Optimization of Bi3+ in Upconversion Nanoparticles Induced

Oct 3, 2016 - Low-temperature molten-salt synthesis and upconversion of novel hexagonal NaBiF 4 :Er 3+ /Yb 3+ micro-/nanocrystals. Xinyang Huang , Lia...
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Optimization of Bi3+ in Upconversion Nanoparticles Induced Simultaneous Enhancement of Near-Infrared Optical and X‑ray Computed Tomography Imaging Capability Pengpeng Lei,†,‡ Peng Zhang,§ Shuang Yao,† Shuyan Song,† Lile Dong,†,‡ Xia Xu,†,‡ Xiuling Liu,† Kaimin Du,†,‡ Jing Feng,*,† and Hongjie Zhang*,† †

State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, 5625 Renmin Street, Changchun 130022, China ‡ University of Chinese Academy of Sciences, Beijing 100049, China § Department of Radiology, The Second Hospital of Jilin University, Changchun 130041, China S Supporting Information *

ABSTRACT: Bioimaging probes have been extensive studied for many years, while it is still a challenge to further improve the image quality for precise diagnosis in clinical medicine. Here, monodisperse NaGdF4:Yb3+,Tm3+,x% Bi3+ (abbreviated as GYT-x% Bi3+, x = 0, 5, 10, 15, 20, 25, 30) upconversion nanoparticles (UCNPs) have been prepared through the solvothermal method. The near-infrared upconversion emission intensity of GYT-25% Bi3+ has been enhanced remarkably than that of NaGdF4:Yb3+,Tm3+ (GYT) with a factor of ∼60. Especially, the near-infrared upconversion emission band centered at 802 nm is 150 times stronger than the blue emission band of GYT-25% Bi3+ UCNPs. Such high ratio of NIR/blue UCL intensity could reduce the damage to tissues in the bioimaging process. The possibility of using GYT-25% Bi3+ UCNPs with strong near-infrared upconversion emission for optical imaging in vitro and in vivo was performed. Encouragingly, the UCL imaging penetration depth can be achieved as deep as 20 mm. Importantly, GYT-25% Bi3+ UCNPs exhibit a much higher X-ray computed tomography (CT) contrast efficiency than GYT and iodine-based contrast agent under the same clinical conditions, due to the high X-ray attenuation coefficient of bismuth. Hence, simultaneous remarkable enhancement of NIR emission and X-ray CT signal in upconversion nanoparticles could be achieved by optimizing the doping concentration of Bi3+ ions. Additionally, Gd3+ ions in the UCNPs endow GYT-25% Bi3+ UCNPs with T1weighted magnetic resonance (MR) imaging capability. KEYWORDS: Bi3+ ion, upconversion nanoparticles, NaGdF4, signal enhancement, trimodal imaging

1. INTRODUCTION

feature of upconversion luminescence (UCL) is that the lowenergy light radiation could be translated to high-energy light radiation through multiphoton absorption processes.23 Compared with the conventional semiconductor quantum dots24,25 and organic dyes,26,27 Ln3+-doped UCNPs possess many superior features, such as low toxicity, long lifetimes, weak autofluorescence, high penetration depth, low radiation damage, and photobleaching.28−37 As a conventional UCL matrix, there is a significant progress in studying the application of Ln3+-doped NaGdF4 UCNPs.38−41 Gd3+ possesses seven unpaired electrons and can effectively accelerate the relaxation time of water protons. Thus, Gd3+-containing nanoparticles have been widely studied for MR imaging diagnosis of clinical disease.42 Moreover, gadolinium also possesses X-ray attenuation properties.43,44 Although the Gd3+-containing UCNPs have the optical, CT, and MR imaging abilities, the relative low UCL intensity and CT signal of UCNPs limits the practical

With the rapid development of modern medicine, novel nanoparticulate-based bioimaging probes have great applications in diagnostic medicine due to the noninvasive diagnosis and visualizing the structural of living objects or systems. Several imaging techniques including X-ray computed tomography (CT) imaging, magnetic resonance (MR) imaging, as well as optical imaging have been studied intensively.1−6 CT imaging could provide excellent image details of hard-tissue contrast but suffers from limited sensitivity at the cellular level.7−9 MR imaging is often used to identify soft tissue lesions; however, it is insensitive to low signal areas.10−12 Optical imaging could achieve single molecule sensitivity but it could not obtain anatomical details.13−15 By taking the advantages of different imaging techniques, nanoparticulatebased multimode imaging has great potential to improve the accuracy for diagnosis in clinic.16−18 Over the past few years, lanthanide (Ln3+)-doped upconversion nanoparticles (UCNPs) have evoked considerable attention in many fields, including biological imaging and labeling technology in the biological specimens.19−22 The main © XXXX American Chemical Society

Received: July 8, 2016 Accepted: October 3, 2016

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DOI: 10.1021/acsami.6b08335 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Research Article

ACS Applied Materials & Interfaces application for multimode imaging.45,46 In the previous reports, great effort has been directed toward enhancing UCL for biological applications, especially for bioimaging. Doping is an effective method to obtain the desired properties and functions of the material.30 Zhang’s group reported the enhancement of UCL of Er3+ by incorporating Li+ into Y2O3:Yb,Er nanoparticles.47 Wu’s group verified the UCL of NaYF4:Yb/Er nanocrystals could be enhanced by Mo3+ doping.48 Xiao’s group showed the enhancement of UCL in Zn2SiO4:Yb3+,Er3+ by codoping with Li+ or Bi3+.49 For in vivo imaging probes, high doses of contrast agent are usually required to get distinct signal, which may induce side effects. The effective way is to improve the imaging contrast signal and reduce the dosage. The preparation of optimal contrast agents with high contrast efficiency is urgent for clinical use. Whereas, it is difficult to improve or enhance different kinds of imaging signals in one kind of UCNPs simultaneously. As far as we know, the UCL of the fluorides UCNPs can be enhanced after doping Bi3+ ions by tailoring the host lattice and local the crystal field.50 Meanwhile, bismuth possesses good X-ray attenuation properties (Z = 83, K-edge value = 90.5 keV, X-ray attenuation coefficient at 100 keV = 5.74 cm2 g−1).51 However, the low doping concentration of Bi3+ ions in the UCNPs could only enhance the UCL intensity but have little effect on CT contrast efficiency. Therefore, optimizing the doping concentration of Bi3+ ions in NaGdF4 UCNPs could acquire both efficient UCL and CT signals, which is important for the further bioimaging application. Herein, monodisperse NaGdF4:Yb3+,Tm3+,x% Bi3+ (abbreviated as GYT-x% Bi3+, x = 0, 5, 10, 15, 20, 25, 30) upconversion nanoparticles (UCNPs) have been prepared through the solvothermal method. For GYT-25% Bi3+ UCNPs, not only the near-infrared (NIR) UCL intensity but also the Xray CT contrast efficiency can be enhanced remarkably. Compared with NaGdF4:Yb3+,Tm3+ (GYT) UCNPs, the NIR emission intensity of GYT-25% Bi3+ UCNPs could be enhanced by a factor of ∼60. Moreover, GYT-25% Bi3+ UCNPs with high ratio of NIR/blue UCL intensity could reduce the damage to tissues in bioimaging process. More importantly, the CT contrast efficiency could be improved significantly by doping Bi3+ ions. Gadolinium element exists in GYT-25% Bi3+ UCNPs further endows them with MR imaging capability. After functionalizing citric acid molecules on the surface, GYT-25% Bi3+ UCNPs show the low cytotoxicity through the MTT assay on HeLa cells. Then, the performances of citrate-coated GYT-25% Bi3+ UCNPs as efficient trimodal imaging probe in vivo were investigated.

was added to the solution of EG (20 mL) containing a certain quantity of NH4F under vigorous stirring. The resulting mixture was stirred for 1 h at room temperature, then transferred to a 50 mL Teflon-lined autoclave, and subsequently heated at 200 °C for 7 h. Then, the system was cooled down to room temperature naturally. The UCNPs were collected through centrifugation and washed with ethanol and water for three times, and then dried in vacuum at 80 °C for 8 h. In addition, monodisperse NaGdF4:20% Yb3+,2% Er3+,x% Bi3+ and NaGdF4:20%Yb3+,2% Ho3+,x% Bi3+ (x = 0, 5, 10, 15, 20, 25, 30) UCNPs were obtained by using the same method. 2.2.2. Preparation of Citrate-Coated GYT-25% Bi3+ UCNPs. To obtain citrate-coated GYT-25% Bi3+ UCNPs, 300 mg of trisodium citrate was dissolved in water (10 mL), then 100 mg of the ligand-free GYT-25% Bi3+ UCNPs was added to the above solution under stirring. After 3 h, the reaction was completed and the citrate-coated GYT-25% Bi3+ UCNPs were obtained through centrifugation and washed with water. The citrate-coated GYT UCNPs were obtained by using the same method. 2.3. Characterization. The crystal structures and phase purities of the products were investigated by powder X-ray diffraction (XRD) with a D8 Focus diffractometer (Bruker) with Cu Kα radiation (λ = 1.5418 Å) with an operation voltage and current maintained at 40 kV and 40 mA. Fourier transform-infrared spectroscopy (FT-IR) was recorded on a PerkinElmer 580B IR spectrophotometer using the KBr pellet technique. The composition of the samples were studied using a field-emission scanning electron microscope (FE-SEM, S-4800, Hitachi) equipped with an energy-dispersive X-ray (EDX) spectrometer. Low-/high-resolution transmission electron microscopy (TEM) were carried out on a FEI Tecnai G2S-Twin instrument with a fieldemission gun operating at 200 kV. X-ray photoelectron spectroscopy (XPS) measurements were conducted with a VG ESCALAB MKII spectrometer. The UCL spectra were recorded by using a 980 nm laser diode and a triple grating monochromator (Spectra Pro-2758, Acton Research Corporation) equipped with a photomultiplier (Hamamatsu R928). The decay curve measurements were performed and analyzed with a LeCroy WaveRunner 6100 1 GHz oscilloscope. All the measurements were performed at room temperature. 2.4. Cytotoxicity Assay. HeLa cells were cultured in a 96-well plate overnight under the conditions of 37 °C and 5% CO2. The citrate-coated GYT-25% Bi3+ UCNPs suspensions with different concentration (0, 15.63, 31.25, 62.5, 125, 250, 500, 1000 μg/mL) were added to the culture medium and incubated for 24 h. Then, 20 μL of MTT solution in PBS (5 mg/mL) was added to each well and the plates were incubated at the same condition for an additional 4 h. Subsequently, the medium containing MTT was removed and DMSO (150 μL) was added to each well to dissolve the MTT formazan crystals formed. Finally, the plates were shaken for 10 min and the absorbance of formazan was recorded at 490 nm by a microplate reader. 2.5. Animal Experiments. Kunming mice were purchased from the Laboratory Animal Center of Jilin University (Changchun, China). Animal care and handing procedures were in agreement with the guidelines of the Regional Ethics Committee for Animal Experiments. The tumor models were established by subcutaneous injection of H22 cells in the left axilla of each mouse. The mice were used for experiments when the tumors had grown to reach the size of around 200 mm3. 2.6. In Vivo NIR-to-NIR UCL Imaging. First, the tumor-bearing mouse was anesthetized with 10% chloral hydrate through intraperitoneal injection. Then, the solution of citrate-coated GYT-25% Bi3+ UCNPs was injected into the tumor site of the Kunming mouse. After that, NIR-NIR UCL imaging of the tumor area was acquired on an in vivo Maestro whole-body imaging system. Moreover, the wholebody UCL imaging was also performed after intravenous injection of GYT-25% Bi3+ UCNPs solution (8 mg/mL, 200 μL) for 60 min. Then, the internal organs were dissected for ex vivo UCL imaging. 2.7. In Vitro and in Vivo X-ray CT Imaging. For in vitro CT imaging, the solutions of citrate-coated GYT-25% Bi3+, citrate-coated GYT, and iobitridol with the concentrations of (Gd + Yb + Bi), (Gd + Yb), and I ranging from 0 to 30 mg/mL, respectively, were scanned to

2. EXPERIMENTAL SECTION 2.1. Materials. Analytical grade Bi(NO3)3·5H2O (99%) and NaNO3 (99%) were purchased from Aladdin Reagents (Shanghai, China). Gd(NO3)3·6H2O (99%), Yb(NO3)3·5H2O (99.9%), Tm(NO3)3·5H2O (99.9%), Er(NO3)3·5H2O (99.9%), and Ho(NO3)3· 5H2O (99.9%) were purchased from Alfa. Trisodium citrate (99%), ethylene glycol (EG), and NH4F (98%) were obtained from Beijing Chemical Reagents (Beijing, China). All reagents were used without further purification. 2.2. Synthesis of Trimodal Imaging Bioprobes. 2.2.1. Preparation of Monodisperse NaGdF4:Yb3+,Tm3+,x% Bi3+ (abbreviated as GYT-x% Bi3+, x = 0, 5, 10, 15, 20, 25, 30) UCNPs. The UCNPs were synthesized by using the solvothermal method. In a typical synthesis route, the solution of EG (30 mL) containing Gd(NO3)3· 5H2O ((79.5 − x)% mol), Yb(NO3)3·5H2O (20% mol), Tm(NO3)3· 5H2O (0.5% mol), Bi(NO3)3·5H2O (x% mol), and NaNO3 (1 mmol) B

DOI: 10.1021/acsami.6b08335 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

indicated more and more Bi3+ ions incorporated into the NaGdF4 matrix. Furthermore, the incorporation of Bi3+ ions into NaGdF4 lattice was proved by EDX (Figure S2, Supporting Information) and XPS analysis (Figure S3, Supporting Information). The two peaks centered at 160.2 and 165.4 eV in XPS spectrum (Figure S3b, inset, Supporting Information) are assigned to the Bi 4f7/2 and Bi 4f5/2 peaks of Bi3+ ions, respectively. The morphology of as-prepared UCNPs were characterized by TEM. Figure 2a−g show the typical TEM images of GYT-x % Bi3+ (x = 0, 5, 10, 15, 20, 25, 30) UCNPs. As demonstrated in Figure 2a, the size of GYT UCNPs is about 95 nm. It could be observed that the size of Bi3+ ions doped UCNPs almost unchanged compared with GYT UCNPs (Figure 2d−g). Figure 2h shows the hexagonal phase (110) crystal plane with interplanar spacing of 0.302 nm. 3.2. Upconversion Luminescence Properties. To study the effect of Bi3+ ions on UCL intensity, the UCL spectra of GYT-x% Bi3+ (x = 0, 5, 10, 15, 20, 25, 30) were measured under 980 nm excitation. The UCL spectra of all the UCNPs exhibit characteristic UC emissions of Tm3+, including a strong NIR emission band (802 nm, 3H4 → 3H6) and a very weak blue emission band (476 nm, 1G4 → 3H6). With increasing Bi3+ ions concentration from 0 to 25%, the UCL intensity enhanced drastically and achieved the maximum when the Bi3+ ions concentration reach at 25% (Figure 3a). The UCL intensity decreases with further increasing the Bi3+ ions doping concentration, indicating that the best doping concentration of Bi3+ ions is 25%. Figure 3b shows the intensities of NIR (3H4 → 3H6) and blue (1G4 → 3H6) emissions as a function of the Bi3+ ions doping concentration. As can be seen, the changing trend of both emissions is identical. Especially, for GYT-25% Bi3+ UCNPs, the intensity of NIR emission centered at 802 nm is distinctly enhanced by a factor of ∼60, when comparing with GYT UCNPs. Importantly, the strong NIR emission of GYT25% Bi3+ UCNPs was almost 150 times than the blue emission. Compared with Bi3+ ions doped GYT, the UCL intensity of Bi3+ ions doped NaGdF4:Yb3+,Er3+(Ho3+) (Figures S4 and S5, Supporting Information) have similar changing trend with the change of Bi3+ ions concentration and the optimal doping concentration is also 25%. Although the UCL intensity of 25% Bi3+ ions doped NaGdF4:Yb3+,Er3+(Ho3+) have been improved than Bi3+-free sample, the multiple of the enhanced UCL intensity is smaller than GYT-25% Bi3+. Therefore, such strong NIR UCL and high ratio of NIR/blue UCL intensity makes GYT-25% Bi3+ UCNPs potential as a probe for deep tissue NIR-to-NIR UCL imaging. Furthermore, the decay curves of 3H4 → 3H6 transition (802 nm) of Tm3+ in GYT-x% Bi3+ (x = 0, 5, 10, 15, 20, 25, 30) UCNPs were measured to prove the enhancement of UCL theoretically. Figure 3c shows the normalized decay profiles of 3 H4 → 3H6 transition in GYT-x% Bi3+. All the decay curves could be well-fitted by double exponential function: I (t) = I0 + A1 exp(−t/τ1) + A2 exp(−t/τ2),52 where A1 and A2 are constants; I (t) and I0 represents the luminescence intensities at time t and 0, τ1 and τ2 refer to two components of the luminescence lifetime, which correspond to the rapid and slow lifetimes for exponential components, respectively. The lifetimes of the 3H4 states of Tm3+ are presented in Table S1 (Supporting Information). The average decay lifetimes of Bi3+ doping samples are all longer than that of Bi3+-free sample. Figure 3d shows the lifetimes of 3H4 states of Tm3+ as a function of Bi3+ ions doping concentrations. The prolonged

assess CT contrast efficacy of these contrast agents. For in vivo CT imaging, the tumor-bearing mouse was anesthetized and scanned before and after intratumoral injection of citrate-coated GYT-25% Bi3+ UCNPs solution (50 μL). The time-dependent biodistribution of citrate-coated GYT-25% Bi3+ UCNPs in the mouse was tracked by the CT contrast signals before and after injection of the UCNPs (30 mg (Gd + Yb + Bi) mL−1, 150 μL) through the tail vein. X-ray CT images were acquired on a Philips 256-slice CT scanner. The operating parameters are as follows: 120 kVp, 300 mA; thickness, 0.9 mm; pitch, 0.99; field of view, 350 mm; gantry rotation time, 0.5 s; table speed, 158.9 mm s−1. 2.8. In Vitro and in Vivo T1-Weighted MR Imaging. For in vitro MR imaging in solution, citrate-coated GYT-25% Bi3+ UCNPs were dispersed in water with Gd concentrations ranging from 0 to 5 mM. The longitudinal relaxivity values (r1) were acquired on a Huantong 0.5 T MR scanner. For in vivo MR imaging, the tumor bearing mouse was anesthetized and scanned before and after intratumoral injection of citrate-coated GYT-25% Bi3+ UCNPs by using a 1.5 T human clinical scanner (Siemens Medical System).

3. RESULTS AND DISCUSSION 3.1. Phase and Morphology. First, XRD technique was used to analyze the structure and composition of the asprepared UCNPs. The diffraction patterns of GYT-x% Bi3+ (x = 0, 5, 10, 15, 20, 25, 30) can be indexed to the standard hexagonal phase of NaGdF4 (JCPDS 27-0699) (Figure 1). No

Figure 1. XRD patterns of GYT-x% Bi3+ UCNPs (x = 0, 5, 10, 15, 20, 25, 30).

extra impurity diffraction peaks are observed in all samples even the Bi3+ ions concentration reach 30%. Compared with hexagonal phase of NaGdF4, the diffraction peaks of Bi3+-free sample shift slightly to higher-angle direction due to the substitution of Gd3+ (r = 1.193 Å) by the smaller Yb3+ (r = 1.125 Å) (Figure S1, blue dots line, Supporting Information). Compared with the Bi3+-free sample, the diffraction peaks of GYT-x% Bi3+ UCNPs shift slightly to the lower-angle side results from the increasing of the unit-cell volume, because of the substitution of Gd3+ by Bi3+ (r = 1.31 Å) in the host lattice (Figure S1, red dots line, Supporting Information). In addition, the shifting trend of peaks increases with the Bi3+ ions concentration increasing from 5 to 30%, which could be C

DOI: 10.1021/acsami.6b08335 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Figure 2. TEM images of GYT-x% Bi3+ UCNPs. (a) x = 0, (b) x = 5, (c) x = 10, (d) x = 15, (e) x = 20, (f) x = 25, (g) x = 30. (h) HRTEM image of GYT-25% Bi3+ UCNPs.

Figure 3. UCL properties of GYT-x% Bi3+ UCNPs. (a) UCL spectra of GYT-x% Bi3+ UCNPs under a 980 nm laser excitation. (b) The NIR and blue UCL intensities of GYT-x% Bi3+ UCNPs as a function of Bi3+ ions doping concentrations (Inset: the amplifying of the blue UCL intensity). (c) Decay profiles of the 3H4 → 3H6 transition in the GYT-x% Bi3+ UCNPs under a 980 nm laser excitation. (d) The lifetimes of the 3H4 state of Tm3+ as a function of Bi3+ ions doping concentrations. x = 0, 5, 10, 15, 20, 25, and 30.

lifetimes might arise from the tailored local crystal field environment of Tm3+ by doping Bi3+ ions.49 3.3. Biocompatibility assessment of UCNPs. For further application in biological evaluation and imaging, the citric acid molecules were used to modify the UCNPs. The successful modification of the citric acid molecules was confirmed by FTIR spectra (Figure S6a, Supporting Information). TGA curve further verified about 2.7 wt % citric acid molecules on the surface of UCNPs (Figure S6b, Supporting Information). Prior to using the citrate-coated GYT-25% Bi3+ UCNPs for in vivo bioimaging, we carried out the cytotoxicity test on HeLa cell line by using the standard MTT method. After incubation with UCNPs for 24 h, the viability of HeLa cells was still high

(Figure S7, Supporting Information). The cell viability is maintained at 90% at the concentration up to 1000 μg mL−1, suggesting the UCNPs have low cytotoxicity and good biocompatibility. 3.4. In Vivo NIR-to-NIR UCL Imaging. Optical imaging could visualize the morphological details without invading the animal tissue.19 To verify the ability of citrate-coated GYT-25% Bi3+ UCNPs for deep tissue imaging, different thicknesses of lean pork were used to evaluate the quantitative imaging penetration depth of UCNPs excited by 980 nm laser. The citrate-coated GYT-25% Bi3+ UCNPs exhibit stronger UCL signal than the citrate-coated GYT UCNPs at the equivalent thickness of lean pork (Figure 4). Encouragingly, the UCL D

DOI: 10.1021/acsami.6b08335 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

(Figure 6a). Good linear relationship between Hounsfield units (HU) value and the concentration of (Gd + Yb + Bi), (Gd +

Figure 4. In vitro NIR-to-NIR UCL imaging of different thickness of lean pork upon citrate-coated GYT-25% Bi3+ and GYT UCNPs.

signal of GYT-25% Bi3+ UCNPs could be detected even the thickness was increased to 20 mm. The excellent result indicates that citrate-coated GYT-25% Bi3+ UCNPs could be used as a promising NIR-to-NIR UCL probe for deep tissue imaging. To verify this point, the citrate-coated GYT-25% Bi3+ UCNPs were injected into the tumor site of the mouse (Figure 5a). As shown in Figure 5b, intense UCL signal could be

Figure 6. Evaluation of the in vitro CT efficiency of citrate-coated GYT-25% Bi3+, citrate-coated GYT, and iobitridol. (a) In vitro CT images of solutions of citrate-coated GYT-25% Bi3+, citrate-coated GYT, and iobitridol with different concentrations. (b) The CT value (HU) of citrate-coate GYT-25% Bi3+, citrate-coated GYT, and iobitridol as a function of the concentrations of (Gd + Yb + Bi), (Gd + Yb), and iobitridol, respectively. Figure 5. In vivo UCL images of the mouse after intratumoral injection of citrate-coated GYT-25% Bi3+ UCNPs: (a) bright field, (b) UCL, and (c) overlay images.

Yb), or iobitridol can be observed (Figure 6b). Obviously, the HU values of citrate-coated GYT-25% Bi3+ are always much higher than that of citrate-coated GYT and iobitridol at equivalent concentration, indicating citrate-coated GYT-25% Bi3+ UCNPs are efficient contrast agent for CT imaging. The above results prompted us to verifiy the ability of GYT25% Bi3+ UCNPs as the efficient contrast agent for in vivo CT imaging. First, the citrate-coated GYT-25% Bi3+ UCNPs was injected into the tumor site of the mouse. As exhibited in Figure 7, the obvious enhancement of CT signal could be observed at the tumor site. For coronal position, the HU value of the tumor site is 791.9 HU (Figure 7d). However, it could be observed that the HU value is 632.1 HU in the coronal position

observed at the tumor site. This could be further confirmed by the overlay image (Figure 5c). The UCL imaging with high signal-to-noise ratio indicates that the UCNPs are excellent in vivo NIR-to-NIR UCL imaging probe. Furthermore, we also studied whole-body UCL imaging of Kunming mice through intravenous injection of GYT-25% Bi3+ UCNPs. The wholebody images are shown in Figure S8 (Supporting Information), GYT-25% Bi3+ UCNPs were gathered in the liver and spleen were proved in the overlay image. The encouraging results indicate the GYT-25% Bi3+ UCNPs have the ability for the whole-body UCL imaging. 3.5. In Vitro and in Vivo X-ray CT Imaging. X-ray CT imaging is commonly used technique in clinical diagnosis due to the deep penetration and high resolution.53,54 Because Gd, Yb, and Bi elements possess high X-ray absorption coefficients, the high contrast efficiency of citrate-coated GYT-25% Bi3+ UCNPs as the CT contrast agent could be expected. Bismuth (5.74 cm2 g−1) possesses higher mass attenuation coefficient than gadolinium (3.11 cm2 g−1) and iodine (1.94 cm2 g−1) at 100 keV. Moreover, Bi3+ ion is nontoxic and highly costeffective.55−57 When doping Bi3+ ions in the GYT UCNPs, it could be expected that GYT-25% Bi3+ UCNPs exhibit much better CT signal than GYT UCNPs and iobitridol contrast agents. Thus, we compared the CT imaging signal of citratecoated GYT-25% Bi3+ with that of citrate-coated GYT and iobitridol (iodine-based clinical contrast agent) in vitro. Accordingly, with increasing the concentration of imaging elements, the CT signals of the three contrast agents enhanced

Figure 7. In vivo CT images of a tumor-bearing mouse before and after intratumoral injection of the citrate-coated GYT and GYT-25% Bi3+ UCNPs: (a and b) preinjection and (c and d) after intratumoral injection. The tumor site was marked by red circles. E

DOI: 10.1021/acsami.6b08335 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

Figure 8. In vivo CT coronal view images of a mouse after intravenous injection of the citrate-coated GYT-25% Bi3+ UCNPs at timed intervals. (a) Heart (circles) and liver (ellipses). (b) Spleen (rectangles) and kidney. (c,d) The corresponding 3D renderings of in vivo CT images.

nanoparticles were expected to exhibit MR imaging capability.60,61 To examine the capability of the citrate-coated GYT25% Bi3+ UCNPs as MR imaging constrast agent, the T1weighted MR images of UCNPs were recorded in vitro under a 0.5 T MR scanner. Figure S11a (Supporting Information) shows the MR signal enhancement with increasing the Gd3+ concentrations. These brighten T1-weighted MR images indicate the citrate-coated GYT-25% Bi3+ UCNPs could be used as MR imaging agent. The longitudinal relaxivity values (r1) calculated from the slope of Gd3+ concentration-dependent relaxation rate is 0.9427 mM−1 s−1 (Figure S11b, Supporting Information). Furthermore, we demonstrated the T1-weighted MR imaging in a tumor-bearing mouse. After in situ injection of the UCNPs for 30 min, the contrast enhancement could be observed at the tumor site (Figure S11c,d, Supporting Information). The above results suggest that the citrate-coated GYT-25% Bi3+ UCNPs could be potentially applied for T1weighted MR imaging.

after injecting the GYT UCNPs at equivalent concentration (Figure 7c). This result further confirmed that the CT signal could be significantly enhanced by optimizing the doping concentration of Bi3+ in UCNPs. Furthermore, the timedependent in vivo CT imaging of the citrate-coated GYT-25% Bi3+ UCNPs was tracked after intravenous injection. The obvious CT signal appeared in the heart, liver, and spleen, except the kidney (Figure 8). After injection for 5 min, the HU value of heart changed from 53 to 77.3. The CT signals of liver and spleen were increased gradually for 120 min. Moreover, the 3D-renderings of CT images further confirmed the contrast signals in the liver and spleen. Figure S9 (Supporting Information) showed the changing of HU values of these organs over time. The long-term blood circulation of nanoparticles is very important for disease diagnosis. The obvious CT signal could be observed in the liver and spleen organs even at 120 min, which probably reflected the uptake of citrate-coated GYT-25% Bi3+ UCNPs by macrophages and the feature might diagnose the hepatic metastases.58,59 In addition, the citrate-coated GYT-25% Bi3+ UCNPs were intravenously injected into a tumor-bearing mouse. As shown in Figure S10 (Supporting Information), a sustained increase of the CT signal in the tumor site was observed from 0 to 2 h. The obvious CT signal could be observed in the tumor site even at 24 h after intravenous injection, indicating the UCNPs could accumulate in the tumor via enhanced permeability and retention (EPR) effect. These desirable results proved that GYT-25% Bi3+ have great potential as efficient CT imaging contrast agent in vivo. 3.6. In Vitro and in Vivo MR Imaging. MR imaging has a wide range of applications in medical diagnosis. Because Gd3+ ion possesses seven unpaired 4f electrons, the gadolinium-based

4. CONCLUSION In summary, we have successfully synthesized monodisperse GYT-x% Bi3+ (x = 0, 5, 10, 15, 20, 25, 30) UCNPs through the solvothermal method. The NIR UCL intensity has been enhanced about 60 times when doping 25% Bi3+ ions. The citrate-coated GYT-25% Bi3+ UCNPs with strong NIR UCL could be applied as excellent NIR-to-NIR UCL imaging bioprobe for deep tissue imaging. The significant improvement of CT signal and the efficient in vivo CT imaging have also been realized by doping 25% Bi3+ ions in GYT. In addition, the citrate-coated GYT-25% Bi3+ UCNPs could also be employed as the contrast agent for T1-weighted MR imaging. Therefore, F

DOI: 10.1021/acsami.6b08335 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

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simultaneous remarkable enhancement of NIR UCL and CT signals could be achieved by incorporating the optimal concentration of Bi3+ ions in GYT UCNPs. The citrate-coated GYT-25% Bi3+ UCNPs could be applied as safe and efficient probe for trimodal imaging.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b08335. Figure S1−S11, Table S1. Magnified XRD patterns; EDX spectra; XPS survey spectra; the UCL properties of NaGdF4:Yb3+,Er3+ (Ho3+),x% Bi3+ UCNPs; FT-IR spectra and thermogravimetric analysis; in vitro cell viability of HeLa cells; whole-body NIR-to-NIR UCL imaging of a mouse; the HU values of different organs at timed intervals; in vivo CT images of a tumor-bearing mouse; in vitro and in vivo T1-weighted MR imaging; and the lifetimes of the 3H4 states of Tm3+ in GYT-x% Bi3+ (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *Phone: +86 431 85262127. Fax: +86 431 85698041. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful for the financial aid from the National Natural Science Foundation of China (Grants 21371165, 21590794, 51372242, 21210001, 21521092, and 21501167), the Hong Kong, Macao and Taiwan Science and Technology Cooperation Special Project of Ministry of Science and Technology of China (Grant 2014DFT10310), the National Key Basic Research Program of China (Grant 2014CB643802), the Strategic Priority Research Program of the Chinese Academy of Sciences (Grant No. XDB20030300), and the Youth Innovation Promotion Association of Chinese Academy of Sciences (Grant 2015181).

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REFERENCES

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