The C-H stretching vibrations of the peptide, the thioalkyl-triazole, and the CTAB are at 2836 cm-1 and 2930 cm-1 (52, 76). The peptide residues show their strong COO- deformation at 722 cm-1 (77). The signal at 831 cm-1 is assigned to the proline and valine C-C stretch, and the 1031 cm-1 signal is assigned to C-N stretch vibrations from glycine and proline (77). The CTAB capping molecules show strong Raman bands at 1137 cm-1 for the C-C stretch vibrations, 1267 cm-1 for δ (CH) of the CH2-N+(CH3)3 groups and 1450 cm-1 and 1490 cm-1 for CH2 bending vibrations (52). From Figure 2, it can be seen that these bands are still observed in the Raman spectrum of the peptide-conjugated gold nanorods due to the incomplete replacement of the CTAB molecules. In comparison to the Raman spectrum of the antibody-conjugated gold nanorods in which CTAB molecules are not replaced (78), the intensity of these bands in the peptide-conjugated gold nanorods are much weaker due to the replacement of the capping molecules with the thiolated peptide. Light Scattering Images of Peptide-Conjugated Gold Nanorods Inside Cells. Figure 3 shows the dark field light scattering images of cells after incubation with 1nM CTAB

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Figure 3. Dark field light scattering images of cells after incubation with gold nanorods and peptide-conjugated gold nanorods for 2 h. A. HaCat normal cells incubated with gold nanorods. B. HSC cancer cells incubated with gold nanorods. C. HaCat normal cells incubated with peptide-conjugated gold nanorods. D. HSC cancer cells incubated with peptide-conjugated gold nanorods. Peptide conjugation promotes the cellular uptake of gold nanorods. Scale bar: 10 µm.

capped nanorods and 1nM thioalkyl-triazole-peptide conjugated gold nanorods for 2 h. It can be seen clearly that, while

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the CTAB-capped gold nanorods are not efficiently absorbed by the cells, the thioalkyl-triazole-peptide conjugated gold nanorods enter into both cell lines with much higher efficiency. This result indicates that peptide conjugation enhances the cellular uptake of the gold nanorods. Importantly, it is worth noting that the observed enhancement of gold nanorod uptake took place in medium containing a very low concentration of peptide-conjugated nanorods (∼1 nM) within a very short incubation time (2 h). This enhanced gold nanorod uptake may be due to the receptor-mediated endocytosis. Moreover, we observed that the thioalkyl-triazole-peptide conjugated nanorods are distributed into both nucleus and cytoplasm in either normal or cancer cells. However, the nanorods are more concentrated in the nucleus of the cancer cells. Reasons for increased nuclear distribution particles in the malignant cells could reflect increased specific and active uptake of the NLS peptide or decreased regulation of passive uptake of cytoplasmic components due to the disruption of the normal cellular process. It is quite possible that the nuclear pores of the nuclear membrane which routinely restrict entrance into the nucleus may be faulty and thereby facilitate the penetration of the peptideconjugated nanorods into the nucleus. Identification of alterations in cellular pathways, such as nuclear pore function, by this multimodal method suggests further avenues of investigation that may yield novel methods of exploiting cellular defects of carcinogenesis for therapeutic applications. SERS of Peptide-Conjugated Gold Nanorods Inside Cells. The Raman spectra of cells incubated with the peptideconjugated gold nanorods are also measured (Figure 4). The Raman laser spot covers the major part of a single cell. Statistically, over twenty cells for each cell line are measured. They gave similar spectra with slight differences in some peak intensities. Shown in Figure 4A,B are five typical spectra for both normal and cancer cells. Figure 4C compares a typical spectrum of a normal and cancer cell as well as the spectrum when the peptide-conjugated nanorods are outside the cells. Spectra of the peptide-conjugated gold nanorods reveal a difference pre- and post-cellular uptake. New bands at 620, 655, and 1158 cm-1 are observed after cellular translocation of the conjugates. The 620 cm-1 band is due to protein COO- wag vibrations and tyrosine δ (ring) vibrations (79). The 655 cm-1 band is due to an overlapping of a strong tyrosine vibration and the guanine bands (36, 80). The DNA backbone vibrations are reflected most likely at bands in the region between 1030 and 1137 cm-1, such as 1058 cm-1 for the DNA C-O stretch (76) and 1094 cm-1 for O-P-O vibrations (81). The 1155 cm-1 band could be assigned to chromatin and histone octamer Raman signals (80). The intensities around 820 and 850 cm-1 bands are greatly increased inside the cells. This indicates that such residues as phenylalanine (830 cm-1, CH2 rock) and tyrosine (850 cm-1, ring breath) in cell proteins (77) are probably enhanced. The lysine Raman signal at 900 cm-1 (77) in the peptide is shifted to 914 cm-1 when it is present inside the cells, suggesting interaction of this residue with the cell components. The Raman intensity of the thioalkyl-triazole C-N stretch band at 760 cm-1 inside the cell is greatly decreased in comparison to that outside the cells. This suggests a change in linker conformation upon nanorod translocation into the cells. The normal and cancer cells show some differences in their Raman signals as well. The 731 cm-1 line which is assigned to adenine (82) is stronger in the cancer cells than that in the normal cells. In addition, a peak at 398 cm-1 is observed only in cancer cells. All these spectral differences might be useful for cancer diagnostics. Feld, Kneipp, and some other groups have demonstrated that individual and aggregated gold nanospheres could be useful for single cell micro-Raman spectroscopy (32-38). However, the

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regarding the local biochemical environment. Identification of spectral differences, such as an increased adenine signal within the cancer cell nucleus, provides detailed biochemical information at the molecular level with concurrent imaging of the particles in subcellular compartments. This multiomodal and ultrasensitive probe has a significant potential for a range of investigative and medical diagnostic applications.

CONCLUSIONS We have demonstrated in this work that gold nanorods, directly conjugated to a SV40 virus NLS peptide through a thioalkyl-triazole linker, could be efficiently delivered into cells within a very short time. Multimodal optical sensing is available with Raman spectroscopy and dark field light scattering imaging. The peptide-conjugated gold nanorods are translocated into both the cytoplasm and the nucleus portion. Raman spectra reveal an enhanced signal of the NLS peptide as well as molecules from both the cytoplasm and the nucleus. In addition, in this pilot study, the Raman signal distinguishes the malignant from nonmalignant cells. This study demonstrates that gold nanorods can be used for nuclear targeting as a novel type of imagingbased contrast agents and an ultrasensitive Raman probe for intracellular molecular sensing as well.

ACKNOWLEDGMENT M.A.E. thanks the Miller Foundation at the University of California at Berkeley for their hospitality during his tenure as a Miller visiting professor. We thank Professor Paul Edmonds for the use of his cell culture facilities. We also like to thank the support of NCI Center of Cancer Nanotechnology Excellence (CCNE) Award (U54CA119338). Supporting Information Available: 1H NMR spectra of compounds 1-5 and LSMS spectra of compound 5. This material is available free of charge via the Internet at http://pubs.acs.org.

LITERATURE CITED

Figure 4. Raman spectra of peptide-conjugated gold nanorods inside HaCat normal cells (A) and HSC cancer cells (B). The Raman spectrum of the peptide-conjugated gold nanorods outside cells is shown in (C) as the bottom curve for comparison. Raman spectra from five cells for each cell line are shown.

inability of controlled delivery of the particles within the cell has limited their applications. The data in this project suggest that the intracellular distribution of the particles inside the cell can be controlled and simultaneously provides information

(1) Rawat, M., Singh, D., Saraf, S., and Saraf, S. (2006) Nanocarriers: promising vehicle for bioactive drugs. Biol. Pharm. Bull. 29 (9), 1790-1798. (2) Petrak, K. (2006) Nanotechnology and site-targeted drug delivery. J. Biomater. Sci. Polym. Ed. 17 (11), 1209-1219. (3) Rabinow, B., and Chaubal, M. V. (2006) Injectable nanoparticles for efficient drug delivery. Drugs Pharm. Sci. 159, 199-229. (4) Chavanpatil, M. D., Khdair, A., and Panyam, J. (2006) Nanoparticles for cellular drug delivery: mechanisms and factors influencing delivery. J. Nanosci. Nanotechnol. 6 (9/10), 2651-2663. (5) Hayat, M. A. (1970) Principles and techniques of electron microscopy: biological applications. Van Nostrand Reihold, New York. (6) Bruchez, M., Jr., Moronne, M., Gin, P., Weiss, S., and Alivisatos, A. P. (1998) Semiconductor nanocrystals as fluorescent biological labels. Science 281, 2013-2016. (7) Chan, W. C. W., and Nie, S. (1998) Quantum dot bioconjugates for ultrasensitive nonisotopic detection. Science 281, 2016-2018. (8) Wu, X., Liu, H., Liu, J., Haley, K. N., Treadway, J. A., Larson, J. P., Ge, N., Peale, F., and Bruchez, M. P. (2003) Immunofluorescent labeling of cancer marker Her2 and other cellular targets with semiconductor quantum dots. Nat. Biotechnol. 21, 41-46. (9) Santra, S., Dutta, D., Walter, G. A., and Moudgil, B. M. (2005) Fluorescent nanoparticle probes for cancer imaging. Technol. Cancer Res. Treat. 4 (6), 593-602. (10) Brigger, I., Dubernet, C., and Couvreur, P. (2002) Nanoparticles in cancer therapy and diagnosis. AdV. Drug DeliVery ReV. 54 (5), 631-651. (11) Holm, B. A., Bergey, E. J., De, T., Rodman, D. J., Kapoor, R., Levy, L., Friend, C. S., and Prasad, P. N. (2002) Nanotechnology in biomedical applications. Mol. Cryst. Liq. Cryst. 374, 589-598.

1496 Bioconjugate Chem., Vol. 18, No. 5, 2007 (12) Moghimi, S. M., Hunter, A. C., and Murray, J. C. (2005) Nanomedicine: current status and future prospects. FASEB J. 19 (3), 311-330. (13) Jain, K. K. (2005) Nanotechnology in clinical laboratory diagnostics. Clin. Chim. Acta 358 (1-2), 37-54. (14) Hirsch, L. R., Stafford, R. J., Bankson, J. A., Sershen, S. R., Rivera, B., Rrice, R. E., Hazle, J. D., Halas, N. J., and West, J. L. (2003) Nanoshell-mediated near-infrared thermal therapy of tumors under magnetic resonance guidance. Proc. Natl. Acad. Sci. U.S.A. 100, 13549-13554. (15) O’Neal, D. P., Hirsch, L. R., Halas, N. J., Payne, J. D., and West, J. L. (2004) Photo-thermal tumor ablation in mice using near infrared-absorbing nanoparticles. Cancer Lett. 209 (2), 171-176. (16) Loo, C., Lin, A., Hirsch, L., Lee, M. H., Barton, J., Halas, N., West, J., and Drezek, R. (2004) Nanoshell-enabled photonics-based imaging and therapy of cancer. Technol. Cancer Res. Treat. 3 (1), 33-40. (17) Loo, C., Lowery, A., Halas, N., West, J., and Drezek, R. (2005) Immunotargeted nanoshells for integrated cancer imaging and therapy. Nano Lett. 5 (4), 709-711. (18) Hainfeld, J. F., Slatkin, D. N., and Smilowitz, H. M. (2004) The use of gold nanoparticles to enhance radiotherapy in mice. Phys. Med. Biol. 49, N309-N315. (19) Zharov, V. P., Galitovskaya, E. N., Johnson, C., and Kelly, T. (2005) Synergistic enhancement of selective nanophotothermolysis with gold nanoclusters: potential for cancer therapy. Lasers Surg. Med. 37, 219-226. (20) El-Sayed, I. H., Huang, X., and El-Sayed, M. A. (2006) Selective laser photo-thermal therapy of epithelial carcinoma using anti-EGFR antibody conjugated gold nanoparticles. Cancer Lett. 239 (1), 129135. (21) Khlebtsov, B., Zharov, V., Melnikov, A., Tuchin, V., and Khlebtsov, N. (2006) Optical amplification of photothermal therapy with gold nanoparticles and nanoclusters. Nanotechnology 17, 51675179. (22) Pissuwan, D., Valenzuela, S., and Cortie, M. B. (2006) Therapeutic possibilities of plasmonically heated gold nanoparticles. Trends Biotechnol. 24 (2), 62-67. (23) Huang, X., El-Sayed, I. H., and El-Sayed, M. A. (2006) Cancer cell imaging and photothermal therapy in the near-infrared region by using gold nanorods. J. Am. Chem. Soc. 128 (6), 2115-2120. (24) Takahashi, H., Niidome, T., Nariai, A., Niidome, Y., and Yamada, S. (2006) Gold nanorod-sensitized cell death: microscopic observation of single living cells irradiated by pulsed near-infrared laser light in the presence of gold nanorods. Chem. Lett. 35 (5), 500501. (25) Huff, T. B., Tong, L., Zhao, Y., Hansen, M. N., Cheng, J. X., and Wei, A. (2007) Hyperthermic effects of gold nanorods on tumor cells. Nanomedicine 2 (1), 125-132. (26) Sokolov, K., Robinson, C., Collier, T., Richards-Kortum, R., Follen, M., and Lotan, R. (2002) Metal nanoparticles as biospecific contrast agents for cancer imaging. Trends Opt. Photonics 71, 376378. (27) Sokolov, K., Follen, M., Aaron, J., Pavlova, I., Malpica, A., Lotan, R., and Richards-Kortum, R. (2003) Real-time vital optical imaging of precancer using anti-epidermal growth factor receptor antibodies conjugated to gold nanoparticles. Cancer Res. 63 (9), 1999-2004. (28) El-Sayed, I. H., Huang, X., and El-Sayed, M. A. (2005) Surface plasmon resonance scattering and absorption of anti-EGFR antibody conjugated gold nanoparticles in cancer diagnostics: Applications in oral cancer. Nano Lett. 5 (5), 829-834. (29) Loo, C., Hirsch, L., Lee, M. H., Chang, E., West, J., Halas, N., and Drezek, R. (2005) Gold nanoshell bioconjugates for molecular imaging in living cells. Opt. Lett. 30 (9), 1012-1014. (30) Stoeva, S. I., Lee, J. S., Smith, J. E., Rosen, S. T., and Mirkin, C. A. (2006) Multiplexed detection of protein cancer markers with biobarcoded nanoparticle probes. J. Am. Chem. Soc. 128 (26), 83788379. (31) Hainfeld, J. F., Slatkin, D. N., Focella, T. M., and Smilowitz, H. M. (2006) Gold nanoparticles: a new X-ray contrast agent. Br. J. Radiol. 79 (939), 248-253. (32) Manfait, M., Morjani, H., Millot, J. M., Debal, V., Angiboust, J. F., and Nabiev, I. (1990) Drug-target interactions on a single living cell. An approach by optical microspectroscopy. Proc. SPIE Int. Soc. Opt. Eng. 1403, 695-707.

Oyelere et al. (33) Nabiev, I., Morjani, H., and Manfait, M. (1991) Selective analysis of antitumor drug interaction with living cancer cells as probed by surface-enhanced Raman spectroscopy. Eur. Biophys. J. 19, 311316. (34) Manfait, M., Morjani, H., and Nabiev, I. J. (1992) Molecular events on single living cancer cells as studied by microspectrofluorometry and micro-SERS Raman spectroscopy. J. Cell. Pharmacol. 3, 120-125. (35) Morjani, H., Riou, J. F., Nabiev, I., Lavelle, F., and Manfait, M. (1993) Molecular and cellular interactions between intoplicine, DNA, and Topoisomerase II studied by surface-enhanced Raman scattering spectroscopy. Cancer Res. 53, 4784-4790. (36) Kneipp, K., Haka, A. S., Kneipp, H., Badizadegan, K., Yoshizawa, N., Boone, C., Shafer-Peltier, K. E., Motz, J. T., Dasari, R. R., and Feld, M. S. (2002) Surface-enhanced Raman spectroscopy in single living cells using gold nanoparticles. Appl. Spectrosc. 56 (2), 150154. (37) Kneipp, J., Kneipp, H., Rice, W. L., and Kneipp, K. (2005) Optical probes for biological applications based on surface-enhanced Raman scattering from indocyanine green on gold nanoparticles. Anal. Chem. 77, 2381-2385. (38) Schluecker, S., Kuestner, B., Punge, A., Bonfig, R., Marx, A., and Stroebel, P. (2006) Immuno-Raman microspectroscopy: in situ detection of antigens in tissue specimens by surface-enhanced Raman scattering. J. Raman Spectrosc. 37 (7), 719-721. (39) Turkevich, J., Stevenson, P. C., and Hillier, J. (1951) The nucleation and growth processes in the synthesis of colloidal gold. Discuss. Faraday Soc. (11), 55-75. (40) Hayat, M. A. (1989) Colloidal gold: principles, methods and applications, Vol 1, Academic Press, San Diego. (41) Mie, G. (1908) Contributions to the optics of turbid media, especially colloidal metal solutions. Ann. Phys. 25, 377-445. (42) Link, S., and El-Sayed, M. A. (2000) Shape and size dependence of radiative, non-radiative and photothermal properties of gold nanocrystals. Int. ReV. Phys. Chem. 19 (3), 409-453. (43) Link, S., and El-Sayed, M. A. (2003) Optical properties and ultrafast dynamics of metallic nanocrystals. Annu. ReV. Phys. Chem. 54, 331-366. (44) Lee, K. S., and El-Sayed, M. A. (2006) Gold and silver nanoparticles in sensing and imaging: sensitivity of plasmon response to size, shape, and metal composition. J. Phys. Chem. B 110, 19220-19225. (45) Connor, E. E., Mwamuka, J., Gole, A., Murphy, C. J., and Wyatt, M. D. (2005) Gold nanoparticles are taken up by human cells but do not cause acute cytotoxicity Small 1 (3), 25-327. (46) Tkachenko, A. G., Xie, H., Coleman, D., Glomm, W., Ryan, J., Anderson, M. F., Franzen, S., and Feldheim., D. L. (2003) Multifunctional gold nanoparticle-peptide complexes for nuclear targeting. J. Am. Chem. Soc. 125, 4700-4701. (47) Tkachenko, A. G., Xie, H., Liu, Y., Coleman, D., Ryan, J., Glomm, W. R., Shipton, M. K., Franzen, S., and Feldheim, D. L. (2004) Cellular trajectories of peptide-modified gold particle complexes: comparison of nuclear localization signals and peptide transduction domains. Bioconjugate Chem. 15, 482-490. (48) Gans, R. (1915) Form of ultramicroscopic particles of silver. Ann. Phys. 47, 270-284. (49) Link, S., Mohamed, M. B., and El-Sayed, M. A. Simulation of the optical absorption spectra of gold nanorods as a function of their aspect ratio and the effect of the medium dielectric constant. J. Phys. Chem. B 103, 3073-3077. (50) Link, S., and El-Sayed, M. A. (2005) Additions and corrections to “Simulation of the optical absorption spectra of gold nanorods as a function of their aspect ratio and the effect of the medium dielectric constant”. J. Phys. Chem. B 109, 10531-10532. (51) Link, S., and El-Sayed, M. A. (1999) Spectral properties and relaxation dynamics of surface plasmon electronic oscillations in gold and silver nanodots and nanorods. J. Phys. Chem. B 103, 84108426. (52) Nikoobakht, B., Wang, J., and El-Sayed, M. A. (2002) Surfaceenhanced Raman scattering of molecules adsorbed on gold nanorods: off-surface plasmon resonance condition. Chem. Phys. Lett. 366, 17-23. (53) Nikoobakht, B., and El-Sayed, M. A. (2003) Surface-enhanced Raman scattering studies on aggregated gold nanorods. J. Phys. Chem. A 107 (18), 3372 -3378.

Bioconjugate Chem., Vol. 18, No. 5, 2007 1497

Peptide-Au Nanorods for Nuclear Targeting (54) Gole, A., Orendorff, C. J., and Murphy, C. J. (2004) Immobilization of gold nanorods onto acid-terminated self-assembled monolayers via electrostatic interactions. Langmuir 20 (17), 71177122. (55) Suzuki, M., Niidome, Y., Terasaki, N., Inoue, K., Kuwahara, Y., and Yamada, S. (2004) Surface-enhanced nonresonance Raman scattering of rhodamine 6G molecules adsorbed on gold nanorod films. Jpn. J. Appl. Phys. 43 (4B), L554-L556. (56) Laurent, G., Fe´lidj, N., Aubard, J., Le´vi, G., Krenn, J. R., Hohenau, A., Schider, G., Leitner, A., and Aussenegg, F. R. (2005) Evidence of multipolar excitations in surface enhanced Raman scattering. Phys. ReV. B 71, 045430-045436. (57) Laurent, G., Fe´lidj, N., Aubard, J., Le´via, G., Krenn, J. R., Hohenau, A., Schider, G., Leitner, A., and Aussenegg, F. R. (2005) Surface enhanced Raman scattering arising from multipolar plasmon excitation. J. Chem. Phys. 122, 011102-011104. (58) Fe´lidj, N., Laurent, G., Grand, J., Aubard, J., Le´vi, G., Hohenau, A., Aussenegg, F. R., and Krenn, J. R. (2006) Far-field Raman imaging of short-wavelength particle plasmons on gold nanorods. Plasmonics 1 (1), 35-39. (59) Orendorff, C. J., Gearheart, L., Jana, N. R., and Murphy, C. J. (2006) Aspect ratio dependence on surface enhanced Raman scattering using silver and gold nanorod substrates. Phys. Chem. Chem. Phys. 8, 165-170. (60) Liao, H., and Hafner, J. H. (2005) Gold nanorod bioconjugates. Chem. Mater. 17, 4636-4641. (61) Niidome, T., Yamagata, M., Okamoto, Y., Akiyama, Y., Takahashi, H., Kawano, T., Katayama, Y., and Niidome, Y. (2006) PEGmodified gold nanorods with a stealth character for in vivo applications. J. Controlled Release 114, 343-347. (62) Takahashi, H., Niidome, Y., Niidome, T., Kaneko, K., Kawasaki, H., and Yamada, S. (2006) Modification of gold nanorods using phosphatidylcholine to reduce cytotoxicity. Langmuir 22, 2-5. (63) Chen, C. C., Lin, Y. P., Wang, C. W., Tzeng, H. C., Wu, C. H., Chen, Y. C., Chen, C. P., Chen, L. C., and Wu, Y. C. (2006) DNAgold nanorod conjugates for remote control of localized gene expression by near infrared irradiation. J. Am. Chem. Soc. 128, 3709-3715. (64) Chan, T. R., Hilgraf, R., Sharpless, K. B., and Fokin, V. V. (2004) Polytriazoles as copper(I)-stabilizing ligands in catalysis. Org. Lett. 6, 2853-2855. (65) Shon, Y., Kelly, K. F., Halas, N. J., and Lee, T. R. (1999) Fullerene-terminated alkanethiolate SAMs on gold generated from unsymmetrical disulfides. Langmuir 15, 5329-5332. (66) Nikoobakht, B., and El-Sayed, M. A. (2003) Preparation and growth mechanism of gold nanorods (NRs) using seed-mediated growth method. Chem. Mater. 15, 1957-1962. (67) Kalderon, D., Roberts, B. L., Richardson, W. D., and Smith, A. E. (1984) A short amino acid sequence able to specify nuclear location. Cell 39, 499-509. (68) Kalderon, D., Richardson, W. D., Markham, A. F., and Smith, A. E. (1984) Sequence requirements for nuclear location of simian virus 40 large-T antigen. Nature (London) 311, 33-38. (69) Goldfarb, D. S., Gariepy, J., Schoolnik, G., and Kornberg, R. D. (1986) Synthetic peptides as nuclear localization signals. Nature (London) 322, 641-644.

(70) Feldherr, C. M., Lanford, R. E., and Akin, D. (1992) Signalmediated nuclear transport in simian virus 40-transformed cells is regulated by large tumor antigen. Proc. Natl. Acad. Sci. U.S.A. 89, 11002-11005. (71) Dietz, G. P., and Bahr, M. (2004) Delivery of bioactive molecules into the cell: the Trojan horse approach. Mol. Cell. Neurosci. 27, 85-131. (72) Numerous examples of click chemistry have appeared in the literature (a comprehensive list is available at www.scripps.edu/chem/ sharpless/click.html). Cited here are two pioneering examples: (i) Rostovtsev, V. V., Green, L. G., Fokin, V. V., and Sharpless, K. B. (2002) A stepwise Huigsen cycloaddition process: copper (I)catalyzed regioselective “ligation” of azides and terminal alkynes. Angew. Chem., Int. Ed. 41, 2596-2599. (ii) Tornoe, C. W., Christensen, C., and Meldal, M. (2002) Peptidotriazoles on solid phase: [1,2,3]-triazoles by regiospecific copper (I)-catalyzed 1,3dipolar cycloadditions of terminal alkynes to azides. J. Org. Chem. 67, 3057-3064. (73) Dujardin, E., Hsin, L. B., Wang, C. R. C., and Mann. S. (2001) DNA-driven self-assembly of gold nanorods. Chem. Commun. 1264-1265. (74) Nikoobakht, B., and El-Sayed, M. A. (2001) Evidence for bilayer assembly of cationic surfactants on the surface of gold nanorods. Langmuir 17, 6368-6374. (75) Bryant, M. A., and Pemberton, J. E. (1991) Surface Raman scattering of self-assembled monolayers formed from 1-alkanethiols: behavior of films at gold and comparison to films at silver. J. Am. Chem. Soc. 113 (22), 8284-8293. (76) Shafer-Peltier, K. E., Haynes, C. L., Glucksberg, M. R., and Van Duyne, R. P. (2003) Toward a glucose biosensor based on surfaceenhanced raman scattering. J. Am. Chem. Soc. 125 (2), 588-593. (77) Stewart, S., and Fredericks, P. M. (1999) Surface-enhanced Raman spectroscopy of amino acids adsorbed on an electrochemically prepared silver surface. Spectrochim. Acta, Part A 55, 1641-1660. (78) Huang, X., El-Sayed, I. H., Qian, W., and El-Sayed, M. A. Cancer cells assemble and align gold nanorods conjugated to antibodies to produce highly enhanced, sharp and polarized surface Raman spectra: a potential cancer diagnostic marker. Nano Lett. in press. (79) Grabbet, E. S., and Buck, R. P. (1989) Surface-enhanced Raman spectroscopic investigation of human immunoglobulin G adsorbed on a silver electrode. J. Am. Chem. Soc. 111 (22), 8362-8366. (80) Peticolas, W. L., Patapoff, T. W., Thomas, G. A., Postlewait, J., and Powell, J. W. (1996) Laser Raman microscopy of chromosomes in living eukaryotic cells: DNA polymorphism in vivo. J. Raman Spectrosc. 27, 571-578. (81) Puppels, G. J., Olminkhof, J. H. F., Segers-Nolten, G. M. J., Otto, C., Demul, F. F. M., and Greve, J. (1991) Laser irradiation and Raman spectroscopy of single living cells and chromosomes: sample degradation occurs with 514.5 nm but not with 660 nm laser light. Exp. Cell Res. 195 (2), 361-367. (82) Nabie, I. R., Sokolov, K. V., and Voloshin, O. N. (1990) Surfaceenhanced Raman spectroscopy of biomolecules. Part III: Determination of the local destabilization regions in the double helix. J. Raman Spectrosc. 21, 333-335. BC070132I