Au Hybrid Nanoprobe via a One

Aug 7, 2013 - Yang Zhao , Jing Peng , Jingjin Li , Ling Huang , Jinyi Yang , Kai Huang .... Yuru Wang , Tianren Wang , Xi Chen , Yang Xu , Huanrong Li...
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Fabrication of Multifunctional Gd2O3/Au Hybrid Nanoprobe via a One-Step Approach for Near-Infrared Fluorescence and Magnetic Resonance Multimodal Imaging in Vivo Shao-Kai Sun,† Lu-Xi Dong,† Yang Cao,‡ Hao-Ran Sun,‡ and Xiu-Ping Yan*,† †

State Key Laboratory of Medicinal Chemical Biology (Nankai University), Synergetic Innovation Center of Chemical Science and Engineering (Tianjin), and Research Center for Analytical Sciences, College of Chemistry, Nankai University, 94 Weijin Road, Tianjin 300071, China ‡ Department of Radiology, Tianjin Medical University General Hospital, 154 Anshan Road, Tianjin 300052, China S Supporting Information *

ABSTRACT: Facile fabrication of multimodal imaging probes is highly desired for bioimaging application due to their integrated advantages of several imaging modalities. Here, we report a simple and one-step mild strategy to fabricate a multifunctional Gd2O3/Au hybrid nanoprobe. Bovine serum albumin (BSA) was used as the template in the biomineralization synthesis. The fabricated BSA-Gd2O3/Au nanoprobe showed excellent chemical stability, intense near-infrared (NIR) fluorescence, and good magnetic resonance imaging (MRI) ability. The multimodal imaging potential of the prepared multifunctional nanoprobe was demonstrated by successful NIR fluorescent and magnetic resonance blood pool imaging. Further modification of BSA-Gd2O3/Au with arginine−glycine− aspartic acid peptide c(RGDyK) (RGD) enabled the nanoprobe for targeted tumor imaging in vivo.

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multimodality properties have been mainly fabricated either by combining presynthesized independent monomodal materials16,18−26 or doping metals with additional modal imaging ability in nanoparticles.17,27−29 The former strategy offers the potential to make full use of existing monomodal imaging nanomaterials but has the limitations of increased size, difficulty in controlling size and homogeneity, possible signal loss, and complicated synthetic routes. The latter strategy has the merits of simple procedure, and versatile design based on various elements, but usually requires rigorous synthesis condition and further biocompatible modification. Therefore, the development of one-pot mild strategies for preparing biocompatible multimodal imaging nanoprobes is highly desired to simplify synthesis procedures, avoid rigorous synthesis conditions and improve biocompatibility.30−37 Herein, we report one-step synthesis of multifunctional Gd2O3/Au hybrid nanoprobe at physiological temperature (37 °C) for multimodal imaging (near-infrared (NIR) fluorescence imaging and magnetic resonance imaging (MRI)) in vivo (Scheme 1). Bovine serum albumin (BSA) was used as the template in the biomineralization synthesis as it has plenty of active chemical groups including carboxyl groups in aspartic and glutamate residues and 35 potential thiol groups.34−36 The fabricated BSA-Gd2O3/Au nanoprobe shows excellent chemical

olecular imaging is a rapidly emerging and novel multidisciplinary field due to its great potential for understanding of integrative biology, early detection and characterization of disease, and evaluation of treatment at the cellular and subcellular levels.1,2 Imaging modalities for molecular imaging generally include optical imaging,3 magnetic resonance (MR) imaging,4 computed tomography,5 ultrasound,6 and positron emission tomography7 or single photon emission computed tomography.8 The conundrum of modality selection in molecular imaging is that individual imaging modality has its own advantages and limitations. For example, optical imaging owns the merits of high sensitivity and good resolution for imaging at the cellular level but suffers the shortcomings of low spatial resolution and poor tissue penetration, while MR imaging possesses the advantages of high spatial resolution and no tissue penetrating limit but has the disadvantage of relatively low sensitivity. Multimodal imaging is attractive in early and precise diagnosis of disease and theragnosis as it integrates the advantages of several imaging modalities and provides complementary information from each imaging modality.9,10 The innovation of multimodal imaging probes plays a crucial role in multimodal imaging.11,12 Functional nanoparticles have emerged as attractive transformative multimodal imaging probes for in vivo imaging due to their unique features including multifunctionality, large surface area, structural diversity, and long circulation time in blood. So far, various multifunctional nanoparticles have been fabricated for multimodal imaging.9−17 Such multifunctional nanoparticles with © 2013 American Chemical Society

Received: June 22, 2013 Accepted: August 7, 2013 Published: August 7, 2013 8436

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water; then, 4 mL of 25 mM HAuCl4 and 1 mL of 50 mM Gd(NO3)3 were added to above solution slowly under vigorous stirring. One mL of 2 M NaOH was introduced 5 min later, and the mixture was allowed to react under vigorous stirring at 37 °C for 12 h. The prepared BSA-Gd2O3/Au nanoprobe was dialyzed against ultrapure water for 24 h to remove excess precursors. The solution was then freeze-dried or concentrated to 4 mL through ultrafiltration with a centrifuge filter tube. Synthesis of BSA-Au Nanoclusters and BSA-Gd2O3 Nanoparticles. In a typical experiment for the synthesis of BSA-Au nanoclusters, 250 mg of BSA was dissolved in 6 mL of ultrapure water; 4 mL of 25 mM HAuCl4 was then added to the above solution slowly under vigorous stirring. For the synthesis of BSA-Gd2O3 nanoparticles, 250 mg of BSA was dissolved in 9 mL of ultrapure water; then, 1 mL of 50 mM Gd(NO3)3 was added to the above solution slowly under vigorous stirring. All the other procedures are the same as those for the synthesis of BSA-Gd2O3/Au nanoprobe. Bioconjugation of BSA-Gd2O3/Au Nanoprobe with RGD. RGD peptide was conjugated to the carboxyl groups of BSA-Gd2O3/Au nanoprobe to form RGD-Gd2O3/Au conjugates by using coupling agents (EDC and NHS). Typically, 2 mL of concentrated BSA-Gd2O3/Au solution was added to the mixture of 7 mL of H2O and 0.75 mL of 0.2 M PBS (pH 7.4). EDC (57.5 mg) was added to the mixture to activate BSAGd2O3/Au nanoparticles in the dark for 10 min; then, 34.5 mg of NHS was added to the above solution, and the mixture was stirred vigorously in the dark for 1 h. Then, 5 mL of 4 mg mL−1 c(RGDyK) solution was poured into the solution of activated BSA-Gd2O3/Au, and the mixture was incubated at room temperature in the dark for 12 h with continuous stirring. After centrifugal ultrafiltration (5500 rpm, 20 min) with a centrifuge filter tube, the obtained RGD-BSA-Gd2O3/Au nanoparticles were redissolved with 2 mL of PBS buffer (pH 7.4, 10 mM). Relaxation Time and in Vitro MR Images. The longitudinal relaxation times were measured using a PQ001 MRI system (Niumag Corporation, Shanghai, China). T1weighted MR images were obtained on a MicroMR-25 mini MRI system (Niumag Corporation, Shanghai, China) under the T1-weighted sequence (spin echo, TR/TE = 100.0/18.2 ms, thickness = 3 mm, 0.55 T, 32.0 °C). Animal Model. The adult athymic BALB/c mice (12−14 g) and Kunming mice (20−22 g) were obtained from Beijing HFK bioscience Co., Ltd. (Beijing, China). Nude mice (29−32 g) harboring U87-MG tumors (8 mm) in axillary fossa were obtained from Tianjin Medical University Cancer Institute and Hospital. Animal procedures were in agreement with the guidelines of Tianjin Medical University Cancer Institute and Hospital. Toxicity Test in Mice. The toxicity caused by side-effect on organs of the BSA-Gd2O3/Au nanoprobe in mice was evaluated by monitoring histological changes of several related organs. After intravenous administration of BSA-Gd2O3/Au (200 μL, 0.37 mmol Au per kg mice), adult athymic BALB/c mice (n = 3) were dissected at 7 days and related organs were removed, which were stained with hematoxylin and eosin (H&E) before the tissue damage of the organs was investigated. NIR Fluorescent Blood Pool Imaging and TumorTargeted Imaging in Vivo. In vivo NIR fluorescent imaging was performed on a Berthold NightOWL LB 983 in vivo Imaging System (Bad Wildbad, Germany). The excitation filter was set as 530 nm, and the emission filter was set as 700 nm.

Scheme 1. Fabrication of BSA-Gd2O3/Au Nanoprobe

stability and biocompatibility, intense NIR fluorescence, and good MRI ability. The prepared multimodal imaging nanoprobe was successfully applied in near-infrared fluorescent and magnetic resonance blood pool imaging and tumor targeted imaging in vivo.

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EXPERIMENTAL SECTION Materials and Reagents. All agents were at least analytical grade. Ultrapure water (Hangzhou Wahaha Group Co. Ltd., Hangzhou, China) was used throughout this work. HAuCl4· 4H2O was obtained from Guoyao Chemicals Co. (Shanghai China). Gd(NO3)3·6H2O was purchased from Alfa Aesa (Tianjin, China). BSA was provided by Beijing Dingguo Biotechnology Co., Ltd. (Beijing, China). Arginine−glycine− aspartic acid peptide c(RGDyK) (RGD) was obtained from ChinaPeptide Co., Ltd. (Shanghai, China). 1-Ethyl-3-(3dimethylaminopropy) carbodiimide (EDC) and N-hydroxysulfosuccinimide sodium salt (NHS) were bought from Aladdin Reagent (Shanghai, China). NaOH, NaH2PO4·2H2O, and Na2HPO4·12H2O were all purchased from Guangfu Fine Chemical Research Institute (Tianjin, China). Instrumentation and Characterization. The contents of Au and Gd elements in the prepared BSA-Gd2O3/Au nanoprobe were measured by an X series inductively coupled plasma mass spectrometer (ICPMS) (Thermo Elemental, UK). X-ray photoelectron spectroscopy (XPS) measurements were carried out on an Axis Ultra DLD spectrometer fitted with a monochromated Al Kα X-ray source (hν = 1486.6 eV), hybrid (magnetic/electrostatic) optics, and a multichannel plate and delay line detector (Kratos Analytical, Manchester, UK). The morphology and microstructure of BSA-Gd2O3/Au nanoparticles were characterized by a Philips Tecnai G2 F20 (Philips, Holland) field emission high-resolution transmission electron microscopy (HRTEM). The samples for HRTEM were prepared by drying sample droplets from water dispersion onto a 230-mesh Cu grid coated with a lacey carbon film. The Fourier transform infrared (FT-IR) spectra (of 400−4000 cm−1) were measured with a Nicolet IR AVATAR-360 spectrometer (Nicolet, USA) with pure KBr as the background. All circular dichroism (Jasco J-715) spectra were measured in a PBS buffer solution (5 mM, pH 7.4) in a 1 mm quartz cell at ambient temperature. The fluorescence measurements were performed on an F-4500 spectrofluorometer (Hitachi, Japan) equipped with a plotter unit and a quartz cell (1 cm × 1 cm). The slit width was 10 and 10 nm for excitation and emission, respectively. The photomultiplier tube (PMT) voltage was set at −700 V. The fluorescence quantum yield of BSA-Gd2O3/Au nanoprobe was determined on an FLS920 spectrometer with an integration sphere attachment under excitation at 380 nm (Edinburgh, UK). UV−vis absorption spectra were recorded using a UV-3600 UV−vis-NIR spectrophotometer (Shimadzu, Japan). Synthesis of BSA-Gd2O3/Au Nanoprobe. In a typical experiment, 250 mg of BSA was dissolved in 5 mL of ultrapure 8437

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Fluorescence images were recorded by the CCD camera with constant exposure time. To investigate the potential use of the BSA-Gd2O3/Au probe in NIR fluorescent blood pool imaging, nude mice (n = 3) were anesthetized with 4% chloral hydrate (6 mL kg−1) first, and fluorescent imaging was carried out before and after injection via the tail vein of 200 μL of prepared nanoprobe (0.37 mmol Au per kg mice). A series of images were collected at scheduled intervals of 48 h post injection. To evaluate the targeting ability of the RGD-BSA-Gd2O3/Au nanoprobe toward U87-MG tumors, the U87-MG tumor bearing mice were anesthetized with 4% chloral hydrate (6 mL kg−1), followed with intravenous injection (via the tail vein) of 0.48 mmol Au per kg body weight of the RGD-/BSA-Gd2O3/ Au nanoprobe (500 μL) (n = 3 for each model). A series of images were collected at scheduled intervals of 48 h post injection. To compare the targeting ability between BSAGd2O3/Au and RGD-BSA-Gd2O3/Au, the fluorescent imaging was also performed on the U87-MG tumor bearing mice using the BSA-Gd2O3/Au nanoprobe (500 μL, 0.48 mmol Au per kg body weight). To quantify the distribution and targeting ability of nanoprobes in mice, the average fluorescence intensity in the regions of interest (liver or tumors) was calculated by using the software of in vivo Imaging System and then plotted against time. In Vivo MR Blood Pool Imaging. In vivo MR imaging of Kunming mice (18−20 g, n = 3) was carried out on a 3.0 T MR imaging system (GE Signa Excite). Typically, 300 μL of BSAGd2O3/Au nanoprobe solution (0.38 mmol Au per kg body) was injected by tail vein into the anesthetized Kunming mice with 4% chloral hydrate (6 mL kg−1). Images were obtained using a small animal coil, before and at subsequent intervals following injection, using a fat-saturated 3D gradient echo imaging sequence (TR/TE = 9.7/3.0 ms; inversion time = 5.0 ms; FA = 13°; FOV = 110 mm × 110 mm; matrix = 256 × 256; slice thickness = 1 mm without gap; 176 coronal slices obtained).

Figure 1. (A) HRTEM image of BSA-Gd2O3/Au nanoprobe. (B) XPS spectra of Au 4f for BSA-Gd2O3/Au nanoprobe. Black line, the background; gray line, experimental data points; blue line, data-fitted curve; red curves, Au-components analyzed by XPS software. (C) XPS spectra of Gd 4d for BSA-Gd2O3/Au nanoprobe. Black line, background; gray line, experimental data points; red line, data-fitted (Gd-components analyzed) by XPS software. (D) CD spectra of BSA and BSA-Gd2O3/Au nanoprobe. (E) Absorption and NIR fluorescence spectra of BSA-Gd2O3/Au nanoprobe. Inset: The photographs of asprepared BSA-Gd2O3/Au nanoprobe solution under visible light and UV light. (F) The fluorescence intensity of BSA-Gd2O3/Au nanoprobe under different pH conditions (20 mM PBS).

Gd 4d spectra give a peak at 142.3 eV, corresponding to Gd2O3 (Figure 1C).38,39 The XPS spectra of BSA-Gd2O3/Au nanoprobe are thus similar to those of BSA-Au nanoclusters and BSAGd2O3 nanoparticles (Figure S2 in the Supporting Information). Compared to BSA-Au nanoclusters, the as-prepared BSAGd2O3/Au nanoprobe gives similar fluorescent spectra (Figure S3 in the Supporting Information) and fluorescence lifetime (1289 ± 150 ns for BSA-Gd2O3/Au nanoprobe vs. 1165 ± 228 ns for BSA-Au nanoclusters). The above results indicate that BSA-Gd2O3/Au nanoprobe is the nanocomposite of Gd2O3 nanoparticles and Au nanoclusters. The conformational change of BSA was investigated by circular dichroism spectrometry (CD). The CD spectra of BSA show two negative peaks at 210 and 220 nm (Figure 1D) from the highly α-helical secondary structure, while in the CD spectra of BSA-Gd2O3/Au nanoparticles, the peak at 210 nm in native BSA was shifted to 204 nm, and the peak at 220 nm almost disappeared (Figure 1D). The above results show that the highly basic condition promoted the conformational change of BSA from α-helical to random coil structures, and such unfolded BSA was very efficient in promoting the coordination interaction between BSA and metal ions and controlling the crystal growth. Fluorescence Properties of BSA-Gd2O3/Au Nanoprobe. The size regime of noble metal nanoclusters is comparable to the Fermi wavelength of the conduction electrons. Discrete and size-tunable electronic transitions are present due to the spatial confinement of free electrons in metal nanoclusters, leading to molecular-like luminescent property.35,36 The prepared BSA-Gd2O3/Au nanoprobe emits strong NIR fluorescence at 700 nm with an absolute quantum yield of 6.5% (Figure 1E), and the fluorescence intensity shows

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RESULTS AND DISCUSSION Synthesis and Characterization of BSA-Gd2O3/Au Nanoprobe. The BSA-Gd2O3/Au nanoprobe was prepared via a one-step biomineralization approach. Under alkalic condition, Au nanoclusters were produced via the reduction of Au(III) by tyrosine and possibly other residues with reducing functionality in BSA,36 while Gd2O3 nanoparticles were formed on the basis of precipitation reaction.34,38 The different crystal growth mechanisms facilitate the formation of the nanocomposite of Gd2O3 nanoparticles and Au nanoclusters. The composition, size, and morphology of the prepared BSAGd2O3/Au nanoprobe were characterized by HRTEM, ICPMS, XPS, and FT-IR. The freeze-dried BSA-Gd2O3/Au nanoprobe contains 6.3% Au and 1.9% Gd (w/w) from ICPMS analysis, corresponding to about 23 Au atoms and 9 Gd atoms in one BSA molecule. The HRTEM images of BSA-Gd2O3/Au nanoprobe show a round-like geometry with an average size of 3.3 nm (Figure 1A). The functionalization of the nanoprobe with BSA was confirmed by FT-IR (Figure S1 in the Supporting Information). The XPS spectra of Au 4f7/2 consists of two distinct components at 83.5 and 83.9 eV, corresponding to Au(0) and Au(I) bound to S in BSA, respectively (Figure 1B).36 The Au(0) and Au(I) are important to fabricate the core and the surface shell (via the metal−ligand interaction of Au(I) bound to the S in BSA) of Au nanoclusters, respectively. The 8438

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injection (Figure 3A). Compared to other PEGylated nanomaterials,41,42 the BSA-Gd2O3/Au nanoprobe shows a much

negligible change in the pH range of 5.0−8.3 (Figure 1F). It should be noted that fluorescent nanoparticles with >1% absolute quantum yields are qualified for NIR imaging in vivo.40 The UV−vis spectra of the BSA-Gd2O3/Au nanoprobe shows a peak at 280 nm for aromatic amino acid and a shoulder peak around 510 nm from Au nanoclusters (Figure 1E; Figure S4 in the Supporting Information). The prepared BSA-Gd2O3/Au nanoprobe also possesses good colloidal stability as not any visible precipitation was observed when it was incubated in 20 mM PBS (pH 7.4) for at least 2 months. The above results show that the prepared BSA-Gd2O3/Au nanoprobe has excellent optical properties and chemical stability for in vivo NIR fluorescent imaging. In Vitro Relaxivity Characterization of BSA-Gd2O3/Au Nanoprobe. The developed BSA-Gd2O3/Au nanoprobe was also compared to a commercial MR imaging contrast agent GdDTPA in respect of magnetic property to show the potential of the proposed nanoprobe for MR imaging application. For this purpose, the relaxivity values of the BSA-Gd2O3/Au nanoprobe and Gd-DTPA were determined by measuring longitudinal proton relaxation time (T1) as a function of Gd concentration. The r1 value of the prepared BSA-Gd2O3/Au nanoprobe was 13.1 s−1 mM−1 Gd, almost three times that of Gd-DTPA (4.6 s−1 mM−1 Gd) (Figure 2A). Furthermore, T1-weighted MR

Figure 3. (A) NIR fluorescent blood imaging in vivo using BSAGd2O3/Au nanoprobe. (B) Magnetic resonance blood imaging in vivo using BSA-Gd2O3/Au nanoprobe.

longer circulation time. The circulation of the probe in the vessel was still clear even at 6 h post injection, which is desired as a good contrast agent for blood pool imaging. The fluorescent signal in the superficial vasculature decreased gradually and mainly accumulated in liver. After 24 h post injection, the probe was mostly cleared from the body as the fluorescence nearly disappeared (Figure 3A). To semiquantitatively analyze the distribution of the nanoprobe in mouse, the average fluorescence intensity in the region of liver was determinated at different time intervals (Figure S6 in the Supporting Information). The average fluorescence intensity in the region of liver became stronger after the injection of nanoprobe, reaching a peak at 2 h post injecion and then slowly declining, which further demonstrates that the nanoprobe was mainly accumulated in liver and was cleared by liver at last. The MR images show that the vessel of the whole Kunming mouse body was easily visualized immediately after injecting the probe (300 μL, 0.38 mmol Au per kg mice). At 10 min post injection, the great vessels of abdominal cavity show intensive MR signal with excellent contrast result. At 20 min post injection, the kidney contour and vessels in liver and kidney were seen clearly. The MR signal disappeared after 24 h post injection (Figure 3B), again indicating the clearance of the probe from the body as shown in NIR fluorescent imaging (Figure 3A). It could be seen that Kunming mouse could clear the nanoprobe a little faster than nude mouse; however, both types of mice showed a similar clearance process. The great vessels of abdominal cavity and the kidney contour and vessels in liver and kidney could be seen clearly from the MR images due to high spatial resolution and no tissue penetrating limit, while the partial region where the MR imaging possesses low signal-noise ratio was visible in the NIR images due to high sensitivity. These results demonstrate the great potential of the synthesized BSA-Gd2O3/Au nanoprobe for multimodal imaging to obain complementary information from each imaging modality. Tumor Targeted Imaging in Vivo. To demonstrate the potential application of BSA-Gd2O3/Au imaging probe for tumor targeted imaging in vivo, RGD peptide, a targetable imaging molecule for imaging integrin ανβ3 overexpressed on

Figure 2. (A) r1 relaxivity curves of BSA-Gd2O3/Au nanoprobe and Gd-DTPA. (B) T1-weighted MR images of Gd-DTPA and BSAGd2O3/Au nanoprobe with various concentrations.

images of BSA-Gd2O3/Au nanoprobe and Gd-DTPA with various Gd concentrations are compared (Figure 2B). BSAGd2O3/Au nanoprobe at lower Gd concentration even exhibits much stronger MR signals than Gd-DTPA at higher Gd concentration (Figure 2B). The high r1 values and excellent MR imaging ability indicate the prepared BSA-Gd2O3/Au nanoprobe is a promising positive MR imaging contrast agent. In Vivo Toxicity. The in vivo toxicity of the prepared BSAGd2O3/Au nanoprobe was evaluated via monitoring histological changes of four organs (heart, liver, kidney, and intestine) of the mice after one week post injection of the probe via tail vein. The obtained organs were stained with hematoxylin and eosin for examination. No difference was found between the structures of the studied organs of the post injection mice and those of the control group, indicating no tissue damage happened due to the injected probe (Figure S5 in the Supporting Information). NIR Fluorescent and MR Blood Pool Imaging in Vivo. The prepared BSA-Gd2O3/Au nanoprobe was used for NIR fluorescent and MR blood pool imaging to show its potential for in vivo multimodal imaging. In vivo multimodal imaging was carried out by intravenous injection of the BSA-Gd2O3/Au nanoprobe (200 μL, 0.37 mmol Au per kg mice). A strong fluorescent signal was observed in the superficial vasculature of the whole nude mouse body immediately after tail vein 8439

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the tumor vasculature,43 was conjugated to BSA-Gd2O3/Au nanoprobe successfully, which was confirmed by FT-IR (Figure S1 in the Supporting Information). BSA-Gd2O3/Au and RGDGd2O3/Au nanoprobes were, respectively, injected via tail vein into U87-MG tumor-bearing mice (500 μL, 0.48 mmol Au per kg mice). As shown in Figure 4A and Figure S7 in the

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ASSOCIATED CONTENT

* Supporting Information S

Additional information as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: (86)22-23506075. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Basic Research Program of China (Grant 2011CB707703), the National Natural Science Foundation of China (Grants 21275079, 20935001), and the Fundamental Research Funds for the Central Universities.

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Figure 4. In vivo NIR fluorescence imaging of U87-MG tumor-bearing mice using BSA-Gd2O3/Au nanoprobe (A) and RGD-Gd2O3/Au nanoprobe (B) at 0, 1, 6, 12, and 24 h, respectively. Red dotted circles indicate the regions of tumors.

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Supporting Information, the NIR fluorescence from BSAGd2O3/Au nanoprobe appeared in the tumor at 0.5 h post injection due to the enhanced permeability and retention effect and tumors were readily distinguished from surrounding tissues after 1 h, but the fluorescent intensity in the tumor dramatically decreased after 2 h. In contrast, the NIR fluorescence from RGD-Gd2O3/Au nanoprobe in the tumor was still apparent even after 24 h (Figure 4B; Figure S7 in the Supporting Information). To semiquantitatively analyze the targeting ability of the two probes, the average fluorescence intensity in the regions of tumors was calculated at different time intervals. For the BSA-Gd2O3/Au nanoprobe and RGD-Gd2O3/Au nanoprobe, the average fluorescence intensity in the regions of tumors reached a peak at about 2 h post injection and 12 h post injection (Figure S6 in the Supporting Information), respectively, which indicates that the highly specific targeting of RGD-Gd2O3/Au nanoprobe to integrin ανβ3-positive U87-MG tumor induced the long-term retention of RGD-Gd2O3/Au nanoprobe in the tumor site.

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CONCLUSIONS In summary, we have reported a one-step fabrication of multifunctional BSA-Gd2O3/Au hybrid nanoprobe for in vivo imaging. Our method achieves the goal of one-step mild synthesis of biocompatible nanoparticles with excellent chemical stability, intense NIR fluorescent emission, and good MR signal for multimodality imaging. Conjugation with RGD peptide enables the functional imaging probe for targeted tumor imaging in vivo. Compared to the previous multimodal imaging agents, the proposed nanoprobe integrates the advantages of a one-step synthesis route, mild experimental condition, no toxic agents, and biocompatible product. More importantly, the proposed design provides an innovative strategy for the preparation of biocompatible multifunctional imaging agents using a one-step mild synthesis route. 8440

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Analytical Chemistry

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dx.doi.org/10.1021/ac401879y | Anal. Chem. 2013, 85, 8436−8441