Surfactant-Free Aqueous Synthesis of Novel Ba2GdF7:Yb3+, Er3+@

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Surfactant-Free Aqueous Synthesis of Novel BaGdF:Yb , Er @PEG Upconversion Nanoparticles for in Vivo Trimodality Imaging Yang Feng, Hongda Chen, Lina Ma, Baiqi Shao, Shuang Zhao, Zhenxin Wang, and Hongpeng You ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b03411 • Publication Date (Web): 14 Apr 2017 Downloaded from http://pubs.acs.org on April 15, 2017

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ACS Applied Materials & Interfaces

Surfactant-Free Aqueous Synthesis of Novel Ba2GdF7:Yb3+, Er3+@PEG

Upconversion

Nanoparticles

for

in

Vivo

Trimodality Imaging Yang Feng,†,‡ Hongda Chen,§, ‡ Lina Ma,§ Baiqi Shao,† Shuang Zhao,†, ‡ Zhenxin Wang,*,§ and 5

Hongpeng You *,† †

State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of Applied Chemistry, University of Chinese

Academy of Sciences, Changchun 130022, P. R. China §

State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, University of Chinese Academy

of Sciences, Changchun 130022, P. R. China

10

‡

University of Science and Technology of China, Hefei 230026, P. R. China

ABSTRACT In this work, we develop the surfactant-free aqueous synthesis of novel polyethylene glycol (PEG) coated Ba2GdF7:Yb3+, Er3+ upconversion nanoparticles (named as, Ba2GdF7:Yb3+, Er3+@PEG UCNPs) for in vivo multi-modality imaging including upconversion luminescence (UCL), X-ray computed tomography (CT) , and

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T1-weighted magnetic resonance (MR). The as-prepared Ba2GdF7:Yb3+, Er3+@PEG UCNPs not only present bright UCL and reasonably high CT/MR enhancements, but also exhibit excellent colloidal stability, inappreciable cytotoxicity and negligible organ toxicity. In particular, the Ba2GdF7:Yb3+, Er3+@PEG UCNPs emit red UCL with high intensity in the tumor site after intravenous injection via tail vein of nude mouse. The Ba2GdF7:Yb3+, Er3+@PEG UCNPs as contrast agents exhibit high performance for in vivo trimodality (UCL/CT/MR) imaging of

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tumor during HepG2 tumor bearing nude mouse experiments. 1. INTRODUCTION Bioimaging techniques including fluorescence/luminescence imaging, X-ray computed tomography imaging (CT), and magnetic resonance imaging (MRI) have been extensively employed for diagnosing various kinds of diseases during last decades.1-3 Because these imaging techniques have different sensitivities, spatial resolutions, imaging

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depths and inherent defects rooted in instrument limitation, single-modality imaging method usually cannot provide sufficient information to meet the high demands for the efficiency and accuracy at clinical diagnostics and theranostics.4-6 For instance, in vivo fluorescence/luminescence imaging is limited, due to the low tissue penetration depth of the as-obtained visible light, CT can not provide efficient information on soft-tissues, and the sensitivity of MRI is relatively lower than those of other in vivo imaging methods.7, 8 It is believed that the 1

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nanoparticle-based multimodal imaging probes have great promise to circumvent shortcomings of single-modality imaging.9 In particular, the NaGdF4 host lattice doped with lanthanide combinations of Yb3+, Er3+, or Yb3+, Tm3+ upconversion nanoparticles (UCNPs) have been used for in vivo multi-modality (upconversion luminescence (UCL)/CT/MRI) imaging exhibiting excellent contrast efficiency.10, 11 Under near-infrared (NIR) irradiation, the

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UCNPs produce high quality optical images of deeper tissues.12-14 Moreover, the UCNPs are ideal CT and MRI contrast agents since the Gd element has unique magnetic resonance (MR), high X-ray attenuation and large atomic number properties.15 It is well known that barium (Ba) is one of the most efficient CT contrast agent owing to its high X-ray mass absorption coefficients and large K-edge values.16, 17 There is a particular interest in the field of material science to

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seek an ideal host matrix for avoiding the leaching of Ba2+ and Gd3+ ions and introducing UCL, CT, and MR imaging function in one system since free Ba2+ and Gd3+ are toxic.18 Meanwhile monodisperse inorganic nanomaterials with uniform morphologies possess the potential technological applications in biomedicine and bioanalysis.19 Therefore, some inorganic nanomaterials containing Gd3+ or Ba2+ ions have been investigated in the past decade, including NaGdF4,20 GdPO4,21 BaGdF5,22 etc.. Among a variety of developed upconversion (UC)

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hosts, fluorides are regarded as the most efficient materials for UCL because they possess low phonon energy and stable physicochemical properties.23-25 For instance, BaGdF5 is demonstrated as an efficient optical-magnetic matrix by several groups.26 The BaGdF5:Yb3+, Er3+ UCNPs show highly efficient UCL emission.27 In our previous work, Ba2GdF7 nanoparticle with pseudo-octahedron structure is successfully synthesized via the hydrothermal method.28 Ba2GdF7 is expected to be more efficient matrix for multi-modality bioimaging than that of BaGdF5

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since Ba2GdF7 possesses relative high X-ray mass absorption coefficients and large K-edge values. In this work, we have developed a surfactant-free method to obtain monodisperse and uniform Ba2GdF7: Yb3+, Er3+ UCNPs by a simple and scalable hydrothermal method. After coating with polyethylene glycol (PEG), the as-prepared UCNPs exhibit excellent biocompatibility, bright red UCL emission and strong CT/MR enhancements, which highlight the clinical potential application as trimodal (UCL/CT/MR) contrast agents for tumor diagnosis.

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2. EXPERIMENTAL SECTION 2.1. Materials. The Ln2O3 (Ln = Gd, Yb, Er, and Tm (99.99%) were received from Shanghai Yuelong Non-Ferrous Metals Ltd. (Shanghai, China). Ln(NO3)3 stock slution was obtained by dissolving Ln2O3 in stoikiometric concentrated HNO3 under heating. Fetal bovine serum (FBS) and Dulbecco’s modified Eagle’s medium

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(DMEM)

were

purchased

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium

from

Gibco

bromide

was

Inc. obtained


(New

York,

USA).

from

Beijing

Dingguo 2

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Biotechnology Ltd. (Beijing, China). HepG2 cell line was obtained from the Shanghai Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Other chemicals were all supplied by Beijing Chemical Reagent Co. (Beijing, China). Ultra-purified water was collected from Milli-Q water (18.2 MΩ·cm) in all experiments. 2.2. Characterization. X-ray powder diffraction spectra were recorded with a D8 Focus diffractometer

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(Rigaku Co., Japan). The infrared spectra were obtained by a Perkin-Elmer 580B infrared spectrophotometer (Perkin-Elmer Co., USA) with the KBr pellet technique. The transmission electron microscopy (TEM) and high-resolution transmission electron microscopy (HRTEM) images were carried out with a JEOL-2010 transmission electron microscope (JEOL Co., Japan) operating at 200 kV with an energy dispersive X-ray spectroscopy (EDS) system (Oxford instrument Ltd., UK). The DLS and Zeta potential of the samples were

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recorded on a Zetasizer Nano ZS (Malvern Instruments Ltd., UK). The element analysis was obtained by an ELAN 9000/DRC ICP-MS system (Perkin Elmer, USA). The upconversion luminescent spectra were recorded with a 980 nm laser from an OPO (optical parametric oscillator, Continuum Sunlite, USA) as the excitation source. The field-dependent magnetization curve was obtained from Quantum Design-MPMS-XL7 SQUID magnetometer (Quantum Design, USA). All the measurements were carried out at room temperature.

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2.3. Synthesis of UCNPs. A total 1 mmol of Gd(NO3)3, Yb(NO3)3, and Er(NO3)3 (lanthanide ion molar ratio, Gd/Yb/Er = 80:18:2) were injected into 15 mL of DI water. The solution was stirred at room temperature for 10 min, respectively. Then, several drops of NH3·H2O (25 wt %) were added into the above solution until the pH value was 8. After the solution was stirred for 1 h, 5 mL of deionized water with Barium nitrate (2 mmol) was poured into the reaction mixture and kept for another 30 min. Then adding NaF (20 mmol in 15 mL H2O), the

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reaction mixture was sealed in a 50 mL Teflon-lined autoclave and heated at 180 °C for 24 h. When the vessel was cooled to room temperature in ambient conditions, the white products were centrifuged (8500 rpm for 10 min), and washed three times with DI H2O (35 mL) to remove other remnants. Finally, the Ba2GdF7:Yb3+, Er3+ UCNPs were redispersed in H2O (15 mL). 2.4. Synthesis of UCNPs@PEG. In brief, 15 mL as-obtained Ba2GdF7:Yb3+, Er3+ UCNPs were dispersed into

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25 mL of an aqueous solution with 1g PEG (MW=2000) by vigorous magnetic stirring for 10 min at room temperature. The obtained mixture was sealed in a 50 mL Telfon-lined autoclave and further heated at 180 °C for 24 h. After naturally cooling to room-temperature, the Ba2GdF7:Yb3+,Er3+@PEG UCNPs were obtained by centrifugation, washed by H2O (30 mL, one time), and dried at 65 °C for 12 h. 2.5. Cell viability assay. To quantitatively evaluate the cytotoxicity of the Ba2GdF7:Yb3+, Er3+@PEG UCNPs,

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Human HepG2 cells were used in vitro cytotoxicity test. Briefly, HepG2 cells were cultured in fresh DMEM 3



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medium containing FBS (10%, v/v) under a humidified atmosphere of 5% CO2 at 37 °C and incubated overnight. 8000 cells were seeded in a 96 well plates per well. Then, HepG2 cells were incubated with the desired amounts of Ba2GdF7:Yb3+, Er3+@PEG UCNPs for 24 h. After washed with PBS (300 µL, two times), the relative cell viabilities of HepG2 cells were obtained by the typical MTT assay. The untreated HepG2 cells were used as a

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control sample, the absorbance in each well were determined by using the optical densities of microplate reader at 490 nm. 2.6. MR and CT Imaging of phantom. The 1.5 mL Eppendorf tubes containting various concentrations of Ba2GdF7:Yb3+, Er3+@PEG UCNPs aqueous solutions were prepared, respectively. T1-weighted MRI images were performed on a GE Signa 1.5-T unit (General Electric Co., Milwaukee, WI, USA) operating imaging parameters:

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repetition time, 5000 ms; an echo time, 9 ms; seven inversion recovery times (TI = 75, 150, 300, 600, 1200, 2400, and 4800 ms); field of view, 102×72 mm2; slice thickness, 4.0 mm. The r1 value of Ba2GdF7:Yb3+, Er3+@PEG UCNPs was obtained by the curve fitting of 1/T1 relaxation time (s−1) versus the Gd concentration (mM). CT images were recorded on a 64-detector row CT unit (General Electric Co., Milwaukee, WI, USA) using imaging parameters: thickness, 0.6 mm; pitch, 0.99; 120 kVp, 300 mA; field of view, 103 mm; gantry rotation time, 0.5 s;

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and table speed, 15.9 mm s-1. 2.7. In vitro UCL, CT and MR imaging. The HepG2 cells were seeded, cultured and treated by desired amounts of Ba2GdF7:Yb3+, Er3+@PEG UCNPs as previously description. After washed by 300 µL PBS (pH=7.4, three times), the Ba2GdF7:Yb3+, Er3+@PEG UCNPs stained cells were directly subjected to UCL imaging under an external 980 nm laser (0.6 Wcm−2), and the exposure time was 6 s. For in vitro CT and MR imaging, the

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Ba2GdF7:Yb3+, Er3+@PEG UCNPs stained cells were detached by 1 mL of EDTA (Gibco Co., China). Then, the cells were centrifuged at 1000 rpm for 5 min. Finally, the cells were transferred to 1.5 mL Eppendorf tubes, and immobilized by 1% agarose. The CT and T1-weighted MR images were collected by a GE Signa 1.5-T MR unit and 64-detector row CT unit as previously described, respectively. 2.8. In vivo UCL, CT and MR imaging. Animal experiments were conformed to the guidelines of the

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Regional Ethics Committee for Animal Experiments established by Jilin University Institutional Animal Care and Use. 5 to 6 weeks old of Male nude mice (average weight 25 g) were purchased from Beijing HFK bio-technology Ltd. (Beijing, China). The mice were inoculated subcutaneously by HepG2 cells via hypodermic. For in vivo trimodality (UCL/CT/MR) imaging, the male nude mice with tumor-bearing were first anesthetized with chloral hydrate (5 wt %).

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After that, the nude mice were injected intravenously with NaCl

solutions (0.9 wt %, 200 uL) containing desired amounts Ba2GdF7:Yb3+, Er3+@PEG UCNPs (Gd3+: 2 mg mL-1 for 4


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UCL imaging, 4 mg mL-1 for CT imaging, and 2 mg mL-1 for MR imaging), respectively. The images were collected at appropriate time points post-injection. Excited by a NIR laser (λ=980 nm) at the power density of 0.6 W cm-2 in a dark room, the in vivo UCL imaging was then obtained by a CCD-based digital camera with a suitable filter. After that, in vivo CT images were collected as previously described except 129 mm field of view was used.

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In vivo T1-weighted MRI images were collected using a GE Signa 1.5-T MR unit with imaging parameters: TR, 240ms; TE, 15.9 ms; field of view, 120 mm × 72 mm and slice thickness, 2.0 mm, respectively. 2.9. In vivo toxicity study. Major organs (spleen, head, heart, liver, kidney, lung, and tumor) were harvested from the F1 male mice at 30 d post-intravenous injection with 200 µL Ba2GdF7:Yb3+, Er3+@PEG UCNPs (Gd: 2 mg mL−1). All the major organs immersed in formalin solution (10%). After co-stained by hematoxylin and eosin

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(H&E), the images of the histological sections were taken using an optical microscope. The untreated mice were used as control group. In order to further evaluate the biosafety of Ba2GdF7:Yb3+, Er3+@PEG UCNPs, the blood of mice were extracted for hematology analysis. 3. RESULTS AND DISCUSSION 3.1. Synthesis and characterization of Ba2GdF7:Yb3+, Er3+@PEG UCNPs. The as-prepared uniform

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Ba2GdF7:Yb3+, Er3+ UCNPs were easily synthesized via hydrothermal process without adding any surfactant, organic solvents, and catalyst. The Gd(NO3)3/Ba(NO3)3 feeding ratio plays the critical role in the pure phase controlling of the UCNPs. After Ba2GdF7:Yb3+, Er3+ UCNPs formed, the PEG were employed as coating ligand for improving the colloidal stability and biocompatibility of UCNPs. As expected, the Ba2GdF7:Yb3+, Er3+@PEG UCNPs were well dispersed in aqueous medium, which was in agreement with the previously reported results.29 In

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addition, PEG coating may facilitate tumor accumulation of UCNPs by prolonging their circulation time.30 The EDX spectroscopy and ICP-AES analyses indicate that Ba2GdF7:Yb3+, Er3+@PEG UCNPs contain Gd, Ba and F elements and molar ratio of Ba with Gd is 2/1 (as shown in Figure S1 and table S1). The XRD patterns of Ba2nGdnF7n crystals were selected as references because there is no standard XRD pattern of Ba2GdF7 in the JCPDS reference database. All the observed peaks were well consistent with the cubic Ba0.625Gd0.375F2.375 (JCPDS

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NO.78-1449) without any additional impurity. Thus the obtained sample could be indexed to cubic Ba2GdF7. The characteristic peaks positions and intensities of Ba2GdF7:Yb3+, Er3+@PEG UCNPs show negligible change, indicating that the crystalline structure of UCNPs is not influenced by the PEG molecules. TEM micrograph demonstrates that the Ba2GdF7:Yb3+, Er3+@PEG UCNPs are spherical nanoparticles with reasonable monodispersity (as shown in Figure 1b). The mean size of Ba2GdF7:Yb3+, Er3+@PEG UCNP is 24± 5 nm in

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diameter. The lattice fringes with interplanar spacing of 0.213 nm in HRTEM micrograph corresponds to the (220) 5



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lattice plane of Ba2GdF7 (Figure 1c).The TEM analysis agrees with the previously reported results.31 As shown in Figure S2, FT-IR spectrum of Ba2GdF7:Yb3+, Er3+@PEG UCNPs exhibits the two notable peaks at 2894 and 1105 cm-1. These peaks are due to the C-H stretching and C-O-C stretching vibration of PEG, respectively. This result confirms that PEG is successfully conjugated on the Ba2GdF7:Yb3+, Er3+ UCNPs. Furthermore, the zeta potential

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value (+25.73 mV) of Ba2GdF7:Yb3+, Er3+@PEG UCNPs is lower than that of Ba2GdF7:Yb3+, Er3+ UCNPs (+33.20 mV), indicating that the PEG coating can help to reduce the interactions of UCNPs with negative charged plasma proteins in bloodstream. In addition, the as-prepared Ba2GdF7: Yb3+, Er3+@PEG UCNPs have been incubated in different dispersants including NaCl solution (0.9 wt %) with 10% FBS, PBS (pH 7.4) with 10% FBS and PBS (pH 7.4) for one week, respectively. After centrifugation, negligible Ba and Gd elements were found in the

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supernatants. The result indicates that the Ba2GdF7: Yb3+, Er3+@PEG UCNPs have good chemical stability and do not release Ba2+ and Gd3+ ions over a relative long period of time. As shown in Figure 2a, the as-prepared Ba2GdF7:Yb3+, Er3+ UCNPs display UCL spectrum under 980 nm excitation. The double relatively weak green emission bands at about 523 and 540 nm can be assigned to the 2

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H11/2→4I15/2 and 4S3/2→4I15/2 transitions of the Er3+ ions and the single red emission band with the maximum

wavelength at 651 nm can be assigned to the 4F9/2→4I15/2 transition. As shown in Figure 2b, the UCL intensity of as-prepared Ba2GdF7:Yb3+, Er3+ UCNPs increases proportionally with the excitation power (P), which could be investigated by the following equation: Iuc ∝ (IIR)n.32 Here, n denote the number of absorbed photons, which is required from the ground state to excite state of rare earth ions. For Ba2GdF7:Yb3+, Er3+ UCNPs, the observed slopes of the linear fit of the green and red emissions at 515-530 nm, 540-560 nm, and 640-675 nm are found to be

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2.37, 1.90, 1.89, suggesting that all of emissions undergo a two-photon process. After PEG conjugation, the UCL intensity of UCNPs increases about 1.85 times. The phenomenon may due to that PEG minimize the hydroxyl groups on UCNPs surface which are normally considered as UCL quenching agents. The MR contrast capability of Ba2GdF7:Yb3+, Er3+@PEG UCNPs is performed in a 1.5 T clinic MRI unit. In Figure 3a, the signal intensity of MR is gradually enhanced by increasing the concentration of Ba2GdF7:Yb3+,

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Er3+@PEG UCNPs. The molar relaxivity (r1) (i.e., the slope of the line in Figure 3a) is calculated to be 2.44 mM−1 s−1. This value can be comparable with that of [email protected] The CT phantom images of Ba2GdF7:Yb3+, Er3+@PEG UCNPs and corresponding Hounsfield unit (HU) values are shown in Figure 3b. The CT signal is strongly enhanced by increasing the concentration of Ba2GdF7:Yb3+, Er3+@PEG UCNPs in the solution. The HU value (200) of 0.674 mg L-1 Ba2GdF7:Yb3+, Er3+@PEG UCNPs is amount to that of 8 mg L-1 iodine in Omnipaque

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(a commercial CT contrast agent for clinic applications). The result suggests that Ba2GdF7:Yb3+, Er3+@PEG 6


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UCNPs has high CT contrast capability, i.e., at the same concentration, the attenuation coefficient of the Ba2GdF7:Yb3+, Er3+@PEG UCNPs is approximately 12 times higher than that of the current iodine-based CT contrast agent. These results suggest that Ba2GdF7:Yb3+, Er3+@PEG UCNPs can be used as trimodality imaging contrast agents (UCL/CT/MR).

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3.2. Cellular internalization and cytotoxicity study. The cellular internalization of Ba2GdF7:Yb3+, Er3+@PEG UCNPs is investigated by in vitro UCL/CT/MR imaging. The HepG2 cells are employed as model cell line to interact with Ba2GdF7:Yb3+, Er3+@PEG UCNPs. The Ba2GdF7:Yb3+, Er3+@PEG UCNPs stained HepG2 cells shows strong red UCL when the cells are incubated as low as 10 µg mL-1 Ba2GdF7:Yb3+, Er3+@PEG UCNPs in the culture medium (Figure 4). Furthermore, the UCL intensity of Ba2GdF7:Yb3+, Er3+@PEG UCNP stained

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HepG2 cells is saturated while the concentration of Ba2GdF7:Yb3+, Er3+@PEG UCNP is above 1 mg mL-1. The phenomenon may due to the restriction on the cellular internalization amount of nanoparticles. Figure S3 shows the UCL spectra of HepG2 cells after incubation with 2.5 mg mL-1 Ba2GdF7:Yb3+, Er3+@PEG UCNPs for various times. The UCL intensity of Ba2GdF7:Yb3+, Er3+@PEG UCNPs stained HepG2 cells increases with incubation time and clearly UCL spectrum can be observed after 3 h incubation. The result demonstrates that the

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Ba2GdF7:Yb3+, Er3+@PEG UCNPs can be efficiently uptaken by living cells. In addition, the Ba2GdF7:Yb3+, Er3+@PEG UCNPs stained HepG2 cells exhibit clearly enhanced MR and CT signals (as shown in Figure S4). The results further confirm the cellular internalization of Ba2GdF7:Yb3+, Er3+@PEG UCNPs. The MTT assay of HepG2 cells is carried out to preliminarily evaluate the biocompatibility of Ba2GdF7:Yb3+, Er3+@PEG UCNPs. The viability of HepG2 cells is still above 85% while the cells are incubated with 5 mg mL-1

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Ba2GdF7:Yb3+, Er3+@PEG UCNPs for 24 h (Figure 5S), revealing low cytotoxicity of Ba2GdF7:Yb3+, Er3+@PEG UCNPs. 3.3. In vivo UCL/CT/MR imaging. The nude mice with HepG2 tumor were used to test the in vivo trimodality contrast capability of the Ba2GdF7:Yb3+, Er3+@PEG UCNPs. 200 µL Ba2GdF7:Yb3+, Er3+@PEG UCNPs solutions with desired concentrations (Gd: 2 mg mL-1 for UCL imaging, 4 mg mL-1 for CT, and 2 mg mL-1

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for MRI) injected intravenously into nude mice with HepG2 tumor-bearing. As expected, a time course of signal enhancement was found in the UCL/CT/MR imaging when the Ba2GdF7:Yb3+, Er3+@PEG UCNPs were administrated intravenously to a mouse (as shown in Figures 5-7 and Figure S6). One can see the remarkable positive enhancement of signals of the tumor regions at 1 h post-injection of Ba2GdF7:Yb3+, Er3+@PEG UCNPs. The enhancements of signal values (4.43 times for T1-weighted MR intensity, and 3.61 times for HU value) were

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obtained at 24 h post-injection. To further estimate the passive tumor-targeting ability, the Ba2GdF7:Yb3+, 7



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Er3+@PEG UCNPs solution was injected intravenously into nude mice with small tumor (c.a., 20 mm3 in volume). The UCL/MR signal enhancing processes of tumor sites agree the results of large tumors (as shown in Figures S7 and S8). The experimental result suggests that the Ba2GdF7:Yb3+, Er3+@PEG UCNPs are highly accumulated in tumor because of the enhanced permeability and retention (EPR) effect.

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3.4. In vivo toxicity. The in vivo toxicity of Ba2GdF7:Yb3+, Er3+@PEG UCNPs was investigated by histochemical analysis and hemolysis assay. As shown in Figure 8, the F1 mice show negligible tissue damage of various organs (spleen, heart, lung kidney, and liver) after 30 d intravenous injection of Ba2GdF7:Yb3+, Er3+@PEG UCNPs with the dose of 200 µL (Gd:4.0 mg mL-1). The biocompatibility of Ba2GdF7:Yb3+, Er3+@PEG UCNPs also tested by hemolysis assay (Figure S9). One can see that the Ba2GdF7:Yb3+, Er3+@PEG UCNPs cannot destroy

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blood cells. The preliminary in vivo toxicity results suggest that Ba2GdF7:Yb3+, Er3+@PEG UCNPs present great promise as a non-toxic nanoprobe in the filed of multiple modality bioimaging. 4 . CONCLUSION In summary, the Ba2GdF7:Yb3+, Er3+@PEG UCNPs with reasonable monodispersity have been successfully synthesized, and employed as trimodal bioimaging contrast agents by surfactant-free hydrothermal strategy.

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Systematic studies indicate that the Ba2GdF7:Yb3+, Er3+@PEG UCNPs exhibit negligible toxicity, excellent contrast capability, and good colloidal stability. Further in vivo trimodality imaging results confirm that the Ba2GdF7:Yb3+, Er3+@PEG UCNPs have satisfying tumor passive targeting ability, and present bright red UCL and strong CT/MR contrast enhancement effects in the xenograft tumor model. These results provide a proof of concept for designation of new generation of Ba2GdF7 hosted UCNPs for bioanalytical and biomedical

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applications. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]; [email protected];

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Notes The authors declare no competing financial interest. ACKNOWLEDGEMENTS This study is financially supported by the National Basic Research Program of China (973 Program, Grant No. 2014CB643803), the National Natural Science Foundation of China (Grant No. 51472236) and the Fund for

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Creative Research Groups (Grant No. 21521092), and Key Program of the Frontier Science of the Chinese 8


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Figure 1 (a) XRD patterns of as-prepared UCNPs, and (b) TEM and (c) HRTEM micrographs of Ba2GdF7:Yb3+, Er3+@PEG UCNPs.

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Figure 2 (a) UCL spectra of a-prepared UCNPs, and (b) dependence of UCL intensities (640-675 nm (red), 515-530 nm (light green) and 540-560 nm (dark green)) of Ba2GdF7:Yb3+, Er3+ UCNPs on the pump power.

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Figure 3 (a) T1-weighted MR images and relaxation rate (1/T1) of aqueous solution of Ba2GdF7:Yb3+, Er3+@PEG UCNPs as a function of the molar concentration of Gd3+ in the solution and, (b) CT images and HU value of aqueous solution of Ba2GdF7:Yb3+, Er3+@PEG UCNPs as a function of the molar concentration of Gd3+ in the

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solution, respectively.

Figure 4 UCL microscopy imaging of Ba2GdF7:Yb3+, Er3+@PEG UCNPs stained HepG2 cells. Images (a to f) are acquired under bright field mode and images (g to l) are acquired under dark field mode (red channel of UCL), respectively. The concentrations of Ba2GdF7:Yb3+, Er3+@PEG UCNPs are (a and g) 10 µg mL-1, (b and h) 50 µg

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mL-1, (c and i) 100 µg mL-1, (d and j) 1 mg mL-1, (e and k) 2.5 mg mL-1, and (f and l) 5 mg mL-1 in the cell culture medium, respectively. Scale bars are 20 µm. 14


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Figure 5 In vivo UCL ((a) bright field mode, (b) dark field mode (red channel), and (c) merging mode) images of HepG2 tumor-bearing nude mouse after intravenous injection of Ba2GdF7:Yb3+, Er3+@PEG UCNPs at different timed intervals (The 0 h means pre-injection.), respectively.

5 Figure 6 In vivo coronal MR images (a) and MR images and relative of MR signal of tumor (b) of HepG2 tumor-bearing nude mouse after intravenous injection of Ba2GdF7:Yb3+, Er3+@PEG UCNPs at different timed 15



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intervals (The 0 h means pre-injection.), respectively.

Figure 7. (a) In vivo coronal CT images (a) and MR images and relative of CT signal of tumor (b) of HepG2 tumor-bearing nude mouse after intravenous injection of Ba2GdF7:Yb3+, Er3+@PEG UCNPs at different timed

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intervals (The 0 h means pre-injection.), respectively.

Figure 8. Histological changes of the nude mouse after 30 days post-injection of a single dose of Ba2GdF7:Yb3+, Er3+@PEG UCNPs (Gd: 4.0 mg mL-1) in NaCl solution (0.9 wt%) (a), and the nude mouse without injection of Ba2GdF7:Yb3+, Er3+@PEG UCNPs (b), respectively.

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Surfactant-Free Aqueous Synthesis of Novel Ba2GdF7:Yb3+, Er3+@PEG Upconversion Nanoparticles for in Vivo Trimodality Imaging Yang Feng,†,‡ Hongda Chen,§,‡ Lina Ma,§ Baiqi Shao,† Shuang Zhao,†,‡ Zhenxin Wang,*,§ and Hongpeng You *,† †

State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of

Applied Chemistry, University of

Chinese Academy of Sciences, Changchun 130022,

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State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied

Chemistry, University of

Chinese Academy of Sciences, Changchun 130022, P. R.

te

§

d

P. R. China

University of Science and Technology of China, Hefei 230026, P. R. China

nR

eg

‡

is

China

U

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The novel Ba2GdF7:Yb3+, Er3+@PEG upconversion nanoparticles are successfully synthesized by a facile aqueous synthesis for in vivo UCL, MR, and CT multi-modality imaging.


Surfactant-Free Aqueous Synthesis of Novel Ba2GdF7:Yb3+, Er3+@

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