Multifunctional Gadolinium-Doped Manganese Carbonate

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Multifunctional Gadolinium-Doped Manganese Carbonate Nanoparticles for Targeted MR/Fluorescence Imaging of Tiny Brain Gliomas Chen Shao, Shuai Li, Wei Gu, Ningqiang Gong, Juan Zhang, Ning Chen, Xiangyang Shi, and Ling Ye Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b01639 • Publication Date (Web): 26 May 2015 Downloaded from http://pubs.acs.org on May 30, 2015

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Analytical Chemistry

Multifunctional Gadolinium-Doped Manganese Carbonate Nanoparticles for Targeted MR/Fluorescence Imaging of Tiny Brain Gliomas

Chen Shao ‡ a, Shuai Li ‡ a, Wei Gu a, Ningqiang Gong c, Juan Zhang a, Ning Chen d, Xiangyang Shi* b and Ling Ye* a

a

School of Chemical Biology and Pharmaceutical Sciences, Capital Medical University, Beijing 100069, P. R.

China b

College of Chemistry, Chemical Engineering and Biotechnology, Donghua University, Shanghai 210620, P. R.

China c

School of Traditional Chinese Medicine, Capital Medical University, Beijing 100069, P. R. China

d

Department of Neurosurgery, Beijing Sanbo Brain Hospital, Capital Medical University, Beijing 100093, P. R.

China

* Corresponding authors. E-mail: [email protected], [email protected] Fax: +86 10 83911533 ‡ C. Shao and S. Li contributed equally to this work

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ABSTRACT Manganese (Mn)-based nanoparticles have been proved to be promising MR T1 contrast agents for the diagnosis of brain tumors. However, most of them exhibit a low relaxation rate, resulting in an insufficient enhancement effect on tiny gliomas. Herein, we developed gadolinium (Gd)-doped MnCO3 nanoparticles with a size of 11 nm via the thermal decomposition of Mn-oleate in the presence of Gd-oleate. Owning to the small size and Gd-doping, these Gd-doped MnCO3 NPs, when endowed with excellent aqueous dispersibility and colloidal stability, exhibited a high r1 relaxivity of 6.81 mM-1 s-1. Moreover, the Gd/MnCO3 NPs were used as a reliable platform to construct a glioma-targeted MR/fluorescence bimodal nanoprobe. The high relaxivity, the bimodal imaging capability, and the specificity nominate the multifunctional Gd doped MnCO3 NPs as an effective nanoprobe for the diagnostic imaging of tiny brain glioma with an improved efficacy.

KEYWORDS: Gadolinium (Gd) doping, MnCO3 nanoparticles, MR/fluorescence dual modal imaging, tiny brain gliomas, targeting

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1. INTRODUCTION The accurate detection of early-stage gliomas by contrast-enhanced magnetic resonance (MR) imaging is important for the timely delivery of treatment and better patient outcome.1-3 Paramagnetic gadolinium (Gd)-based contrast agents have been developed to enhance T1-weighted MR contrast for brain tumor diagnosis.4, 5 However, the occurrence of nephrogenic systemic fibrosis (NSF) related to Gd (III) ions restricts widespread application of Gd-based contrast agents in clinical practice.6, 7 Therefore, attention has shifted to alternative agents such as manganese (Mn)-based complexes8-11 and nanoparticles (NPs),12-15 which has produced encouraging results for both in vitro and in vivo MR imaging of cancer.16-18 For instance, MnO NPs have been successfully used for T1-weighted MR imaging of metastatic brain tumors.19 However, compared with gadolinium-based contrast agent,20-23 most of the Mn-based NPs exhibit relatively low r1 relaxivity,24 which may limit their uses in sensitive T1-weighted MR imaging of tiny gliomas. Up to now, several strategies have been put forward to improve the relaxation rate. For instance, Lee’ group prepared the hollow MnO nano-ring via the corrosion of MnO NPs in acidic buffer, and owing to the larger water-accessible surface area, the relaxation rate of MnO nano-ring increased to 1.417 mM-1 s-1,25 a 6-fold enhancement in T1-weighted contrast. Another study conducted by Chen’s et al. showed that the relaxivity of MnO NPs could be boosted by surface modification of HSA, which led to more efficient water-surface interaction and consequently, a higher r1 of 1.97 mM-1 s-1.16 Nonetheless, these relaxivitites are still far less than that of Gd-based contrast agent. Therefore, the development of Mn-based contrast agent with an improved relaxivity, which is highly desirable for the better detection of tiny brain gliomas, remains an ongoing challenge. The Gd ions with seven unpaired electrons have a long electronic relaxation time, which efficiently decreases the spin-lattice and spin-spin relaxation times of water protons in the 3



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proximity.26 As such, Gd doping provides an alternative and straightforward strategy for improving the relaxivity of contrast agents. In this context, we attempted to introduce a Gd-OA complex to the thermal decomposition of Mn-OA with intention to enhance the relaxivity of MnO NPs. Unexpectedly, this led to the formation of OA-capped, Gd-doped MnCO3 (Gd/MnCO3-OA) NPs with a size of 11 nm instead of the predicted Gd-doped MnO NPs. To our best knowledge, such small-sized MnCO3 NPs with high crystallinity have not been prepared by other methods.27,

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MnCO3 particles are an alternative potential Mn-based contrast agent and have been reported to be used as an MR contrast agent for liver imaging.29 Moreover, Shapiro et al. demonstrated that MnCO3 particles could slowly release Mn2+ under an acidic condition, resulting in bright contrast enhancement in the T1-weighted MR images.27 However, the development of MnCO3 particles as a T1 contrast agent is rarely reported so far. The major obstacle is that the size of the reported MnCO3 particles is relatively large, thus yielding an extremely low longitudinal relaxivity.29 The developed Gd-doped MnCO3 NPs in our work may overcome this bottleneck due to the nanoscale size along with the Gd loading. When endowed with high water dispersibility and excellent aqueous stability via ligand exchange of carboxylic acid-terminated silane and conjugation of polyethylene glycol (PEG), the Gd/MnCO3 NPs exhibited a marked high relaxivity of 6.81 mM-1 s-1, which is comparable to that of Gd2O3 NPs. Furthermore, these Gd/MnCO3 NPs confer a reliable platform that allow us to construct a multifunctional nanoprobe by the conjugation of the near infrared (NIR) dye Cy5.5 and the targeting ligand folic acid (FA) (Scheme 1). Such specific nanoprobe that combines the high spatial resolution of MRI and the high sensitive of fluorescence imaging enables an effective detection of tiny brain gliomas with an improved efficacy.

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2. EXPERIMENTAL SECTION Materials Manganese chloride tetrahydrate and sodium oleate were purchased from Sinopharm Chemical Reagent (Beijing, China). Gadolinium chloride hydrate was purchased from Sigma-Aldrich (St. Luis, MO). N-(trimethoxysilylpropyl) ethylene diamine triacetic acid (TETT) silane (45% in water) was supplied by Gelest, Inc. (Tokyo, Japan). Folic acid-poly(ethylene glycol) (FA-PEG) was provided by JenKem Technology Co., Ltd (Beijing, China). All other chemicals and reagents were of reagent grade and used as received. Preparation of Mn-oleate and Gd-oleate complex Mn-oleate complex was synthesized according to the previously reported method.30 Briefly, 40 mL of ethanol, 30 mL of distilled water, 70 mL of n-hexane, 20 mmol of manganese chloride tetrahydrate, and 40 mmol of sodium oleate were added into a round bottom flask. The mixture was heated to 70 ºC for 4 h and then transferred to a separatory funnel. The upper organic layer containing the Mn-oleate complex was washed three times with distilled water and the hexane was evaporated to collect the Mn-oleate complex. Gd-oleate complex was also prepared according to the same procedure described above, except that 60 mmol of sodium oleate was added. Synthesis of oleate-capped Gd/MnCO3 NPs The oleate-capped Gd/MnCO3 (Gd/MnCO3-OA) NPs were synthesized by the thermal decomposition method. Briefly, 9.6 mmol of Mn-oleate complex, 2.4 mmol of Gd-oleate complex, and 150 mL of 1-octadecene were added into a three-necked flask. The mixture was heated to 100 ºC for 15 min under vacuum to remove the water and oxygen, followed by sequential heating at 200 ºC (20 min), 280 ºC (10 min), and 310 ºC (10 min) with the purging of N2. After cooling to room temperature, 40 mL of ethanol was added to precipitate the product. The precipitate was separated by

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centrifugation and washed with ethanol and acetone, respectively. Synthesis of TETT-modified Gd/MnCO3 NPs The Gd/MnCO3-OA NPs were subjected to a silanization step according to a reported method in the literature.31 Typically, 100 mg of Gd/MnCO3-OA NPs were dispersed in anhydrous toluene containing 60 µL of acetic acid. After sonicating for 15 min, 0.6 mL of TETT silane was added and the suspension was stirred at 70 ºC for 48 h. Then, the product was collected and washed with toluene and ethanol, respectively. Finally, the TETT-modified Gd/MnCO3 (Gd/MnCO3-TETT) NPs were dialyzed against deionized water for 24 h using a cellulose dialysis membrane (MWCO = 3500), followed by lyophilization to get the Gd/MnCO3-TETT NPs. Synthesis of multifunctional Gd/MnCO3 NPs 1.25 mg Cy5.5-NHS, 8.86 mg H2N-PEG2000-NH2, and 10 mL of distilled water were stirred at room temperature for 24 h (pH = 8, the molar ratio of Cy5.5-NHS to H2N-PEG-NH2 was 1:4) to yield Cy5.5-PEG-NH2. Gd/MnCO3-TETT NPs (100 mg) were dispersed in 10 mL of distilled water. Then, 3.206 mg of EDC and 4.808 mg of NHS were added for activation (pH = 4.7 - 6.0). Next, 5 mL of Cy5.5-PEG-NH2 and 20 mg of H2N-PEG2240-FA were added. The mixture was stirred at room temperature for 24 h (pH = 8). After the reaction was completed, the solution was dialyzed against deionized water for 24 h using a cellulose dialysis membrane (MWCO = 8000-14000). The targeted Gd/MnCO3 NPs were obtained by lyophilization. The non-targeted Gd/MnCO3 NPs were prepared in the same procedure described above except that 17.86 mg of H2N-PEG2000-COOH was added instead of 20 mg of H2N-PEG2240-FA. Characterization Transmission electron microscopy (TEM) and high resolution TEM (HRTEM) images were obtained on JEM-2100F (JEOL, Tokyo, Japan) at an operating voltage of 200 kV. X-ray diffraction (XRD) 6


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patterns were obtained on a PANalytical X’pert Pro MPD diffractometer at Cu-Kα radiation 40 kV and 40 mA (PANalytical, Holland). Dynamic light scattering (DLS) measurements were performed on a Malvern Nano-ZS90 Zetasizer (Worcestershire, UK). Fourier transform infrared (FTIR) spectra were recorded on an IR Prestige-21 spectrophotometer (Shimadzu, Tokyo, Japan). The content of Mn and Gd was determined on an inductively coupled plasma optical emission spectrometry (ICP-OES, Varian 710-ES, USA). The magnetic properties of NPs were determined on SQUID (MPMSXL-7, Quantum Design, USA). Fluorescence emission spectra were recorded on an F-2500 fluorescence spectrophotometer (Hitachi, Japan). The UV-vis absorption spectra were recorded on a UV-2600 spectrophotometer (Shimadzu, Tokyo, Japan). Relaxivity measurements The non-targeted and targeted Gd/MnCO3 NPs were dispersed in 1% agarose solution with various Mn concentrations (0.03125, 0.0625, 0.125, 0.25 and 0.5 mM, respectively) for relaxivity measurement and MR imaging on a 7T MR scanner (Bruker Pharmascan, Germany) using RARE-T1-map MRI sequence. The measurement parameters were as follows: repetition times (TR) = 200, 400, 800, 1500, 3000, 5000 ms, echo time (TE) = 11.00 ms, field of view (FOV) = 50 mm × 50 mm, matrix = 256 mm × 256 mm, flip angle (FA) = 180o, and slice thickness = 1 mm. Relaxivity (r1) value was obtained from the linear fitting of 1/T1 (s-1) as a function of the Mn concentration (mM). Cell culture C6 glioma cells (or GFP-transfected C6 glioma cells) and NIH 3T3 cells were cultured in DMEM with 10% FBS, 100 µg mL-1 streptomycin and 100 U mL-1 penicillin supplemented at 37 ºC, 5% CO2 atmosphere, and a humid environment. In vitro cytotoxicity assay The cytotoxicity of non-targeted and targeted Gd/MnCO3 NPs was evaluated via MTT 7



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[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] colorimetric assays. C6 glioma and NIH 3T3 cells were seeded in a 96-well plate at a density of 1 × 104 cells per well. After 24 h of incubation, the medium was replaced with a fresh medium containing non-targeted or targeted Gd/MnCO3 NPs at different Mn concentrations and the cells were incubated for another 24 h. Then, cell culture medium was removed and MTT (0.5 mg mL-1, 100 µL) was added into each well. After incubating for another 4 h, MTT solution was removed and 150 µL of DMSO was added into each well to dissolve the formazan crystals. The absorbance was recorded at 570 nm using a Multiskan Spectrum microplate reader (Thermo Electron Corporation, USA) and the cell viability was assessed by the ratio of OD values between the experimental group and the control group. Data were presented as the mean value with standard deviations from triplicate wells. Histology analysis The mice were sacrificed 15 days after injection of the non-targeted or targeted Gd/MnCO3 NPs at a dosage of 24 mg Mn kg-1 body. The major organs (brain, heart, liver, kidney, spleen, and lung) were harvested and fixed in freshly prepared formalin 10% for 72 h. Then, the tissues were dehydrated using a tissue processor and embedded with paraffin, sectioned into 5 µm slices, stained with hematoxylin and eosin (H&E), or Masson trichrome (only for kidney) according to the standard clinical pathology protocol, and observed under an optical microscope. Tiny brain glioma model All animal experiments were performed according to protocols evaluated and approved by the ethical committee of Capital Medical University. Male nude mice were anesthetized with 6% chloral hydrate (0.10 mL/20 g) and placed in a stereotactic frame. A burr hole was drilled into the skull (1.0 mm anterior and 2.0 mm lateral to the bregma). Approximately 5 × 105 GFP-transfected C6 glioma cells (suspended in a total volume of 5 µL) were injected into the burr hole. When the tumors reached 1.5−1.8 mm in diameter, MR imaging was performed. 8


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In vivo MR imaging of the tiny brain glioma The glioma-bearing nude mice were anesthetized by intraperitoneal injection of 6% chloral hydrate (0.10 mL/20 g). The non-targeted or targeted Gd/MnCO3 NPs were dispersed in 0.2 mL of PBS solution and injected via tail vein at a dosage of 10 mg Mn kg-1 body. In vivo MR imaging was conducted on a 7T MR scanner (Bruker Pharmascan, Germany) using MSME MR sequence. The measurement parameters were set as follows: TR = 300 ms, TE = 8 ms, matrix = 256 mm × 256 mm, FOV = 25 mm × 25 mm, slice thickness = 0.8 mm, and number of averages = 3. The MR signal intensity of tumors was obtained at the region of interest (ROI) with the same diameter placed at the tumor site in the same slice on T1-weighted MR images before and after the administration of the NPs. In vivo fluorescence imaging of the tiny brain glioma The brain glioma-bearing nude mice were anesthetized by intraperitoneal injection of 6% chloral hydrate (0.10 mL/20 g). The non-targeted or targeted Gd/MnCO3 NPs were dispersed in PBS solution (0.2 mL) and injected via tail vein at a dosage of 10 mg Mn kg-1 body. Fluorescence signal acquisitions were performed on NightOWL LB 983 in vivo Imaging System before and at 24 h post injection. Additionally, the brains were collected and the ex vivo imaging was also performed. Confocal microscopic imaging of brain slices The glioma-bearing nude mice were fixed by heart perfusion with saline and 4% paraformaldehyde at 24 h post injection of the non-targeted or targeted Gd/MnCO3 NPs. The brains were harvested and fixed in 4% paraformaldehyde overnight and dehydrated with 30% sucrose solution until subsidence. The brains were embedded in optimum cutting temperature compound (OCT) and frozen in liquid nitrogen for several seconds, sectioned at 20 µm. Slides were examined under a confocal microscope. Statistical analysis 9



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The data were expressed as mean ± standard error. Statistical differences were evaluated using the Student’s t test. A difference of P < 0.05 was considered to be statistically significant.

3. RESULTS AND DISCUSSION In a typical thermal decomposition, Gd-OA complex was mixed with the Mn-OA complex at 25 mol % Mn, which was followed by sequential heating steps of 100 °C for 15 min, 200 °C for 20 min, 280 °C for 10 min, and 300 °C for 10 min. Gd-doped MnCO3 NPs were obtained with a Gd doping content of 1.3% estimated by ICP-OES. The Gd-OA precursor clearly played a key role in the production of MnCO3 NPs; however, the underlying mechanism awaits a more detailed study. TEM and XRD were used to characterize the morphology and crystal structure of Gd/MnCO3-OA NPs. A TEM image revealed that Gd/MnCO3-OA NPs have uniform rhomboid shape with a size of 11 nm (Figure. 1A). HRTEM image showed the clear lattice fringes (Figure 1B), indicating the high-quality crystallinity. The polycrystalline diffraction rings in the selected area electron diffraction (SAED) pattern (Figure 1C) were indexed to the featured (012), (104), (006), (110), and (113) planes of the MnCO3 rhombohedral structure, which was verified by corresponding reflections in the XRD pattern (Figure 1D). Elemental analysis by energy-dispersive spectroscopy (Figure 1E) confirmed the presence of Mn, Gd, C, and O atoms in the NPs. Furthermore, the field-dependent magnetization curve (M-H curve) of the Gd/MnCO3-OA NPs assessed using a SQUID displayed no remanence coercivity in zero field at 300 K (red line) and magnetic hysteresis loops (black line) at 5 K (Figure 1F), demonstrating that the Gd/MnCO3-OA NPs were paramagnetic at room temperature but weakly ferromagnetic at extremely low temperature. The OA were replaced by carboxylic acid-terminated TETT silane to render Gd/MnCO3-TETT NPs water-dispersible. TEM and DLS were used to assess particle morphology and size distribution (Figure S1). Ligand exchange likely resulting from corrosion by surface modification made the NP 10


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edges ambiguous. Silanized Gd/MnCO3 NPs have a hydrodynamic diameter of about 59 nm, which is considered as a suitable size for vascular retention and tumor penetration.32 Ligand exchange was confirmed by FTIR spectroscopy (Figure. 2A). The Gd/MnCO3-OA NPs displayed strong, characteristic -CH2- IR bands at 2922, 2852, and 1433 cm−1.33 Upon ligand exchange with Gd/MnCO3-OA NPs displayed strong, characteristic -CH2- IR TETT silane, the bands for OA disappeared and new peaks emerged in the range of 1328 and 1150 cm−1, corresponding to the characteristic Si-C, C-N, and Si-O bonds of TETT silane.30 Following conjugation of PEG-Cy5.5 and PEG-FA to Gd/MnCO3-TETT NPs, Gd/MnCO3-PEG-Cy5.5-FA NPs specifically targeting gliomas and possessing fluorescent properties were obtained. Non-targeted Gd/MnCO3 NPs were also fabricated for comparison. The attachment of PEG was confirmed by the increase in intensity of -CH2- bands at 2939, 2885, and 1465 cm−1 by FTIR spectroscopy (Figure 2A), and FA conjugation was evidenced by the absorbance peak at 280 nm in the ultraviolet-visible (UV-vis) spectrum of Gd/MnCO3-PEG-Cy5.5-FA NPs (Figure 2B, solid lines).34 The presence of Cy5.5 was also confirmed by the absorption peak at 675 nm (Figure 2B, solid lines) and the emission peak at 700 nm (Figure 2B, broken lines) in the UV-vis and fluorescence spectra, respectively, for both targeted and non-targeted NPs. The colloidal stability of multifunctional Gd/MnCO3 NPs in aqueous media must be determined in order for these particles to be useful in biomedical imaging applications. Both targeted and non-targeted multifunctional Gd/MnCO3 NPs were dispersed in water at concentrations as high as 20 mg/mL and remained stable for at least 1 month (Figure 2C). Moreover, upon exposure to 50% fetal bovine serum at a concentration of 5 mg/mL, there was no obvious aggregation or precipitation for at least 1 month (Figure 2C), suggesting that the particles have good colloidal stability. The toxicity of the targeted and non-targeted Gd/MnCO3 NPs was evaluated with the MTT cell viability assay, as well as by histological analysis of mouse organs after intravenous injection of the 11



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particles. The results of the MTT assay (Figure S2) revealed that the viability of NIH 3T3 and C6 cells treated with either type of Gd/MnCO3 NP remained at 92% at a Mn concentration of 160 µM, suggesting low toxicity. A histological analysis of mouse organs showed no evidence of atrophy or necrosis of cardiac tissue, degeneration or necrosis of hepatic and epithelial cells of renal tubules, nor of inflammatory cells in the brain (Figure S3), demonstrating that targeted and non-targeted Gd/MnCO3 NPs have low toxicity to organs. It should be noted that NSF is generally associated with exposure to gadolinium-based MR contrast agents with severe kidney failure. However, in this study, Gd was doped into MnCO3 NPs and the doping percentage is only 1.3% (as determined by ICP-OES). On this occasion, the risk of NSF related to free Gd ions could be greatly minimized. This was supported by the HE staining result, which did not show typical NSF-like lesion at the kidneys of the mice (Figure S3, Supporting Information). To further assay the risk of occurrence of NSF related to the Gd/MnCO3 NPs, we performed Masson trichrome staining on the same batch of samples (Figure S4, Supporting Information). It turned out that no noticeable renal glomerulus fibrosis sign was observed after administration of the Gd/MnCO3 NPs. Nevertheless, a detailed study long-term is still needed to further evaluate the risk of NSF related to the Gd/MnCO3 NPs. The quality of contrast enhancement for MR imaging depends on the relaxivity of the contrast agent. To evaluate the potential of Gd/MnCO3 NPs as a T1-weighted MR contrast agent, the T1 relaxation times of particles at different Mn concentrations were measured with a 7T MR scanner, and the corresponding r1 was calculated from the slope of the linear curve of inverse relaxation time (1/T1) as a function of the Mn concentration (measured by ICP-OES) (Figure 3A). Owning to the smaller size and Gd doping, the Gd/MnCO3-TETT NPs exhibited a high r1 value of 6.08 mM-1s-1. Upon conjugation with PEG-Cy5.5 and PEG (non-targeted NPs), the r1 increased to 6.81 mM−1s−1, probably because the attached PEG moieties made the particles highly hydrophilic and thereby increased the accessibility of paramagnetic ions to water molecules. However, conjugation of 12


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PEG-Cy5.5 and PEG-FA (targeted NPs) yielded an r1 value of 6.58 mM−1s−1. This slight decrease was probably due to the shielding effect of the hydrophobic FA moiety. Nevertheless, this value is comparable to that of ultrasmall Gd2O3 NPs22, 23, 35 and higher than that of MnO NPs prepared by the same thermal decomposition method.30 As the r1 decreases with the increase of magnetic field strength,36 the high r1 obtained at 7T thus inferred the effectiveness of the Gd/MnCO3 NPs in T1-weighted MR imaging. This was further supported by the corresponding T1-weighted MR images, which revealed an increase in brightness corresponding to increases in Mn concentration (Figure. 3B). It should be noted that a fully understanding of the underlying mechanism would help to optimize the composition of Gd/MnCO3 NPs and therefore further improve the r1. On account of high r1 relaxivity and excellent biocompatibility, the feasibility of using Gd/MnCO3-PEG-Cy5.5-FA NPs as targeted nanoprobes for MR imaging of tiny brain gliomas was explored. A significant contrast enhancement in the tumor region was observed at 10 min postinjection of the targeted or non-targeted Gd/MnCO3 NPs (Figure 4), suggesting the rapid accumulation of particles inside the gliomas as a result of the enhanced permeation and retention effect. The contrast continued to increase over time, and then decreased after 120 min due to particle metabolization. However, a significant contrast enhancement was again observed at 24 h postinjection. This can be explained by the gradual release of Mn2+ ions from the Gd/MnCO3 NPs due to the acidic tumor microenvironment.24,

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More importantly, a greater enhancement was

produced by targeted than by non-targeted Gd/MnCO3 NPs, presumably due to the specificity of the FA moiety for FA receptor-overexpressing gliomas. Therefore, it is believed that the MR contrast enhancement in this study originates not only from the MnCO3 NPs but also from the released Mn2+ ions. Moreover, contrast-to-noise ratio (CNR) data, which reflect the contrast enhancement effects of the Gd/MnCO3 particles instead of quantifying their relaxation,29 were obtained at different time points (Figure 4, inset). The CNRs of the tumor region 10 min post-injection were 6.67 and 6.86 for 13



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non-targeted and targeted NPs, respectively; these values were about 5.5 times higher than the pre-injection CNR. The difference in CNR increased over time, highlighting the role of FA-mediated targeting. The conjugation of Cy5.5 to Gd/MnCO3 NPs enabled additional fluorescence imaging and yielded greater sensitivity in the acquisition of molecular information. After injecting non-targeted or targeted NPs, intense fluorescence signals in the kidney and in brain tumors were observed 24 h, suggesting that the particles were retained in the brain and were metabolized by the kidney (Figure 5B). However, the fluorescence intensity differed for the two types of particles within the same organ, resulting in a higher ratio of brain/kidney fluorescence intensity for targeted Gd/MnCO3 NPs, highlighting their higher accumulation in the brain. This was confirmed by ex vivo fluorescence imaging of a dissected brain harboring gliomas (Figure 5C); brain sections showing GFP-transfected C6 gliomas were examined by confocal laser scanning microscopy (CLSM) (Figure 5D, E). In the brain treated with non-targeted Gd/MnCO3 NPs, the Cy5.5 signal was predominantly located at the tumor rim, with negligible signal present in the glioma core. In contrast, in the targeted Gd/MnCO3 NP-treated brain, the red fluorescence was evenly distributed throughout the tumor and had a higher intensity. These results, which are in agreement with the MR imaging data, suggest that targeted Gd/MnCO3 NPs can penetrate more deeply into tumor tissue, permitting enhanced and prolonged imaging of tumor fluorescence.

4. CONCLUSION In summary, Gd-doped MnCO3 NPs with a size of 11 nm were prepared by high-temperature thermal decomposition. The Gd doping played a key role in the formation of Gd/MnCO3 NPs and improved r1 relaxivity. The silanization and subsequent PEGylation not only conferred water dispersibility and colloidal stability to the NPs, but also allowed conjugation of Cy5.5 and the 14


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targeting ligand FA to generate Gd/MnCO3-PEG-Cy5.5-FA NPs. These multifunctional NPs were efficient bimodal nanoprobes for targeted MR/fluorescence imaging of tiny brain gliomas and therefore hold great potential for improved detection of early-stage gliomas.

ACKNOWLEDGEMENTS The authors gratefully acknowledge the financial supports from the Natural Science Foundation of China (81271639, 21273032). X. Shi also thanks the support from the Program for Professor of Special Appointment (Eastern Scholar) at Shanghai Institutions of Higher Learning.

Supporting Information Available: TEM images, hydrodynamic size distribution, cytotoxicity assay, and histology analysis. This information is available free of charge via the Internet at http://pubs.acs.org/.

REFERENCES (1) Cleary, K.; Peters, T. M. Annu. Rev. Biomed. Eng. 2010, 12, 119-142. (2) Hu, F. Q.; Zhao, Y. S. Nanoscale 2012, 4, 6235-6243. (3) Narayana, A.; Kunnakkat, S. D.; Medabalmi, P.; Golfinos, J.; Parker, E.; Knopp, E.; Zagzag, D.; Eagan, P.; Gruber, D.; Gruber, M. L. Int. J. Radiat. Oncol., Biol., Phys. 2012, 82, 77-82. (4) Terreno, E.; Delli Castelli, D.; Viale, A.; Aime, S. Chem. Rev. 2010, 110, 3019-3042. (5) Pishko, G. L.; Astary, G. W.; Mareci, T. H.; Sarntinoranont, M. Ann. Biomed. Eng. 2011, 39, 2360-2373. (6) Sieber, M. A.; Steger-Hartmann, T.; Lengsfeld, P.; Pietsch, H. J. Magn. Reson. Imaging 2009, 30, 1268-1276. (7) Idee, J. M.; Port, M.; Dencausse, A.; Lancelot, E.; Corot, C. Radiol. Clin. N. Am. 2009, 47, 855-869. 15



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(8) Gallez, B.; Bacic, G.; Swartz, H. M. Magn. Reson. Med. 1996, 35, 14-19. (9) Drahos, B.; Lukes, I.; Toth, E. Eur. J. Inorg. Chem. 2012, 1975-1986. (10) Rummeny, E. J.; Marchal, G. Acta Radiol. 1997, 38, 626-630. (11) Ye, Z.; Jeong, E. K.; Wu, X. M.; Tan, M. Q.; Yin, S. Y.; Lu, Z. R. J. Magn. Reson. Imaging 2012, 35, 737-744. (12) Schladt, T. D.; Schneider, K.; Shukoor, M. I.; Natalio, F.; Bauer, H.; Tahir, M. N.; Weber, S.; Schreiber, L. M.; Schroeder, H. C.; Mueller, W. E. G.; Tremel, W. J. Mater. Chem. 2010, 20, 8297-8304. (13) Chen, Y.; Ye, D.; Wu, M.; Chen, H.; Zhang, L.; Shi, J.; Wang, L. Adv.Mater. 2014, 26, 7019-7026. (14) Hu, H.; Dai, A.; Sun, J.; Li, X.; Gao, F.; Wu, L.; Fang, Y.; Yang, H.; An, L.; Wu, H.; Yang, S. Nanoscale 2013, 5, 10447-10454. (15) Xing, R.; Liu, G.; Quan, Q.; Bhirde, A.; Zhang, G.; Jin, A.; Bryant, L. H.; Zhang, A.; Liang, A.; Eden, H. S.; Hou, Y.; Chen, X. Chem. Commun. 2011, 47, 12152-12154. (16) Huang, J.; Xie, J.; Chen, K.; Bu, L.; Lee, S.; Cheng, Z.; Li, X.; Chen, X. Chem. Commun. 2010, 46, 6684-6686. (17) Letourneau, M.; Tremblay, M.; Faucher, L.; Rojas, D.; Chevallier, P.; Gossuin, Y.; Lagueux, J.; Fortin, M. J. Phys. Chem. B 2012, 116, 13228-13238. (18) Chevallier, P.; Walter, A.; Garofalo, A.; Veksler, I.; Lagueux, J.; Begin-Colin, S.; Felder-Flesch, D.; Fortin, M. A. J. Mater. Chem. B 2014, 2, 1779-1790. (19) Na, H. B.; Lee, J. H.; An, K.; Park, Y. I.; Park, M.; Lee, I. S.; Nam, D.; Kim, S. T.; Kim, S.; Kim, S.; Lim, K.; Kim, K.; Kim, S.; Hyeon, T. Angew. Chem., Int. Ed. 2007, 46, 5397-5401. (20) Johnson, N. J. J.; Oakden, W.; Stanisz, G. J.; Prosser, R. S.; van Veggel, F. C. J. M. Chem. Mater. 2011, 23, 3714-3722. 16


Page 16 of 26

Page 17 of 26

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(21) Abdukayum, A.; Yang, C. X.; Zhao, Q.; Chen, J. T.; Dong, L. X.; Yan, X. P. Anal. Chem. 2014, 86, 4096-4101. (22) Fortin, M.; Jr. Petoral, R. M.; Soederlind, F.; Klasson, A.; Engstroem, M.; Veres, T.; Kaell, P.; Uvdal, K. Nanotechnology 2007, 18, 395501-395509. (23) Ahren, M.; Selegard, L.; Klasson, A.; Soderlind, F.; Abrikossova, N.; Skoglund, C.; Bengtsson, T.; Engstrom, M.; Kall, P.; Uvdal, K. Langmuir 2010, 26, 5753-5762. (24) Bennewitz, M. F.; Lobo, T. L.; Nkansah, M. K.; Ulas, G.; Brudvig, G. W.; Shapiro, E. M. ACS Nano 2011, 5, 3438-3446. (25) Shin, J.; Anisur, R. M.; Ko, M. K.; Im, G. H.; Lee, J. H.; Lee, I. S. Angew. Chem., Int. Ed. 2009, 48, 321-324. (26) Tan, M.; Lu, Z. Theranostics 2011, 1, 83-101. (27) Shapiro, E. M.; Koretsky, A. P. Magn. Reson. Med. 2008, 60, 265-269. (28) Wang, X.; Li, Y. D. Mater. Chem. Phys. 2003, 82, 419-422. (29) Wisner, E. R.; Merisko-Liversidge, E.; Kellar, K.; Katzberg, R. W.; Karpinski, P. H.; Amparo, E. G.; Drake, C.; Griffey, S. M.; Brock, J. M. Acad Radiol. 1995, 2, 140-147. (30) Chen, N.; Shao, C.; Qu, Y.; Li, S.; Gu, W.; Zheng, T.; Ye, L.; Yu, C. ACS Appl. Mater. Interfaces 2014, 6, 19850-19857. (31) Qi, Y.; Shao, C.; Gu, W.; Li, F.; Deng, Y.; Li, H.; Ye, L. J. Mater. Chem. B 2013, 1, 1846-1851. (32) Kievit, F. M.; Zhang, M. Adv.Mater. 2011, 23, H217-H247. (33) Deng, Y.; Wang, H.; Gu, W.; Li, S.; Xiao, N.; Shao, C.; Xu, Q.; Ye, L. J. Mater. Chem. B 2014, 2, 1521-1529. (34) Cui, S.; Yin, D.; Chen, Y.; Di, Y.; Chen, H.; Ma, Y.; Achilefu, S.; Gu, Y. ACS Nano 2013, 7, 676-688. 17



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(35) Dixit, S.; Das, M.; Alwarappan, S.; Goicochea, N. L.; Howell, M.; Mohapatra, S.; Mohapatra, S. RSC Adv. 2013, 3, 2727-2735. (36) Caravan, P.; Farrar, C. T.; Frullano, L.; Uppal, R. Contrast Media Mol. Imaging 2009, 4, 89-100.

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FIGURE CAPTIONS Scheme 1. Schematic illustration of the synthesis of multifunctional Gd/MnCO3 NPs. Figure 1. TEM (A), HRTEM (B), SAED (C), XRD (D), EDS (E), and field-dependent magnetization curve (F) of Gd/MnCO3-OA. Inset in (F) shows an enlarged view of the hysteresis loops at 5 and 300 K. Figure 2. (A) FTIR spectra of oleate-capped, TETT-modified, non-targeted, and targeted Gd/MnCO3 NPs. (B) UV-vis (solid lines) and fluorescence (broken lines) spectra of the FA-PEG segment, free Cy5.5 dye, and non-targeted and targeted Gd/MnCO3 NPs. (C) Digital images of TETT-modified, non-targeted, and targeted Gd/MnCO3 NPs dispersed in water and 50% fetal bovine serum. Figure 3. r1 relaxivities (A) and T1-weighted MR images (B) of TETT-modified, non-targeted, and targeted Gd/MnCO3 NPs. Figure 4. MR images of tiny gliomas in mice before and at different time points after intravenous injection of non-targeted (upper panel) or targeted (lower panel) Gd/MnCO3 NPs. The quantitative analysis of CNR is shown in the inset.

Figure 5. In vivo near infrared fluorescence images of tiny gliomas in mice before (A) and 24 h after (B) injection of non-targeted (upper panel) or targeted (lower panel) Gd/MnCO3 NPs. (C) ex vivo fluorescence images of the excised brain harboring gliomas. Brain sections treated with non-targeted (D) or targeted (E) Gd/MnCO3 NPs were imaged by confocal microscopy. Note the GFP signal distributed throughout the tumor tissue.

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Scheme 1

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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For TOC only

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Multifunctional Gadolinium-Doped Manganese Carbonate

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