Direct Synthesis of Rev Peptide-Conjugated Gold Nanoparticles and

Jun 14, 2011 - Genomic Medicine Research Core Laboratory, Chang Gung Memorial Hospital, Taoyuan, Taiwan. ) Department of Health Technology and ...
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Direct Synthesis of Rev Peptide-Conjugated Gold Nanoparticles and Their Application in Cancer Therapeutics Ngoc Thi Thanh Tran,† Tzu-Hao Wang,‡,§ Chiao-Yun Lin,‡ Yi-Chun Tsai,† Chyong-Huey Lai,‡ Yian Tai,*,† and Benjamin, Y. M. Yung|| †

Department of Chemical Engineering, National Taiwan University of Science and Technology, Taipei, Taiwan Department of Obstetrics and Gynecology, Chang Gung Memorial Hospital and Chang Gung University, Taoyuan, Taiwan § Genomic Medicine Research Core Laboratory, Chang Gung Memorial Hospital, Taoyuan, Taiwan Department of Health Technology and Informatics, The Hong Kong Polytechnic University, Hong Kong, China

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bS Supporting Information ABSTRACT: We have developed a simple approach for generating peptideconjugated gold nanoparticles (AuNPs) from the Rev peptide and gold aqueous solution. The peptide functions as both a reducing agent and a capping molecule. AuNPs of various sizes (20 300 nm) and shapes (spheres, triangular plates, and polygons) can be obtained upon modulating the ratio of gold ions to the Rev peptide. Transmission electron microscopy, X-ray diffraction, and UV vis spectroscopy were utilized to characterize these nanoparticles. Fourier-transform infrared and X-ray photoelectron spectroscopy measurements were performed to investigate chemical interactions between the Rev peptide and AuNPs. Lactate dehydrogenase and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays revealed that the Rev peptide AuNP nanocomposites exhibited exceptionally high cytotoxic effects toward mouse ovarian surface epithelial cell lines, relative to the effects of equal doses of the free Rev peptide. Our study suggests a new way of utilizing biomolecule-conjugated AuNPs as potentially effective anticancer drugs.

’ INTRODUCTION Metal nanoparticles possess many properties that are useful in biomedical applications.1,2 In particular, gold nanoparticles (AuNPs) are of tremendous interest due to their unique optical and photothermal properties,1 4 which are due in large part to their localized surface plasmon resonance (LSPR) properties that can be modulated by varying particle size and shape.5 AuNPs are good candidates for medical processes such as labeling,6 sensing and imaging,7 drug delivery,8 and cancer therapeutics.6,9 Among their versatile biomedical applications, cancer therapeutics that utilize AuNPs have attracted much attention since they can provide both localized and targeted therapies, minimizing invasion, thus reducing the severity of side effects10,11 and improving a patient's quality of life. With thiol-derivatized PEG and recombinant human tumor necrosis factor (TNF) directly bound to the AuNP surface, Paciotti et al. demonstrated that a less toxic and more effective reduction in tumor burden can be realized by using AuNPs in preference over native TNF since maximal antitumor response is achievable at lower drug doses.12 The most promising cancer therapy that utilizes AuNPs is hyperthermic tumor cell ablation by optical heating. In AuNP-mediated hyperthermia, nanoparticles are delivered to cancer cells where they agglomerate into larger clusters that form bubbles under laser irradiation, providing effective tumor cell elimination.13,14 Such improvements benefit from the high photon-energy absorption r 2011 American Chemical Society

profile of AuNPs for which minimal irradiation energy is required. Additionally, selective targeting of AuNPs to biomarkers on cancer cells is a promising cancer therapy technique. By binding HER2, a protein often overexpressed in breast cancer cells, with gold nanoshell conjugated antibodies, Halas et al. successfully performed precision photodamage of cancer cells in vitro using a NIR laser.15 Moreover, a method using AuNPs has been applied in a noninvasive radio wave cancer treatment. Tumor ablation using a variable power RF signal, after direct injection of citrate-AuNPs into a target tumor to focus the radio wave for selective heating, has been reported for both in vitro and in vivo investigations 16 One of the prerequisites for AuNPs in cancer therapy applications is their biocompatibility. These nanoparticles are typically functionalized with biomolecules post-synthesis to avoid any undesired effects on live cells.17,18 Efforts have been made for direct synthesis of AuNPs using various biological entities, such as plant extracts,19 fruit extracts,20 fungi,21 and peptides.22,23 In each case, the biospecies functioned as both reducing agent and capping molecule.22 Among the list of biological entities, peptides have attracted significant attention due to their versatility in Received: March 10, 2011 Revised: June 7, 2011 Published: June 14, 2011 1394

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Bioconjugate Chemistry cancer therapy.24 26 For example, the Rev peptide has been reported as a promising candidate as an anticancer drug;27 Nucleophosmin/B23, which is often overexpressed in proliferating cells,28 has been implicated as a marker for ovarian,29 gastric,30 colon,31 prostate,32 and bladder 33 cancers. Using small molecules to bind to cancer-specific targets is a potentially powerful anticancer strategy. Since the Rev peptide exhibits high binding affinity toward nucleophosmin/B23, it might prove useful as a powerful anticancer technology. Herein, we report a simple, direct, and reproducible method for fabricating water-dispersible Rev peptide-conjugated AuNPs (Rev_AuNPs) for cancer therapy applications. Various sizes and shapes of Rev_AuNPs can be generated by manipulation of the concentration of Au ion solution or the temperature during the synthetic process. Lactate dehydrogenase (LDH) and 3-(4,5dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT) assays revealed that Rev_AuNPs exhibited remarkably higher in vitro cytotoxic effects on mouse ovarian surface epithelial cell (MOSEC) lines relative to the effect of an equal dose of the pure Rev peptide.

’ EXPERIMENTAL SECTION Materials. The Rev peptide, C17H128N36O18S (Figure S1, Supporting Information), was provided by Kelowna International Scientific Inc. Hydrogen tetrachloroaurate hydrate (HAuCl4 3 3H2O), sodium hydroxide (NaOH), and potassium bromide (KBr, IR grade) were purchased from Acros Organics (USA). Deionized (DI) water (18 MΩ) was obtained from a Milli Q reagent water system. The colorimetric MTT assay was acquired from Roche Applied Science (Mannheim, Germany); the LDH assay was purchased from Roche Applied Science (Indianapolis, IN, USA). Synthesis of Rev_AuNPs. In a standard experiment, aqueous HAuCl4 (0.001 M, 2 mL) was added dropwise into a solution of Rev (5 mM, 1 mL) and NaOH (0.01 M, 1 mL), and then the mixture was stirred (500 rpm) for 4 h at room temperature. After the addition of DI water (4 mL), the mixture was left undisturbed, for one day to complete of the reaction. The mixture was centrifuged (24,000 rpm, 30 min) and the precipitate collected and redispersed in DI water (4 mL) using an ultrasonic bath to form a solution; the supernatant was collected separately. Both solutions were passed through a membrane filter (mesh size: 400 nm) and quantified (UV vis spectrometry) prior to in vitro testing. The shape-modulation experiments of the peptide-conjugated AuNPs were performed by varying the ratio of Au3+ ions to Rev peptide or by altering the temperature during synthesis, while maintaining other conditions constant. Synthesis of Reference AuNPs. Reference AuNPs for the in vitro tests were fabricated using a previously reported standard procedure.34 Aqueous sodium citrate solution (3.88  10 2 M, 10 mL) was added to boiling HAuCl4 solution (1  10 3 M, 100 mL), and the mixture was boiled for a further 10 min. As the AuNPs formed, the solution color changed from light yellow to wine red. Characterization. Rev_AuNPs UV vis absorbances were measured using UV vis NIR spectroscopy (Jasco V-670). The sizes and shapes of the fabricated AuNPs were determined using a Philips Tecnai F30 field-emission-gun transmission electron microscope, equipped with selected area electron diffraction (SAED) attachment. FTIR spectra were recorded in transmittance mode, on a Nicolet iS10 instrument using KBr

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plates. The synthesized AuNP crystallinity was determined by X-ray diffraction (XRD, Rigaku D/MAX-B) with CuKR radiation (λ = 1.54 Å). X-ray photoelectron spectroscopy (XPS) was performed in an analysis chamber equipped with an X-ray photoelectron spectrometer (VG ESCA Scientific Theta Probe; takeoff angle, 53°) and an aluminum KR (1486.5 eV) anode, operated at a power of 200 W (20 kV). XPS spectra were recorded using a hemispherical energy analyzer operated at a pass energy of 50.0 eV for survey scans. For practical XPS data analyses, spectral calibration was performed by employing Au 4f peaks at either 83.9 or 84.1 eV as internal standards. Quantification. Two calibration plots were constructed to quantify the amount of Rev peptide bonded to the AuNPs prior to conducting in vitro tests. One plot was based on UV vis absorbance measurements of the reaction solutions to quantify the concentration of excess Rev peptide after the synthetic reaction. The plot (Figure S2a, Supporting Information) was constructed using the pure Rev peptide absorption band at 276 nm, the absorbance of which changed with concentration of the peptide solution (Figure S2b, Supporting Information). Rev_AuNP exhibits a characteristic absorption at 550 nm, and its intensity changes according to its concentration, thus providing the basis for a calibration graph. Figure S3a (Supporting Information) presents the calibration plot monitored at three wavelengths: the band at 550 nm, the first base wavelength at 490 nm, and the second at 620 nm, respectively; Figure S3b (Supporting Information) shows variation in absorbance with changes in Rev_ AuNP concentration. Cytotoxicity and Viability Assays. The MOSEC line was cultured in RPMI 1640, supplemented with 10% fetal bovine serum and antibiotics, at 37 °C with 5% CO2. Approximately 5000 cells (200 μL of a suspension of 2.5  104 cells/mL) were placed in each well of a 96-well culture plate for 24 h. For the cytotoxicity and viability experiments, cancer cells in serum-free medium were treated with the AuNPs, the Rev peptide, or Rev_AuNPs for various periods. After gentle agitation, the culture medium (100 μL) was transferred into fresh wells in a 96-well plate for the LDH assay, performed according to the manufacturer’s protocol. For the colorimetric MTT assay, MTT (5 mg/mL, 25 μL) was added into each well containing treated cells; after 4 h, the supernatant was discarded, and DMSO (100 μL) was added to each well; the mixture was shaken and measured at 570 nm using an ELISA reader scanning multiwell spectrophotometer (PerkinElmer VICTOR 2, GMI, Minnesota, USA). Transmission Electron Microscopy Images of Cells. Cells were fixed in phosphate buffer containing 3% glutaraldehyde, postfixed in 1% OsO4, dehydrated in graded alcohol, and finally embedded in Epon 812. Ultrathin (80 nm) sections were stained with 4% uranyl acetate and lead citrate, then examined using transmission electron microscopy (TEM) (H-7500, Hitachi, Tokyo, Japan).

’ RESULTS AND DISCUSSION AuNPs with an average size of 25 nm (Figure 1a) were fabricated by the dropwise addition of 0.001 M aqueous HAuCl4 into 5 mM Rev peptide solution under constant stirring at room temperature. The nanostructures exhibited characteristic UV vis absorption centered at 550 nm (Figure 1b), which can be assigned to the surface plasmon resonance (SPR) band of AuNPs.2,5 The dispersion of these AuNPs in water produced a light-purple color (Figure 1b, inset). The presence of CdO, NHx, and SH functional groups within the Rev peptide molecular 1395

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Figure 1. (a, c, and e) TEM images and (b, d, and f) UV vis spectra (the insets are photographs of aqueous dispersions) of the Rev_AuNPs synthesized from HAuCl4 at concentrations of (a,b) 0.001 M, (c,d) 0.002 M, and (e,f) 0.005 M.

structure presumably caused it to act as a mild reductant, reducing Au3+ ions to metal Au, thereby forming the AuNPs.35 Moreover, the AuNPs were protected and stabilized by the conjugated Rev peptide backbones.22,35 Larger triangular plate (100 200 nm) AuNPs can be prepared by increasing the aqueous HAuCl4 concentration to 0.002 M (Figure 1c). The UV vis absorption spectrum of this sample features an absorption band at around 550 nm, and a broad band appeared in the higher wavelength region (Figure 1d). The band centered at 550 nm possibly results from a mixture of the SPR band of small AuNPs as shown in Figure 1b and an out-of-plane quadrupole resonance of the triangular plates.36 The Figure 1d inset shows a photograph of an aqueous dispersion of these nanostructures, which is dark red in color. We suspect that variation in Rev_AuNPs size and shape occurs upon varying the ratio of the Rev peptide to HAuCl4. Our explanation for this is that strong relationships exist between the capping agent to metal ion concentration ratio, and the sizes and shapes of metal NPs.37,38 Indeed, a large

abundance of anisotropic Rev_AuNPs nanoplates, including triangular, hexagonal, and truncated polygonal plates form upon increasing the concentration of Au ions to 0.005 M (Figure 1e), possibly due to the increased HAuCl4 concentration which provided more Au ions for further growth and led to larger nanoplates. The aqueous dispersion of these Rev peptideconjugated nanoplates was colored brown (Figure 1f, inset). The UV vis absorption of these particles (Figure 1f) is similar to the spectrum shown in Figure 1d. In addition to manipulation of the ratio between Au ions and Rev peptide, Rev_AuNPs of various sizes and shapes can be generated by carrying out the reaction at elevated temperatures (Figure S4, Supporting Information). With the purpose of application in cancer therapeutics, we selected the 25-nm Rev_AuNPs, as shown in Figure 1a, for further investigation since the most suitable particle size for biomedical applications is 20 70 nm.8 1396

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Figure 2. XRD and SAED patterns of AuNPs synthesized using the Rev peptide.

XRD analysis revealed that these AuNPs were polycrystalline (Figure 2), with characteristic peaks at 38.3, 44.6, 64.9, and 77.8° that correspond to the (111), (200), (220), and (311) crystal planes, respectively, implying a face-centered cubic crystal structure.39 Moreover, selected area electron diffraction (SAED) (Figure 2, inset) reveals a random distribution of bright spots together with ring patterns, further confirming the polycrystalline structure of Au atoms in Rev_AuNPs. The Rev_AuNPs FTIR spectra were recorded to verify that the Rev peptide had conjugated to AuNP as a capping agent. Figure 3a and b shows FTIR spectra of the pure Rev peptide and the Rev_AuNPs, respectively. The intense broad bands in the region between 3308 and 3401 cm 1 in Figure 3a represent N H stretching vibrations, indicating the presence of amino groups.40 We assign two sharp absorption bands at 1536 and 1669 cm 1 to the amide II and amide I groups of the Rev peptide, respectively.40 By contrast, the Rev_AuNPs absorption bands for amide II and amide I groups appeared at 1544 and 1676 cm 1, respectively (Figure 3b). The shifts of these two amide absorption bands to the higher wavenumber suggest that conformational changes occurred to the Rev peptide upon binding to the Au surface.40 The major vibrational absorption bands corresponding to the CdO groups were not identifiable, possibly because of overlap with the amide I bands.41 Nevertheless, the signals at 1436 cm 1 in Figure 3a and 1451 cm 1 in Figure 3b correspond to the symmetrical stretching vibrations of carboxylate groups, indicating the presence of CdO units in the free Rev peptide and in Rev_AuNPs.40 Again, this shift to a higher wavenumber suggests that a conformational change occurred to the Rev peptide upon binding to AuNPs.41,42 To further understand the nature of the chemical interactions between the Rev peptide and the AuNP, XPS has been carried out. The signals in the Rev_AuNPs Au 4f spectrum (Figure 4a) indicate the presence of Au atoms.20The low intensity of the signal seen for the Au 4f energy level resulted from attenuation caused by an abundance of Rev peptide units on the AuNP surfaces. We attribute the slight shift in the Au 4f signal toward higher binding energy to the Au atoms attached to organic molecules (i.e., the Rev peptide).20 Moreover, the presence of C 1s, O 1s, and N 1s photoelectron signals confirmed the existence

Figure 3. FTIR spectra of the (a) free Rev peptide and (b) Rev_AuNPs.

of Rev peptide units anchored to the AuNP surfaces. Figure 4b presents the C 1s spectra of both the free Rev peptide and the Rev_AuNPs. The XPS signals in both spectra are represented by three individual peaks, 284.9 285.1, 286.0 286.2, and 288.6 288.8 eV, which we assign to aliphatic and aromatic hydrocarbon species, CNHx and CdO groups, respectively.20 Notably, the relative intensity of the high binding energy peak in the range 288.6 288.8 eV to the hydrocarbon signal (284.9 285.1 eV) in the spectrum of the Rev_AuNPs [Figure 4b(ii)] was lower than that of the free Rev peptide [Figure 4b(i)]. This possibly arose through conversion of a carbonyl (CdO) functional group in the free Rev peptide to a C O Au moiety when the Rev peptide bound to the AuNPs surface. Since AuNPs are electron reach species, they donate electrons to the oxygen atom via the O Au bond, thus decreasing oxygen’s inductive electron withdrawing effect toward the attached carbon atoms. As a consequence, the signal of these carbon atoms move to lower binding energy and overlap with that for the hydrocarbon units, leading to the observed decrease in high binding energy signal and increase in the low binding energy intensity. The oxygen spectra shown in Figure 4c further confirm our proposed mechanism. The O 1s peak of the free Rev peptide [Figure 4c(i)] shifts to lower binding energy for Rev_AuNP [Figure 4c(ii)]), presumably because of the conversion of the oxygen atoms from CdO units to C O Au moieties upon binding of the Rev peptide to the Au surface. The signal for the N 1s energy level remains unchanged in the spectra of the free Rev peptide [Figure 4d(i)] and Rev_AuNPs [Figure 4d(ii)], suggesting that no (or very few) 1397

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Figure 4. XPS spectra of (a) the Au 4f energy level of the Rev_AuNPs and (b d) the (b) C 1s, (c) O 1s, and (d) N 1s energy levels for the (i) free Rev peptide and (ii) Rev_AuNPs.

CNHx groups were involved in the formation of Rev_AuNPs. Moreover, the full-width-at-half-maximum height (fwhm) of the XPS peaks for Rev_AuNPs were noticeably narrower than those of the free Rev peptide, implying that the Rev peptide molecules possessed higher configurational order when bound to the AuNPs’ surfaces compared to that in their free form. The Rev peptide (primary sequence: ARRNRRRRWREYC) has a high binding affinity for nucleophosmin/B23 within tumor cells.27 In principle, conjugation to AuNP should result in the Rev peptide being retained for longer durations with improved stability within a cellular environment.43,44 Therefore, we employed the MOSEC line to test the Rev_AuNPs as Rev peptide carriers in vitro.45 The cells were incubated individually with both Rev_AuNPs and free Rev peptide at the same dose for comparison. Additionally, AuNPs of similar size and shape to Rev_ AuNPs, but lacking the bound Rev peptide (Figure S5, Supporting Information), were incubate separately as a reference. For all samples, cell incubation was performed for 24 or 48 h, and then

cell viability and toxicity were determined using MTT and LDH assays. The results, summarized in Figure 5, are presented as mean viability ( standard deviation (SD) from three independent experiments, each of which were performed in triplicate. Figure 5a shows that both the reference AuNPs and free Rev peptide exhibited MOSEC survival rates close to 100% after incubation for 24 h (MTT assay), suggesting no cytotoxic effects. By contrast, the survival rate (MTT assay) decreased significantly to 56.1% for MOSEC incubated with Rev_AuNPs. Moreover, Figure 5b reveals that LDH release from this cell line reached 76.5%, nearly twice that from the cell line incubated with the free Rev peptide (39.0%). Furthermore, the incubated MOSEC death rate for the reference AuNPs was only 15.9%, implying a very low cytotoxic effect by AuNP itself. Notably, a significantly lower MOSEC survival rate (24.2%, MTT assay) occurred when the Rev_AuNPs incubation time was increased to 48 h (Figure 5c). Similar results from the LDH assay were obtained. A remarkably high death rate of 92.3% is seen (Figure 5d). Once more, the free 1398

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Figure 5. Cytotoxicity data after MOSEC exposure to the reference AuNPs, the free Rev peptide, and the Rev_AuNPs for (a, b) 24 and (c, d) 48 h. (a, c) MTT assays; (b, d) LDH assays.

a confluent MOSEC monolayer with the Rev_AuNPs. After 24 h of exposure, the cells were fixed and examined by TEM. Figure 6 compares the degree of Rev_AuNPs internalization at different periods. Initially (0 h), no Rev_AuNPs were present within the cells (Figure 6a). After 24 h, however, AuNP internalization occurred in the cells (Figure 6b). Notably, Rev_AuNPs were localized in the intracellular membrane-bound vesicles and were not freely dispersed in the cytoplasm. These results are consistent with those of previous investigations performed with HeLa cells.46 By using TEM, Chithrani et al. found that AuNPs having diameters between 14 and 74 nm entered cultured HeLa tumor cells where they became trapped in vesicles of the cytoplasm. Thus, we suspect that Rev_AuNP’s enhanced cytotoxicity might be mediated through internalization of the particles into the mouse ovarian cells.

Figure 6. TEM images of MOSEC internalization of Rev_AuNPs after (a) 0 h and (b) 24 h.

Rev peptide and the reference AuNPs had no dramatic effect on the MOSECs after 48 h of incubation, suggesting that Rev_ AuNPs had a significantly greater cytotoxic effect on the MOSEC cell line than equal doses of either the free Rev peptide or the reference AuNPs did. To investigate whether the enhanced Rev_AuNP cytotoxicity was due to internalization of Rev_AuNPs by the cells, we incubated

’ SUMMARY In summary, we have developed a simple, one-pot method for the direct synthesis of AuNPs in the presence of the Rev peptide in aqueous solution. Varying either the concentration of auric ions or the reaction temperature influenced the sizes and shapes of the synthesized Rev_AuNPs. In vitro testing (LDH and MTT assays) using 25-nm size Rev_AuNPs produced significantly greater cytotoxicity toward the MOSEC line than the pure Rev peptide exhibited. These results suggest that Rev_AuNP might be more effective than the free Rev peptide when used in cancer therapies. We anticipate that our new route toward the direct synthesis of biomolecule-conjugated Au nanocomposites will pave the way toward new effective therapeutic applications. 1399

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’ ASSOCIATED CONTENT

bS

Supporting Information. Chemical structure of the Rev peptide, UV vis calibration curve for the Rev peptide and Rev_AuNPs, TEM images of Rev_AuNPs synthesized at elevated temperature, and TEM image of reference AuNPs. This material is available free of charge via the Internet at http://pubs. acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Dr. Yian Tai, Department of Chemical Engineering, National Taiwan University of Science and Technology, 43 Keelung Road, Sec. 4, Taipei 106, Taiwan. Phone: +886-2-2737-6620. Fax: +886-2-2737-6644. E-mail: [email protected].

’ ACKNOWLEDGMENT We are grateful to Mr. Tsung-Ching Wu for technical assistance, Taiwan Tech (NTUST) for providing support of the TEM and XPS instruments, and Professor Thomas C.-K. Yang (NTUT) for providing support of the XRD instrument. This study was partially funded by the National Science Council (NSC98-2113-M-011-002-MY2). ’ REFERENCES (1) Giljohann, D. A., Seferos, D. S., Daniel, W. L., Massich, M. D., Patel, P. C., and Mirkin, C. A. (2010) Gold nanoparticles for biology and medicine. Angew. Chem., Int. Ed. 49, 3280–3294. (2) Jain, P. K., Huang, X., El-Sayed, I. H., and El-Sayed, M. A. (2008) Noble metals on the nanoscale: Optical and photothermal properties and some applications in imaging, sensing, biology, and medicine. Acc. Chem. Res. 41, 1578–1586. (3) Cao, Y. C., Jin, R., and Mirkin, C. A. (2002) Nanoparticles with Raman spectroscopic fingerprints for DNA and RNA detection. Science 297, 1536–1540. (4) Skrabalak, S. E., Chen, J., Sun, Y., Lu, X., Au, L., Cobley, C. M., and Xia, Y. (2008) Gold nanocages: Synthesis, properties, and applications. Acc. Chem. Res. 41, 1587–1595. (5) Orendorff, C. J., Sau, T. K., and Murphy, C. J. (2006) Shapedependent plasmon-resonant gold nanoparticles. Small 2, 636–639. (6) 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, 8378–8379. (7) Chuang, Y. C., Li, J. C., Chen, S. H., Liu, T. Y., Kuo, C. H., Huang, W. T., and Lin, C. S. (2010) An optical biosensing platform for proteinase activity using gold nanoparticles. Biomaterials 31, 6087–6095. (8) Han, G., Ghosh, P., and Rotello, V. M. (2007) Functionalized gold nanoparticles for drug delivery. Nanomedicine 2, 113–123. (9) Huang, X., El-Sayed, I. H., Qian, W., 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, 2115–2120. (10) Bhattacharyya, S., Kudgus, R. A., Bhattacharya, R., and Mukherjee, P. (2011) Inorganic nanoparticles in cancer therapy. Pharm. Res. 28, 237–259. (11) Kennedy, L. C., Bickford, L. R., Lewinski, N. A., Coughlin, A. J., Hu, Y., Day, E. S., West, J. L., and Drezek, R. A. (2011) A new era for cancer treatment: Gold-nanoparticle-mediated thermal therapies. Small 7, 169–183. (12) Paciotti, G. F., Myer, L., Weinreich, D., Goia, D., Pavel, N., McLaughlin, R. E., and Tamarkin, L. (2004) Colloidal gold: A novel nanoparticle vector for tumor directed drug delivery. Drug Delivery: J. Delivery Targeting Ther. Agents 11, 169–183.

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Bioconjugate Chemistry

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dx.doi.org/10.1021/bc2001215 |Bioconjugate Chem. 2011, 22, 1394–1401