Core–shell BaYbF₅:Tm@BaGdF₅:Yb,Tm nanocrystals for in vivo trimodal UCL/CT/MR imaging

Abstract

Lanthanide-doped nanocrystals have been researched extensively and used for bioimaging because of their optical properties, magnetic properties and X-ray absorption. Core–shell-structured lanthanide-doped nanocrystals have been developed and characterized by TEM and XRD analysis. The nanocrystals are composed of BaYbF₅:0.5% Tm as the core and BaGdF₅:20% Yb, 0.5% Tm as the shell. Apart from characterization of the nanocrystals, evaluation of both their cytotoxicity via MTT assays and their long-term toxicity via histological analysis showed that their cytotoxicity was low, indicating the possibility of further in vivo imaging. This work combined the functions of trimodal imaging into one nanoplatform and then UCL, CT, and MR imaging with core–shell-structured nanocrystals were investigated both in vitro and in vivo. Taking into consideration its structural characteristics and trimodal imaging abilities, it is expected that the developed multifunctional nanoplatform may be potentially useful for diagnosing diseases at an early stage.

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1. Introduction

In the past twenty years, lanthanide-doped nanocrystals have been researched extensively because of their optical properties, such as long luminescence lifetime (μs–ms range), large Stokes shift (up to 500 nm), and sharp emission bandwidths (<10 nm). These nanocrystals have been successfully applied in laser sources, biological labeling, and so on.^1–6 Moreover, lanthanide-doped nanocrystals have attracted much interest in diagnostics because of their X-ray absorption and magnetic properties.^7–12 Recently, lanthanide-doped nanoprobes have become a fascinating area, especially for multimodal imaging.^12–18

Many imaging techniques have been used for disease diagnosis such as ultrasound imaging, magnetic resonance imaging (MRI), computed tomography (CT), positron emission tomography (PET), and so on.^19–23 Among these imaging modalities, CT imaging has been recognized as one of the most useful because of its high spatial resolution, deep tissue penetration and powerful post-processing techniques such as 3D volume-rendering techniques. Nanoprobes composed of lanthanide-doped elements with atomic number Z > 50 usually present excellent effects during CT imaging.^24–27 In spite of the advantages of CT, this imaging technique is not sensitive for diagnosing diseases of soft tissue. However, MR imaging has advantages for examining soft tissue in the musculoskeletal system or brain diseases. MRI has been widely used in the diagnosis of diseases because of its admirable soft-tissue contrast and deep tissue penetration. For early or differential diagnosis of diseases, contrast materials are usually required. Recently, lanthanide-doped nanocrystals have generated a great deal of interest for CT or MR imaging.^28–37 Although CT and MR imaging have the above advantages, these two techniques have similar shortcomings: the resolutions of CT and MR are about 50 μm and 10–100 μm, respectively.³⁸ This resolution is incapable of cellular imaging. Optical imaging, on the other hand, has high resolution and sensitivity for imaging at the cellular level; for example, the resolution of fluorescence reflectance imaging is about 2–3 nm.³⁸ The above problems have aroused much attention of researchers in the synthesis of lanthanide-doped nanocrystals for fluorescence imaging.^39–43 However, optical imaging could not provide deep tissue penetration or 3D information. Therefore, multimodal imaging is attracting a great deal of attention, because it can integrate the merits of different techniques and improve the efficiency of diagnosis.

With this in mind, we report the synthesis of multifunctional lanthanide-doped nanocrystals that are composed of BaYbF₅:0.5% Tm (hereinafter abbreviated as BaYbF₅:Tm) as the core and BaGdF₅:20% Yb, 0.5% Tm (hereinafter abbreviated as BaGdF₅:Yb,Tm) as the shell. The core–shell structure was characterized by powder X-ray diffraction (XRD), transmission electron microscopy (TEM), and so on. The cytotoxicity and tissue toxicity of the nanocrystals were described in detail. Finally, this work combined the functions of trimodal imaging into one nanoplatform and then UCL, CT, and MR imaging with core–shell-structured nanocrystals were investigated both in vitro and in vivo.

2. Materials and methods

2.1 Chemicals

Analytical grade Ba(OH)₂·8H₂O was purchased from Sinopharm Chemical Reagent Co. Ltd (Shanghai, China). Yb₂O₃ (99.9%), Gd₂O₃ (99.99%) and Tm₂O₃ (99.99%) were obtained from Aladdin Reagents (Shanghai, China). Oleic acid (OA, >90%), 1-octadecene (ODE, >90%), and CF₃COOH were purchased from Sigma-Aldrich. Other chemicals were of analytical grade and used as received without further purification.

2.2 Preparation of BaYbF₅:Tm nanocrystals

Initially, 2 mmol Ba(OH)₂ was dissolved in 2 mL deionized water under vigorous magnetic stirring. CF₃COOH was added to the above mixture at room temperature and the solution was heated to 100 °C at a pH of 7 and then dried in a vacuum drying oven at 60 °C for 24 h. Tm(CF₃COO)₃ and Yb(CF₃COO)₃ were also prepared by the above procedure.

For the synthesis of BaYbF₅:Tm nanocrystals, 1 mmol Ba(CF₃COO)₂, 0.98 mmol Yb(CF₃COO)₃ and 0.02 mmol Tm(CF₃COO)₃ were added to a 100 mL three-necked round-bottom flask and then 10 mL oleic acid and 10 mL octadecene were added. The mixture was heated to 100 °C with magnetic stirring for 0.5 h under an argon protective atmosphere and then heated to 300 °C for 1 h. After reacting completely, the system was naturally cooled to room temperature. The prepared nanocrystals were isolated by centrifugation, washed with ethanol several times, and then redispersed in cyclohexane.

2.3 Synthesis of BaYbF₅:Tm@BaGdF₅:Yb,Tm nanocrystals

Initially, 1 mmol Ba(CF₃COO)₂, 0.78 mmol Gd(CF₃COO)₃, 0.2 mmol Yb(CF₃COO)₃, and 0.02 mmol Tm(CF₃COO)₃ were added to a 100 mL three-necked round-bottom flask and then 10 mL oleic acid and 10 mL octadecene mixed with 1 mmol BaYbF₅:Tm were added. The mixture was heated to 100 °C with magnetic stirring for 0.5 h under an argon protective atmosphere and then heated to 300 °C for 1 h. After reacting completely, the system was naturally cooled to room temperature. The prepared nanocrystals were isolated by centrifugation, washed with ethanol several times, and then redispersed in cyclohexane.

2.4 Surface modification of BaYbF₅:Tm@BaGdF₅:Yb,Tm

BaYbF₅:Tm@BaGdF₅:Yb,Tm nanocrystals in chloroform (10 mL, 5 mg mL⁻¹) were mixed with a chloroform solution of DSPE-PEG2000 (20 mL, 10 mg mL⁻¹) under stirring at room temperature for 10 minutes. The mixture was evaporated and the resulting solution was heated to 60 °C for 1 h under vacuum. After cooling, 10 mL deionized water was added. The solution was dispersed with ultrasound and then centrifuged at 10 000 rpm for 20 minutes. The PEG-modified nanocrystals were redispersed in deionized water for future use.

2.5 Measurements and characterization

The morphology and composition of the BaYbF₅:Tm@BaGdF₅:Yb,Tm nanocrystals were determined by a field emission scanning electron microscope (FESEM, Hitachi S-4800). The concentrations of nanocrystals were obtained by inductively coupled plasma-mass spectrometry (ICP-MS). X-ray powder diffraction (XRD) patterns were obtained on a D8 ADVANCE (Germany) using Cu Kα (0.15406 nm) radiation. Transmission electron microscope (TEM) measurements were made with a JEOL JEM-2010EX TEM. X-ray photoelectron spectroscopy (XPS) measurements were made on an ESCALAB-MKII spectrometer (VG Co., United Kingdom). UCL spectra were recorded using a 980 nm laser diode and a triple-grating monochromator (Spectra Pro-2758, Acton Research Corporation, USA) equipped with a photomultiplier (Hamamatsu R928). MRI images were obtained with a 1.5 T scanner (Achieva, Siemens, Germany). CT images were acquired using a 256-slice multidetector CT scanner (Brilliance iCT, Philips Healthcare, Cleveland, Ohio, USA).

2.6 In vitro cytotoxicity studies

HepG2 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin in a humidified incubator at 37 °C under 5% CO₂. The HepG2 cells were cultured and put back into fresh complete medium before plating.

The in vitro cytotoxicity of nanocrystals was evaluated via the viability and proliferation of HepG2 cells using methyl thiazolyl tetrazolium (MTT) reduction assays. In a typical procedure, HepG2 cells were seeded into 96-well plates for 12 h to allow the cells to attach. Subsequently, a DMEM mixture containing BaYbF₅:Tm@BaGdF₅:Yb,Tm–PEG nanocrystals of different concentrations (from 0 to 500 μg mL⁻¹) was added to the wells. The HepG2 cells were incubated in the incubator for another 24 h and washed with the medium twice. Thereafter, MTT (10 μL, 5 mg mL⁻¹) was added to the samples for another 4 h and then dimethylsulfoxide (DMSO) was added to the wells to dissolve the formazan crystals. Finally, an enzyme-linked immunosorbent assay reader was applied to measure the absorbance at a wavelength of 570 nm.

2.7 Animal protocol and histopathology analysis

Kunming mice were obtained from the Laboratory Animal Center of Jilin University (Changchun, China). Animal care and handing procedures were in agreement with the guidelines of the Institutional Animal Care and Use Committee.

Kunming mice with and without the injection of BaYbF₅:Tm@BaGdF₅:Yb,Tm–PEG were sacrificed after 30 days. Tissues (kidney, heart, liver, spleen, lungs) were collected from the above two groups and then fixed in 10% neutral buffered formalin. Then, the well-prepared tissues were embedded in paraffin and then sectioned to a thickness of 4 μm and stained with hematoxylin and eosin (H&E). The histological sections were analysed using an optical microscope.

2.8 UCL imaging

To perform upconversion luminescence imaging, chloral hydrate (10 wt%) was injected into a mouse intraperitoneally and then 200 μL BaYbF₅:Tm@BaGdF₅:Yb,Tm–PEG aqueous solution with a concentration of 10 mg mL⁻¹ was injected into the mouse subcutaneously. After administration, upconversion luminescence imaging was carried out using an in vivo Maestro whole-body imaging system equipped with an external 980 nm laser as the excitation source. Upconversion luminescence imaging was obtained with an exposure time of 5 s.

2.9 CT imaging

For in vitro CT imaging, BaYbF₅:Tm@BaGdF₅:Yb,Tm–PEG nanocrystals were dispersed in deionized water at different concentrations (0–236 mM). Eppendorf tubes with different concentrations of the nanocrystals and iobitridol were scanned using a Philips CT imaging system.

For in vivo CT imaging, after anesthetizing them by intraperitoneal injection of 10 wt% chloral hydrate, mice were intravenously injected with BaYbF₅:Tm@BaGdF₅:Yb,Tm–PEG nanocrystals (20 mM mL⁻¹) and iobitridol (20 mM mL⁻¹), respectively.

CT images were obtained using a Philips iCT scanner. The scanning parameters included 120 kVp (tube voltage), 300 mA (tube current), and 0.9 mm (thickness). The post-processing techniques of multiplanar reconstruction (MPR) and volume rendering (VR) were used to obtain coronal images.

2.10 MR imaging

For in vitro MR imaging, nanocrystals were dispersed in deionized water at different concentrations (0–2.4 μM mL⁻¹). Eppendorf tubes with different concentrations of nanocrystals and gadolinium–DTPA (Gd–DTPA) were scanned using a clinical MRI instrument for T₁-weighted imaging (T₁WI).

For in vivo MR imaging, after anesthetizing them by intraperitoneal injection of 10 wt% chloral hydrate, Kunming mice were subcutaneously injected with nanocrystals (20 mM mL⁻¹).

MR images were obtained from a 1.5 T Achieva scanner (Siemens). The scanning parameters (T₁WI) included TR (time of repetition) = 450 ms and TE (time of echo) = 14 ms. The post-processing techniques of multiplanar reconstruction (MPR) and volume rendering (VR) were used to obtain coronal images.

3. Results and discussion

3.1 Characterization of BaYbF₅:Tm@BaGdF₅:Yb,Tm nanocrystals

OA-stabilized BaYbF₅:Tm nanocrystals were synthesized using a high-temperature solvent thermal method. As shown in a transmission electron microscopy (TEM) image, these nanocrystals were regular quadrilaterals with a mean diameter of 6 nm (Fig. 1a). A TEM image of BaYbF₅:Tm@BaGdF₅:Yb,Tm nanocrystals showed quadrilaterals with a narrow size distribution and an average diameter of 9 nm (Fig. 1b). High-resolution TEM images clearly showed lattice fringes with an observed d-spacing of 0.29 nm, which was highly consistent with the lattice spacing of the (010) planes of BaYbF₅:Tm (Fig. 1a, inset). X-ray diffraction (XRD) analysis further indicates a crystalline structure in Fig. 2. All the diffraction peaks could be indexed to pure cubic-phase BaGdF₅ (JCPDS no. 24-0098) very well, and no trace of other phases and impurities could be observed. The successful modification of PEG on the surface of the BaYbF₅:Tm@BaGdF₅:Yb,Tm nanocrystals was confirmed by FTIR spectroscopy (Fig. 3). Two new bands at 1737 and 1109 cm⁻¹ in the FTIR spectrum of PEG–UCNPs were assigned to the stretching vibrations of carboxyl esters and the ether bonds of PEG chains, respectively. Fig. 4 shows UCL spectra of BaYbF₅:Tm (core) and BaYbF₅:Tm@BaGdF₅:Yb,Tm (core–shell) nanocrystals. BaYbF₅:Tm nanocrystals displayed no obvious emission in the visible region and one weak emission in the NIR region. However, compared with the core nanocrystals, the core–shell nanocrystals exhibited two emissions in the visible region and one obvious emission in the NIR region upon excitation by a 980 nm laser. BaYbF₅:Tm@BaGdF₅:Yb,Tm nanocrystals displayed excellent NIR upconversion luminescence, of which the intensity was very high and more than five times the intensity of UCL at 475 nm.

Fig. 1 (a) TEM image of BaYbF₅:Tm nanocrystals. The inset shows a HRTEM image with a d-spacing of about 0.29 nm. (b) TEM image of BaYbF₅:Tm@BaGdF₅:Yb,Tm. The inset shows a HRTEM image with a d-spacing of about 0.29 nm.

Fig. 2 XRD patterns of BaYbF₅:Tm and BaYbF₅:Tm@BaGdF₅:Yb,Tm nanocrystals. The line spectrum corresponds to the standard data for BaGdF₅ (JCPDS no. 24-0098).

Fig. 3 FTIR spectra of OA-stabilized BaYbF₅:Tm@BaGdF₅:Yb,Tm and BaYbF₅:Tm@BaGdF₅:Yb,Tm–PEG nanocrystals.

Fig. 4 UCL spectra of BaYbF₅:Tm (core) and BaYbF₅:Tm@BaGdF₅:Yb,Tm (core–shell) nanocrystals dissolved in water and excited with a 980 nm laser. The power of the 980 nm laser was 800 mW cm⁻².

3.2 Toxicology investigation

Encouraged by the high efficiency of the NIR upconversion luminescence imaging, the in vitro and in vivo toxicity of BaYbF₅:Tm@BaGdF₅:Yb,Tm–PEG nanocrystals were investigated. The in vitro cytotoxicity was evaluated on HepG2 cells using an MTT assay and the in vivo cytotoxicity was evaluated via the histological changes in several tissues after the injection of nanocrystals for one month.

The MTT assay results are illustrated in Fig. 5. The viability of HepG2 cells treated with BaYbF₅:Tm@BaGdF₅:Yb,Tm–PEG nanocrystals still remained at approximately 90%, even at the highest tested dose (500 μg mL⁻¹). No significant differences in the proliferation of the cells were observed in the presence of concentrations of 0–500 μg mL⁻¹.

Fig. 5 In vitro cell viability of HepG2 cells incubated with BaYbF₅:Tm@BaGdF₅:Yb,Tm–PEG nanocrystals at different concentrations.

The results of histopathology analysis are shown in Fig. 6. Tissues from the kidney, heart, liver, spleen and lungs were normal in the control group, which was not injected with nanocrystals. Compared with the control group, no tissue damage or any other side effect was observed in mice injected with BaYbF₅:Tm@BaGdF₅:Yb,Tm–PEG nanocrystals.

Fig. 6 Histological changes in the control group and imaging group. Hematoxylin and eosin (H&E)-stained histological images of tissues of kidney, heart, liver, spleen and lungs with and without injection of nanocrystals at 400× magnification.

3.3 UCL imaging

Because BaYbF₅:Tm@BaGdF₅:Yb,Tm–PEG nanocrystals exhibited obvious emissions in both the visible region and the NIR region, we investigated the feasibility of in vivo UCL imaging using the nanocrystals (980 nm, 0.8 W cm⁻²). Fig. 7 shows the in vivo imaging of mice with a subcutaneous injection of the BaYbF₅:Tm@BaGdF₅:Yb,Tm–PEG nanocrystals. As shown in Fig. 7, an intense UC signal was observed after the injection (Fig. 7b), whereas there were no signals in white-light imaging (Fig. 7a). The overlay image shows an excellent match of white-light and UC emission bioimaging. This result indicated that the nanocrystals were perfect for in vivo UCL bioimaging.

Fig. 7 In vivo upconversion luminescence imaging of a mouse with the injection of BaYbF₅:Tm@BaGdF₅:Yb,Tm–PEG nanocrystals: the left panel is white-light imaging (a), the middle panel is UC emission imaging (b), and the right panel is an overlay image (c). The wavelengths collected for small-animal imaging are from 795 nm to 805 nm.

3.4 Computed tomography imaging

Our previous study showed that bimetallic nanomaterials composed of elements with a high atomic number displayed excellent CT imaging effects.^39–42 Compared with iobitridol (iodine) (53), BaYbF₅:Tm@BaGdF₅:Yb,Tm–PEG nanocrystals contain Ba (56), Yb (70), Gd (64), and Tm (69) elements and may hold great promise as novel CT contrast agents.

To evaluate the feasibility and efficiency of the BaYbF₅:Tm@BaGdF₅:Yb,Tm–PEG nanocrystals, in vitro CT imaging was performed by scanning Eppendorf tubes containing nanocrystals with different concentrations (0–236 mM). The effect of X-ray absorption was calculated by measuring the CT attenuation value of the nanocrystals in Hounsfield units (HU). As shown in Fig. 8, the nanocrystals displayed excellent X-ray absorption compared with iobitridol, which was clinically used for many years (Fig. 8a and b). The CT attenuation value (HU) increased with an increase in the concentration of the nanocrystals (Fig. 8c), and the linear correlation between these values was good. Furthermore, the absorption efficiency of the nanocrystals was higher than that of iobitridol at the same molar concentration. Based on the above results, the BaYbF₅:Tm@BaGdF₅:Yb,Tm–PEG nanocrystals provided higher absorption efficiency than that of elemental I.

Fig. 8 (a) CT images of BaYbF₅:Tm@BaGdF₅:Yb,Tm–PEG nanocrystals at different concentrations. (b) X-ray CT images of iobitridol at different concentrations. (c) CT value (HU) of different concentrations of the nanocrystals (red) and iobitridol (black), respectively.

For in vivo CT imaging, Kunming mice were scanned using iCT several times before and after the injection of nanocrystals and iobitridol, respectively. The scanning times were pre-injection and 3 min, 30 min, 1 h and 2 h after injection. After the administration of nanocrystals, 3 min later the liver (Fig. 9 row a) and spleen (Fig. 9 row b) were enhanced and the enhancement increased gradually; there was no further enhancement 2 h after injection. The phenomenon was easily recognized using a volume-rendering technique (Fig. 9 row c). Compared with the nanocrystals, iobitridol was discharged from the urinary system (Fig. 10). After the administration of iobitridol, 3 min later the kidneys were enhanced (Fig. 10 row b) and the bladder was enhanced after 30 min; the liver and spleen were not enhanced after 3 min. The detection of hepatic metastases may be improved by long-lasting enhancement of liver signals.

Fig. 9 CT images of mice after intravenous injection of 1 mL BaYbF₅:Tm@BaGdF₅:Yb,Tm–PEG nanocrystals (118 mM mL⁻¹) solution at timed intervals. (a) Liver. (b) Spleen and kidney. (c) Volume-rendering technique of CT images.

Fig. 10 CT images of mice after intravenous injection of 1 mL iobitridol (118 mM mL⁻¹) solution at timed intervals. (a) Liver. (b) Spleen and kidney. (c) Volume-rendering technique of CT images.

3.5 MR imaging

The Gd(III) ions that were present in BaYbF₅:Tm@BaGdF₅:Yb,Tm–PEG nanocrystals enabled the enhancement of the T₁ MR imaging of mice. In vitro MR imaging was performed by scanning Eppendorf tubes containing nanocrystals with different concentrations (0–2.4 μM mL⁻¹). As shown in Fig. 11a and b, the MR signal exhibited a similar effect compared with Gd–DTPA. For in vivo T₁WI MR imaging, Kunming mice were scanned using a 1.5 T clinical scanner. Fig. 11c shows the muscle signal without injection (arrow) and Fig. 11d shows an oval area of high signal after the subcutaneous injection of nanocrystals (arrow).

Fig. 11 (a) T₁WI MR images of Gd–DTPA with different concentrations. (b) T₁WI MR images of BaYbF₅:Tm@BaGdF₅:Yb,Tm–PEG nanocrystals with different concentrations. (c) MR image showing muscle signal before the injection of nanocrystals (arrow). (d) MR image showing high signal after the injection of nanocrystals (arrow).

4. Conclusions

In conclusion, BaYbF₅:Tm@BaGdF₅:Yb,Tm–PEG core–shell-structured nanocrystals have been successfully constructed. The nanocrystals exhibit a narrow size distribution and a diameter of less than 10 nm. Apart from characterization of the nanocrystals, evaluation of both their cytotoxicity via MTT assays and long-term toxicity via histological analysis showed that their cytotoxicity was low, indicating the possibility of further in vivo imaging. These multifunctional nanocrystals have been used as a nanoplatform for the upconversion luminescence, computed tomography and magnetic resonance trimodal imaging of some major organs in mice. The Gd(III) ions enable the enhancement of T₁ MR imaging and the shell of BaGdF₅:Yb,Tm exhibits perfect UCL emission and enhancement in CT imaging. Taking into consideration its structural characteristics and trimodal imaging abilities, it is expected that the developed multifunctional nanoplatform may be especially useful for diagnosing diseases at an early stage.

Acknowledgements

Financial support was provided by the Technology Development Project of the National Development and Reform Commission of Jilin province (No. JF2012c007-1).

Notes and references

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