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Gd–Al co-doped mesoporous silica nanoparticles loaded with Ru(bpy)32+ as a dual-modality probe for fluorescence and magnetic resonance imaging† Dan Zhang,a Ai Gao,a Yang Xu,a Xue-Bo Yin,*a Xi-Wen Hea and Yu-Kui Zhangab Mesoporous silica nanoparticles (MSNs) were co-doped with Gd3+ and Al3+ and then loaded with Ru(bpy)32+ by ion-exchange to prepare Ru/Gd–Al@MSNs. The as-prepared Ru/Gd–Al@MSNs were applied as contrast agents for in vivo fluorescence and magnetic resonance (MR) dual-modality imaging with a mouse as a model. The effects of Al3+ and MSNs on longitudinal relaxivity (r1) and fluorescence were investigated using a series of Gd-containing silica nanoparticles, including Gd@MSNs, Gd– Al@MSNs, and Ru/Gd–Al@nonporous silica nanoparticles. Co-doping with Al3+ improved the loading of Gd3+; the mesoporous structure improved the water exchange rate. The improvement enhanced the MR imaging efficiency of the Ru/Gd–Al@MSN probe. A higher relaxivity (19.2 mM1 s1) was observed compared to that from a commercial contrast agent, Gd-diethylene triamine pentaacetic acid (GdDTPA). Importantly, the mesoporous structure provided a large specific surface area for the loading of

Received 7th May 2014 Accepted 25th June 2014

Ru(bpy)32+ by a simple ion-exchange procedure. Intense red fluorescence was observed from Ru/Gd–

DOI: 10.1039/c4an00816b

Al@MSN probes. The versatility of Ru/Gd–Al@MSNs for dual-modality imaging was demonstrated using in vivo fluorescence imaging and T1-weighted MR imaging with a mouse model. The nanoparticles are

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biocompatible and may be attractive for clinical applications.

Introduction Molecular imaging aims at monitoring biological processes in humans and other organisms at cellular and molecular levels and is becoming increasingly important in clinical diagnosis.1–4 One of the most important areas of molecular imaging is the design and development of multifunctional imaging probes.2,5 Over the past two decades, nano-probes have been successfully applied for in vivo biomedical imaging and therapy.6–8 Among all of the available imaging techniques, magnetic resonance (MR)9 and uorescence imaging10,11 are the most convenient and widely used strategies as they are noninvasive.12 Fluorescence imaging shows high sensitivity, while MR provides depth penetration and spatial visualization. Because of their complementary advantages, MR and uorescence strategies could be combined to obtain comprehensive diagnostic and imaging information.

a

State Key Laboratory of Medicinal Chemical Biology, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), and Research Center for Analytical Sciences, College of Chemistry, Nankai University, Tianjin, 300071, China. E-mail: [email protected]

b

National Chromatographic R. & A. Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, 116011, China † Electronic supplementary 10.1039/c4an00816b

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available.

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DOI:

Ruthenium complexes have a high uorescence efficiency with infrared or near infrared emission,13 which facilitates the circumvention of auto-uorescence and scattering light from biological samples. Gadolinium (Gd) is one of the most important MR contrast elements.14 Multifunctional nanoparticles with a luminescent Ru(bpy)32+ core and a paramagnetic Gd complex layer have been fabricated for multimodality MR and uorescence imaging using layer-by-layer selfassembly.15 However, the assembly procedure is complex. Moreover, Gd3+ in the deep layer of these structures shows low contrast efficiency because of the low effect of Gd on the 1H protons of ambient water molecules. Therefore, a simple procedure of fabrication of Gd-doped nanomaterials is required to provide high Gd-loading while the high relaxation efficiency is maintained, i.e., the contrast agents inuence 1H protons in the water molecules easily.16,17 Mesoporous silica nanoparticles (MSNs) have uniform pore sizes, large surface areas, and highly accessible pore volumes.18 Their low toxicity and high biocompatibility make them useful as delivery vehicles for imaging probes and small-molecule drugs.19–21 Doping of MSNs with paramagnetic Gd, with easy interaction between Gd and water protons, may enhance the MR signal.22 Co-doping with Al3+ was reported to enhance the uorescence yield of rare-earth ion distribution in sol–gel glasses by increasing dispersal of the rare-earth ions.23 Multiple functionalization of MSNs showed an improved and controllable release of a drug.24 The enhancement or improvement was

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obtained because Al3+ ions have the same charge as Gd3+ ions and a similar size to Si4+; Al3+ ions can therefore ameliorate the mismatch between Gd3+ and Si4+ to increase the loading of Gd3+ doped into MSNs.25 Herein, we reported monodisperse hybrid MSNs loading Gd ions and Ru(bpy)32+. Co-doping with Al3+ was used to improve Gd loading. Aer Gd3+ and Al3+ co-doped MSNs (Gd–Al@MSNs) were prepared by a sol–gel process,26,27 the Ru(bpy)32+ probe was introduced into Gd–Al@MSNs through a simple ion-exchange procedure to obtain Ru/Gd–Al@MSNs. Their MR and uorescence dual-modality imaging efficiency was conrmed with a mouse model. This work provides a simple method to prepare MR and uorescence dual-modality imaging probes with high relaxivity by the high loading of Gd and strong uorescence from the adsorbed Ru(bpy)32+.

Experimental section

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were lyophilized to get the Ru/Gd–Al@MSNs, which can be dissolved again for imaging applications. As a comparison, Gd@MSNs and Gd–Al@MSNs with the different content ratios of Gd and Al were prepared to validate the efficiency of Ru/Gd–Al@MSNs. The synthesis process was similar to that of Ru/Gd–Al@MSNs. The relative amounts of Gd3+ and Al3+ and the comparison of the performance of these nanoparticles are summarized in Tables S2 and S3.† Synthesis of Ru(bpy)32+, Gd3+ and Al3+ co-doped nonmesoporous silica nanoparticles (Ru/Gd–Al@NSNs) 15 mL ethanol, 1 mL NH4OH, 3 mg Ru(bpy)3Cl2$6H2O and 0.25 mL TEOS were mixed and reacted by sonication in a 50 mL beaker. Aer 10 min, 50 mL solutions of GdCl3 (1.0 M) and AlCl3 (0.5 M) were added into the milky mixture. Aer a total reaction time of 2 h, the product was collected by centrifugation and washed with ethanol and ultrapure water four times.

Chemicals and materials

Cytotoxicity measurement of Ru/Gd–Al@MSNs

Cetyltrimethyl ammonium bromide (CTAB, 99%) and diethanolamine (DEA, 99%) were purchased from J&K Chemical, Beijing, China. Ammonia (NH4OH, 25%) and tetraethoxysilane (TEOS, 98%) were obtained from Concord Reagent Co., Tianjin, China. Tris(2,20 -bipyridine) ruthenium(II) chloride hexahydrate (Ru(bpy)3Cl2$6H2O) was purchased from Sigma-Aldrich, Shanghai, China. Aluminum chloride (anhydrous, AlCl3, 99%) and gadolinium oxide (Gd2O3, 99.99%) were purchased from Aladdin Chemistry Co. Ltd., Shanghai, China. Gadolinium chloride (GdCl3) was prepared by the reaction of excess hydrochloric acid and Gd2O3, and then dried by solvent evaporation. Ultrapure water was prepared with an Aquapro system (18.25 MU).

HepG-2 cancer cells were cultured with fresh Dulbecco's modied Eagle's medium (DMEM, GIBCO), supplemented with 10% heat-inactivated fetal bovine serum (FBS, GIBCO), 100 U mL1 penicillin and 100 mg mL1 streptomycin at 37  C in a humidied atmosphere containing 5% CO2. The cytotoxicity of Ru/Gd–Al@MSNs was measured by performing methyl thiazolyl tetrazolium (MTT) assays on HepG-2 cells. Cells were seeded in a 96-well cell culture plate at 1  104 cells per well under a humidied atmosphere containing 5% CO2 at 37  C for 24 h. Samples with different concentrations of nanoparticles (concentrations of 0, 50, 100, 200 and 500 mg mL1 in PBS) were then introduced into the wells. The cells were subsequently incubated for 24 h at 37  C with 5% CO2. Then, 20 mL of MTT solution in PBS with a concentration of 5 mg mL1 was added and the plates were incubated for another 4 h at 37  C, Aer that, the medium containing MTT was removed and 150 mL of DMSO was added to each well to dissolve the MTT formazan crystals. Finally, the plates were shaken for 10 min and colorimetric measurements were performed at 570 nm using a microplate reader (Thermo).

Synthesis of Gd3+–Al3+ co-doped and Ru(bpy)32+ loaded mesoporous silica nanoparticles (Ru/Gd–Al@MSNs) Mesoporous silica nanoparticles were synthesized by a cocondensation process.26,28 Briey, CTAB (0.29 g, 0.79 mmol) was dissolved in the mixture of ultrapure water (6.4 mL, 0.36 mol) and ethanol (0.92 mL, 0.02 mol) by sonication for 10 min. Aer adding DEA (0.05 g, 0.48 mmol), the mixture solution was heated to 60  C. Then, TEOS (0.73 mL, 3.16 mmol) was added drop-wise under vigorous stirring for 10 min. 0.15 mL solution of GdCl3 (0.10 mmol) and AlCl3 (0.05 mmol) was then added. Aer reacting for a total time period of 2 h, the mixture was cooled to room temperature and the nanoparticles were collected and washed with ethanol three times aer centrifugation. Aer the particles were reuxed in an acidic ethanol solution for 6 h, the template, CTAB, was removed. Subsequently, the white product was washed with ethanol and ultrapure water four times and then was collected by centrifugation at 12 000 rpm. The precipitate nally dispersed into 10 mL ultrapure water. Then, 5 mg Ru(bpy)3Cl2$6H2O was added into the solution of Gd–Al@MSNs and stirred at room temperature for 12 h under the dark. Aer centrifugation and washing with ultrapure water four times, orange nanoparticles

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Luminescence spectra and in vivo uorescence imaging The steady-state uorescence experiments were performed on a Hitachi FL-4500 Fluorescence Spectrometer. Female nude mice (6–8 weeks old, BALB/c-nu) were anesthetized with 4% chloral hydrate using a dose of 8.25 mL g1. Then, 200 mL of Ru/Gd– Al@MSN saline solution was injected intraperitoneally or 50 mL for subcutaneous injection as contrast agents. In vivo and in vitro uorescence imaging was performed using a Kodak multimodal living imaging system (Kodak in vivo FX). All procedures using animals were approved by the Institutional Animal Care Committee of Nankai University. Fluorescence images were analyzed using Kodak Molecular Imaging Soware. Excitation was provided at 480 nm and the emission signals were collected at 700 nm. The uorescent images with better visualization were achieved by superimposing uorescence images of the black and white pictures. Aer imaging, the mice

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were sacriced and dissected for the imaging of their different organs.

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Relaxivity measurement and in vitro and in vivo MR imaging Proton relaxation time (T1 and T2) was measured in a 1.2 T (50 MHz, 30  C) MRI scanner system (HT-MRSI60-60 KY, Huantong, Shanghai, China) using the standard inversionrecovery (IR) method. Relaxation rates (1/T1 and 1/T2) were plotted against Gd concentrations and the relaxivities (r1 and r2) of the as-prepared probe were calculated from the slope of these curves. In vitro T1-weighted phantom imaging and in vivo MR imaging by the conventional spin-echo method were performed at 30  C using the 1.2 T (50 MHz) MRI scanner system. In vivo experiments were performed with anesthetized Kunming mice (eight-week-old with the weight of 45 g) in compliance with the institution's guidelines for the use of laboratory animals in Nankai University. 300 mL of Ru/Gd–Al@MSN (10 mg mL1) saline solution was injected intraperitoneally to enterocoelia or intravenously to tail vein into the mice as a contrast agent. MRI was conducted using a T1-weighted sequence with TR/TE ¼ 200 ms/14 ms, FOV ¼ 75 mm  75 mm, ip angle ¼ 60 , number of excitations ¼ 3, and acquisition matrix size ¼ 256  256  128.

Results and discussion Synthesis and characterization of Ru/Gd–Al@MSNs To construct an MR and uorescence dual-modality imaging probe, MSNs were prepared and then loaded with Gd3+ and Ru(bpy)32+, which were chosen for their strong MR and uorescence responses, respectively. To improve the probe efficiency, Al3+ and Gd3+ were co-doped into the MSNs (Gd– Al@MSNs), using a sol–gel process with tetraethyl orthosilicate (TEOS) as a precursor in the presence of surfactant cetyltrimethyl ammonium bromide (CTAB). Al3+ was used to mend the inharmonious between Gd3+ and Si4+. Al3+ doped in the silica framework of MSNs enables high loading of Gd3+ in the material.29 Diethanolamine was used as a base catalyst in the reaction system, where it was also used as the passivated agent to inhibit the growth and aggregation of the particles.30 Dye molecules, Ru(bpy)32+, were then introduced onto the silanolrich surfaces of the as-prepared MSNs by the ion exchange procedure. Various nanoparticles, including Gd@MSNs, Gd– Al@MSNs and Ru(bpy)32+, Gd3+ and Al3+-doped nonporous silica nanoparticles (Ru/Gd–Al@NSNs), were prepared to conrm the efficiency of Ru/Gd–Al@MSNs. X-ray photoelectron spectroscopy illustrated that Gd3+ was successfully incorporated into the nanoparticle framework (Fig. S1†). The change of the zeta-potential of the nanoparticles conrmed the adsorption of Ru(bpy)32+ (Table S1†). EDX patterns indicated that Gd, Al, and Ru were successfully incorporated into the MSNs (Fig. S2†). Elemental analysis results from inductively coupled plasma-atomic emission spectroscopy (ICP-AES) conrmed that the doped Al3+ improved the Gd loading in MSNs.27 Aer storage in PBS (pH 7.4) at 37  C for 1 month, no Gd or Al and only less than 1% Ru were leaked from the MSNs, showing the good storage stability

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of Ru/Gd–Al@MSNs. The optimum amount of Gd3+/Al3+/ Ru(bpy)32+ was 0.1 mmol/0.05 mmol/0.008 mmol and the Gd/ Al/Ru molar ratio was found to be 2.56/2.87/0.56 in the asprepared probe. The morphology and microstructure of the as-prepared nanoparticles were investigated by transmission electron microscopy (TEM). All of the nanoparticles were uniform and non-aggregated spheres (Fig. 1). Ru/Gd–Al@MSNs, 4.4% Gd– Al@MSNs and Ru/Gd–Al@NSNs have the average diameters of about 60 nm, while Gd@MSNs were approximately 70 nm in size. Mesopores were clearly observed in all of the MSN materials, whereas the Ru/Gd–Al@NSNs were solid. The morphology and mesoporous structure of the intragranular network in the particles were clearly observed in the inset of Fig. 1C. Aer loading Ru(bpy)32+, the size and appearance of the nanoparticles did not change. By contrast, the loading of Al3+ makes the nanoparticles smaller possibly due to the change in the pH of the reaction system aer addition of Al3+. Lower pH usually results in the decreased nanoparticle size.26 The effect of Gd and Al on the mesoporous structure of the nanoparticles was investigated by X-ray diffraction of Gd@MSNs (Gd mass fraction of 3.4%) and various Gd– Al@MSNs (with the mass fractions, 2.9% Gd and 0.4% Al, 4.4% Gd and 0.8% Al, 5.2% Gd and 2.3% Al) (Fig. S3†). Only one broad Bragg peak at low angles was observed and indexed as (100) diffractions in Gd@MSNs. In contrast, the signal for the MSNs co-doped with Gd3+ and Al3+ became smaller and the intensity decreased as the amounts of Gd3+ and Al3+ increased. Usually, mesoporous nanoparticles are long-range order, such as hexagonal MCM-41, and have several sharp peaks in XRD patterns.15,20 As the nanoparticle size decreases or the mesoporous structure becomes disordered, the XRD peaks become broadened or disappear.26 Therefore, the decreasing peak intensity in XRD conrmed the reduced nanoparticle size with Al doping as seen in TEM images in Fig. 1. In addition, these XRD results show that Gd and Al disrupted the order of the structure, which is similar to the result in earlier Gd3+-doped mesoporous silica materials.27,31

TEM images of (A) Gd@MSNs, (B) Gd–Al@MSNs, (C) Ru/Gd– Al@MSNs and (D) Ru/Gd–Al@NSNs. Scale bars represent: (A) 100 nm; (B–D) 200 nm. Insets: magnified images of the same samples with a scale bar of 10 nm.

Fig. 1

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(A) N2 adsorption–desorption isotherms and (B) pore-size distribution curves obtained from the adsorption branch of the N2 physisorption isotherms of (a) Gd@MSNs, (b) Gd–Al@MSNs and (c) Ru/ Gd–Al@MSNs.

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Fig. 2

The textural properties of Gd@MSNs, Gd–Al@MSNs and Ru/ Gd–Al@MSNs were investigated by N2 adsorption–desorption tests (Fig. 2). All the samples showed the type IV isotherms with an obvious hysteresis loop, indicating the formation of mesoporous structures, which were in agreement with the TEM results shown in Fig. 1A–C. The small particle size was attributed to the textural porosity and demonstrated by the hysteresis at high relative pressures. The high surface areas calculated from the Brunauer–Emmett–Teller test are 879 m2 g1, 905 m2 g1 and 867 m2 g1 for Gd@MSNs, Gd– Al@MSNs and Ru/Gd–Al@MSNs, respectively. Total pore volumes of the three MSNs are 1.1 cm3 g1, 1.0 cm3 g1 and 1.0 cm3 g1. The Barrett–Joyner–Halenda results exhibited in Fig. 2B show the maximum pore size distributions at 2.5, 2.9 and 2.8 nm for Gd@MSNs, Gd–Al@MSNs and Ru/Gd– Al@MSNs, respectively, which was in the range (2–8 nm) of the most frequently used pore sizes in the eld of molecular imaging.32 The results implied that a large number of Ru(bpy)32+ can be loaded not only on the surface of nanoparticles but also in the pores. The amount of Ru loaded in Ru/Gd–Al@MSNs was 56.1 mmol g1, which is obviously higher than 19.5 mmol g1 in Ru/Gd–Al@NSNs (Table S2†). As the amount of Gd increased and Al3+ was introduced, the pore size increased slightly, which conrmed that the introduction of Gd ions disrupts the mesoporous structure, as shown by the XRD result. The agreement between XRD and N2 adsorption–desorption results evidenced that the co-doped Al ions intensied the effect of Gd doping on the mesoporous structure, thus improved MR and uorescence properties of the nano-probe.

Fig. 3 Photographs of Ru/Gd–Al@MSNs under (A) daylight and (B) UV light. (C) Photoluminescence spectra of 1 mg mL1 (a) Ru/Gd– Al@MSNs and (b) Ru/Gd–Al@NSNs with the same concentration (lex ¼ 475 nm).

surface area of the mesoporous structure in MSNs compared with the NSNs, which enhanced Ru(bpy)32+ loading (56.1 mmol g1 vs. 19.5 mmol g1, Table S2†). Emission of the two samples likely arose from the parent dye Ru(bpy)32+ (lem ¼ 621 nm) because of their same emission prole. The MR performance of these nanoparticles was tested in ultrapure water at 1.2 T (30  C). A commercially available control, Gd-diethylene triamine pentaacetic acid (Gd-DTPA), was compared with Ru/Gd–Al@MSNs, Gd@MSNs and Gd–Al@MSNs. All of the nano-materials prepared in this study showed higher relaxivity than Gd-DTPA (Table S2†). Fig. 4A illustrates that the r1 relaxivity of Ru/Gd–Al@MSNs (19.2 mM1 s1) was 4.6-fold higher than that of Gd-DTPA (4.19 mM1 s1). Similarly, Ru/Gd–Al@MSNs increased the positive contrast compared with Gd-DTPA at the same Gd content (Fig. 4B). This result agreed well with the relaxivity data. The relaxation of Gd-containing MSNs is affected by the Gd content and water exchange rate around the Gd probe. Co-doped Al3+ improved Gd3+ loading and the mesoporous

Fluorescence and relaxation properties of Ru/Gd–Al@MSNs The uorescence and relaxation properties of Ru/Gd–Al@MSNs were tested to illustrate their dual-modality imaging potential and efficiency. Ru/Gd–Al@MSNs appear orange under sunlight and show a bright orange-red when illuminated by 365 nm UV light at a concentration of 1 mg mL1 (Fig. 3A and B). High optical transparency of silica enables the probe to be efficiently used for imaging.33 Under 475 nm excitation, emission with the maximum wavelength of 621 nm was observed for the two samples (Fig. 3C), but the uorescence intensity of Ru/Gd– Al@MSNs was much higher than that of Ru/Gd–Al@NSNs at the same concentrations. This could be explained by the high

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Fig. 4 (A) The r1 relaxivity curves of Ru/Gd–Al@MSNs and Gd-DTPA. (B) T1-weighted MR images of Ru/Gd–Al@MSNs and Gd-DTPA with various Gd concentrations.

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structure of Ru/Gd–Al@MSNs and therefore enhanced the water exchange rate compared with that of the NSNs. These factors contributed to the higher relaxivity of the Ru/Gd–Al@MSN probe. The MSN probe had a higher relaxation rate than NSNs (19.2 mM1 s1 vs. 7.91 mM1 s1 for r1, Table S2†). The porous structure enables bulk water to interact with Gd3+ easily and promotes relaxivity through the enhanced diffusion rate. On incorporation of Gd and Al, as shown by XRD and nitrogen adsorption–desorption, the disordered porosity was found in MSNs and also facilitated the higher relaxivity. Similarly, the materials with a disordered pore arrangement were reported to show a higher MRI contrast efficiency than an ordered P6mm geometry.31 The disordered pore structure exhibits more interconnection of pores, which facilitates water diffusion, and enhanced the relaxation by increasing water exchange.34 The highest contrast enhancement was achieved with the Gd dose in the mass fraction of 4.03% (Table S2†). With high Gd loading, the dipole–dipole interaction between Gd ions becomes more important than the interaction between the proton and electron of the paramagnetic substance. Thus, the electronic relaxation becomes short.27 The r2/r1 ratio (1.1) was observed with the Ru/Gd–Al@MSNs at 1.2 T eld strength and indicated that the probe was suitable for MR applications as a T1 contrast agent. Generally, the r2/r1 ratio of T1 agents is less than 2, but the ratios of T2 agents are as high as 10 or more, such as iron oxide particles.35 The r1 of a material for the highly sensitive T1 MR contrast agent should be as large as possible. Gd@MSNs were used as Al-free silicate control samples to investigate the effect of the Al3+ co-doping. The enhanced relaxivity of the samples doped with Al could be attributed to their positive role in ameliorating the mismatch between Gd3+ and Si4+ (Table S2†). Thereby Gd could be homogeneously and directly incorporated into the large inorganic host matrix, which would affect the parameters that control the proton relaxation in a paramagnetic system.36,37 Cytotoxicity of Ru/Gd–Al@MSNs The toxicity of Ru/Gd–Al@MSNs was evaluated by performing a cytotoxicity assay towards HepG-2 cells. Various concentrations of Ru/Gd–Al@MSNs were incubated with the cells for 24 h and then the cell viability was assessed. Within the tested concentration range, no signicant cytotoxicity was observed. The cell viability still remained above 90% as high as 200 mg mL1 of the nanoprobes (Fig. S4†). Ru/Gd–Al@MSNs as contrast agents for FL and MR dualmodality imaging The hybrid nanoparticles were tested for in vivo imaging to illustrate their bio-applications and bio-effects.5,38,39 Because the Ru/Gd–Al@MSNs have high luminescence and relaxation rates, we anticipate that the hybrid MSNs will be effective as a dualmodality imaging probe. Mice were injected with a colloidal saline solution of the probe at a concentration of 10 mg mL1 to evaluate its uorescence imaging efficiency. The emission from the probe was clearly illustrated as shown in Fig. 5A. The mice

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In vivo fluorescence images of mice with (A) intraperitoneal injection of Ru/Gd–Al@MSNs. (B) Ex vivo optical imaging of resected organs during necropsy at 24 h after injection.

Fig. 5

were monitored in the period between 30 min and 24 h aer injection (Fig. 5A). The hybrid probe was observed to have a rapid diffusion rate aer intraperitoneal injection. Strong uorescence was observed from the abdomen of the mouse body. Most of the Ru/Gd–Al@MSNs accumulated gradually in the mononuclear phagocyte system, including the liver and spleen. Some of the probes were excreted through renal routes, which illustrated that the route of absorption and metabolism of Ru/Gd–Al@MSNs. The bio-distribution was conrmed by imaging of the single organ aer dissection (Fig. 5B). No emission was found from the heart and lung, but high intensity emission was observed from the liver and spleen. Among all the organs, the kidney was the brightest, which illustrated that the nanoparticles could be naturally eliminated by renal excretion. ICP-AES results showed that less than 1% of free Ru and no Gd3+ or Al3+ were released from Ru/Gd–Al@MSNs. These results indicate that Ru/Gd–Al@MSNs are promising as a probe for in vivo uorescence imaging. MR imaging further illustrated the distribution of the probe in the mouse model (Fig. 6). Aer Kunming mice were injected with 300 mL of 10 mg mL1 Ru/Gd–Al@MSN solution, the T1-weighted MR efficiency at different time points was recorded. The MR images were acquired before and aer intraperitoneal injection of the mice at 30 min, 2 h and 24 h or aer intravenous injection at 15 min, 1.5 h and 24 h. The MR signal of the liver clearly showed the enhanced T1-weighted imaging and illustrated that the liver was the main uptake organ for the probe.40 The basal lamina of the kidneys has ca. 10 nm pore, so the large materials in the blood cannot enter into the lamina. The way of injection plays an important role in the bio-distribution of injected probes.41 Thus, an intravenous injection was carried out with Ru/Gd–Al@MSNs (Fig. 6B). The enhanced MRI signal in the liver was observed, which was similar to intraperitoneal injection. The results in different injection modes validated that a few Ru/Gd–Al@MSNs enter into the kidney by blood circulation but most of them enter into the kidney by affinity and

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Fig. 6 In vivo T1-weighted images of mice (coronal plane) with 300 mL of Ru/Gd–Al@MSNs recorded at different time points. (A) Intraperitoneal injection and (B) intravenous injection (the marked area and orange arrow showed the liver).

tissue diffusion. The results were consistent with the transfer and metabolism of the Ru/Gd–Al@MSN probe in uorescence images and validated that the probe was sufficient stability for in vivo dual-modality applications.

Conclusions Hybrid mesoporous silica nanoparticles doped with Gd3+ and Ru(bpy)32+ were prepared and used as magnetic resonance and uorescence dual-modality imaging probes. Co-doped Al3+ improved the loading of Gd3+ in the nanoparticles. The MR performance of the Ru/Gd–Al@MSNs was enhanced compared with Gd-DTPA and other structures prepared in this study (Table S2†). This enhancement was attributed to an integration of Gd3+ within the mesoporous matrices and the high water exchange rate in the mesopore. Moreover, the high surface area of MSNs allows for high loading of Ru(bpy)32+ through a simple ion-exchange procedure. These results demonstrate the potential of the probe as optical and magnetic dual-modality imaging agents.

Acknowledgements This work was supported by the National Basic Research Program of China (973 Program, no. 2011CB707703), the Natural Science Foundation of China (no. 21375064), and the Research Fund for the Doctoral Program of Higher Education (no. 20130031110016).

Notes and references 1 J. Culver, W. Akers and S. Achilefu, J. Nucl. Med., 2008, 49, 169–172. 2 A. Louie, Chem. Rev., 2010, 110, 3146–3195. 3 B. J. Pichler, A. Kolb, T. N¨ agele and H.-P. Schlemmer, J. Nucl. Med., 2010, 51, 333–336.

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Analyst, 2014, 139, 4613–4619 | 4619

Gd-Al co-doped mesoporous silica nanoparticles loaded with Ru(bpy)₃²⁺ as a dual-modality probe for fluorescence and magnetic resonance imaging.

Mesoporous silica nanoparticles (MSNs) were co-doped with Gd(3+) and Al(3+) and then loaded with Ru(bpy)3(2+) by ion-exchange to prepare Ru/Gd-Al@MSNs...
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