Incorporation of Computed Tomography and Magnetic Resonance

Nov 15, 2013 - ... Tianjin Medical University General Hospital, 154 Anshan Road, ... Medical Center, Peking Union Medical College, Beijing 100021, Chi...
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Incorporation of Computed Tomography and Magnetic Resonance Imaging Function into NaYF4:Yb/Tm Upconversion Nanoparticles for in Vivo Trimodal Bioimaging Ji-Wei Shen,† Cheng-Xiong Yang,† Lu-Xi Dong,† Hao-Ran Sun,‡ Kai Gao,§ and Xiu-Ping Yan*,† †

State Key Laboratory of Medicinal Chemical Biology (Nankai University), Collaborative 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 § Key Laboratory of Human Disease Comparative Medicine, Ministry of Heath, Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences & Comparative Medical Center, Peking Union Medical College, Beijing 100021, China S Supporting Information *

ABSTRACT: Rational design and fabrication of multimodal imaging nanoprobes are of great significance for in vivo imaging. Here we report the fabrication of a multishell structured NaYF4:Yb/Tm@NaLuF4@NaYF4@NaGdF4 nanoprobe via a seed-mediated epitaxial growth strategy for upconversion luminescence (UCL), X-ray computed tomography (CT), and magnetic resonance (MR) trimodal imaging. Hexagonal phase NaYF4:Yb/Tm is used as the core to provide UCL, while the shell of NaLuF4 is epitaxially grown on the core not only to provide an optically inert layer for enhancing the UCL but also to serve as a contrast agent for CT. The outermost NaGdF4 shell is fabricated as a thin layer to give the high longitudinal relaxivity (r1) desired for MR imaging. The transition shell layer of NaYF4 not only provides an interface to facilitate the formation of NaGdF4 shell but also inhibits the energy transfer from inner upconversion activator to surface paramagnetic Gd3+ ions. The fabricated multishell structured nanoprobe shows intense nearinfrared UCL, high r1 value of 3.76 mM−1 s−1, and in vitro CT contrast effect. The multishell structured nanoprobe offers great potential for in vivo UCL/CT/MR trimodal imaging. Further covalent bonding of folic acid makes the multishell structured nanoprobe promising for in vivo targeted UCL imaging of tumor-bearing mice.

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fabricated for UCL/MR imaging by doping paramagnetic Gd3+/Mn2+ ions20,21 or epitaxial growth of the NaGdF4/FexOy shell.22−25 However, the low contrast CT efficiency limits their in vivo CT application without further modification.7,8 Small and uniform β-phase NaGdF4 based UCNPs have also been reported as dual-modal UCL/MR26,27 and even trimodal UCL/ CT/MR imaging nanoprobes.28 However, surface quenchers make NaGdF4 less efficient as upconversion host material; very high excitation light density is thus needed for in vivo UCL imaging of small animals.27 The surface quenching effect can be reduced by covalently linking NaGdF4 based UCNPs onto methylphosphonate functionalized silica nanospheres, but the hydrodynamic diameter of the resulting probe increases to a great extent.28 NaLuF4 based UCNPs not only provide higher UCL efficiency even than NaYF4 based UCNPs,29 but also powerful CT contrast ability from large atomic number Lu3+ ions.9−11

ultimodal imaging is promising for accurate and reliable disease diagnosis due to the integrated merits of several imaging modalities and complementary information from each imaging modality.1−3 Design and fabrication of efficient multimodal nanoprobes is one of the keys to successful multimodal imaging. Rare-earth upconversion nanoparticles (UCNPs) have been widely used in small-animal imaging, as they emit visible-near-infrared (vis-NIR) fluorescence under continuous-wave NIR light excitation and show lack of autofluorescence and high tissue penetration.4−6 UCNPs are also attractive as potential multimodal nanoprobes for the forceful trend to integrate the multifunction necessary for optical imaging, X-ray computed tomography (CT), and magnetic resonance (MR) imaging into one nanoprobe.4,7−13 NaYF4, NaGdF4, NaLuF4, and NaYbF4 are the most commonly used upconversion host materials for multimodal nanoprobes.4 Hexagonal (β) phase NaYF4 is one of the most efficient host materials for UCNPs.14−17 Recently, great efforts have been made to prepare small and uniform NaYF4 based UCNPs as upconversion luminescence (UCL) nanoprobes for bioimaging.18,19 Dual-modal NaYF4 based UCNPs have been © 2013 American Chemical Society

Received: October 28, 2013 Accepted: November 15, 2013 Published: November 15, 2013 12166

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However, the quite narrow β-phase transformation temperature range makes controllable solvothermal synthesis of uniform NaLuF4 based UCNPs with high UCL efficiency difficult.30−33 NaYbF4 based UCNPs also show powerful CT ability from large atomic number Yb3+ ions, but solvo(hydro)thermally synthesized NaYbF4 (or BaYbF5) based UCNPs are generally in the cubic phase.8,13 Doping paramagnetic Gd3+ ions into NaYbF4 or NaLuF4 based UCNPs facilitates the control of their phase and morphology but leads to the decrease of UCL.12,34 It is, therefore, imperative and significant to prepare the controllable morphology of β-phase UCNPs with good performance of UCL, CT, and magnetic resonance imaging (MRI) for trimodal bioimaging. Here we report the fabrication of a multifunctional multishell structured NaYF4:Yb/Tm@NaLuF4@NaYF4@NaGdF4 nanoprobe (MUCNP) for in vivo trimodal imaging via a seedmediated epitaxial growth strategy.35−41 Rational structure design to integrate UCL, CT, and MRI capacity into one nanoparticle organically is described for the fabrication of the in vivo trimodal imaging nanoprobe. The prepared MUCNP shows attractive performance for in vivo small-animal UCL/ CT/MR trimodal imaging.

4500 spectrofluorometer (Hitachi, Tokyo, Japan) equipped with a plotter unit and a quartz cell (1 cm × 1 cm). Photoluminescent measurements were taken at an excitation wavelength of 370 nm. Upconversion fluorescence spectra were obtained on a PTI spectrofluorometer (Photon Technology International, Birmingham, NJ, USA) with an external 0−0.5 W adjustable 980 nm laser source (SDL-980-LM-500MFL, Shanghai DreamLasers Technology Co., China) as the excitation source. Dynamic light scattering (DLS) experiments and ζ potential determination were carried out on a Malvern Zetasizer (Nano series ZS, Worcestershire, U.K.). The fluorescence images of the mice were obtained with a NightOWL LB 983 in vivo Imaging System (Berthold, Bad Wildbad, Germany) with an external and adjustable continuous-wave infrared laser (980 nm, BWT Beijing Ltd., Beijing, China) as the excited source. The emission filter was set as 790 ± 20 nm, and fluorescence images were recorded by the CCD camera with constant exposure time. Synthesis of OA Capped β-NaYF4:Yb/Tm@NaLuF4@ NaYF4@NaGdF4 Nanoparticles. The core β-NaYF4:Yb/Tm (20/0.5 mol %) nanoparticles and multishell layers were prepared by a solvothermal method with corresponding rareearth acetate as precursors.18,35−41 For the synthesis of β-NaYF4:Yb/Tm nanoparticles, 3 mL of OA and 7 mL of 1-octadecene were added to a 50 mL flask containing 0.4 mmol of Ln(CH3COO)3 (Ln: 79.5 mol % Y + 20 mol % Yb + 0.5 mol % Tm). The solution was heated at 150 °C with magnetic stirring for 1 h to obtain a transparent yellow solution and then cooled down to room temperature. NH4F (1.6 mmol) and NaOH (1 mmol) in methanol (5 mL) were added dropwise, and the solution was stirred at 50 °C for 30 min. After degassing at 100 °C, the solution was heated at 300 °C for 1 h under argon atmosphere and then cooled down to room temperature. The resulting nanoparticles were precipitated with ethanol and collected by centrifugation. After washing with ethanol for several times, the β-NaYF4:Yb/Tm nanoparticles were finally redispersed in 3 mL of cyclohexane for shell coating. For the synthesis of β-NaYF4:Yb/Tm@NaLuF4 nanoparticles, the shell stock solution was prepared by mixing Lu(CH3COO)3 (0.4 mmol) with 3 mL of OA and 7 mL of 1octadecene in a 50 mL flask. The mixture was heated at 150 °C for 1 h and then cooled down to 50 °C. The prepared βNaYF4:Yb/Tm core nanoparticles were added and stirred for 10 min. NH4F (1.6 mmol) and NaOH (1 mmol) in methanol (5 mL) were added dropwise and stirred at 50 °C for 30 min. Subsequent procedures were similar to those in preparing βNaYF4:Yb/Tm nanoparticles. The prepared β-NaYF4:Yb/Tm@ NaLuF4 nanoparticles were also redispersed in 3 mL of cyclohexane for shell coating. The procedures for subsequent epitaxial growth of NaYF4 and NaGdF4 shell layers on the β-NaYF4:Yb/Tm@NaLuF4 nanoparticles were similar to those for NaLuF4 shell growth except that 0.2 mmol of corresponding rare-earth acetate and 3 mL of methanol solution containing NH4F (0.8 mmol) and NaOH (0.5 mmol) were used. In addition, the final reaction temperature was kept at 280 °C for 1.5 h instead of 300 °C for 1 h. Synthesis of OA Capped β-NaYF 4:Yb/Tm@NaYF 4 Nanoparticles. For comparison, the β-NaYF4:Yb/Tm@ NaYF4 nanoparticles were also prepared based on the same procedures for preparing β-NaYF4:Yb/Tm@NaLuF4 nano-



EXPERIMENTAL SECTION Reagents and Materials. All reagents used were of at least analytical grade unless otherwise stated. Y(CH3COO)3·4H2O (99.9%), Lu(CH 3 COO) 3 ·xH 2 O (x ≈ 4, 99.9%), Yb(CH3COO)3·xH2O (99.9%), Gd(CH3COO)3·xH2O (99.9%), Tm(CH3COO)3·xH2O (99.9%), 1-octadecene (technical grade, 90%), and oleic acid (OA, technical grade, 90%) were purchased from Alfa Aesar (Tianjin, China). 1-Ethyl-3-(3dimethylaminopropyl)carbodiimide hydrochloride (EDC), Nhydroxysuccinimide sodium (NHS), 4-morpholineethane sulfonic acid (MES) monohydrate, and folic acid (FA) were purchased from Aladdin Reagent (Shanghai, China). Poly(acrylic acid) (PAA) and diethylene glycol (DEG) were purchased from Sigma-Aldrich (St. Louis, MO, USA). NaOH and NH4F were purchased from Guangfu Fine Chemical Research Institute (Tianjin, China). Instrumentation and Characterization. The X-ray diffraction (XRD) spectra were collected on a Rigaku D/max2500 X-ray diffractometer (Rigaku, Tokyo, Japan) with Cu Kα radiation (λ = 0.15418 nm). The morphology and microstructure of the upconversion nanocrystals were characterized by transmission electron microscopy (TEM) using a JEOL100CX-II microscope (JEOL, Japan) and high-resolution TEM (HRTEM) on Philips Tecnai G2 F20 microscope (Philips, Eindhoven, The Netherlands) operating at an acceleration voltage of 200 kV. Energy-dispersive X-ray analysis (EDXA) of the samples was also performed during HRTEM measurements to obtain the elements of samples. X-ray photoelectron spectroscopy (XPS) measurements were carried out on a 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, U.K.). Fourier transform infrared (FTIR) spectra were obtained on a Magna-560 spectrometer (Nicolet, Madison, WI) in KBr plates. The NIR absorption spectra were recorded on a UV-3600 UV−vis-NIR spectrophotometer (Shimadzu, Japan). The content of Gd3+ ions was measured with an inductively coupled plasma mass spectrometer (ICP-MS, Thermo Elemental X7, U.K.). The photoluminescence measurements of FA were performed on an F12167

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HFK bioscience Co., Ltd. (Beijing, China). In vivo CT imaging for Kunming mouse (∼20 g) was performed on a Siemens Inveon CT system (Siemens Healthcare, Erlangen, Germany) under the conditions as follows: 80 kV, 400 μA; exposure time, 800 ms; field of view, 50 mm × 70 mm. An aqueous solution of 15 mg of PAA-MUCNP was administered to a Kunming mouse via tail vein injection under isoflurane anesthesia, followed by CT imaging. Three-dimensional CT images were reconstructed with the processing workstation. In Vitro Relaxivity Measurements and T1-Weighted MR Imaging. The T1-weighted MR images were acquired on a small Niumag MR imaging instrument with a 0.5 T magnetic field using pure water as a control at room temperature. Aqueous dilutions of PAA-MUCNP were placed in a series of 1.5 mL tubes for T1-weighted MR imaging. The parameters adopted were as follows: TR/TE, 200.0/13.6 ms; matrix, 170 × 384; FOV, 100; slice thickness, 1 mm. Furthermore, relaxation time values (T1) of the PAA-MUCNP samples were also measured on the Niumag MRI instrument by inversion recovery pulse sequence to determine longitudinal relaxivity (r1). A plot of relaxation rates (1/T1) verse Gd3+ concentration gave a fitting straight line with a slope of r1. In Vivo MR Imaging. In vivo T1-weighted MR imaging of Kunming mice (∼20 g) was carried out on a 3.0-T GE Signa Excite scanner (GE Medical Systems, Milwaukee, WI, USA). The MR imaging parameters were as follows: T1-weighted FSE sequence; TR, 360 ms; TE, 10 ms; FOV, 80 × 80; matrix, 256 × 256; slice thickness, 1.0 mm. Typically, 80 μL of PAAMUCNP solution (3 mg mL−1) was injected via tail vein into the anesthetized Kunming mouse with 4% chloral hydrate (400 mg kg−1). Images were obtained using a small animal coil, before and at 40 min following injection.

particles but with Y(CH3COO)3 shell stock solution. The resulting nanoparticles were redispersed in cyclohexane. Synthesis of Hydrophilic PAA Capped MUCNP (PAAMUCNP). The surface OA was replaced by PAA via a ligand exchange process.42 Typically, PAA (0.3 g) and 10 mL of DEG were mixed in a flask and the mixture was heated to 110 °C under vigorous stirring to form a clear solution under argon atmosphere. OA capped MUCNP (OA-MUCNP) dispersed in toluene was added, and the mixture was then heated at 240 °C for 1.5 h. After the reaction mixture was cooled to room temperature, the resulting PAA-MUCNP nanoparticles were precipitated out with ethanol, and collected by centrifugation, and washed three times with water. Synthesis of FA-MUCNP Conjugate. An EDC/NHS coupling strategy was used to prepare FA-MUCNP conjugate.43,44 Briefly, 5 mg of PAA-MUCNP was dissolved in 2.5 mL of MES buffer (10 mM, pH 5.5); 2.5 μmol of EDC and an equal amount of NHS were added and stirred for 0.5 h to activate the carboxylic acid of PAA. After adjusting the pH value of the reaction solution to ∼7.0 with dilute NaOH solution, 50 μL of FA (0.025 M solution in dimethyl sulfoxide (DMSO)) was added and stirred for 10 h at room temperature. The product (FA-MUCNP) was then collected by centrifugation, washed with DMSO and water for several times, and redispersed in water. Cytotoxicity Assay. Hela cells (human cervical carcinoma cell lines, folate receptor (FR) positive) were cultured with FAMUCNP to measure their in vitro cytotoxicity by the methyl thiazolyl tetrazolium (MTT) assay. The Hela cells were cultured in a 96-well plate (4000 cells/well) in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine plasma in 5% CO2 at 37 °C overnight. Then, fresh DMEM medium with different concentrations of FA-MUCNP (0, 50, 100, 200, 400, 600 μg mL−1) was introduced to replace the old culture medium, followed by incubation for 24 h. MTT solution (20 μL, 0.1 mg) was added to each well, and the cells were incubated for another 4 h. Subsequently, DMSO (150 μL) was added to each well and the absorbance at 545 nm of each well was measured. The cell viability was defined as the ratio of the absorbance of each sample in the presence of FA-MUCNP to that in the absence of FA-MUCNP. In Vivo UCL Imaging. Nude mice harboring Hela tumors (8 mm) in the right lateral thigh were obtained from the Institute of Hematology & Hospital of Blood Diseases, Chinese Academy of Medical Sciences & Peking Union Medical College (License Number: SCXK-2004-001, Tianjin, China). Animal procedures were in agreement with the guidelines of the regional ethic committee for animal experiments. The mice were anesthetized with intraperitoneal administration of 4% chloral hydrate at a dosage of 400 mg kg−1. For in vivo UCL imaging, PAA-MUCNP (200 μL, 2 mg mL−1) was first administered to normal nude mice via tail vein injection before imaging. FA-MUCNP (200 μL, 2 mg mL−1) was administered to Hela tumor-bearing mice via tail vein injection. In an additional control experiment, PAA-MUCNP (200 μL, 2 mg mL−1) was administered to a HeLa tumor-bearing nude mouse via intravenous injection. The fluorescence profiles in Hela tumor-bearing mice were imaged. X-ray Attenuation Measurements and In Vivo CT Imaging. Aqueous dilutions of PAA-MUCNP in a series of 1.5 mL tubes were arranged on a plastic shelf for X-ray attenuation measurements using GE discovery CT750 HD (GE Healthcare, Milwaukee, WI). Kunming mice (∼20 g) were obtained from



RESULTS AND DISCUSSION The design and fabrication of the multishell structured MUCNP is illustrated in Scheme 1. The OA capped Scheme 1. Schematic Protocol for the Preparation of Multishell Structured MUCNP

NaYF4:Yb/Tm UCNPs are designed as the core to provide highly efficient UCL and as seeds to grow a uniform β-phase NaLuF4 shell. The thick NaLuF4 shell is epitaxially grown on the core not only to give a CT functional layer but also to offer an optically inert layer for remarkable enhancement of the UCL. Subsequently, a thin NaYF4 shell is coated on the surface of NaLuF4 as a friendly interface to facilitate the generation of a new functional layer,22 that is, a MR functional shell of NaGdF4. A thin shell of NaGdF4 is coated as the outermost layer to enhance the longitudinal relaxation rate of water protons in tissues, as the Gd3+ ions buried deeply in the lattice will lose their ability.23,24 It is worth mentioning that the transition layer of NaYF4 can inhibit the energy transfer from inner up-conversion activator to outer NaGdF4 layer, thereby reducing the negative effect of Gd3+ doping on UCL efficiency.40 12168

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The OA-MUCNP was synthesized by a solvothermal method in the presence of OA and 1-octadecene.18 NaYF4:Yb/Tm UCNPs were used as the core to fully utilize their excellent properties of UCL and facile morphology control. An epitaxial growth strategy35−41 was employed for controllable coating of β-phase NaLuF4, NaYF4, and NaGdF4 shells. The thickness of the functional layers was adjusted by changing the amount of the precursors used. The size and morphology evolution of the OA-MUCNP in the synthetic process was monitored by TEM. Multishell coating of individual NaLuF4, NaYF4, and NaGdF4 shells led to an increase in the average diameter of the UCNPs from ∼24 nm (core, spherical) to ∼29 nm × 34 nm (elliptical), ∼34 nm × 36 nm (elliptical) and ∼38 nm × 39 nm (elliptical), respectively (Figure 1A−D). No significant phase separation

Figure 2. (A) XRD patterns: (a) NaYF4:Yb/Tm; (b) NaYF4:Yb/Tm@ NaLuF4; (c) NaYF4:Yb/Tm@NaLuF4@NaYF4; (d) NaYF4:Yb/Tm@ NaLuF4@NaYF4@NaGdF4. (B) UCL spectra of as-prepared MUCNP under continuous-wave excitation at 980 nm (power, 500 mW). Inset of part B: The bright field and UCL photos of the as-prepared MUCNP dispersed in cyclohexane. Figure 1. TEM images: (A) NaYF4:Yb/Tm; (B) NaYF4:Yb/Tm@ NaLuF4; (C) NaYF4:Yb/Tm@NaLuF4@NaYF4; (D) NaYF4:Yb/ Tm@NaLuF4@NaYF4@NaGdF4. HRTEM image of a typical nanoparticle (E) in (D) and its corresponding FFT (inset).

stronger. Subsequent coating of the NaGdF4 shell resulted in characteristic peaks of Gd 3d3/2 (1185 eV) and Gd 4d (140 eV), but the decay of the Y 3d peak. The variation in the element composition of OA-MUCNP with the epitaxial growth steps was traced by EDXA (Figure S2 in the Supporting Information). The above results prove the successive growth of the functional layers of NaLuF4, NaYF4, and NaGdF4 as illustrated in Scheme 1. The morphology evolution of OA-MUCNP reveals the potential difference in the crystal growth orientation of NaLuF4 and NaYF4/NaGdF4. NaYF4:Yb/Tm@NaLuF4 is elliptical while NaYF4:Yb/Tm@NaYF4 is square-like plate though both of them were synthesized under similar conditions (Figure S3 in the Supporting Information). Sequential growth of NaYF4 and NaGdF4 shell layers gradually tuned the nanoparticles from elliptical (Figure 1B) into spherical (Figure 1C and D), likely due to the faster growth rate of the NaLuF4 layer in the [001] than the [100] direction while the growth rate was slower of the NaYF4 and NaGdF4 layers in the [001] than the [100] direction.18 The above explanation for the morphology evolution of the MUCNP is also supported by the differentia of the XRD patterns (Figure 2A).47 To render OA-MUCNP dispersible in aqueous solution for biological evaluation and application, the surface OA was replaced by PAA. PAA-MUCNP still showed bright UCL in water (Figure S4 in the Supporting Information) with a mean hydrodynamic diameter of ∼55 nm (Figure S5 in the Supporting Information) and a ζ potential of about −21 mV in Tris-HCl (10 mM, pH 7.4) buffer solution. PAA-MUCNP was further functionalized by tumor-targeting molecule (FA)48,49 through an EDC/NHS coupling strategy to form FA-MUCNP conjugate. The FA functionalization was supported by FTIR spectra (Figure S6 in the Supporting Information) and revealed by comparing photoluminescence characteristic spectra before and after FA-functionalization of

was observed, demonstrating the Ostwald ripening mechanism for the shell growing process39,45 and the possible formation of the designed multishell structured MUCNP. The HRTEM image of the MUCNP shows the typical d-spacing values of 0.52 nm in the central region and 0.30 nm at the edge, corresponding to the (100) and (110) planes of β-phase NaLuF4 and NaYF4/NaGdF4 layers, respectively (Figure 1E). Furthermore, the fast Fourier transform (FFT) of the HRTEM image (Figure 1E, inset) and XRD patterns of the prepared OA-UCNPs (Figure 2A) agree well with that of β-phase NaYF4 (JCPDS: 16-0334). The NaLuF4 shell significantly enhanced the UCL of the core. About 4-fold enhancement of the UCL intensity at 800 nm was achieved by growing a NaLuF4 layer, comparable to that by growing a NaYF4 shell layer (Figure 2B). The marked enhancement of the UCL intensity after epitaxial growth also confirms the formation of the NaLuF4 shell on NaYF4:Yb/Tm. Subsequent shell layers of NaYF4 and NaGdF4 also suppressed the surface quenching effect to further improve the UCL intensity (Figure 2B). The UCL enhancement agrees with the absorption enhancement of the UCNPs in the NIR region after shell coating (Figure S1 in the Supporting Information). The multishell structure of the OA-MUCNP was further confirmed by XPS because the 2−5 nm shell thickness is in good agreement with the sensitive detection depth of XPS.46 The characteristics of shell composition change were reflected by XPS scanning on Y, Yb, Lu, and Gd (Figure 3). A new strong Lu 4d peak (195 eV) appears, and the Y 3d peak (160 eV) becomes much weaker after coating NaLuF4. Further coating of NaYF4 on the surface of the NaLuF4 shell makes the peak of Lu 4d much weaker but the peak of Y 3d much 12169

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Figure 3. XPS spectra: (A) NaYF4:Yb/Tm; (B) NaYF4:Yb/Tm@NaLuF4; (C) NaYF4:Yb/Tm@NaLuF4@NaYF4; (D) NaYF4:Yb/Tm@NaLuF4@ NaYF4@NaGdF4.

contribution of Gd3+. The T1-weighted MR signal increased as the concentration of Gd3+ increased (Figure 4B). The thin outermost layer of NaGdF4 (2 nm × 1.5 nm) gave an r1 of 3.76 mM−1 s−1 (Figure 4B). The above results show the possibility of the MUCNP as a promising contrast agent for CT and T1weighted MR imaging. The PAA-MUCNP was employed for in vivo UCL and CT imaging. To demonstrate the capacity of the PAA-MUCNP for in vivo UCL imaging, PAA-MUCNP (200 μL, 2 mg mL−1) was administered to a normal nude mouse via tail vein injection, followed by whole-body UCL imaging (Figure 5A). A significant UCL signal of the PAA-MUCNP appeared in the mouse at 0.5 h postinjection. The PAA-MUCNP distributed

PAA-MUCNP (Figure S7 in the Supporting Information). The cytotoxicity of the as-prepared FA-MUCNP is negligible (Figure S8 in the Supporting Information). To show the potential of PAA-MUCNP for CT and MR imaging application, X-ray CT and T1-weighted MRI of a phantom were performed at various concentrations of PAAMUCNP. As the concentration of PAA-MUCNP increased, the CT images became brighter and the Hounsfield unit values increased due to the high attenuation of X-ray caused by the rare-earth ions with larger atomic number (Lu3+, Yb3+, and Gd3+) in PAA-MUCNP (Figure 4A). Similarly, PAA-MUCNP also possesses good contrast effect for MR imaging due to the

Figure 5. In vivo UCL image of a nude mouse at 0.5 h after intravenous injection with PAA-MUCNP (A) and representative UCL image of dissected organs of the mouse sacrificed at 24 h postinjection with PAA-MUCNP (B). CT coronal (left, heart and liver; middle, spleen and kidney) and sagittal view (right) images of a Kunming mouse before (C) and after intravenous injection of the PAA-MUCNP (D). 1, liver; 2, stomach; 3, spleen; 4, kidney; 5, intestines; 6, lung; 7, heart.

Figure 4. (A) CT images (inset) and CT values of PAA-MUCNP with different mass concentrations. (B) Plot of relaxation rate (1/T1) against the Gd3+ concentration and T1-weighted MR images (inset) of PAA-MUCNP dispersed in water. 12170

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multishell structured trimodal UCL/CT/MR imaging nanoprobe design. The β-phase NaLuF4 shell layer with uniform morphology is achieved by a seed-mediated epitaxial growth strategy. Compared with several previous non-multi-shell structured trimodal imaging probes,7,10,11 the NaLuF4 shell design in the prepared MUCNP provides greater enhancement of UCL along with a simultaneous significant contrast effect for CT, while a thin outermost NaGdF4 shell structure results in a remarkable T1-weighted enhancement for MR imaging. The designed multishell structured nanoprobe shows promise for in vivo multimodal imaging.

mainly in the liver and spleen (Figure 5B). Relatively slow excretion rate and accumulation mainly in the liver and spleen are classical behavior of in vivo nanoparticles. PAA-MUCNP was further employed for in vivo CT application. Aqueous solution of the MUCNP was administered to a Kunming mouse, followed by CT imaging. A significant CT signal enhancement in the liver and spleen was observed at 2 h postinjection, and the main organs of the mouse can be clearly distinguished due to the excellent CT contrast effect of the MUCNP (Figure 5C vs Figure 5D). For in vivo MR imaging, the PAA-MUCNP was administered to a Kunming mouse via tail vein injection. T1-weighted MR signal enhancement was clearly observed at 40 min postinjection in the liver and spleen (Figure 6A vs Figure 6B).



ASSOCIATED CONTENT

S Supporting Information *

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



AUTHOR INFORMATION

Corresponding Author

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

The authors declare no competing financial interest.



ACKNOWLEDGMENTS This work was supported by the National Basic Research Program of China (No. 2011CB707703), the National Natural Science Foundation of China (Nos 21275079, 20935001), and the Fundamental Research Funds for the Central Universities.

Figure 6. In vivo T1-weighted MR coronal images of a Kunming mouse before (A) and after intravenous injection of the PAA-MUCNP (B) (white arrow, liver; red arrow, spleen).



To further demonstrate the capacity of FA-MUCNP conjugate for in vivo active targeted UCL imaging of FR positive tumors, FA-MUCNP dispersion (200 μL, 2 mg mL−1) was administered to a nude mouse bearing HeLa tumor via tail vein injection, followed by UCL imaging. In an additional control experiment, PAA-MUCNP (200 μL, 2 mg mL−1) was administered to a HeLa tumor-bearing nude mouse by intravenous injection. The UCL signal of FA-MUCNP appeared in the tumor at 3 h postinjection (Figure 7A),

REFERENCES

(1) Louie, A. Chem. Rev. 2010, 110, 3146. (2) Lee, D. E.; Koo, H.; Sun, I. C.; Ryu, J. H.; Kim, K.; Kwon, I. C. Chem. Soc. Rev. 2012, 41, 2656. (3) Lee, N.; Cho, H. R.; Oh, M. H.; Lee, S. H.; Kim, K.; Kim, B. H.; Shin, K.; Ahn, T. Y.; Choi, J. W.; Kim, Y. W.; Choi, S. H.; Hyeon, T. J. Am. Chem. Soc. 2012, 134, 10309. (4) Zhou, J.; Liu, Z.; Li, F. Y. Chem. Soc. Rev. 2012, 41, 1323. (5) Haase, M.; Schäfer, H. Angew. Chem., Int. Ed. 2011, 50, 5808. (6) Wang, F.; Banerjee, D.; Liu, Y. S.; Chen, X. Y.; Liu, X. G. Analyst 2010, 135, 1839. (7) Xing, H. Y.; Bu, W. B.; Zhang, S. J.; Zheng, X. P.; Li, M.; Chen, F.; He, Q. J.; Zhou, L. P.; Peng, W. J.; Hua, Y. Q.; Shi, J. L. Biomaterials 2012, 33, 1079. (8) Xing, H. Y.; Bu, W. B.; Ren, Q. G.; Zheng, X. P.; Li, M.; Zhang, S. J.; Qu, H. Y.; Wang, Z.; Hua, Y. Q.; Zhao, K. L.; Zhou, L. P.; Peng, W. J.; Shi, J. L. Biomaterials 2012, 33, 5384. (9) Zhu, X. J.; Zhou, J.; Chen, M.; Shi, M.; Feng, W.; Li, F. Y. Biomaterials 2012, 33, 4618. (10) Xia, A.; Chen, M.; Gao, Y.; Wu, D. M.; Feng, W.; Li, F. Y. Biomaterials 2012, 33, 5394. (11) Zhou, J.; Zhu, X. J.; Chen, M.; Sun, Y.; Li, F. Y. Biomaterials 2012, 33, 6201. (12) Liu, Y. L.; Ai, K. L.; Liu, J. H.; Yuan, Q. H.; He, Y. Y.; Lu, L. H. Angew. Chem., Int. Ed. 2012, 51, 1437. (13) Xing, H. Y.; Zheng, X. P.; Ren, Q. G.; Bu, W. B.; Ge, W. Q.; Xiao, Q. F.; Zhang, S. J.; Wei, C. Y.; Qu, H. Y.; Wang, Z.; Hua, Y. Q.; Zhou, L. P.; Peng, W. J.; Zhao, K. L.; Shi, J. L. Sci. Rep. 2013, 3, 1751. (14) Krämer, K. W.; Biner, D.; Frei, G.; Güdel, H. U.; Hehlen, M. P.; Lüthi, S. R. Chem. Mater. 2004, 16, 1244. (15) Heer, S.; Kömpe, K.; Güdel, H. U.; Haase, M. Adv. Mater. 2004, 16, 2102. (16) Hu, H.; Xiong, L. Q.; Zhou, J.; Li, F. Y.; Cao, T. Y.; Huang, C. H. Chem.Eur. J. 2009, 15, 3577. (17) Mai, H. X.; Zhang, Y. W.; Sun, L. D.; Yan, C. H. J. Phys. Chem. C 2007, 111, 13730.

Figure 7. (A) In vivo UCL imaging of Hela tumor-bearing nude mice at 3 h after intravenous injection with FA-MUCNP. (B) In vivo UCL imaging of Hela tumor-bearing nude mice at 3 h after intravenous injection with PAA-MUCNP. The red circle indicates the tumor site.

whereas that of PAA-MUCNP was not observed in the tumor site (Figure 7B). The above results demonstrate the specific in vivo targeting capacity of the FA-MUCNP to FR positive tumor.



CONCLUSIONS In summary, we have reported the design, fabrication, and application of a multishell structured multifunctional nanoprobe for trimodal bioimaging (UCL (NIR-to-NIR)/CT/MR). The efficient NaYF4:Yb/Tm UCNPs are incorporated into 12171

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Analytical Chemistry

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(18) Li, Z. Q.; Zhang, Y. Nanotechnology 2008, 19, 345606. (19) Wang, F.; Han, Y.; Lim, C. S.; Lu, Y. H.; Wang, J.; Xu, J.; Chen, H. Y.; Zhang, C.; Hong, M. H.; Liu, X. G. Nature 2010, 463, 1061. (20) Kumar, R.; Nyk, M.; Ohulchanskyy, T. Y.; Flask, C. A.; Prasad, P. N. Adv. Funct. Mater. 2009, 19, 853. (21) Tian, G.; Gu, Z. J.; Zhou, L. J.; Yin, W. Y.; Liu, X. X.; Yan, L.; Jin, S.; Ren, W. L.; Xing, G. M.; Li, S. J.; Zhao, Y. L. Adv. Mater. 2012, 24, 1226. (22) Guo, H.; Li, Z. Q.; Qian, H. S.; Hu, Y.; Muhammad, I. N. Nanotechnology 2010, 21, 125602. (23) Chen, F.; Bu, W. B.; Zhang, S. J.; Liu, X. H.; Liu, J. N.; Xing, H. Y.; Xiao, Q. F.; Zhou, L. P.; Peng, W. J.; Wang, L. Z.; Shi, J. L. Adv. Funct. Mater. 2011, 21, 4285. (24) Chen, F.; Bu, W. B.; Zhang, S. J.; Liu, J. N.; Fan, W. P.; Zhou, L. P.; Peng, W. J.; Shi, J. N. Adv. Funct. Mater. 2013, 23, 298. (25) Xia, A.; Gao, Y.; Zhou, J.; Li, C. Y.; Yang, T. S.; Wu, D. M.; Wu, L. M.; Li, F. Y. Biomaterials 2011, 32, 7200. (26) Zhou, J.; Sun, Y.; Du, X. X.; Xiong, L. Q.; Hu, H.; Li, F. Y. Biomaterials 2010, 31, 3287. (27) Liu, C. Y.; Gao, Z. Y.; Zeng, J. F.; Hou, Y.; Fang, F.; Li, Y. L.; Qiao, R. R.; Shen, L.; Lei, H.; Yang, W. S.; Gao, M. Y. ACS Nano 2013, 7, 7227. (28) Liu, F. Y.; He, X. X.; Liu, L.; You, H. P.; Zhang, H. M.; Wang, Z. X. Biomaterials 2013, 34, 5218. (29) Liu, Q.; Sun, Y.; Yang, T. S.; Feng, W.; Li, C. J.; Li, F. Y. J. Am. Chem. Soc. 2011, 133, 17122. (30) Shi, F.; Wang, J. S.; Zhai, X. S.; Zhao, D.; Qin, W. P. CrystEngComm 2011, 13, 3782. (31) Yang, T. S.; Sun, Y.; Liu, Q.; Feng, W.; Yang, P. Y.; Li, F. Y. Biomaterials 2012, 33, 3733. (32) Gai, S. L.; Yang, G. X.; Li, X. B.; Li, C. X.; Dai, Y. L.; He, F.; Yang, P. P. Dalton Trans. 2012, 41, 11716. (33) Wang, J.; Song, H. W.; Xu, W.; Dong, B.; Xu, S.; Chen, B. T.; Yu, W.; Zhang, S. Nanoscale 2013, 5, 3412. (34) Zeng, S. J.; Xiao, J. J.; Yang, Q. B.; Hao, J. H. J. Mater. Chem. 2012, 22, 9870. (35) Qian, H. S.; Zhang, Y. Langmuir 2008, 24, 12123. (36) Abel, K. A.; Boyer, J. C.; van Veggel, F. C. J. M. J. Am. Chem. Soc. 2009, 131, 14644. (37) Wang, F.; Wang, J. A.; Liu, X. G. Angew. Chem., Int. Ed. 2010, 49, 7456. (38) Wang, F.; Deng, R. R.; Wang, J.; Wang, Q. X.; Han, Y.; Zhu, H. M.; Chen, X. Y.; Liu, X. G. Nat. Mater. 2011, 10, 968. (39) Johnson, N. J. J.; Korinek, A.; Dong, C. H.; van Veggel, F. C. J. M. J. Am. Chem. Soc. 2012, 134, 11068. (40) Su, Q. Q.; Han, S. Y.; Xie, X. J.; Zhu, H. M.; Chen, H. Y.; Chen, C. K.; Liu, R. S.; Chen, X. Y.; Wang, F.; Liu, X. G. J. Am. Chem. Soc. 2012, 134, 20849. (41) Li, X. M.; Shen, D. K.; Yang, J. P.; Yao, C.; Che, R. C; Zhang, F.; Zhao, D. Y. Chem. Mater. 2013, 25, 106. (42) Zhang, T. R.; Ge, J. P.; Hu, Y. X.; Yin, Y. D. Nano Lett. 2007, 7, 3203. (43) Chen, L. N.; Wang, J.; Li, W. T.; Han, H. Y. Chem. Commun. 2012, 48, 4971. (44) Gabrielson, N. P.; Cheng, J. J. Biomaterials 2010, 31, 9117. (45) Peng, X. G.; Wickham, J.; Alivisatos, A. P. J. Am. Chem. Soc. 1998, 120, 5343. (46) Dou, Q. Q.; Idris, N. M.; Zhang, Y. Biomaterials 2013, 34, 1722. (47) Liang, X.; Wang, X.; Zhuang, J.; Peng, Q.; Li, Y. D. Adv. Funct. Mater. 2007, 17, 2757. (48) Xiong, L. Q.; Chen, Z. G.; Yu, M. X.; Li, F. Y.; Liu, C.; Huang, C. H. Biomaterials 2009, 30, 5592. (49) Ma, J. B.; Huang, P.; He, M.; Pan, L. Y.; Zhou, Z. J.; Feng, L. L.; Gao, G.; Cui, D. X. J. Phys. Chem. B 2012, 116, 14062.

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