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Sub-10 nm monoclinic Gd2O3:Eu3+ nanoparticles as dual-modal nanoprobes for magnetic resonance and fluorescence imaging Jun Liu, Xiumei Tian, Ningqi Luo, Chuan Yang, Jun Xiao, Yuanzhi Shao, Xiaoming Chen, Guo Wei Yang, Dihu Chen, and Li Li Langmuir, Just Accepted Manuscript • DOI: 10.1021/la503228v • Publication Date (Web): 07 Oct 2014 Downloaded from http://pubs.acs.org on October 10, 2014

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Sub-10 nm monoclinic Gd2O3:Eu3+ nanoparticles as dual-modal nanoprobes for magnetic resonance and fluorescence imaging Jun Liu,†,‡,# Xiumei Tian,§,# Ningqi Luo,† Chuan Yang,‡ Jun Xiao,† Yuanzhi Shao,† Xiaoming Chen,§ Guowei Yang,† Dihu Chen,†,* Li Li,‡,* †

State Key Laboratory of Optoelectronic Materials and Technologies, Sun Yat-sen

University, Guangzhou 510275, P.R. China ‡

State Key Laboratory of Oncology in South China, Sun Yat-sen University Cancer Center,

Collaborative Innovation Center for Cancer Medicine, Guangzhou 510060, P.R. China §

Department of Biomedical Engineering, Guangzhou Medical University, Guangzhou

510182, P.R. China KEY WORDS: Dual-modal nanoprobes; Laser ablation in liquids (LAL); Fluorescence imaging (FI); Magnetic resonance imaging (MRI); Nanotoxicity

ABSTRACT: Monoclinic Gd2O3:Eu3+ nanoparticles (NPs) possess favorable magnetic and optical properties for biomedical application. However, how to obtain its small enough NPs still remains a challenge. Here, we combined the standard solid state reaction with the laser ablation in liquids (LAL) technique to fabricate sub-10 nm monoclinic Gd2O3:Eu3+ NPs and explained their formation mechanism. The obtained Gd2O3:Eu3+ NPs exhibit bright red fluorescence emission, and can be successfully used as fluorescence probe for cells imaging. 1

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In vitro and in vivo magnetic resonance imaging (MRI) studies show that the product can also serve as MRI good contrast agent. Then, we systematically investigated the nanotoxicity including cell viability, apoptosis in vitro, as well as the immunotoxicity, and pharmacokinetics assays in vivo. This investigation provides a platform for the fabrication of ultrafine monoclinic Gd2O3:Eu3+ NPs and evaluating their efficiency and safety in preclinical application.

1. INTRODUCTION In recent years, nanomaterials applied in bioimaging to improve the precision of the malignant disease diagnosis and therapy, have been attracted intensive attention.1-7 However, information obtained from single-modal technique usually cannot satisfy the high requirement on the efficiency and accuracy for diagnosis and prognosis due to their limitations.8-10 For instance, magnetic resonance imaging (MRI) provides an excellent spatial resolution and exceptional anatomic information, but suffers from limited sensitivity. Fluorescence imaging (FI) provides the high sensitivity, but still lacks the spatial resolution to obtain anatomical and physiological details. The use of dual-modal nanoprobes combining the merits of MRI and FI, which can provide the high sensitive and spatial resolution, is one of the most useful approaches in bioimaging investigation to obtain comprehensive information.10-13 There is no doubt that dual-modal nanoprobes will play a vital role in diagnosing diseases in the near future. The lanthanide doped gadolinium oxide (Gd2O3) NPs have both excellent magnetic and fluorescent properties, which are useful for dual-modal imaging.14-18 In this conformation, 2

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Gd2O3 portion serves as MRI contrast agent while doped lanthanide ions as the fluorescence emitting center. Among these lanthanide ions, Eu3+ exhibits much higher emitting efficiency, and the emission in monoclinic Gd2O3:Eu3+ has a red shift (about 12 nm) than its cubic phase, which is much closer to the optical transition window.19 Therefore, the monoclinic Gd2O3:Eu3+ NPs will be extremely useful for dual-modal imaging. Meanwhile, the clinic translation requires that nanomaterials injected into the human body must be small enough (sub-10 nm) to be completely filtered out in a reasonable amount of time.20-22 Conventional techniques (precipitation, sol-gel, hydrothermal method, etc.) for preparing Gd2O3-based nanomaterials generally require sufficiently high reaction temperatures, annealing treatment at high temperature, or prolonged annealing treatment.23-31 And the structure transition of Gd2O3 from cubic to monoclinic phase occurs at ≥1250 ºC.19,32 Clearly, these processes are associated with significant aggregation and enlarged size. Therefore, how to obtain the monoclinic Gd2O3:Eu3+ NPs smaller than 10 nm for in vivo bioimaging still remains a challenge. Laser ablation in liquids (LAL) technique, a ‘top-down’ strategy, has been demonstrated the ability to produce small-sized NPs.33-35 For bioimaging application, Xiao et al. synthesized Mn3O4 NPs with approximately 9 nm in diameters by LAL approach and applied as MRI contrast agents.36 Our previous work also reported the synthesis of small-sized and ligand-free Gd2O3 NPs as MRI contrast agents by LAL technique.37,38 Currently, none of previous articles report the fabrication of dual-modal nanoprobes for bioimaging by LAL technique. In this contribution, we combined the standard solid state reaction with LAL 3

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technique, to prepare sub-10 nm monoclinic Gd2O3:Eu3+ NPs and applied as efficient dual-modal nanoprobes for MRI and FI. Furthermore, we perform a systematic pre-clinical study on the nanotoxicology of the Gd2O3:Eu3+ NPs, which include MTT, apoptosis, immunotoxicity and pharmacokinetics assays. 2. EXPERRIMENTAL SECTION 2.1 Materials Gd2O3 (99.9%) and Eu2O3 (99.9%) were purchased from Aladdin Chemistry Co. Ltd (China), polyvinyl alcohol (PVA, AR) from Guangdong Shantou Xilong Chemical Co. Ltd (China). Magnevist (Gd-DTPA) was product of Berlex Laboratories (USA), Dulbecco's Modified Eagle Media (DMEM) and phosphate buffered saline (PBS) from Gibco (Switzerland), lipopolysaccharide

(LPS),

dimethyl

sulfoxide

(DMSO),

and

[3-4,5-dimethyl

thiazol-2-yl]-2,5-diphenyltetrazolium bromide succinate (MTT) from Sigma-Aldrich Corp (USA), Annexin V-FITC, propidium Iodide (PI), anti-mouse CD206-PE from eBioscience (USA), anti-mouse CD11b-FITC, anti-mouse CD69-FITC, and anti-mouse F4/80 APC from Becton Dickinson Pharmingen (USA), anti-mouse CD71-FITC, anti-mouse CD25-FITC, and anti-mouse CD3-PE from Tianjin Sungene Biotech Co. Ltd (China), IL-1β, IL-2 and IL-4 ELISA kits from RayBiotech Inc. (China). Four to six-week-old Balb/c nude mice (weight of 16±2 g), Kunming white mice (weight of 22±2 g), and cell lines (Raw267.4 cells, S18 cells, and PC12 cells) were purchased from the animal experiment center of the Medical College, Sun Yat-sen University (China). 2.2 Target and nanoparticles preparation 4

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The Gd2O3:Eu3+ target was prepared via solid state reaction technique. Gd2O3 and Eu2O3 powders with a stoichiometric ratio of 95:5 were milled in an agate mortar for 30 min, and then PVA (7 wt%) solution was added as the binder. After that, the powders were dried at 50 ºC for 10 min, and then sieved by a 60 mesh filter. The obtained powders were uniaxially pressed into cylinders with 15 mm in diameter under 15 MPa, sintered at 1500 ºC for 10 h in the air at a heating rate of 10 ºC/min. Finally, the Gd2O3:Eu3+ target was obtained after cooling down to room temperature. The details of LAL technique have been reported in our previous works (Fig. S1).37,38 The Gd2O3:Eu3+ target was fixed on the bottom of the container and immersed in deionized water. A microsecond laser of Nd:YAG with the wavelength of 1064 nm, pulse duration of 6 µs, repetition of 100 Hz, and power of 70 mJ/pulse, was focused onto the surface of Gd2O3:Eu3+ target. The ablation process lasted for 15 min. The ablated colloids were aged for 24 h, and then the upper clear liquid were collected for further measurement. 2.3 Characterization of Gd2O3:Eu3+ NPs Transmission electron microscope (TEM, FEI Tecnai G2 Spirit, Netherlands), X-ray diffractometer (XRD, D-MAX2200 VPC, Japan), and X-ray photoelectron spectrometer (XPS, ESCALab250, USA) were employed to identify the morphology, structure, and component of the product, respectively. The fluorescence spectrum of the colloid was measured by Edinburgh spectrofluorophotometer (FLS920, UK) at the room temperature. The concentration of Gd3+ was measured by inductively coupled plasma mass spectrometry (ICP-MS, Thermo Instrument System Inc. USA). 5

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2.4 Fluorescence imaging of live cells Cell lines were grown in DMEM in 96-well plates at 37 ºC, 5% CO2. Cells in logarithmic growth period were used for live cells FI and toxicity assay. The Raw264.7 cells were incubated with the Gd2O3:Eu3+ NPs for 2 h. After co-incubation, the cells were washed with PBS to remove the remaining particles and dead cells, and then observed under a confocal fluorescence microscope (Leica TCS SP8, Germany). 2.5 In vitro and in vivo magnetic resonance imaging In vitro and in vivo MRI data were acquired at 3.0-T clinical MRI system (Siemens Medical Solutions, Germany). The sequence are TSE T1 axial (5% dist. Factor, FOV 64 mm, slice thickness 2.0 mm, TR 600 ms, TE 12 ms, six averages). All the dates are analyzed by picture archiving and communications system (PACS). In the in vitro MRI study, various samples with different Gd3+ concentrations (0-0.05 mM) were measured. Animal experiments were performed in accordance with the National Institutes of Health guidelines on the rules of animal’s research and our Institution’s Animal Board. The Balb/c nude mice were subcutaneously injected with 5 × 106 nasopharyngeal carcinoma (NPC) CNE-2 cells with 100 µL PBS. Ten days after tumor cell inoculation, mice with xenografted tumor (approximately 60 mm3) were induced anesthesia by intraperitoneal injection of 0.1% mebumalnatrium (10 µL per g weight), then injected with the Gd2O3:Eu3+ NPs (Gd3+, 15 µmolkg-1) in 100 µL of PBS (2× buffer) via the tail vein, and scanned on the 3.0-T MRI system using a surface coil constructed specifically for small animals (3 inch in

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diameter). To be not biased toward aberrantly enhanced regions, the entire tumor is generated the normalized histograms of signal intensity. 2.6 Cytotoxicity assay The Raw267.4 cells, S18 cells, and PC12 cells were incubated with different Gd3+ concentrations (10 µM, 1 µM, and 0.1 µM) of the Gd2O3:Eu3+NPs in 96-well plates, treated with culture media (DMEM) as negative control and LPS as positive control, all groups were cultured for 48 h. After co-incubation, added 20 µL of MTT for another 4 h of incubation. Then, all the culture medium was removed and 100 µL DMSO was added to dissolve the formazan crystals for 10 min. The absorbance at 490 nm was measured by a microplate reader (Bio-Rad, USA). 2.7 Apoptosis assay The Raw264.7 cells were incubated with PBS (negative control), LPS (positive control), Gd-DTPA, and Gd2O3:Eu3+ NPs in 6-well plants for 48 h, then washed twice in cold PBS by gentle shaking, and resuspended cell pellet with 200 µL Binding Buffer (1×) at 4×105 cells/ml, added 5 µL Annexin V-FITC

into 195 µL cell suspension, mixed and incubated for

10 min at room temperature, washed cells twice in 200 µL Binding Buffer (1×), and resuspended in 190 µL Binding buffer (1×), then added 10 µL PI, (20 µg/mL), finally the samples were measured on a FACScan (Becton Dickinson, Mountain View, CA). 2.8 Pharmacokinetic characterizations 2.81 Half-life of the Gd2O3:Eu3+ NPs in the blood circulation. The half-life in blood circulation was determined by 30 clean Kunming white mice (50% males and 50% females), 7

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and divided into 10 groups at random. Blood was drawn by the tail veins at 5, 15, 30, 60, 120, 180, 240, 360, 480, and 720 min, respectively, after administration of the Gd2O3:Eu3+ NPs (Gd3+, 15 µmolkg-1) via tail vein. 2.82 Biodistribution of the Gd2O3:Eu3+ NPs at the organ and subcellular level. At the organ lever, liver, lung, spleen, heart, kidney, and tumor are collected at 4, 24, and 48 h, respectively, after nanoprobes injection (Gd3+, 15 µmolkg-1). At the subcellular level, liver, lung, kidney, and tumor are obtained at 4 h after injection. Samples were measured by TEM. 2.83 Excretion of the Gd2O3:Eu3+ NPs. Faces and urine of mice are collected every week (n = 3) for 12 weeks after injection (Gd3+, 15 µmolkg-1). 2.9 Immunotoxicity assays in vivo Twenty Male Balb/c mice were divided into 4 groups at random: (a) PBS (100 µL, Negative control), (b) LPS (positive control), (c) Gd-DTPA (15 µmolkg-1), and (d) Gd2O3:Eu3+ NPs (Gd3+, 15 µmolkg-1). Samples (peripheral blood, lymphocyte) were collected after 48 h post injection, stained and measured by flow cytometry. The regulator of immunity (ROS, reactive oxygen species), CD markers of innate immune including CD206 and CD11b of monocytes/macrophages in peripheral blood, as well as CD makers of adaptive immune including CD69, CD71, and CD25 in lymphocyte cells of the peripheral blood were determined by flow cytometry. The cytokines analysis also included interleukin (IL-1β, IL-2, and IL-4) assessment. 3. RESULTS AND DISCUSSION 3.1 Formation mechanism and characterization of Gd2O3:Eu3+ NPs 8

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3.11 Morphology, structure, and component of Gd2O3:Eu3+ NPs The typical TEM image of the Gd2O3:Eu3+ NPs is shown in Fig. 1(a). Clearly, the NPs prepared by LAL technique exhibit good uniformity and monodispersibility. The corresponding size distribution histogram (Fig. 1(b)) shows that the mean size of the NPs is about 7.4 nm. As shown in the HRTEM image (Fig. 1(c)), the interplanar distance of Gd2O3:Eu3+ nanocrystal is about 0.310 nm, which is in agreement with that of Gd2O3 in the

Fig. 1 (a) Typical TEM image; (b) Size distribution histogram; (c) HRTEM image; (d) SAED pattern; (e) XRD pattern; and (f) Gd 3d and Eu 3d XPS spectra of the Gd2O3:Eu3+ NPs.

database (d111 = 0.316 nm, PDF#42-1465). The SAED pattern (Fig. 1(d)) of the NPs is consistent with diffraction pattern of the polycrystalline. XRD pattern (Fig. 1(e)) shows that all the peaks match well with the monoclinic Gd2O3 (PDF#42-1465), and no impurity phase is observed. The strong and narrow peaks are evidence of good crystallinity of the NPs. As shown in XPS spectra (Fig. 1(f)), peaks at 1220.5, 1188.5, and 1135.0 eV denote Gd3d3/2, 9

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Gd3d5/2, and Eu3d5/2, respectively. The peak positions are in agreement with the energy level reported for Gd3+ in Gd2O3 and Eu3+ in Eu2O3.39,40 Existence of Eu3d5/2 peak shows that Eu3+ has been successfully doped into the Gd2O3. 3.12 Formation mechanism of sub-10 nm monoclinic Gd2O3:Eu3+ NPs by LAL technique The above results show that the sub-10 nm monoclinic Gd2O3:Eu3+ NPs are successfully prepared by LAL technique. Fig. 2 shows the schematic illustration of formation mechanism of ultrafine Gd2O3:Eu3+ NPs by LAL technique. The process starts with the absorption

Fig. 2 Schematic illustration of formation mechanism of Gd2O3:Eu3+ NPs by laser ablation in liquid.

of the laser pulse by Gd2O3:Eu3+ target, then a high-temperature and high-pressure plasma plume containing the ablated material is produced at the target/liquid interface, and subsequent ultrasonic adiabatic expands into surrounding liquid, accompanied by the emission of a shockwave.41 During the expansion, the plasma plume quickly cools down and releases energy to the deionized water, resulting in the formation of Gd2O3:Eu3+ NPs. This process also generates a cavitation bubble, which expands in the liquid and then collapses.42,43 The quenching time of the pulse laser induced plasma is very short because of 10

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the cooling effect of confining liquid. Thus, the nucleation and growth processes are transient processes, which are the most important factors to fabricate the ultrafine Gd2O3:Eu3+ NPs by LAL technique. Meanwhile, the ultrafast quenching from hot plasma plume can make the Gd2O3:Eu3+ NPs remain its monoclinic structure and good crystallinity. 3.13 Fluorescence properties and fluorescence imaging of live cells The luminescence spectrum of Gd2O3:Eu3+ NPs colloid were obtained under the excitation at 275 nm. As shown in Fig. 3(a), the visible emission of the colloid exhibit two main bands centered at 590 and 623 nm. The insets are the corresponding excitation spectrum, schematic illustration of fluorescence emission, and the fluorescence photograph of the NPs colloid. Fig. 2(b) also shows the energy-level diagram and proposed fluorescence emission mechanism scheme of the Gd2O3: Eu3+ NPs. Under excitation at 275 nm, the ground state (8S7/2) electrons of the Gd3+ absorb the photons and populate the excited state (6IJ), then transfer the energy to Eu3+. The high energy states electrons of Eu3+ are unstable resulting in electrons back to the ground states through photons emission. The emission peaks centered at 590 and 623 nm are assigned to the transitions from the 5D0 state to the ground states of 7F1 and 7F2, respectively. The most intense peak centered at 623 nm (5D0→7F2) has a red-shift compared with the reported cubic counterpart.25,44 To verify the feasibility of using Gd2O3:Eu3+ NPs as FI probes, the Raw264.7 cells incubated with the Gd2O3:Eu3+ NPs were imaged under the laser confocal fluorescence microscope. As shown in Fig. 3(c), red fluorescence is clearly observed from the Raw264.7 cells, which demonstrated that the Gd2O3:Eu3+ NPs are useful for live cell

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imaging. But, the aggregation of the NPs in cells is also observed from the fluorescence image.

Fig. 3 (a) Emission spectrum, excitation spectrum, schematic illustration of fluorescence emission, and fluorescence images of the as-prepared Gd2O3:Eu3+ NPs colloids. (b) Schematic energy level diagram of the Eu3+ and Gd3+, and possible fluorescence emission process. (c) Bright field, fluorescence, and overlay images of the Raw 264.7 cells incubated with Gd2O3:Eu3+ NPs.

3.14 In vitro MR images It is known that the key parameter of T1-weighted contrast agents, the longitudinal relaxivity (r1), is proportional to the number of water molecules that coordinate with the unpaired electrons of Gd3+.45 The commercial clinical Gd-DTPA only offers one hydration position 12

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since its other six electrons are coordinated by chelates, which results in low r1 value. The surface Gd3+ of Gd2O3:Eu3+ NPs can offer its seven unpaired electron for water hydration (see Fig. 4(a)) results in larger r1 values than that of Gd-DTPA. Fig. 4(b) show the in vitro T1-weighted MR images of Gd2O3:Eu3+ NPs and Gd-DTPA. Clearly, the MR images brightened with the increase of the Gd3+ concentration (from 0 to 0.05 mM), and the Gd2O3:Eu3+ NPs possess the better contrast enhancement compared with Gd-DTPA. As shown in Fig. 4(c), the r1 value of Gd2O3:Eu3+ NPs is calculated to be 34.26 s-1mM-1, which is over 7 times higher than that of Gd-DTPA (4.22 s-1mM-1).

Fig. 4 (a) Schematic illustration of interaction between the surface Gd atom of Gd2O3:Eu3+ NPs and water. (b) In vitro T1-weighted MR images of the Gd2O3:Eu3+ NPs and Gd-DTPA. (c) Plot of the relaxation rate (1/T1) as a function of Gd3+ concentration, whose slope provides the longitudinal relaxivity (r1).

3.15 In vivo MR images The T1-weighted MR images and dynamic enhancement curve of NPC CNE-2 xenografted tumor (white arrow) in Balb/c nude mice are showed in Fig. 5(a-f) and 5(g), respectively. The 13

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control group is the uninjected mice. T1-weighted images in the axial orientations are obtained at 0, 10, 20, 35, 50, and 70 min after intravenous administration of the Gd2O3:Eu3+ NPs, which clearly show a high contrast enhancement of the tumor after injecting the Gd2O3:Eu3+ nanocrystals at 35 min. Therefore, both the in vitro and in vivo MRI investigations indicate that the Gd2O3:Eu3+ NPs may be a promise T1-weighted MRI contrast agent for biomedical applications.

Fig. 5 (a-f) In vivo MR images of a NPC CNE-2 xenografted tumor after intravenous administration of the Gd2O3:Eu3+ NPs (Gd3+, 15 µmolkg-1) at 0, 10, 20, 35, 50, and 70 min, respectively. (g) Dynamic enhancement curve of xenografted tumor.

3.2 In vitro and in vivo biocompatibility assessment of Gd2O3:Eu3+ NPs 3.21 Toxicity assays in vitro After co-incubation, the viability of Raw267.4 cells, S18 cells and PC12 cells were determined by MTT assay. As shown in Fig. 6(a), there is no significant difference between 14

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Gd-DTPA and Gd2O3:Eu3+ NPs. The results indicate that the Gd2O3:Eu3+ NPs have no effect on the cells survival. In addition, death and apoptosis of the Raw267.4 cells are evaluated by flow cytometry stained with Annexin-V/PI. The results are show in Fig. 6(b) and Supplementary Fig. S3, which suggest the cytotoxicity of the Gd2O3:Eu3+ NPs is negligible, and in vitro biocompatibility is satisfactory. 3.22 Immunotoxicity assay in vivo To further investigate the in vivo toxicity of the NPs, the immunotoxicity are evaluated in Balb/c mice. The data are shown in Fig. 6(c, d) and Supplementary Fig. S4. The generation level of ROS and the expression levels of CD11b and CD206 on the Gd2O3:Eu3+ NPs are much higher than those of Gd-DTPA, which indicated that the generation of ROS in peripheral blood neutrophils, the counts of mononuclear cells and macrophages in peripheral blood were increased. No significant differences are observed in the expression of early T-lymphocyte activation antigen (CD69 and CD71) between the Gd2O3:Eu3+ NPs and Gd-DTPA. However, the late T-lymphocyte activation antigen (CD25) of the NPs expresses a higher level than that of Gd-DTPA. Collectively, the Gd2O3:Eu3+ NPs might slightly stimulate the innate and adaptive immune cell proliferation in mice after injection.

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Fig. 6 (a) Cell viability of Raw264.7, S18 and PC12 cells after incubation with different concentrations of Gd2O3:Eu3+ NPs (Gd3+ concentrations, 10µM, 1µM and 0.1µM) for 48 h. (b) Apoptosis rate of Raw264.7 cells was measured by flow cytometry at 48 h after incubation of PBS, LPS, Gd-DTPA and Gd2O3:Eu3+ NPs. (c) The generation level of ROS on the neutrophils in peripheral blood. (d) Immunotoxicity assay in vivo. The expression levels of CD206 and CD11b on monocytes/macrophages, as well as CD69, CD71 and CD25 on lymphocyte cells of peripheral blood. (e) The secretion levels of cytokines including IL-1β, IL-2, and IL-4 in serum.

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The interleukins are the cytokines activated and mediated the immune cells, which play an important role in the differentiation and function of T cells, as well as in the inflammatory diseases. To some extent, the interleukins assessment reflects the level of the immunotoxicity. The results (Fig. 6(e)) show that our Gd2O3:Eu3+ NPs do stimulate the immune system. Interestingly, the results also show that the interleukins express no obvious differences between Gd2O3:Eu3+ NPs and Gd-DTPA. Compare with the Gd-DTPA, the Gd2O3:Eu3+ NPs did not stimulate the immune cells to secret the cytokines. This suggests that the Gd2O3:Eu3+ NPs might have minimal immunotoxicity, and could be regulated within normal control of homeostasis. 3.23 Pharmacokinetics assays including half-time, biodistribution, and excretion Pharmacokinetics characteristic is also one of the indexes of the nanotoxicity assessment in biomedicine. Fig. 7 shows the pharmacokinetics assays results of the LAL-derived Gd2O3:Eu3+ NPs. The half-life (Fig. 7(a)) of the Gd2O3:Eu3+ NPs is 67.16 (±11.91) min in blood, which is much longer than that of Gd-DTPA (20 min).46 The quantitative analysis of Gd concentration in typical organs, xenografted tumor tissue, feces and urine of mice is measured to investigate the distribution and excretion of the Gd2O3:Eu3+ NPs by ICP-MS. From Fig. 7(b), the nanoprobes accumulate gradually in the liver, spleen, and lung, but few are found in the heart, kidney, and tumor. The corresponding biodistribution of NPs at the subcellular level in the kidney, liver, lung, and tumor tissue are observed in TEM images marked with black arrows (Fig. 7(d)). According to Fig. 7(c), the excretion of the Gd-NPs is a slow process.

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Fig. 7 Pharmacokinetic characterizations of Gd2O3:Eu3+ NPs. (a) Half-life of the nanoprobes in the blood circulation. (b) Concentrations of Gd in the heart, liver, spleen, lung, kidney and tumor tissues. (c) Excretion of Gd was assayed in feces and urine of mice every week. (d) TEM images of liver, lung, spleen and xenografted tumors in nude nice at 4 h after intravenous administration of Gd2O3:Eu3+ NPs (Gd3+ concentrations, 15µmol/kg-1).

3.24 Mechanism analysis of nanotoxicity It is a crucial role for nano-based biomaterials to explore the risk reduction strategies in their preclinical research. To date, the scientific basis on nanotoxicity of most manufactured nanomaterials remains unclear.47,48 The unique physicochemical properties of nanostructure and nanoscale particles are closely related to the toxic mechanisms on the intravenously injected nanomedicine.47,49 Their specific physical parameters, including the size, surface charge, surface area to volume, morphology, might elicit the toxic responses in body.48,50 On the other hands, Adsorption and 18

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recognition are associated with their retention and clearance time, which are depended on the physicochemical properties of the particle. Injected particles with optimal clearance characteristics will minimize toxicity risk.20 Renal clearance is capable of rapidly removing some certain particles (6-8 nm with positive charge or less than 6 nm) from the body unaltered from their original form and minimizing the involvement of intracellular catabolism, while hepatobiliary system represents the primary route of excretion for particles that do not undergo renal clearance, which is associated with endocytosis and enzymatic breakdown of foreign particles.20,51 For our Gd2O3:Eu3+ NPs, the size is ultra-small with large surface area-to-volume, and the surface charge is negative (as shown in Supplementary Fig. S5). Thus, the Gd2O3:Eu3+ NPs might be endocytosed by neutrophil and monocyte in the blood or macrophages in organs after injection, and the particles with negative charge are much easier to accumulate than the ones with positive charge.52,53 TEM images of organs suggested that few Gd2O3:Eu3+ NPs were localized in the phagolysome of cytoplasm by the endocytic pathway. The NPs were exhibited little aggregation which might induce the micro-structural damage in the cells. Moreover, it is difficult for Gd2O3:Eu3+ NPs to be excreted by renal clearance. The renal clearance is thought to be the preferred clearance route to minimize agent retention and toxicity. According to our results, aggregation and absorption can be obviously found in the fluorescence image of living cells (Fig. 4(c)) and TEM images of tissues (Fig. 7(d)). The formation of aggregation is not typically capable of kidney filtration, while the liver serves as an important site for elimination them. Meanwhile, studies have shown that filtration is 19

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greatest for cationic molecules, followed by neutral molecules, while anionic molecules are least readily filtered through the glomerular capillary wall.20 Thus, the injected NPs main accumulate in liver, few are found in the kidney, and experience prolonged circulatory times due to slow transport across the endothelium. Consequently, the long-term damage of cells might further stimulate the over-generation of reactive oxygen species (ROS). The generation of ROS are essential factors for the host’s immune response.54,55 With these in mind, we propose that aggregation and absorption of our products in vivo may be the main factor responsible for the immunotoxicity. The prolonged blood half-life increase their duration of exposure to immune system. The immune system recognizes them as foreign substances and stimulates immune response against them, and extremely enlarges the toxicity risk. 4. CONCLUSION In summary, we successfully prepared the sub-10 nm monoclinic Gd2O3:Eu3+ NPs, and demonstrated their potential as dual-modal nanoprobes for MRI and FI. As MRI contrast agent, the Gd2O3:Eu3+ NPs show ultra-high r1 value and the T1-weighted contrast enhancement of xenografted tumor in mice. As FI nanoprobe, the product exhibits bright fluorescence emission. The toxicity assessments show the Gd2O3:Eu3+ NPs without surface modification cause no significant in vitro cytotoxic effect, while the in vivo immunotoxicity is higher than that of Gd-DTPA. The main reason of the induced immunotoxicity can be ascribed to the negative surface charge and aggregation of NPs. The above results provide a

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novel platform to the growing field of diagnosis, and motivate future investigations into the use of the rare earth doped Gd2O3 NPs as dual-modal nanoprobes for bioimaging application.

ASSOCIATED CONTENT Supporting information Schematic diagram of the laser ablation in liquid technique, calculation of the longitudinal relaxivity value (r1), in vivo MR images, apoptosis assay, in vivo immunotoxicity assay, and Zeta potential of the Gd2O3:Eu3+ NPs.

AUTHOR INFORMATION Corresponding Author *

E-mail: [email protected], [email protected]. Mobile: 86-20-13600452080, Fax:

86-20-84113398. Author Contributions #

Jun Liu and Xiumei Tian contributed equally to this work.

ACKNOWLEDGMENTS This work was supported by the National Basic Research Program of China (2014CB931700), the National Natural Science Foundation of China under Grant No. 81071264 and 81271622, and Guangdong Province of Higher School "Thousand Hundred Ten Talents Project".

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