Carbon Quantum Dot Stabilized Gadolinium Nanoprobe Prepared via

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Carbon Quantum Dot Stabilized Gadolinium Nanoprobe Prepared via a One-Pot Hydrothermal Approach for Magnetic Resonance and Fluorescence Dual-Modality Bioimaging Yang Xu,† Xiao-Hua Jia,‡ Xue-Bo Yin,*,† Xi-Wen He,† and Yu-Kui Zhang†,§ †

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 ‡ Key laboratory of Molecular Imaging, Institute of Automation, Chinese Academy of Sciences, Beijing, 100190, China § Dalian Institute of Chemical Physics, National Chromatographic R&A Center, Chinese Academy of Sciences, Dalian, 116011, China S Supporting Information *

ABSTRACT: Magnetic resonance imaging (MRI) is used extensively for clinical diagnoses. It is critical to design and develop highly efficient MR contrast agents with simple preparation procedure, low toxicity, and high biocompatibility. Here, we report a carbon quantum dots (CQDs)-stabilized gadolinium hybrid nanoprobe (Gd−CQDs) prepared via a one-pot hydrothermal treatment of the mixture of citrate acid, ethanediamine, and GdCl3 at 200 °C for 4 h. In vitro and in vivo tests confirmed their low toxicity and high biocompatibility. Gd−CQDs were observed to have a higher MR response than gadopentetic acid dimeglumine (Gd−DTPA) because of their high Gd content and hydrophilicity. Moreover, the fluorescence of CQDs was remained in Gd−CQDs. The in vivo MR and fluorescence dual-modality imaging of Gd−CQDs was confirmed with zebrafish embryo and mice as models. The modification of Gd−CQDs with arginine-glycine-aspartic acid (RGD) tripeptide provided a high affinity to U87 cancer cells for targeted imaging. Whereas the MR response showed a depth penetration and spatial visualization, fluorescence revealed the fine distribution of Gd−CQDs in tissues because of its high resolution and sensitivity. We found that Gd−CQDs distributed in the tissues in a heterogeneous mode: they entered into the tissue cells but were observed less in the extracellular matrix. The MR and fluorescence dual-modality imaging of Gd−CQDs makes them a potential contrast agent for clinic applications because of their simple preparation procedure, ease of functionalization, high contrast efficiency, low toxicity, and high biocompatibility. biocompatibility is expected. Although Au−CNMs and Fe3O4− CNMs hybrid materials have been widely reported,30−33 few examples of Gd 3+ −CNMs hybrid materials have been reported.34,35 Huang et al. prepared a Gd2O3/C core−shell material by carbonizing a mixture of Gd and citrate acid at high temperature.34 A carbon nanoshell stably covered on the Gd2O3 spheres and endowed the hybrid materials with near-IR absorbance properties. Nonetheless, carbonization decreased the hydrophilicity of the material, making further modification of the probe difficult.34 Herein, we report a carbon quantum dots (CQDs)-stabilized Gd3+ hybrid nanomaterial (Gd−CQDs) for MR and fluorescence dual-modality applications. Compared with the carbonization at high temperatures,34 our one-pot hydrothermal treatment of citrate acid, ethanediamine, and GdCl3 at 200 °C is much simpler. In vitro and in vivo tests confirmed the low toxicity and high biocompatibility of Gd−CQDs because of the stable coordination between Gd3+ ions and CQDs. Gd−CQDs

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agnetic resonance imaging (MRI) has been widely used as a noninvasive imaging technology with high spatial resolution in clinical diagnoses.1,2 Gadolinium (Gd) is confirmed to exhibit excellent contrast efficiency because of its unique magnetic property, but gadolinium ions (Gd3+) are highly toxic because they inhibit calcium channels, induce change in intracellular reactive oxygen species (ROS) levels, and cause cardiovascular and neurologic toxicity.3−8 Preventing leakage of Gd3+ is the key for Gd-based MR contrast agents.7,9,10 Gdcontaining nanoparticles and chelates have been developed as effective probes for MR diagnosis.2,11−19 Some Gd chelates are commercially available, but they are expensive and their contrast efficiency needs to be improved.10 Therefore, one challenge for MRI contrast agents is the design of Gd-containing agents with high contrast efficiency, high stability, low toxicity, and simple preparation procedure.10,20 Carbon nanomaterials (CNMs) have been explored as biosensing and imaging probes and as drug carriers because of their high mechanical strength, good optical properties, high biocompatibility, and low toxicity.21−29 If a Gd3+−CNM composite can be prepared via a simple procedure, a viable MRI contrast agent with improved MR efficiency and high © XXXX American Chemical Society

Received: August 11, 2014 Accepted: November 10, 2014

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dialyzed against water for 1 day. The solid product was collected after freeze-drying and could be dissolved again for further applications. Bioconjugation of Gd−CQDs with RGD. RGD tripeptide was conjugated with Gd−CQDs in the presence of coupling agents (SA, DDC, and NHS). Briefly, 10 mg of Gd−CQDs was added to 10 mL of 4.0 mg mL−1 SA−DMF solution, followed by refluxing for 3 h. Gd−CQDs/SA was collected by precipitation with ether.37 Then, the as-prepared Gd−CQDs/SA was dissolved again into DMF. The coupling agents (60 mg of DDC and 11.5 mg of NHS) were added to the mixture to activate Gd−CQDs, and the mixture was stirred at 30 °C overnight. Then, 6 mg of RGD was added to the activated Gd−CQDs solution (pH = 8.5) in an ice bath and reacted for 20 min. The product (Gd−CQDs−RGD) was collected by centrifugation and washed with ethanol and ultrapure water for four times.38 Quantum Yields of Gd−CQDs. The quantum yields of Gd−CQDs and Gd−CQDs−RGD were calculated by a slope method with quinine sulfate in 0.1 M H2SO4 as a reference. The quantum yield was calculated with the following equation:

were observed to have a higher MR response than a commercially available contrast agent, Gd−DTPA. Interestingly, the fluorescence of CQDs was remained. Therefore, fluorescence− magnetic resonance dual responses were observed from the hybrid nanomaterial and confirmed by the use of zebrafish embryos and mice as models. Whereas a depth penetration and spatial visualization is achieved with their MR response, the fine distribution of the Gd−CQDs in tissues was revealed by the high resolution and sensitivity of fluorescence. Tumor-targeted imaging was demonstrated with arginine-glycine-aspartic acid tripeptide-functionalized Gd−CQDs, which crossed the blood− brain barrier and showed high affinity toward target cancer tissue. Gd−CQDs not only provides a nontoxic Gd-based MR contrast agent with simple preparation procedure, ease of functionalization, high contrast efficiency, and high biocompatibility, but also promotes the extensive application of CQDs.

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EXPERIMENTAL SECTION Materials and Instrumentation. GdCl3 was prepared from Gd2O3 (Aladdin, Shanghai, China) after acidification with excess HCl. Gadopentetic acid dimeglumine salt injection (0.5 M Gd− DTPA, Magnevist) was obtained from BayerSchering Pharma AG, Berlin, Germany. Arginine-glycine-aspartic acid (RGD) tripeptide was obtained from China Peptide Co., Ltd., Shanghai, China. Succinic anhydride (SA) was purchased from J&K Co., Beijing, China. N,N′-Dicyclohexyl-carbodiimide (DDC) was obtained from Medpep. Co. Ltd., Shanghai, China. NHydroxysul-fosuccinimide sodium salt (NHS) was bought from Sigma, Shanghai, China. Ether, ethanol, and dimethylformamide (DMF) were obtained from Tianjin chemical reagent Co., Tianjin, China. All of the reagents were used without any purification. Ultrapure water was prepared with an Aquapro system (18.25 MΩ). Transmission electron microscopy (TEM) images and energydispersive X-ray spectroscopy (EDX) chemical mapping were recorded with a Tecnai G2 F20, (FEI Co. U.S.A.) operated at an accelerating voltage of 200 kV. The hydrodynamic size and ζpotentials of Gd−CQDs were tested at 25 °C and recorded with a Zetasizer Nano ZS (Malvern, British) with a 633 nm He−Ne laser. The UV−vis absorption spectrum was recorded by a UV2450-visible spectrophotometer (Shimadzu, Japan). The fluorescent spectrum was obtained with a Hitachi FL-4500 fluorescence spectrometer. The infrared spectrum was measured with a Bruker TENSOR 27 Fourier transform infrared spectrometer. The content of Gd was measured by inductively coupled plasma atomic emission spectroscopy (ICP-AES), IRIS advantage, Thermo, U.S.A. X-ray photoelectron spectroscopy (XPS) analysis was performed by a Kratos Axis Ultra DLD spectrometer fitted with a monochromated Al Kα X-ray source (hν 1486.6 eV), hybrid (magnetic/electrostatic) optics, a multichannel plate, and delay line detector. X-ray diffraction (XRD) patterns were recorded by a D/max-2500 diffractometer (Rigaku, Japan) using Cu Kα radiation (λ = 1.5418 Å). Fluorescence images were acquired by a confocal microscope (Leica Tcs sp5, Germany). The MR images were conducted using a spectroscope (1.2 T, Huantong, Shanghai, China). Preparation of Gd−CQDs. Gd−CQDs were synthesized according to previous a CQDs method with fine modification.36 Briefly, 0.42 g of citrate acid, 0.5 mL of ethanediamine, and 0.1 g of GdCl3 were dissolved into 10 mL of water with ultrasonic mixing for 10 min. The homogeneous solution was transferred into poly(tetrafluoroethylene) (Teflon)-lined autoclave (30 mL). After being heated at 200 °C for 4 h, the solution was

Φx = Φst(Kx /K st)(ηx /ηst)2

where Φ is quantum yield, K is the slope of curves, and η is the refractive index. The subscript “st” refers to the referenced fluorophore (quinine sulfate in 0.1 M H2SO4) with known quantum yield and “x” refers as the samples (Gd−CQDs and Gd−CQDs−RGD in this work) for the determination of quantum yield. In order to minimize reabsorption, absorption was kept below 0.05 at the excitation wavelength of 360 nm. In Vitro Cytotoxicity of Gd−CQDs. The cell viability of Gd−CQDs was tested on the HepG2 cell line, using CCK8 assay. Briefly, the cells were incubated to 96-well culture plates at a density of 5 × 105 cells per well in culture medium. Gd−CQDs, Gd−DTPA, Gd2O3, and GdCl3 at the concentration of 0.2 mM were introduced to the medium for incubation of 24 h after HepG2 cells reached 90−95% confluences. An amount of 10 μL of CCK-8 was added to each well, and then the cells were incubated for 1 h. All of the incubation process was carried out at 37 °C with 5% CO2. The absorbance at 490 nm was used to calculate the cell survival rate. The experiment was performed under identical conditions for six times. In Vitro MR Imaging with Gd−CQDs as a Probe. In vitro MR imaging of Gd−CQDs with different concentrations (0, 0.2, 0.4, 0.6, 0.8, and 1.0 mM) was carried out with a 1.2 T MR imaging system (Huantong, Shanghai, China). As a comparison, Gd−DTPA was tested with the same procedure. Images were obtained using a 50 mm animal coil, using a fat-saturated 3D gradient echo imaging sequence. The MR imaging parameters were described as follows: spin−echo T1-weighted MRI sequence, TR/TE = 100.0/8.8 ms, FOV = 100 × 50 mm2, matrix = 256 × 256, slice thickness = 1 mm, 30.0 °C. Animal Models. The embryos and larva of AB strain zebrafish were obtained from Key Laboratory of Animal Models and Degenerative Neurological Diseases, Nankai University. Zebrafish were incubated in aquaria at 28.5 °C. Embryos were collected after natural spawns. All embryos and zebrafish were grown in 0.003% 1-phenyl-2-thiourea (PTU, Sigma-Aldrich, St. Louis, MO, U.S.A.) to block pigmentation and mediate visualization. Nude mice (18−25 g) were purchased from Beijing HFK Bioscience Co., Ltd., Beijing, China. The subcutaneous U87-MG model nude mice were obtained from Key Laboratory of Molecular Imaging, Chinese Academy of B

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Scheme 1. Schematic Illustration for the One-Pot Synthesis of Gd−CQDs as a Dual-Modal Imaging Probe

(PBST). After being incubated with 20% normal sheep serum (NSS) in PBST, the sections were incubated overnight at 4 °C in rabbit anti-integrin β3 antibody (1:500; Abcam, U.S.A.) diluted in 2% NSS-PBST. After washing with PBST, the sections were incubated for 1.5 h at room temperature in PBST containing 2% NSS and Cy 3-conjugated secondary antibody (Millipore, Billerica, MA). The slides were observed under a fluorescence microscope (Olympus BX51, Japan) with an excitation of 470− 490 nm and emission at 515 nm long pass.

Science, Beijing, China. All animal procedures were in agreement with the guidelines of the Institutional Animal Care Committee of Nankai University. In Vivo Toxicity of Gd−CQDs in Mice. In vivo toxicity of Gd−CQDs was tested with mice (18−25 g, n = 3 per group). Typically, Gd−CQDs solution (20 μmol Gd per kg) was injected by tail vein into the mice, which were anesthetized with 4% chloral hydrate (6 mL kg−1). Hematoxylin and eosin (H&E) stained images were used to investigate the difference between experimental group (injected with Gd−CQDs) and control group (injected with normal saline). The body weight of the mice was assessed with a counter balance during 28 days. In Vivo Fluorescence and MR Dual-Modality Images of Zebrafish Embryos with Gd−CQDs as Probe. AB strain fertilized embryos were added into 24-well culture plates (5−7 zebrafish per well) with regular tank water at 28.5 °C. Gd−CQDs were added to the wells with different concentrations: 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mg mL−1. After being cultured for 4 h, embryos were rinsed with tank water three times to remove excess Gd− CQDs, followed by imaging with a fluorescence microscope (Olympus BX51, Japan) with an excitation of 470−490 nm and emission at 515 nm long pass. The MR imaging was carried out with embryos treated with different concentrations of Gd− CQDs on a 1.2 T MRI System with a 50 mm animal coil, using a fat-saturated 3D gradient echo imaging sequence. The cuvette was 2 mL of pointed centrifuge tube. Biodistribution of Gd−CQDs in Mice and TumorTargeted Imaging. Gd−CQDs solution (20 μmol Gd per kg) was injected via tail vein into the mice anesthetized with 4% chloral hydrate. MR imaging was performed on a 1.2 T MRI system with a 50 mm animal coil, using a fat-saturated 3D gradient echo imaging sequence. The parameters were described as follows: spin−echo T1-weighted MRI sequence, TR/TE = 100.0/8.8 ms, FOV = 100 × 50 mm2, matrix = 256 × 256, slice thickness = 1 mm, 30.0 °C. To evaluate the targeting ability of Gd−CQDs−RGD toward U87 tumor, U87 tumor-bearing mice were injected with Gd− CQDs or Gd−CQDs−RGD solution via tail vein (20 μmol Gd per kg). The biodistribution of Gd−CQDs and Gd−CQDs− RGD was tested after the mice were sacrificed 0.5 h post administration, and the organs and tumors were collected, followed by frozen sections (n = 3). The immunohistochemistry was performed to illustrate the efficiency of Gd−CQDs−RGD.39 Briefly, the sections were rinsed in 0.1 M phosphate-buffered saline and 0.5% Triton X-100

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RESULTS AND DISCUSSION

Synthesis and Characterization of Gd−CQDs Hybrid Material. Various carbon-stabilized metal nanoparticles have been prepared using molecular organic precursors.30−34,40 Similarly, we attempted to synthesize carbon-passivated gadolinium nanoparticles as an MRI contrast agent via one-pot hydrothermal treatment of the mixture of ethanediamine, citrate acid, and GdCl3 because citrate acid is easily polymerized in the presence of ethanediamine.36 A transparent yellow solution was obtained. After being subjected to dialysis and lyophilization, the solid product can be dispersed again in aqueous solution (Supporting Information Figure S1, parts A and B). With the same purification procedure, the content of as-prepared yellowish product was larger than that of carbon quantum dots (CQDs) because of the introduction of Gd (Supporting Information Figure S1, parts C and D). Different from carbonpassivated metal nanoparticles, the product was found to be CQDs-stabilized gadolinium ions (Gd3+−CQDs) as confirmed by subsequent analysis. The preparation and dual-modality application of Gd−CQDs are illustrated in Scheme 1. Transmission electron microscopy (TEM) images showed that the hybrid material was well-monodispersed with a flowerlike structure, and the average diameter was 47 nm (Figure 1, parts A and B). High-resolution TEM image showed that the hybrid material contained small dots with the diameters ranging from 2 to 8 nm and a lattice spacing of 0.25 nm, which corresponds to the (100) facet of graphite.41 The small dots exhibit graphitic properties and fluorescence as will be discussed later, similar to CQDs. Energy-dispersive X-ray spectroscopy chemical mapping showed characteristic L and M emission signals of Gd and a homogeneous distribution of Gd dispersed in the hybrid material (Figure 1D). XPS results confirmed the presence of Gd in the product with 37.99% C, 24.47% O, 3.84% N, and 33.70% Gd (Supporting Information Figure S3). No Cl ions were detected by XPS analysis, indicating that unreacted C

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different from those of Gd2O3 (141.7 eV) (Supporting Information Figure S3, parts C and D). Thus, Gd existed in the form of Gd3+ and coordinated with CQDs.35,42 The XRD patterns of Gd−CQDs did not exhibit well-defined diffraction peaks, ascribable to an amorphous carbon structure (Supporting Information Figure S4).43 Compared with the XRD pattern of GdCl3, no diffraction peaks that corresponded to free Gd3+ were observed, suggesting that Gd3+ was stably chelated with the CQDs (Supporting Information Figure S4) and the coordination between Gd3+ and CQDs led to the formation of Gd−CQDs. All of the results confirmed that the hybrid material was CQD-passivated Gd3+ (Gd−CQDs) through the coordination between Gd3+ and the surface groups on CQDs. Formation of Gd−CQDs. To demonstrate the formation process of Gd−CQDs, the size, TEM images, and MR response of the products at different incubation times were tested. The size of the products increased dramatically from 4 nm at 1 h to approximately 47 nm at 4 h and then aggregated at reaction times in excess of 5 h (Figure 2). Similarly, the T1-weighted MR response of the products had a time-dependent behavior (Supporting Information Figure S5). A weak MR signal was observed from the product less than 2 h. We speculated that the size of Gd−CQDs was too small, whereas Gd ions were removed by dialysis as shown in Figure 2A. The longitudinal relaxation rate (R1) increased as the reaction time increased from 2 to 4 h (Supporting Information Figure S5A). After 4 h, the growth of regular Gd−CQDs was uncontrollable, and a portion of Gd precipitated to diminish the MR signal. Therefore, the formation of Gd−CQDs was divided into three stages and reflected the dramatic process of nucleation, growth, and aggregation. Before the hydrothermal procedure, Gd3+ was stabilized by citrate acid and ethanediamine to form Gd3+ complex; therefore, no precipitation was observed. In stage 1, the tiny carbon nuclei emerge through the polymerization between citrate acid and ethanediamine to form CQDs (Figure 2A), whereas some of the Gd3+ ions coordinate with the oxygenand nitrogen-containing groups on the surface of CQDs.36 In stage 2, with the increased amount of CQDs, Gd3+ is chelated with oxygen and nitrogen on the surface of neighboring CQDs to

Figure 1. (A) TEM image of Gd−CQDs after hydrothermal reaction for 4 h. Inset: size distribution of Gd−CQDs obtained from TEM images. (B) A higher-magnification TEM image of a Gd−CQD. (C) Highresolution TEM images of Gd−CQDs with the amplified lattice spacing of CQDs in Gd−CQDs (the white round frames show tiny CQDs in the Gd−CQDs with a lattice spacing of 0.25 nm). (D) EDX chemical mapping of Gd−CQDs on a TEM copper grid. Inset: an image of Gd− CQDs captured with a CCD camera during EDX analysis; the images of the characteristic L and M emission signals of Gd in Gd−CQDs.

GdCl3 was removed completely. The Gd 4d peak was deconvoluted into two peaks centered at 142.2 and 147.1 eV,

Figure 2. TEM images of the hydrothermal products after different incubation times: (A) 1, (B) 3, and (C) 5 h, and schematic illustration of the process of Gd−CQD formation. D

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with CQDs, similar to the case of the Gd3+−PEI complex.7 Moreover, the Gd centers in Gd−CQDs were easily accessed by water molecules because oxygen exists in the form of carboxyl and hydroxyl groups. High oxygen content (24.47%, data from XPS) contributes to the high hydrophilicity and solubility of Gd−CQDs (Supporting Information Figure S1A). High hydrophilicity and Gd content achieve the high T1-weighted MR performance of Gd−CQDs. UV−Visible and Fluorescence Spectra of Gd−CQDs. UV−vis and fluorescence spectra illustrated the optical properties of Gd−CQDs (Figure 4). Gd−CQDs have the similar UV−

form the Gd−CQDs hybrid material (Figure 2B).10 Correspondingly, the nanoparticles grow from 4 to 47 nm with the incubation time increasing from 1 to 4 h (Figure 1A and Figure 2), accompanied by the formation of Gd3+−CQDs and an enhancement of the T1-weighted MR signal.44,45 In stage 3, the insoluble substance becomes visible after hydrothermal reaction beyond 4 h because of the carbonization and aggregation (Figure 2C). However, the direct reaction between Gd3+ and CQDs to form a monodisperse Gd−CQD complex is difficult (Supporting Information Figure S6). Thus, Gd3+ ions are transferred from the Gd3+−citrate acid/ethanediamine complex to Gd−CQDs for the effective formation of Gd−CQDs hybrid material during the hydrothermal procedure. The MR response of the products was used to optimize the amount of ethanediamine for the preparation of Gd−CQDs. The products exhibited the highest R1 intensity when 0.5 mL of ethanediamine was used (Supporting Information Figure S5B), whereas precipitation was observed when the ethanediamine content was less than 0.25 mL (Supporting Information Figure S7), indicating that ethanediamine is critical to stabilize the Gd3+ ions. The reaction time was tested again with 0.5 mL of ethanediamine, and the MR signal still exhibited the highest intensity at 4 h. Therefore, Gd−CQDs were prepared using the optimal ratio of 0.42 g citrate acid/0.1 g GdCl3/0.5 mL ethanediamine in 10 mL of water with an incubation time of 4 h at 200 °C in subsequent experiments. MR Property of Gd−CQDs. The longitudinal (r1) and transverse relaxivity (r2) of the Gd−CQDs prepared under optimal conditions was measured at 1.2 T and 30 °C. Commercially available MRI agent, Gd−DTPA, was tested to illustrate the MR performance of Gd−CQDs. The r1 and r2 relaxivity curves showed an apparent enhancement of relaxation with the concentration of Gd3+ increased (Figure 3 and

Figure 4. (A) UV−vis absorption spectra of Gd−CQDs and CQDs. (B) Fluorescence spectra of Gd−CQDs with different excitation wavelengths. (C) The bright-field and fluorescence images of Gd−CQDs at different Gd concentrations (0, 0.2, 0.4, 0.6, 0.8, 1.0 mM). (D) The fluorescence spectra of Gd−CQDs and CQDs excited at 360 nm.

vis absorption spectra to CQDs prepared from the hydrothermal reaction between citrate acid and ethanediamine (Figure 4A). The peak at 350 nm is the characteristic absorption of the CQDs.36,46 The enhanced absorption peak at 290 nm in the spectrum of Gd−CQDs confirmed the strong interaction between Gd3+ and CQDs via n−π* transition (Figure 4A) because Gd3+ disturbed the energy band of the CQDs.33,47 An excitation-dependent fluorescence was observed from Gd− CQDs with emission wavelength ranging from 470 to 525 nm under 340−460 nm excitation, the same as that from CQDs (Figure 4B and Supporting Information Figure S9). A concentration-dependent bright-blue emission was observed under 365 nm excitation (Figure 4C).36 No apparent wavelength shift between Gd−CQDs and CQDs indicated that the emission properties of CQDs were remained in the hybrid material (Figure 4D). Gd−CQDs have a quantum yield of 0.21, showing their high fluorescence efficiency (Supporting Information Figure S10 and Table S1). Compared with the original CQDs, Gd3+ disrupted the radiative recombination of the CQDs surface, to decrease their fluorescence efficiency (Supporting Information Figure S10 and Table S1), which also confirmed the formation of hybrid material by the chelation between Gd3+ and CQDs.48,49 Cytotoxicity and Leakage of Gd−CQDs. The toxicity of Gd−CQDs is a major concern for bioimaging. Comparative cell viability was tested with HepG2 cells as a model using the CCK8 assay (Figure 5A). GdCl3 exhibits the highest toxicity because

Figure 3. (A) The r1 relaxivity curve of Gd−CQDs and Gd−DTPA. (B) In vitro MR images of (a) Gd−CQDs and (b) Gd−DTPA prepared using a series of Gd concentrations: 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mM.

Supporting Information Figure S8). The r1 relaxivity of Gd− CQDs was 7.36 mM−1 s−1, higher than that from Gd−DTPA (4.23 mM−1 s−1) under the same tested conditions (Figure 3A). In vitro MR images further demonstrated the excellent MR performance of Gd−CQDs (Figure 3B), where Gd−CQDs show greater brightness than Gd−DTPA at their same Gd concentration. The Gd content in Gd−CQDs determined using HNO3 digestion and ICP-AES was 34.6 ± 4.7%. Approximately 33% of total Gd3+ ions fed into the reaction were coordinated E

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whereas no MR response was observed from the control group (Figure 6B). Cross-validation between fluorescence and MR results confirmed that Gd−CQDs were embedded into the embryos and the fluorescence and MR efficiency of Gd−CQDs remained. Thus, Gd−CQDs have the potential as in vivo MR and fluorescence dual-response probes. In Vivo Fluorescence−MR Imaging and Distribution of Gd−CQDs in Mice. The Gd−CQDs distribution in tissue is vital to evaluate their imaging efficiency and side effects.54 In vivo MR imaging and the tissue distribution of Gd−CQDs were tested with mice as model. Gd−CQDs entered the blood circulation of mice immediately after injection of Gd−CQDs (Figure 7, parts A and B). The liver was highly visualized by the

Figure 5. (A) Viability of HepG2 cells incubated with Gd−CQDs, Gd− DTPA, GdCl3, and Gd2O3 (0.2 mM Gd). The control was incubated with PBS solution. The error bars indicate the standard deviations for six independent experiments. (B) Released free Gd3+ from Gd−CQDs incubated in serum at 37 °C. The error bars indicate the standard deviations for three independent tests.

Gd3+ has the same biophysical properties to Ca2+ and can replace Ca2+ in tissue.4,5,8 Gd3+ interferes with intracellular calciumdependent stretch-activated channels and induces cell apoptosis.4,5,8 Figure 5A shows that 90% cell viability was observed after the cells were treated with Gd−DTPA. Gd−CQDs exhibited the lowest cytotoxicity toward HepG2 cells among the species investigated under the same conditions. The cell viability of 96% indicated that Gd3+ was trapped and passivated stably by the nontoxic CQDs. To verify this hypothesis, the leakage of free Gd3+ was tested after Gd−CQDs were incubated in serum at 37 °C for 0−2 weeks. The samples were centrifuged to collect the supernatant, and the Gd3+ released into the supernatant was detected by ICP-AES. The results in Figure 5B indicate a small amount of Gd3+ in the supernatant probably due to incomplete centrifugation or by the release of weakly bound Gd3+ in Gd− CQDs,7 because Gd leakage did not increase even after Gd− CQDs were incubated in serum for 2 weeks. CQD chelation is effective in minimizing the leakage of Gd3+, so Gd−CQDs are stable sufficiently to be used as probes because of their low toxicity. In Vivo Fluorescent and MR Imaging of Zebrafish Embryos with Gd−CQDs as Probe. Zebrafish have been widely used to investigate pattern formation, developmental mechanism, and disease progression because of their welldefined developmental stages and amenability to optical imaging.50−53 Zebrafish embryos were first used to confirm the fluorescence and MR dual response of Gd−CQDs. After the embryos were incubated with Gd−CQDs, their yolk sac became brighter in a concentration-dependent manner (Figure 6A), indicating that Gd−CQDs crossed the chorion of embryos and deposited primarily at their yolk. Similarly, the MR images were gradually brightened with increased Gd−CQD concentrations,

Figure 7. In vivo MR images of mice (A) before and (B) after tail-vein injection of Gd−CQDs (20 μmol Gd3+ per kg). (C) The fluorescence images of the liver slices of (top) the mice injected with saline solution and (bottom) the mice injected with Gd−CQDs. (D) The quantification of Gd3+ content in heart, spleen, lung, liver, and kidney after the mice were injected with Gd−CQDs (n = 3).

excellent contrast MR signal and was substantially distinguished from other organs after 0.5 h post injection. To obtain the accurate information on Gd−CQDs in the tissue,38 fluorescence imaging assays were performed. Green fluorescence was observed from Gd−CQDs in liver slices and illustrated the distribution of Gd−CQDs clearly in a heterogeneous mode (Figure 7C): they can enter into the liver cells but cannot be observed in the extracellular matrix as illustrated by the highresolution of fluorescence images. The distribution of Gd−CQDs in heart, spleen, lung, liver, and kidney was quantified by ICP-AES after the organs were removed from the mice at 0.5, 1, 4, 12, and 24 h after the mice were injected with Gd−CQDs. Amounts of 52% and 29.6% of injected dose (ID) per gram tissue of the Gd−CQDs accumulated in the liver and spleen at 1 h post injection, respectively, whereas low content was observed in the kidney; this result is consistent with the fluorescence images in Figure 7C. After 1 h, the content of Gd−CQDs were decreased in the liver and spleen (Figure 7D). Assessment of Biotoxicity of Gd−CQDs. The toxicity of Gd−CQDs was assessed via the histological changes of different organs (heart, liver, spleen, kidney, and intestine) after staining with H&E 1 week post intravenous injection of Gd−CQDs. No difference was observed between the control and experiment groups, indicating that no tissue damage derived from the administration of Gd−CQDs (Figure 8A). The weight change of the mice was used to evaluate the long-term in vivo toxicity of

Figure 6. (A) Fluorescence and (B) MR images of zebrafish embryos incubated with different concentrations of Gd−CQDs. Scale bar: 250 μm. F

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Figure 8. (A) Histological changes of the heart, liver, kidney, spleen, and intestine of mice (7 days) after being injected with (top) saline solution and (bottom) Gd−CQDs (20 μmol Gd3+ per kg). (B) The body weight changes of the mice injected with Gd−CQDs (20 μmol Gd3+ per kg) and the control injected with saline solution (n = 3).

Gd−CQDs. None of the mice treated with Gd−CQDs died, and the weight of the mice kept increased 4 weeks post injection (Figure 8B).The almost same weight trend was observed between the Gd−CQDs treated and untreated groups. Thus, Gd−CQDs did not cause pathological damage to the tissues and inhibit the growth of mice. In Vivo Tumor-Targeted Imaging with Functionalized Gd−CQDs. To demonstrate the in vivo tumor-targeted imaging potential of Gd−CQDs, a targetable tripeptide, RGD, was integrated into Gd−CQDs (Gd−CQDs−RGD) to image integrin ανβ3-positive U87 tumors. Gd−CQDs−RGD have a little bigger hydrodynamic diameter and a lower quantum yield than Gd−CQDs (Supporting Information Figures S12 and S13). Gd−CQDs and Gd−CQDs−RGD were injected into U87 tumor-bearing mice to illustrate the targeted imaging of Gd− CQDs−RGD. Gd−CQDs−RGD was found to be easily transported to the targeted tumor tissue 0.5 h post injection and to brighten the tumor tissue such that it was easily distinguished from surrounding tissues in the MR images (Figure 9, parts A and B). In contrast, few MR-enhanced signals were observed at tumor tissue in Gd−CQDs group (Supporting Information Figure S14). Similarly, green fluorescence was observed in tumor slices fluorescence images after the mice were injected with Gd−CQDs−RGD (Figure 9C). To further confirm that Gd−CQDs−RGD bound to the tumor, immunofluorescent studies were performed by using integrin β3 staining (Supporting Information Figure S15). A good correlation between Gd− CQDs−RGD and positive integrin β3 staining was observed from the merge map, indicating the position of Gd−CQDs− RGD on the tumor section (Supporting Information Figure S15). In combination with the cross-validated results from MR and fluorescence imaging, Gd−CQDs−RGD was confirmed to cross the blood−brain barrier (BBB) and to reach the tumor tissue in the head of the mice. Because no apparent MR contrast enhancement was observed in the other parts of the head, Gd− CQDs−RGD indeed exhibited a high affinity toward U87 tumor. Additionally, the accumulation of Gd in the tumor was in the range of 9−14% ID/gram tissue during 24 h post injection, and reached a peak at 4 h post injection (Figure 9D). Thus, Gd−

Figure 9. In vivo MR images of mice (A) before and (B) after intravenous injection of Gd−CQDs−RGD (20 μmol Gd3+ per kg). (C) The fluorescence images of the tumor slices of (top) the mice injected with normal saline and (bottom) those injected with Gd−CQDs. (D) The quantification of the Gd3+ content in major organs (tumor, heart, spleen, lung, liver, and kidney) after the mice were injected with Gd− CQDs−RGD (n = 3).

CQDs−RGD has the potential for diagnostic application by its high biocompatibility, good stability in serum, and ease of functionalization for target affinity.

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CONCLUSION In summary, a simple one-pot synthesis method was proposed to prepare the biocompatible CQD-stabilized Gd3+ (Gd−CQDs) hybrid nanomaterial with fluorescence and MR dual response. While CQDs passivated Gd3+ ions to decrease their leakage and toxicity, the fluorescence of CQDs was remained. The strong coordination between the Gd3+ and CQD led to a stable structure of Gd−CQDs. The dual response was confirmed with zebrafish embryo and mice as models. After being functionalized with RGD tripeptide, the hybrid material was used for the in vivo U87 tumor-targeted imaging. Whereas the MR response indicates a depth penetration and spatial visualization, fluorescence images revealed the fine distribution of the probes in tissue because of its high sensitivity. The simple preparation procedure, ease of functionalization, high stability, and low toxicity of Gd−CQDs could promote the development of further applications of this composite material.

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ASSOCIATED CONTENT

S Supporting Information *

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

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: +86-22-23503034. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Basic Research Program of China (973 Program, No. 2011CB707703), Natural Science Foundation of China (Nos. 21435001 and 21375064), and Research Fund for the Doctoral Program of Higher Education (No. 20130031110016).

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dx.doi.org/10.1021/ac503002c | Anal. Chem. XXXX, XXX, XXX−XXX