ZnS Quantum Dots Conjugating Gd(III) Chelates for Near

Jun 28, 2017 - The longitudinal relaxivity (r1) of the resulted QDs@DTDTPA-Gd nanoparticles ... (20-23) Directly doping Gd ions could minimize the siz...
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CuInS2/ZnS Quantum Dots Conjugating Gd(III) Chelates for NearInfrared Fluorescence and Magnetic Resonance Bimodal Imaging Yongbo Yang,†,§ Li Lin,† Lijia Jing,† Xiuli Yue,*,† and Zhifei Dai*,‡ †

School of Municipal and Environmental Engineering, Harbin Institute of Technology, Harbin 150080, China Department of Biomedical Engineering, College of Engineering, Peking University, Beijing 100871, China § State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China ‡

S Supporting Information *

ABSTRACT: A bimodal contrast nanoagent was developed by chelating gadolinium ions to 2-[bis[2-[carboxymethyl-[2-oxo-2-(2sulfanylethyl-amino)ethyl]amino]ethyl]amino]acetic acid (DTDTPA)-modified CuInS2/ZnS quantum dots (QDs). The longitudinal relaxivity (r1) of the resulted QDs@DTDTPA-Gd nanoparticles (NPs) was calculated to be 9.91 mM−1 s−1, which was 2.5 times as high as that of clinically approved Gd-DTPA (3.9 mM−1 s−1). In addition, the in vivo imaging experiments showed that QDs@DTDTPA-Gd NPs could enhance both near-infrared fluorescence and T1-weighted magnetic resonance (MR) imaging of tumor tissue through passive targeting accumulation. Moreover, the high colloidal and fluorescence stabilities and good biocompatibility indicate that QDs@DTDTPA-Gd NPs have a great potential for use as an efficient nanoagent to integrate the extremely high sensitivity of fluorescence imaging to the high resolution of MR imaging. Integration of bimodal detectability in the same agent of QDs@DTDTPA-Gd NPs can avoid extra stress on the blood clearance mechanisms as the administration of multiple dose of agents. KEYWORDS: magnetic resonance imaging, near-Infrared fluorescence imaging, bimodal contrast agent, quantum dots, Gd(III) chelate

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been widely used for NIR fluorescence imaging.12−15 In our previous study, magnetic Prussian blue nanoparticles (MPB NPs) were conjugated with ZCIS QDs to achieve NIR fluorescence and T2-weighted MR bimodal imaging. Nevertheless, the size of the obtained MPB and ZCIS composite NPs increased significantly after conjugation.16 Furthermore, clinical applications of T2 MR imaging have been limited due to the inherent disadvantage of negative (darker) contrast enhancement.17−19 Thus, the integration of ZCIS QDs and T1-weighted MR contrast agents would be highly desirable. The paramagnetic metal gadolinium is commonly used to achieve T1-weighted MR enhancement of QDs by chelate or ion doping.20−23 Directly doping Gd ions could minimize the size of bifunctional QDs, which is beneficial for fast excretion of administered agents, and further improve the biosafety.24 However, doping QDs with paramagnetic metal ions always causes a reduction in the QD fluorescence quality. A kind of T1weighted MR and fluorescence bimodal nanoprobe was prepared by coating hydrophobic ZCIS QDs with amphiphilic

INTRODUCTION In recent years, the rapid development of nanotechnology has been the major power to promote the innovation of contrast agents used in biomedical imaging. Compared with the single imaging mode, multimodal imaging could improve the accuracy of diagnosis and provide more comprehensive biomedical information.1,2 Optical imaging, especially fluorescence imaging, possesses many advantages, such as high sensitivity and selectivity, superb contrast, and ability of separating the absorption spectrum from the emission spectrum by virtue of Stokes’ shift, making it suitable for the early diagnosis of cancer.3,4 However, low tissue penetration depth and poor resolution have greatly limited the application of fluorescence imaging.5 On the contrary, magnetic resonance (MR) imaging offers strong tissue penetration and excellent spatial resolution but low sensitivity and long scan time.6−11 Therefore, integrating fluorescence imaging and MR imaging has attracted wide attention due to the capability of allying the high sensitivity of fluorescence imaging to the high spatial resolution of MR imaging. Owing to good photostability, high fluorescence efficiency, deep tissue penetration, and low toxicity, near-infrared (NIR) luminescence CuInS2/ZnS quantum dots (ZCIS QDs) have © 2017 American Chemical Society

Received: April 26, 2017 Accepted: June 28, 2017 Published: June 28, 2017 23450

DOI: 10.1021/acsami.7b05867 ACS Appl. Mater. Interfaces 2017, 9, 23450−23457

Research Article

ACS Applied Materials & Interfaces

Figure 1. Fabrication procedure and functional description of QDs@DTDTPA-Gd NPs. trimethylamine were added into 15 mL of the DMF solution to obtain solution B. Then, solution A is mixed with solution B. After magnetic stirring at 70 °C overnight, the reaction solution was treated with ice water for 1 h and allowed to cool down to room temperature. After the removal of the white precipitate by filtration, the obtained filtrate was concentrated by distillation in vacuum, followed by addition into chloroform to produce a white precipitate. After filtration, the precipitate was washed with chloroform and dried under vacuum to obtain DTDTPA (90% yield). The as-prepared DTDTPA was characterized by Bruck 400 proton nuclear magnetic resonance (1H NMR) spectroscopy and API 3000 liquid chromatography−tandem mass spectrometry (LC/MS/MS). 1H NMR (300 MHz, D2O, 298 K): δ 3.551 (s, 4H, −N−CH2−CO−N−), 3.406 (s, 4H, −N−CH2−COOH), 3.312 (s, 2H, −N−CH2−COOH), 3.154− 3.097 (m, 12H, −N−CH2−CH2−SH, −N−CH2−CH2−N−), 2.698 (t, 4H, N−CH2−CH2−SH) (Figure S2); MS: m/z 512.4285 [M + H] + (Figure S3). Preparation of QDs@DTDTPA NPs. ZCIS QDs were synthesized according to our reported method.28 Then, 0.3 mmol ZCIS QDs were dissolved in 2 mL of chloroform and added dropwise into 20 mL of the DTDTPA methanol solution (0.6 mmol, pH = 10.0). After 4 h of magnetic stirring at room temperature, chloroform and methanol were removed through reduced-pressure distillation. The residue was dispersed in phosphate-buffered saline (PBS) and washed three times using a 30 kDa Millipore ultrafiltration centrifuge tube by centrifugation (5000 rpm for 30 min) to obtain QDs@DTDTPA NPs. Preparation of QDs@DTDTPA-Gd NPs. QDs@DTDTPA NPs (10 μmol) were dispersed in PBS, and then 10 mmol gadolinium chloride was added under magnetic stirring. After 2 h, the obtained QDs@DTDTPA-Gd NPs were filtrated and washed three times using a 30 kDa Millipore ultrafiltration centrifuge tube by centrifugation (5000 rpm for 30 min). Characterization of NPs. The optical properties of QDs@ DTDTPA-Gd NPs were acquired by a Cary 4000 UV−vis spectrophotometer (Varian) and Cary Eclipse fluorescence spectrophotometer (Varian). The morphology of QDs@DTDTPA-Gd NPs was observed by a H-7650 transmission electron microscope (TEM; Hitachi). The hydrodynamic diameter and surface potential of QDs@ DTDTPA-Gd NPs were tested by a Holtsville PALS/90 Plus Particle Sizing and Potential Analyzer (Brookhaven). The gadolinium concentration of QDs@DTDTPA-Gd NPs was determined by a PerkinElmer 3300 inductively coupled plasma−optical emission spectroscope (ICP-OES; PerkinElmer). The crystal structures of ZCIS QDs and QDs@DTDTPA@Gd NPs were clarified by D8 ADVANCE A25 X-ray diffraction (XRD, Bruker) patterns. The element compositions of ZCIS QDs, QDs@DTDTPA NPs, and QDs@DTDTPA-Gd NPs were analyzed by ESCALAB 250Xi X-ray

poly(maleic anhydride-alt-1-octadecene) (PMO), followed by conjugating with DTPA-Gd and folic acid.20 Yet, the process of preparation is complicated, which might affect the fluorescence quality of the nanoprobe. Recently, a dithiolated derivative of 2[bis[2-[carboxymethyl-[2-oxo-2-(2-sulfanylethyl-amino)ethyl]amino]ethyl]amino]acetic acid (DTDTPA) has been synthesized and applied for the preparation of gadolinium-chelatecoated gold NPs for X-ray computer tomography (CT) and MR bimodal imaging.25 Because of water solubility and strong affinity between thiol groups and metal atoms, DTDTPA has the potential to be used for surface ligand exchange of hydrophobic ZCIS QDs and to be further chelated with gadolinium. In addition, it has been demonstrated that some of the DTDTPA molecules use only one thiol group to bind with the surface metal atoms of NPs and the other one tends to form disulfide bonds with neighboring DTDTPA.25,26 It could provide more gadolinium chelating sites compared with other dithiolated ligands, whose both thiolated ends are bound with the metal atoms on NPs.27 In this article, we reported the fabrication of ZCIS QDs conjugating the gadolinium complex (QDs@DTDTPA-Gd) for NIR fluorescence and T1-weighted MR bimodal imaging by depositing DTDTPA onto the surface of ZCIS QDs followed by chelating Gd3+ (Figure 1). The morphology, hydrodynamic diameter, and optical properties of the obtained QDs@ DTDTPA-Gd NPs were characterized. The in vivo NIR fluorescence and T1-weighted MR enhancement capabilities were evaluated using HeLa tumor-bearing BALB/c nude mice. The biocompatibility of such a bimodal contrast agent was also assessed both in vitro and in vivo.

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MATERIALS AND METHODS

Materials. Diethylenetriaminepentaacetic dianhydride (DTPA-BA) (98%) was purchased from TCI. Gadolinium trichloride, calcein acetoxymethyl ester (calcein AM), methylthiazolyldiphenyl-tetrazolium bromide (MTT), and 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI) were obtained from Sigma-Aldrich. All reagents and chemicals were used without further purification unless specified otherwise. Synthesis of DTDTPA. DTDTPA was synthesized according to the literature with a slight modification.25 DTPA-BA (2.8 mmol) was added into dimethylformamide (DMF) (20 mL) under magnetic stirring and then heated to 70 °C for 10 min to obtain solution A. In another flask, 7.65 mmol 2-aminoethanethiol and 0.87 mL of 23451

DOI: 10.1021/acsami.7b05867 ACS Appl. Mater. Interfaces 2017, 9, 23450−23457

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ACS Applied Materials & Interfaces

Figure 2. TEM micrograph (A) and dynamic light scattering measurements (B) of QDs@DTDTPA-Gd NPs,(C) UV−vis adsorption spectra of ZCIS QDs and QDs@DTDTPA-Gd NPs, and (D) fluorescence spectra of ZCIS QDs and QDs@DTDTPA-Gd NPs. The fluorescence intensities in the region of interest (ROI) were quantified using IndiGO Imaging Software. MR Imaging Study. For in vitro MR imaging, QDs@DTDTPAGd NPs with the gadolinium concentration from 0 to 0.25 mM in PBS were prepared, T1-weighted images of QDs@DTDTPA-Gd NPs were acquired using a Philips whole-body MR imaging scanner, and the parameters were applied with an echo time (TE) of 60 ms, a repetition time of 2000 ms, and a slice thickness of 1.5 mm. The specific relaxivity value (r1, mM−1 s−1) was acquired from the slopes of the relaxation rate (1/T1) versus gadolinium concentration curve. For in vivo MR imaging, the mice were i.v. administered with 100 μL of the QDs@DTDTPA-Gd NP suspension (4 mg/mL in saline). T1weighted MR images were captured at 0, 2, 8, 24, and 48 h post injection. Biocompatibility Evaluation. For the in vitro biocompatibility study, human umbilical vein endothelial cells (HUVEC) and HeLa cells were seeded into 96-well plates with the density of 5 × 104 cells for each well, respectively. After overnight incubation at 37 °C, the cells were washed thrice with 1× fresh PBS buffer. Then, 200 μL of QDs@DTDTPA-Gd NPs with different concentrations was added into each well for 24, 48, and 72 h incubation at 37 °C. Afterward, the standard MTT assay was used to study the cell viability. For the in vivo biocompatibility study, healthy BALB/c mice were randomly divided into treated and control groups (n = 9). For the treated group, mice were i.v. treated with 100 μL of the QDs@ DTDTPA-Gd NP suspension (8 mg/mL, n = 9), whereas the other group without any treatment was kept as control. The mice were sacrificed by cervical dislocation at days 1 and 30 post treatment, and the main organs, including the heart, liver, spleen, lungs, and kidneys, were collected for histopathological investigation. Biodistribution and Pharmacokinetics Evaluation. For the biodistribution study, tumor-bearing BALB/c mice were i.v. injected with QDs@DTDTPA-Gd NPs at a dose of 20 mg/kg (n = 6 for each

photoelectron spectroscopy (XPS; Thermo). The photoluminescence quantum yields (QYs) of QDs@DTDTPA-Gd NPs were determined using rhodamine 6G as reference (QY = 95% in absolute ethanol).20,29 Cellular Uptake of QDs@DTDTPA-Gd NPs. The human cervical carcinoma cells (HeLa) cells were seeded into a six-well plate with the density of 5 × 104 cells for each well and cultured at 37 °C. When cell confluence reached about 40%, 0.1 mg/mL QDs@DTDTPA-Gd NPs were added into each well for 2, 6, 12, and 24 h incubation. Then, the cells were washed thrice with 1× fresh PBS buffer. Afterward, the nuclei were stained with DAPI for observation of the cellular uptake of QDs@DTDTPA-Gd NPs by Carl Zeiss 510 confocal laser scanning microscopy (CLSM, Carl Zeiss) using excitation wavelength of 430 nm. Xenograft Tumor Models. All animal experiments were in agreement with the guidelines of the Harbin Institute of Technology Institutional Animal Use Committee. BALB/c nude mice and BALB/c mice (female, 5−7 weeks) were purchased from Beijing Vital River Laboratories for the study. The xenograft tumor model was developed by subcutaneously injecting the HeLa cells (5 × 106 suspended in 100 μL PBS) into the back of each mouse. The tumor size was recorded post injection and calculated according to the reported formula.16 The NIR fluorescence and MR imaging, biodistribution, and pharmacokinetics evaluation were started when the tumor volume increased to about 300 mm3. NIR Fluorescence Imaging Study. The tumor-bearing BALB/c nude mice were intravenously (i.v.) administered with 100 μL of the QDs@DTDTPA-Gd NP suspension (4 mg/mL in saline). The in vivo NIR fluorescence images were acquired at 0, 2, 8, 24, and 48 h post injection using a Berthold LB 983 small-animal imaging system, the excitation and excitation wavelength were set as 500 and 700 nm, respectively. Afterward, the mice were sacrificed by cervical dislocation and the tumor and main organs, including the liver, spleen, lungs, kidneys, and heart, were collected for ex vivo fluorescence imaging. 23452

DOI: 10.1021/acsami.7b05867 ACS Appl. Mater. Interfaces 2017, 9, 23450−23457

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Figure 3. CLSM images of HeLa cells after incubation with QDs@DTDTPA-Gd NPs and PBS for 2, 6, 12, and 24 h. Scale bar = 20 μm; n = 3.

Figure 4. (A) T1-weighted MR images of QDs@DTDTPA-Gd NPs with various gadolinium concentrations; (B) linear relationship between the T1 relaxation rate (1/T1) and gadolinium concentrations for QDs@DTDTPA-Gd NPs; and in vivo T1-weighted MR images (C) and MR signal intensities (D) of HeLa-bearing nude mice at 0, 8, 24, and 48 h after tail vein injection of QDs@DTDTPA-Gd NPs. Data shown as mean standard deviation (SD), n = 6 (*p < 0.05, **p < 0.01). time point). The tumor and main organs were excised at 6 h, 12 h, 24 h, 48 h, 4 day, and 10 day post injection and digested with nitric acid and perchloric acid, and then zinc contents in each organ were determined by ICP-OES. For pharmacokinetics study, QDs@DTDTPA-Gd NPs were i.v. injected into tumor-bearing BALB/c mice at a dose of 20 mg/kg. The orbital venous blood (500 μL for each mice) of mice was collected at 0.5, 1, 2, 4, 8, 12, and 24 h (n = 6 for each time point). Plasma was isolated from the collected blood samples by centrifugation (2000 rpm for 10 min), and analyzed by ICP-OES to determine the content of zinc. The kinetic parameters of QDs@DTDTPA-Gd NPs in the blood were calculated according to the following formula:30 the terminal elimination rate constant (k) = −2.303 × slope (the slope of the log Cp versus time curve, Cp means the concentration of the sample in plasma); the half-life (t1/2) = 0.693/k. Statistical Analysis. The results of statistical significance between the experiment data were analyzed by one-way analysis of variance and t-tests, which were performed using SPSS 17.0 software. The statistical significance level was defined as *p < 0.05 or **p < 0.01.

prepared by depositing DTDTPA onto the surface of ZCIS QDs followed by chelating Gd3+. The obtained QDs@ DTDTPA NPs were well-dispersed in PBS and presented bright red fluorescence under UV irradiation (Figure S4). The TEM image revealed that the obtained QDs@DTDTPA-Gd NPs were well-dispersed and uniform (Figure 2A). The hydrodynamic diameter and zeta potential of QDs@ DTDTPA-Gd NPs in PBS were measured to be 24.7 ± 2.7 nm and −15.91 ± 2.60 mV, respectively (Figure 2B). The UV− vis absorption spectra of QDs@DTDTPA-Gd NPs showed no obvious change in comparison to that in native ZCIS QDs (Figure 2C), whereas the emission peak had a red shift of about 55 nm, accompanied by a slight decrease in the fluorescence intensity and a narrowed half-peak width (Figure 2D). The QY of QDs@DTDTPA NPs and QDs@DTDTPA-Gd NPs was decreased from 29.1% of native ZCIS QDs to 18.7 and 18.5%, respectively. This phenomenon is because the surface band gap of ZCIS QDs was changed and more defects were formed during the surface ligand exchange process.31,32 The XRD analysis was used to evaluate the crystal structures of ZCIS QDs and QDs@DTDTPA@Gd NPs. As shown in Figure S5, the reflection peaks of ZCIS QDs and QDs@ DTDTPA@Gd NPs correspond to (111), (220), and (311), indicating a typical zinc blende structure.33,34 The XPS analysis was carried to test the element composition change of the NPs

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RESULTS AND DISCUSSION Preparation and Characterization of QDs@DTDTPAGd NPs. NIR fluorescent ZCIS QDs were prepared by our reported method.28 DTDTPA was synthesized according to the literature25 (Figure S1). The two sulfhydryls of DTDTPA were activated in the base condition to exchange the native ligands on the surface of ZCIS QDs. QDs@DTDTPA-Gd NPs were 23453

DOI: 10.1021/acsami.7b05867 ACS Appl. Mater. Interfaces 2017, 9, 23450−23457

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animal imaging systems. As shown in Figure 5, no fluorescence signal was observed at the tumor site before intravenous

during preparation. The results are shown in Table S1 and Figure S6. After the native ligand of ZCIS QDs has been exchanged by DTDTPA, a new signal peak at 398.28 eV (N 1s) was observed. Furthermore, the XPS spectra of QDs@ DTDTPA NPs showed an increase in the C 1s signal, which could further prove the successful DTDTPA modification. In the XPS spectra of QDs@DTDTPA-Gd NPs, a new peak at 153.95 eV (Gd 4d) was detected, which is attributed to the successful chelation of the gadolinium ion. The colloidal stability and fluorescence stability of QDs@ DTDTPA-Gd NPs have been evaluated. QDs@DTDTPA-Gd NPs (8 mg) were dispersed in 1 mL of three kinds of physiologic medium including saline, plasma, and RPMI-1640 medium, respectively (Figure S7). After 7 days of storage at 4 °C, no deposition or aggregation of NPs was observed. Furthermore, the hydrodynamic diameter and the fluorescence intensity (emission at 710 nm) of QDs@DTDTPA-Gd NPs in the physiologic medium had no significant changes during the 7 day storage period (p > 0.05) (Table S2, Figure S8). The good colloidal and fluorescence stabilities of QDs@DTDTPA-Gd NPs are mainly attributable to the two sulfhydryls of DTDTPA. It has been proved that the two sulfhydryls could provide enhanced colloidal stability for gold NPs through increased binding sites of the dithiolated compound on gold atoms within a wide pH range.26,27 Moreover, the negative charge of QDs@ DTDTPA-Gd NPs will further ensure colloidal stability and make it available for biomedical applications. Cellular Uptake Study. To evaluate the tumor cellular uptake ability of QDs@DTDTPA-Gd NPs, a HeLa cell line was selected as a tumor cell model. After various incubation time periods (2, 6, 12, and 24 h) of HeLa cells with QDs@ DTDTPA-Gd NPs, the nuclei of HeLa cells were stained by DAPI as blue fluorescence, and red fluorescence was emitted from ZCIS QDs. As shown in Figure 3, the red fluorescent signal in the cytoplasm was increased with the incubation time extension. The strongest fluorescent signal was observed at 24 h incubation. It revealed that QDs@DTDTPA-Gd NPs could be taken in by the HeLa cells, which could be used for further in vivo study. MR Imaging Study. The gadolinium content of QDs@ DTDTPA-Gd NPs was determined to be 4.73 ± 0.61 wt % by ICP-OES (Table S3). The MR images of QDs@DTDTPA-Gd NPs at different gadolinium concentrations were obtained to study the T1-weighted MR imaging enhancement effect of NPs in vitro. The results are shown in Figure 4A; the MR signal of QDs@DTDTPA-Gd NPs was gradually enhanced with the increasing gadolinium concentration. The longitudinal relaxivity, r1, of QDs@DTDTPA-Gd NPs was calculated to be 9.91 mM−1 s−1 (Figure 4B), which was 2.5 times as high as that of clinically approved Gd-DTPA (3.9 mM−1 s−1) at 3.0 T.35 These results indicated that QDs@DTDTPA-Gd NPs have potential for T1 weighted MR imaging study in vivo. The in vivo MR images of mice were captured before and after i.v. injection of QDs@DTDTPA-Gd NPs at different time points. The results are shown in Figure 4C,D; the grayscale image of the tumor tissue became brighter as the time prolonged. More specifically, the MR signal intensity was increased significantly in 24 h post injection and the increased rate has slowed down over the next 24 h. This result suggested that QDs@DTDTPA-Gd NPs could efficiently accumulate at the tumor site after i.v. injection. Fluorescence Imaging Study. The fluorescence imaging of QDs@DTDTPA-Gd NPs was then conducted using small-

Figure 5. In vivo fluorescence images (A) and quantification analysis (B) of HeLa-bearing nude mice at 0, 2, 8, 24, and 48 h after tail vein injection of QDs@DTDTPA-Gd NPs, n = 6.

injection of QDs@DTDTPA-Gd NPs and the signal became stronger with increasing time. The photon counts at the ROI reached to about 1.47 × 106 at 24 h post injection and further increased to about 1.64 × 106. The results are in good agreement with the in vivo MR imaging result. The main organs and tumor were excised and imaged after in vivo fluorescence imaging at 48 h post treatment. As shown in Figure 6, an obvious fluorescence signal in tumor was detected. Furthermore, strong fluorescence was observed in the liver, spleen, and lungs, which indicated that QDs@DTDTPA-Gd NPs could be taken in by the reticuloendothelial system (RES). Overall, both the MR imaging and fluorescence imaging results suggested that QDs@DTDTPA-Gd NPs could act as an excellent agent for T1-weighted MR and fluorescence bimodal imaging. Biodistribution and Pharmacokinetics Evaluation. The biodistribution of QDs@DTDTPA-Gd NPs was further evaluated through ICP-OES measurements. QDs@DTDTPAGd NPs were i.v. administered into tumor-bearing BALB/c mice (20 mg/kg of body weight). As the main component of QDs@DTDTPA-Gd NPs (Table S1), zinc contents in the major organs and tumor at different time points (n = 6 for each time point) were measured post injection. As shown in Figure S9, the zinc content in 1 g of tumor tissue was gradually increased to 138.6 ± 11.3 μg at 48 h post administration of QDs@DTDTPA-Gd NPs, which was significantly higher than that of PBS-injected mice (12.7 ± 4.9 μg) (p < 0.01), indicating that QDs@DTDTPA-Gd NPs could effectively accumulate in the tumor tissue by the enhanced permeability and retention effect. It could be found that a number of QDs@DTDTPA-Gd NPs were deposited in the liver, lungs, and spleen in 4 days and most of the NPs were removed from the body after 10 days. The accumulation in RES organs (liver, lungs, and spleen) 23454

DOI: 10.1021/acsami.7b05867 ACS Appl. Mater. Interfaces 2017, 9, 23450−23457

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Figure 6. Ex vivo fluorescence images (A) and quantification analysis (B) of the main organs from HeLa-bearing nude mice after 48 h tail vein injection of QDs@DTDTPA-Gd NPs, n = 6.

Figure 7. Viabilities of HUVEC (A) and HeLa (B) cells incubated with different concentrations of QDs@DTDTPA-Gd NPs for 24, 48, and 72 h. Data shown as mean SD, n = 3. (C) Body weight change of mice after intravenous administration of QDs@DTDTPA-Gd NPs. (D) Histological sections of the heart, liver, spleen, lungs, and kidneys stained with hematoxylin and eosin (H&E) at days 1 and 30 post injection; the untreated group was used as control. Scale bar = 40 μm. Data shown as mean SD, n = 9.

could be attributed to the small size and negative charge of QDs@DTDTPA-Gd NPs.36−39 To study the pharmacokinetics of QDs@DTDTPA-Gd NPs, the zinc contents in the blood were measured by ICP-OES after i.v. injection of QDs@DTDTPA-Gd NPs (20 mg/kg) at

various time points (n = 6 for each time point), and the content of zinc in the control group (PBS injection) was subtracted. The kinetic parameters were calculated according to the literature.30 As shown in Figure S10, the terminal elimination rate constant (k) and the half-life (t1/2) were calculated to be 23455

DOI: 10.1021/acsami.7b05867 ACS Appl. Mater. Interfaces 2017, 9, 23450−23457

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ACS Applied Materials & Interfaces 0.107 ± 0.003 and 6.5 ± 0.2 h, respectively. This result revealed that QDs@DTDTPA-Gd NPs exhibited relatively long circulation and could therefore be used for in vivo imaging. Biocompatibility Evaluation. To investigate the in vitro cytotoxicity of QDs@DTDTPA-Gd NPs, HeLa cells and HUVEC were incubated with various concentrations of NPs for 24, 48, and 72 h, respectively. The cell viabilities were evaluated by the standard MTT method. The results are shown in Figure 7A,B; in both HeLa cells and HUVEC, the cell viabilities showed no decrease within 24 h incubation. When prolonging the incubation time to 48 h, the viabilities of HUVEC obviously decreased more than those of HeLa cells at various concentrations. After 72 h incubation with 40 mg/mL QDs@DTDTPA-Gd NPs, the viability of HUVEC was reduced to 63.52%, whereas the viability of HeLa cells was 84.77%. It is noteworthy that 40 mg/mL concentration of NPs was 10 times higher than that used in the bimodal imaging study (4 mg/mL), when no apparent in vitro cytotoxicity was observed for both HUVEC and HeLa cells. Subsequently, in vivo toxicity analyses were carried out by comparing the body weight change and histopathological examination of the mice injected with 0.8 mg (double imaging dose) of QDs@DTDTPA-Gd NPs and the untreated mice. As shown in Figure 7C, the body weight change curve of the NPinjected group was well in agreement with that of the control group. Histological sections of the major organs were carried out for both the QDs@DTDTPA-Gd NP-injected group and the control group at 1 and 30 day post injection and then stained with H&E. As shown in Figure 7D, in both the groups, the muscle fibers of heart were arranged radially and nucleus structures were clear. The liver cells were normal, no edema, venous congestion, or fatty degeneration was observed. The boundaries between the white and the red pulp in the spleen were clear, and lymphocytes were arranged tightly. The alveolar structures were clear, and no inflammatory cell infiltration was observed in the alveolar walls. The renal tubule epithelial cells were arranged radially, and the renal capsule thickness was normal. The histological analysis showed no histological injury of major organs in the QDs@DTDTPA-Gd NP-injected mice until 30 days. The low toxicity of QDs@DTDTPA-Gd NPs can therefore be attributed to the cadmium-free ZCIS QDs and low toxicity of DTDTPA.40,41 Overall, these results revealed that QDs@DTDTPA-Gd NPs showed reliable biosafety and could be further developed for biomedical applications.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (X.Y.). *E-mail: [email protected]. http://bme.pku.edu.cn/ ~daizhifei/ (Z.D.). Author Contributions

All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Key Research and Development Program of China (No. 2016YFA0201400), National Natural Science Foundation of China (No. 81371580), State Key Program of the National Natural Science Foundation of China (Grant No. 81230036), and the Foundation for Innovative Research Groups of the National Natural Science Foundation of China (No. 81421004).

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CONCLUSIONS A bimodal contrast agent was successfully developed by chelation of the gadolinium ion to DTDTPA-modified CuInS2/ZnS QDs. The obtained QDs@DTDTPA-Gd NPs were well-dispersed in aqueous solution and showed good stability. Both in vitro and in vivo experiments indicated that QDs@DTDTPA-Gd NPs could be accumulated into HeLa cells via the passive targeting effect. Furthermore, the in vivo study proved that QDs@DTDTPA-Gd NPs had excellent capability to enhance both NIR fluorescence and T1-weighted MR imaging. Therefore, QDs@DTDTPA-Gd NPs could operate as a promising bimodal contrast agent, which could hopefully improve the diagnostic accuracy of cancer.

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Synthesis route, 1H NMR and mass spectrum of DTDTPA, XRD patterns, ICP-OES data, XPS spectra, colloidal and fluorescence stability analyses, biodistribution profile, and plasma kinetics of QDs@DTDTPA-Gd NPs (PDF)

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S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b05867. 23456

DOI: 10.1021/acsami.7b05867 ACS Appl. Mater. Interfaces 2017, 9, 23450−23457

Research Article

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