Sub-10 nm Water-Dispersible Î²-NaGdF4:X% Eu3+ Nanoparticles with

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Sub-10 nm Water-dispersible #-NaGdF:X% Eu Nanoparticles with Enhanced Biocompatibility for In vivo X-ray Luminescence Computed Tomography Wenli Zhang, Yingli Shen, Miao Liu, Peng Gao, Huangsheng Pu, Li Fan, Ruibin Jiang, Zong-Huai Liu, Feng Shi, and Hongbing Lu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b11295 • Publication Date (Web): 24 Oct 2017 Downloaded from http://pubs.acs.org on October 27, 2017

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ACS Applied Materials & Interfaces

Sub-10 nm Water-dispersible β-NaGdF4:X% Eu3+ Nanoparticles with Enhanced Biocompatibility for In vivo X-ray Luminescence Computed Tomography Wenli Zhang,‡⊥ Yingli Shen,†⊥ Miao Liu,† Peng Gao,‡ Huangsheng Pu,‡ Li Fan,§ Ruibin Jiang,† Zonghuai Liu,† Feng Shi*† and Hongbing Lu*‡ †Shaanxi

Key Laboratory for Advanced Energy Devices; Shaanxi Engineering Lab for Advanced Energy Technology; Key Laboratory of Applied Surface and Colloid Chemistry, National Ministry of Education; School of Materials Science and Engineering, Shaanxi Normal University, Xi'an 710119, P. R. China. ‡Department

of Computer Application, School of Biomedical Engineering, Fourth Military Medical University, 169 Changle W Road, Xi'an 7100032, P. R. China. §Department

of Pharmaceutical Analysis, School of Pharmacy, Fourth Military Medical University, 169 Changle W Road, Xi'an 7100032, P. R. China. ⊥These

authors contribute equally to this work.

E-mail: [email protected], [email protected].

ABSTRACT As a novel molecular and functional imaging modality, X-ray luminescence computed tomography (XLCT) has shown its potentials in biomedical and pre-clinic applications. However, there are still some limitations of X-ray excited luminescent materials, such as low luminescence efficiency, poor biocompatibility and cytotoxicity, making in vivo XLCT imaging challenging. In this study, for the very first time, we present on using sub-10 nm -NaGdF4:X% Eu3+ nanoparticles with poly-(acrylic acid) (PAA) surface modification, which demonstrate outstanding luminescence efficiency, uniform size distribution, water-dispersity and biosafety, as the luminescent probes for in vivo XLCT application. The pure hexagonal phase (-) NaGdF4 has been successfully synthesized and characterized by XRD and TEM, and then the results of XPS, EDX and elemental mapping further confirm Eu3+ ions doped into NaGdF4 host. Under X-ray excitation, the -NaGdF4 nanoparticles with a doping

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level of 15% Eu3+ exhibited the most efficient luminescence intensity. Notably, the doping level of Eu3+ has no effect on the crystal phase and morphology of the NaGdF4-based host. Afterward, -NaGdF4:15% Eu3+ nanoparticles were modified with PAA to enhance the water dispersity and biocompatibility. The compatibility of in vivo XLCT imaging using such nanoparticles was systematically studied via in vitro cytotoxicity, physical phantom and in vivo imaging experiments. The ultra-low cytotoxicity of PAA-modified nanoparticles, which is confirmed by over 80% cell viability of SH-SY5Y cells when treated by high nanoparticles concentration of 200 μg/mL, overcome the major obstacle for in vivo application. In addition, the high luminescence intensity of PAA-modified nanoparticles enables the location error of in vivo XLCT imaging less than 2 mm, which is comparable to that using commercially available bulk material Y2O3: 15%Eu3+. The proposed nanoparticles promote XLCT research into an in vivo stage. Further modification of these nanoparticles with bio-functional molecules could enable the potential of targeting XLCT imaging. KEYWORDS: XLCT imaging, PAA--NaGdF4:X% Eu3+ NPs, ultra-small size, surface modification, biocompatibility, high accuracy.

1. INTRODUCTION Due to the penetrating and high-resolution imaging of human tissues since its discovery in 1895, X-ray has been widely applied in clinical and biomedical research imaging systems, such as X-ray computed tomography (XCT). Recently, with the fast development of X-ray excited materials, X-ray luminescence computed tomography (XLCT) has been proposed as a new molecular and functional imaging technique.1, 2 In principle, when an object is irradiated with X-rays, X-ray excitable particles in the imaged object will produce luminescence emission at specific wavelengths over the visible to short-wave infrared (Vis-to-SWIR) spectral domain,3-6 which can propagate through tissue and be efficiently collected by sensitive light-based detection systems. By solving an inverse problem, the three-dimensional (3D) distribution of the phosphors can be reconstructed. XLCT shows its advantages in biomedical imaging


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and clinical diagnosis due to the combination of high sensitivity of optical detection and high spatial resolution of X-ray imaging. In addition, comparing with other optical imaging techniques, such as fluorescence molecular tomography (FMT) or bioluminescence tomography (BLT),7-10 XLCT can avoid tissue autofluorescence and increase imaging depth due to the use of X-ray excitation, making it a promising way for in vivo and clinical imaging.11, 12 For XLCT imaging, X-ray excited luminescent phosphors of high quality is imperative. In the past years, a great variety of materials have been utilized for XLCT imaging, such as rare earth oxides (Gd2O3: Eu, Eu2O3) 2, 13 and oxysulfides (Gd2O2S: Eu).1, 14-16 However, high toxicity and poor biocompatibility of these XLCT probes represent major obstacles to clinical implementation, resulting in that few in vivo XLCT studies have been reported so far. New materials with enhanced biosafety and luminescence emission are in great need for successful in vivo XLCT applications. As an important class of luminescent materials, rare earth-based functional nanophosphors impart the ability to be used in biomedical applications owing to their high photochemical stability, low photo-bleaching, sharp emission bands, and long luminescence lifetime.17 Among these materials, rare earth fluoride-based NaREF4:Ln3+ (RE = rare earth, Ln = lanthanide) nanoparticles, which demonstrate chemically stable with low cytotoxicity, has been considered as one of the most efficient downconversion phosphors.18, 19 It is well known that low band gap materials offers better scintillation efficiency. This kind of nanophosphors has relatively low phonon energy and low band gap of 9-10 eV, which can decrease the non-radiative relaxation probability and consequently increase the luminescent quantum yield.18, 19 In addition, fluoride-based NaREF4:Ln3+ materials offer other advantages on the basis of radiation hardness and high density,20, 21 enabling them to be applied in XLCT imaging efficiently. Recently, Gd3+ serves as an excellent UV-excited downconversion photosensitizer for lanthanide emitters.22 The best-studied Gd3+-Eu3+ host-dopant combination can be excited by low UV irradiation because the emission energy transitions within Gd3+



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resonantly couple to the excited state of Eu3+ ions. Notably, the K-edge value of 50.23 keV and high X-rays mass absorption coefficient of Gd element make it absorb the X-ray energy efficiently and transfer to the active center of the phosphor for higher luminescence emission.6, 23 Based on the above established science, NaGdF4:X% Eu3+ nanoparticles (NPs) can provide high X-ray induced luminescence efficiency and appear to be good candidates for in vivo XLCT imaging. Considering that the luminescence efficiency of such NPs is related not only to their purity and dopant, but also to the morphology, particle size, and size distribution, systemic investigation on the controlled synthesis of NaGdF4:X% Eu3+ nanoparticles for enhanced luminescence and biosafety is extremely urgent for in vivo XLCT application.23 In this work, we present a simple and effective method to obtain ultra-small, monodisperse, and highly uniform -NaGdF4:X% Eu3+ NPs by a co-precipitation route. After surface functionalized with poly-(acrylic acid) (PAA), the as-obtained PAA-NaGdF4:15% Eu3+ NPs exhibits intense X-ray luminescence efficiency and low cytotoxicity. The XLCT imaging performance of the obtained NPs was evaluated with phantom and in vivo experiments. More importantly, with boosted luminescence intensity and enhanced biosafety, for the first time, such NPs were applied for in vivo XLCT imaging. The result verifies the feasibility of PAA-NaGdF4:X% Eu3+ NPs in in vivo XLCT imaging and demonstrates their potentials in biomedical research and clinical diagnosis.

2. EXPERIMENTAL SECTION 2.1 Materials Rare earth chloride hexahydrate (GdCl3·6H2O 99.99% and EuCl3·6H2O 99.99%) were obtained from Shandong Yutai Chemical Reagent Co., Ltd. Ammonium fluoride (NH4F, 98%), sodium hydroxide (NaOH, 98%), anhydrous ethanol (EtOH) and methanol (MeOH) were supplied by Sinopharm Chemical Reagent Co., Ltd. 1-octadecene (ODE, 90%), oleic acid (OA, 90%), Poly-(acrylic acid) (PAA, Mw = 1800), tetrahydrofuran (THF, 99.9%), MTT assay kit, and Fetal


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Bovine Serum (FBS) were purchased from Sigma-Aldrich. Phosphate Buffered Saline (PBS, pH = 7.4), 2-(N-morpholino) ethanesulfonic acid (MES) Monohydrate Free acid (pH=6.5), Dulbecco's Modified Eagle Media (DMEM), and Roswell Park Memorial Institute-1640 (RPMI-1640) were purchased from Solarbio. All the chemical reagents used in this work were of analytical grade and used without further purification.

2.2 Synthesis of OA-stabilized -NaGdF4:X% Eu3+ NPs OA-capped -NaGdF4:1% Eu3+ NPs were synthesized via a solvothermal route adapted from the literature.24 In brief, GdCl3·6H2O (0.99 mmol) and EuCl3·6H2O (0.01mmol) was mixed with 6 mL OA and 15 mL ODE in a 100 mL three-neck round flask, then the resultant mixture was heated to 160 C for 60 min under argon gas protection to form a homogeneous solution. After the solution was cooled down to room temperature, 10 mL methanol solution containing NH4F (4 mmol) and NaOH (2.5 mmol) was added drop by drop into the mixture. The reaction mixture was stirred for another 60 min, slowly heated to 60 C for 60 min to remove methanol, and then degassed at 120 °C for 20 min. After that, the solution was further heated to 290 C at an increasing rate of 15 C/min, and then maintained at this temperature for 60 min under argon gas protection. The final solution was cooled to room temperature and the product was precipitated by the addition of an excess amount of ethanol, collected by centrifugation at 9000 rpm for 10 min. The precipitate was washed with an ethanol/hexane mixture (3:1 v/v) three times, finally the OA-stabilized NPs (OA-NPs) was dispersed in hexane for further use. To evaluate the influence of Eu3+ doping on luminescence efficiency, -NaGdF4:X% Eu3+ NPs (X = 5, 10, 15, 20 and 25 mol%, respectively) were further synthesized by using procedures similar to those described above, except that different amounts of EuCl3·6H2O were added.



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2.3 Surface modification of -NaGdF4:X% Eu3+ NPs The surface PAA-functionalization of the as-prepared -NaGdF4:X% Eu3+ NPs was carried out by using a modified ligand exchange strategy. 25 Typically, PAA (0.25 g) was mixed with OA-NPs (15 mg) in tetrahydrofuran (THF, 10 mL) in a 100 mL round flask and this mixture was tenderly stirred at room temperature at least for 24 h to make the solution homogeneous. THF was then evaporated under 50 C and some white solid was obtained. Subsequently, 10 mL hexane was added to the white solid, and ultrasonicated for 10 min and then violently stirred for 30 min to dissolve the replaced OA molecules. Finally, the as-prepared PAA-NPs were washed with an ethanol/hexane mixture (1:3 v/v) three times, and were dispersed in deionized water for further experiments.

2.4 Characterization The crystalline structures of the as-synthesized samples were recorded by X-ray powder diffraction (XRD) on a D/Max-3c diffractometer (DX-2700) with Cu Ka radiation (λ = 1.5406 Å) at 40 kV and 30 mA. The morphologies and sizes were characterized by transmission electron microscopy (TEM) using a JEOL JEM-2100 microscope at an acceleration voltage of 200 kV. The X-ray excited luminescence spectrum was recorded by a Zolix Fluorescent Spectrometer (Zolix Omni-λ) equipped with an X-ray source irradiating at room temperature. STEM images, elemental mapping and Energy Dispersive X-Ray Spectroscopy(EDX) were measured using a Tecnai G2 F20 (FEI, Japan) at 200 kV. X-ray photoelectron spectroscopy (XPS) was carried out using an AXIS ULTRA (Kratos Analytical Ltd.) with Kα radiation (1486.6 eV) as an excitation source. The Dynamic light scattering (DLS) measurements were performed using Malvern Zeta Seizer (ZEN3690) instrument and all the scattered photons are collected at 90°scattering angle. A Perkin-Elmer SCIEX ELAN6100DRC (dynamic reaction cell) ICP-MS instrument with the outer and intermediate ICP gas flow rate fixed at 13 and 1.3 L/min was used.


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2.5 SH-SY5Y cell culture and MTT cell viability assay In vitro cytotoxicity of the obtained water-dispersible nanophosphors was measured by performing MTT assays on the human neuroblastoma cell line SH-SY5Y cells. SH-SY5Y cells were grown in RPMI Medium 1640 basic supplemented with 10% FBS, 500 U/mL penicillin and 500 μg/mL streptomycin. The cells were maintained in a humidified, 5% carbon dioxide atmosphere at 37 °C. For viability studies, 104 SH-SY5Y cells were seeded in 96-well plates (Corning) and cultured for 48 h. Different concentrations (0, 50, 100, 150, 200 μg/mL) of PAA--NaGdF4:Eu3+ NPs were added to the wells. 24 hours later, the wells were washed with PBS for three times to remove exceeded nanoparticles. Cell viability was evaluated using MTT assays by following the vendor’s protocols. The viability of the untreated SH-SY5Y cells was set as 100%, and then the viability of the PAA-NPs-treated SH-SY5Y cells was calculated. For each NPs concentration, 8 samples were prepared and student’s t-test was performed to find significant intergroup differences with p < 0.05.

2.6 Configuration of the XLCT imaging system To evaluate the feasibility of XLCT using the obtained water-dispersible X-ray excited PAA--NaGdF4:Eu3+ NPs, a custom-made cone-beam XLCT system was developed, as shown in Figure S1 (SI†). The system consists of a whole X-ray spectrum of micro-focus X-ray source (Source-Ray, SB-80-500, N.Y.), an X-ray flat panel detector (2923, Dexela, United Kingdom), a motorized rotation stage, and an electron-multiplying CCD (EMCCD) camera (iXon DU-897, Andor, United Kingdom) coupled with a Nikon 50-mm f/1.8D lens (Nikon, Japan). The X-ray source was placed 26.5 cm away from the rotation centre, with the maximal power of 40 W, while the X-ray detector positioned at the opposite side of the imaging object, with pixel size of 74.8m covering a 3888  3072 digital image matrix. To collect luminescence photons emitted from the imaged object, the EMCCD camera was positioned at 90 toward the X-ray axis, with the protection of a lead shield.



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2.7 Phantom XLCT experiments A physical phantom was made to evaluate the emitted luminescence of the synthesized NPs. The configuration of the phantom is shown in Figure 5. A transparent glass cylinder (3.0 cm in outer diameter, 4.0 cm in height) was fixed on the rotation stage. It was filled with 1%vol intralipid and water. A small transparent glass tube (3.0 mm in outer diameter) filled with 0.1 mL of 10%wt PAA--NaGdF4: Eu3+ NPs aqueous solution was used as the X-ray luminescent target. The voltage of the X-ray source was set as 80 kV with tube current of 0.5 mA. The phantom was rotated from 0°to 360°, and the luminescence photons emitted from the target were acquired every 15°by the EMCCD camera. The exposure time of EMCCD was set to 3 s, and the electron multiplying (EM) gain was set to 260. Totally 24 projections were acquired for each rotation. For performance comparison, a phantom experiment with a kind of bulk materials, i.e. one kind of rare earth oxide Y2O3: 15%Eu3+ (bought from Jiangxi Illuma Fluorescent Materials Co., Ltd., China) was also performed by filling the small glass tube with 5 mm height of solid Y2O3: 15%Eu3+. The XLCT acquisition procedure was the same as that described above.

2.8 In vivo X-ray excited luminescence imaging and XLCT experiments To evaluate the feasibility of the obtained NPs for in vivo imaging, an in vivo X-ray luminescence bioimaging was first performed by using a female five-week-old nude mouse model with weight of 20 g, whose four limbs were subcutaneously injected with 0.1 mL PAA-NaGdF4:15% Eu3+ NPs of different concentrations (0, 30, 70 and 100 μg/mL dispersed in phosphate buffered saline (PBS)), respectively. Then a female eight-week-old nude mouse with weight of 22 g was used for in vivo XLCT imaging. The mouse was anesthetized by an abdominal cavity injection of 1.5% Pelltobarbitalum Natricum, with a dose of 80 mg/kg recommended for nude mouse. To evaluate the imaging performance of the proposed PAA-NPs, a transparent


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tube with inner diameter of 2.0 mm, which was filled with 0.1 mL 10%wt. of PAA--NaGdF4:15% Eu3+ NPs, was implanted into the mouse to simulate a luminescent target, as shown in Figure 6a. The XLCT acquisition procedure was the same as that of the phantom experiment described above. Similarly, for performance comparison, in vivo XLCT imaging was further performed by replacing the luminescent target with the bulk material Y2O3: 15% Eu3+. After each XLCT scan, the X-ray projections were also acquired with the developed system to get the anatomical information of the phantom and the mice. The X-ray projections were obtained with an angular increment of 1° and totally 360 projections were acquired for each scan. The traditional Feldkamp-Davis-Kress (FDK) algorithm was used for XCT reconstruction,26 while XLCT images were reconstructed using algebraic reconstruction technique (ART) with the nonnegative constraint.27

3. RESULTS AND DISCUSSION 3.1 Structure and morphology of the obtained NPs A series of NaGdF4:X% Eu3+ NPs with increased Eu3+ doping were fabricated utilizing a method adapted from the literature.24 To examine the crystal structure and phase purity of the as-prepared samples, the XRD patterns of NaGdF4:X% Eu3+ NPs (X = 1, 5, 10, 15, 20, and 25 mol%, respectively) are shown in Figure 1. All the diffraction peaks match well with the values for (100), (110), (101), (200), (111), (201), (210), (211), (102), and (112) reflections of -NaGdF4 (JCPDS No. 27-0699). No extra Eu-rich phases were detected and thus this is compatible with the Eu incorporation into the NaGdF4 nanoparticles. In order to confirm the successful incorporation of Eu3+ ions into the NaGdF4 host, X-ray photoelectron spectroscopy (XPS) is acted as a convenient method to use. The XPS spectrum shows the presence of Na, F, Gd, and Eu elements which corresponds well to the host composition (Figure S2). This reveals that pure hexagonal phase (-) NaGdF4 was fabricated and Eu3+ ions were successfully incorporated into NaGdF4 host. In addition, XRD patterns of other as-prepared NaGdF4:X% Eu3+ NPs are shown in Figure S3a (SI†). Notably,



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with the increase of Eu3+ contents, the phase of the samples was still maintained owing to the matched radii of Eu3+ and Gd3+ (the radius of Eu3+ and Gd3+ is 1.30 Å and 1.247 Å for CN = 9, respectively), indicating that different Eu3+ contents have no influence on the crystal phase of the NaGdF4-based host. To reveal the slight differences caused by Eu3+ ions doping, selected areas of the diffraction peak (100) were magnified as shown in Figure S3b. It can be seen from Figure S3b that these diffraction peaks gradually shift toward lower angles with increasing Eu3+ dopant concentrations (3%, 7%, 12% and 22%). This phenomenon of peak shifting further indicates that Eu3+ ions can be doped into host lattice. By using the Scherrer's equation, the average crystallite size of the grain was determined to be  9 nm for the entire Eu3+ doping range, while the interplanar spacing was found to be 0.520  0.0015 nm corresponding to (100) plane, which was further supported by high-resolution TEM characterization.

Figure 1 XRD patterns of the NaGdF4:X% Eu3+ NPs with different Eu3+ doping contents (X = 1, 5, 10, 15, 20 and 25 mol%, respectively), in reference to the standard diffraction pattern of the -NaGdF4 (JCPDS No. 27-0699).

To further provide insight into the morphology and microstructure of the prepared samples with varying doping concentrations of Eu 3+, the obtained NPs were characterized by TEM. Figure 2 shows typical TEM images of different contents Eu3+ doped NaGdF4 NPs, from which we can see that all of these nanoparticles are excellent uniform and monodispersity. It is clearly found that


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the

as-obtained

-NaGdF4

NPs

without

Eu3+

doping

presents

the

quasi-spherical NPs with an average diameter of about 9 nm as shown in Figure S4 (SI†). However, with increasing Eu3+ content from 0 to 5, 10, 15, 20 and 25 mol%, the average grain sizes of the NPs did not change. It is noteworthy to mention that the particle size remained the same in all the samples indicating that the size of NPs was not influenced by variation in the dopant concentration. It can also be noticed that the NaGdF4 nanostructures with Eu3+ does not lead to significant changes in the morphology or size of the NPs. An additional high-resolution TEM (HRTEM) image of an individual particle of the NaGdF4 doped with 15 mol% Eu3+ sample shown in Figure 2d displays clear lattice fringes of the hexagonal phase (100) crystal plane with a d-spacing of 0.520 nm. The histograms of the size distribution of the synthesized NPs indicate that the average diameters are about 9 nm, which are in good agreement with the TEM images. The data for the size distribution were obtained from the TEM images of over 100 NPs (Figure S5, SI†). Therefore, sub-10 nm ultra-small and uniform NaGdF4 NPs with simultaneously controlled shape and size can be achieved by only adjusting the doped Eu3+ contents, which provides a facile route for synthesis of sub-10 nm NaGdF4 NPs. Moreover, the atomic compositions of NaGdF4:X% Eu3+ NPs were further confirmed by scanning transmission electron microscopy (STEM), elemental mapping, energy dispersive X-ray spectroscopy (EDX) and line-profile elemental (F, Na, Gd, Eu) analysis. These results indicate that the successful doping of Eu3+ ions in our prepared NPs and that all these elements are uniformly distributed throughout the NPs (Figure S6, SI†). Unfortunately, the obtained NaGdF4:X% Eu3+ NPs bearing hydrophobic surface ligand oleic acid (OA) are not suitable for biomedical applications. Thus, the surface PAA-functionalization of the as-prepared -NaGdF4:X% Eu3+ NPs was performed by using a modified ligand exchange strategy.28 The successful exchange with PAA can be further confirmed by FT-IR spectroscopy (Figure S7, SI†). The peaks at 2923 and 2854 cm-1 are attributed to the asymmetric and symmetric stretching vibrations of



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methylene (CH2) in the long alkyl chain of the OA molecules. In addition, there are two peaks at 1563 cm-1 and 1458 cm-1, corresponding to symmetric and asymmetric stretching vibration of the carboxylic group (COOH) of the bound OA, respectively. After modification with PAA, a strong peak at 1728 cm-1 is observed which is attributed to the carbonyl group (C=O) stretching mode of protonated carboxylate groups (COOH) of PAA,29 indicating that ligand exchange is successful. Furthermore, the as-obtained PAA-NPs have good dispersity and stability in deionized water, the inset (a) and (b) in Figure S7 are digital photographs of OA-NPs and PAA-NPs which dispersed in hexane and deionized water, respectively.

Figure 2 TEM images of the -NaGdF4:X% Eu3+ NPs with different contents of Eu3+: (a-f) X = 1, 5, 10, 15, 20, and 25 mol%, respectively. The inset in (d) is the HRTEM image of a single -NaGdF4:15% Eu3+ NPs. All scale bars are 50 nm.

3.2 X-ray excited luminescence properties Due to the gadolinium component of the rare earth fluoride acts as an effective X-ray absorber with a K-edge at 50.23 keV, which makes the energy transfer from the host lattice to luminescent center is easily happened. To assess X-ray excited luminescence properties of the as-prepared samples, spectra of the emissions under X-ray excitation are shown in Figure 3 and Figure S3c. It can be seen that these luminescence peaks still show the characteristic emissions of Eu3+, which are similar to the


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photoluminescence spectra in the previous report.30 The characteristic peaks centered at 592, 613, 653, and 695 nm are clearly observed, which should be ascribed to the 5

D0  7FJ (J = 1, 2, 3, and 4) transitions and other emission peaks centered at 535 and

555 nm which correspond to 5D1  7F1 and 5D1  7F2 transitions of Eu3+, respectively. The strongest emission is located in the red light range of 600-625 nm, which is good accordance with the Judd-Ofelt theory.31, 32 Generally speaking, the intensity ratio of the electric dipole to magnetic dipole transitions is used to determine the symmetry of the local environment. It can be seen that the relative intensity of the electric dipole transition 5D0  7F2 (613 nm) is higher than that associated with the magnetic dipole transition 5D0  7F1 (592 nm) in all cases, suggesting a crystallographic site without inversion symmetry for Eu3+.33 Thereby, the red emission is dominant in the emission spectra. This phenomenon is quite different from that of NaGdF4:X% Eu3+ (i.e. X% = 0.01-0.1%) with ultra-low doping concentration.34 Because that when the doping concentration of Eu3+ is low enough, Eu3+ may emit not only from lower energy level 5

D0, but also from higher excited state 5D1. However, with increasing Eu3+ doping

content, the higher-level 5D1 emission is quenched gradually owing to the cross relaxation occurring between two neighbouring Eu3+, and which ultimately lead to the lower energy level 5D0 emission dominating. In addition, it can be seen from the inset that the doping content of Eu3+ markedly influences the intensity of emission and the optimum doping content is 15% for 5D0 emission in the NaGdF4: X% Eu3+ NPs. As well known that the Eu3+ emission increases firstly and then decreases when the concentration of Eu3+ is above 15% as a result of a self-quenching effect due to the interactions between Eu3+ ions. For X-ray excited luminescence, in contrast to the direct optical excitation wherein an electron within the luminescent chromophore is directly excited from the 4f n  4f n-15d1 levels, the X-ray excitation process is mainly defined as follows: the secondary electrons are firstly generated in the host lattice by absorbing the X-ray energy, which directly or indirectly excites luminescent centers, and then X-ray luminescence (visible or near-infrared (NIR) emission) is produced.23 The more detail information on the physical processes can be found in previous studies.6, 35



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Figure 3 X-ray luminescence spectra of -NaGdF4:X% Eu3+ NPs with different Eu3+ doping contents (X = 1, 5, 10, 15, 20 and 25 mol%), respectively. The inset shows the integrated intensity of

5D

0

7F 1

and

5D

0

7F 2

emissions as a function of Eu3+ doping content. Nanoparticle

concentration in all the luminescence measurements was 10 mg/mL, and dispersed in cyclohexane.

3.3 Cytotoxicity of PAA-β-NaGdF4:15% Eu3+ NPs For in vivo applications, the cytotoxicity of the obtained PAA-NPs should be considered with higher priority. In this study, based on the X-ray luminescence spectra of NPs with different Eu3+ doping, in vitro cytotoxicity of the most efficient PAA-β-NaGdF4:15% Eu3+ NPs was measured by performing MTT assays on SH-SY5Y cells. As shown in Figure 4a, SH-SY5Y cells were exposed to NPs with different concentrations of 50, 100, 150, and 200 μg/mL. After 24 h, cell viabilities were 96.03%, 92.21%, 88.79%, and 84.19%, respectively. It indicates that the cytotoxicity of the nanoparticles increased with the concentration. Though statistical analysis indicates that the cell viability of SH-SY5Y cells treated with NPs decreased significantly comparing with those untreated (p = 0.00005, 0.021) for NPs concentrations of 150 and 200 g/mL, respectively. Plus, the cell viability drop significantly of treated with 200 g/mL NPs comparing with which of all other samples with p = 0.00005, 0.0076, 0.006 and 0.031, respectively, which indicated that the cell viability did not drop significantly with the increase of NPs exposure up to


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150 μg/mL. For NPs with concentration less than 100 μg/mL, the cell viability is higher than 92.21  3.78%, which is comparable with that of the silica coated NaYF4 upconversion nanoparticles reported previously.36 These results demonstrate that PAA-β-NaGdF4:15% Eu3+ NPs prepared by the ligand exchange synthesis are uniformly dispersed, water soluble, and low cytotoxicity, making them promising candidates for in vivo functional imaging. In addition, the hydrodynamic size distribution of PAA-modified NPs dispersed in different medium has also been studied. From dynamic light scattering (DLS) study, the average hydrodynamic size of such NPs was approximate 60 nm in DMEM and 1640 medium, 110 nm in DI water, and 140 nm in PBS pH 7.4 and 50 mM MES buffer pH 6.5, which were suitable for biological applications, shown in Figure S8a. Notably, the hydrodynamic size of such NPs increased and became stable in each medium after 3 h, illustrating in Figure S8b-8f. In order to further understand insight into the kinetics of PAA-β-NaGdF4:15% Eu3+ in vivo, the bio-distribution of the NPs were quantitative analyzed by ICP-MS, and Gd amount were presented for the NPs in the major organs. Specific time points (1 h, 6 h, 12 h, 24 h, and 48 h) were chosen to study the NPs biodistribution and circulation time (Figure 4b). A rapid accumulation of NPs in organs from the blood circulation was observed at 1 h post-injection, and became stable. The major accumulation of NPs was in liver up to 40% of injection dose (NPs over certain size easily scavenged by RES system and tend to accumulate in liver in a significant proportion) and approximate 70% total of injected NPs was accumulated in the heart, liver, spleen, lung and kidney. Notably, no decreasing of NPs concentration was found in all five organs over time until 48 h due to the PAA-modified surfaces, which was beneficial for imaging and therapy applications to avoid multiple drug injection. It is worth to note that no obvious acute toxic or adverse health effects was observed after 48 h post-injection at a dose level of 100 μg/mice. The rat exhibited normal behaviors, such as eating patterns based on the standards set by International Animal Care and Use Committee.



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Figure 4 (a) In vitro cell viabilities of SH-SY5Y cells with PAA-NaGdF4:15% Eu3+ NPs of different concentrations (0, 50, 100, 150 and 200 g/mL) for 24 h. Standard deviation of mean and p values are shown. (b) In vivo biodistribution of PAA-NaGdF4:15% Eu3+ NPs in heart, liver, spleen, lung, and kidney of rats harvested after 1 h, 6 h, 12 h, 24 h, and 48 h after intravenous injection through tail vein.

3.4 Phantom validation of PAA-NPs for XLCT imaging Physical phantom experiments were carried out to evaluate the performance of PAA-NaGdF4:15% Eu3+ NPs for XLCT imaging. Figure 5a and Figure 5b give an X-ray projection and an optical image of the phantom acquired at the same angle. Figure 5c-5f show tomographic XCT and XLCT slices of the phantom reconstructed by using the FDK algorithm and ART algorithm, respectively. To evaluate the positioning accuracy of XLCT for the imaging target, two tomographic slices of XCT and XLCT in Figure 5c and Figure 5d were merged, as shown in Figure 5e. Figure 5f shows the 3D visualization of the reconstructed XLCT tomographic images. For comparison, the imaging results using Y2O3: 15%Eu3+ as the luminescent target are depicted in Figure S9 (SI†). It indicates that due to intense luminescence emission of nanoparticles upon X-ray excitation, the target can be located and reconstructed with relatively high accuracy. For XLCT using the proposed PAA-NPs, the location error between the centers of the reconstructed target in XCT and XLCT is 1.72 mm, which is larger than the error of 1.10 mm using bulk material Y2O3: 15%Eu3+ but still less than 2 mm. Considering that this is a first attempt to use water dispersible nanoparticles of ultra-small size ( 9 nm) for XLCT imaging, the result is comparable with those reported in literatures, in which traditional X-ray excited bulk-materials,


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such as rare earth oxide (Gd2O3:Eu, Eu2O3)

2, 13

and oxysulfides (Gd2O2S: Eu)

1, 14-16

were used as light emitting sources.

Figure 5 The XLCT reconstruction results of the physical phantom experiment using PAA-NaGdF4:15% Eu3+ NPs. (a) and (b) depict the projection images acquired by the X-ray detector and the EMCCD camera at the same angle, respectively. The region between the red line (Z = 1.5 cm) and the green line (Z = 0 cm) was used for XLCT reconstruction. (c) depicts the XCT tomographic image at Z = 0.7 cm. (d) depicts the corresponding XLCT image. The red curves in (d) depicts the phantom boundary obtained by back-projecting the 72 white light images. (e) shows the fusion image of the XLCT and XCT images. (f) shows the 3D visualization of the imaging target reconstructed by XLCT.

3.5 In vivo validation of PAA-NPs for X-ray luminescence imaging Figure 6 gives in vivo luminescence imaging result using the water-dispersible PAA-β-NaGdF4:15% Eu3+ NPs. As shown in Figure 6, luminescent signals were detected in all three regions injected with 30, 70, 100 mg/mL of nanoparticles, respectively. By contrast, no luminescent signal was detected from the negative control region (0 mg/mL). Notably, there was no autofluorescence from tissues detected, which indicates an improved signal-to-noise ratio for bioimaging. This observation confirms the intense luminescent emission of the proposed ultra-small NPs.



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Figure 6 In vivo luminescence bioimaging of a nude mouse with subcutaneous injection on four limbs of the water-dispersible PAA-NaGdF4:15% Eu3+ NPs: (a) bright field, (b) X-ray luminescence imaging and (c) overlaid image.

3.6 In vivo validation of PAA-NPs for XLCT imaging The results of in vivo X-ray luminescence imaging verify the feasibility of PAA-NaGdF4:15% Eu3+ NPs as luminescent nanoprobes. Please note that for in vivo XLCT imaging, a transparent tube was used instead of intravenous injection. That was to keep the luminescent target fixed during the long scanning process of XLCT and to avoid the influence of NPs metabolism on the XLCT reconstruction. Since no targeting function was modified on NP’s surface in this study, the synthesized NPs would not accumulate in any organ of the imaged mouse specifically after intravenous injection. The luminescence emissions upon X-ray excitation, therefore, would be too low for high-quality XLCT reconstruction. Figure 7 presents the results of in vivo XLCT imaging using the proposed water dispersible NPs. Figure 7b gives an optical image of the mouse indicating the penetration of luminescent emission of NPs through body tissues. As shown in Figure 7e, with the fused image of the reconstructed XCT slice in Figure 7c and XLCT slice in Figure 7d, the location error of the implanted tube is estimated as 1.92 mm. Compared to the location error of 1.85 mm using bulk material Y2O3: 15%Eu3+, which is shown in Figure S10 (SI†), the result is relatively close and is comparable to limited in vivo studies reported previously,16, 37 which is acceptable for localizing the fluorescent targets.38 From 3D visualization of the reconstructed target illustrated in Figure 7f, it is observed that the light emitted from water-dispersible PAA-β-NaGdF4:15% Eu3+ NPs could be recovered accurately.


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As indicated previously, this study is a first attempt to use biocompatible NPs for in vivo XLCT imaging. Both phantom and in vivo studies indicate that the luminescence intensity emitted from β- NaGdF4:15% Eu3+ NPs is strong enough for X-ray luminescence imaging and XLCT imaging. Considering the sub-10 nm size distribution, low cytotoxicity, and excellent water dispersity of the proposed NPs, the obtained-PAA-NPs appear to be good candidates for in vivo XLCT application.15, 39, 40 It is worth noting that such surface modification nanoparticles can be further combined with bio-functional molecules to achieve functional molecular imaging, for instance, modified with tumor targeting molecules for tumor targeting XLCT imaging.

Figure 7 The X-ray (a) and luminescence (b) projection images of the nude mouse using PAA-NaGdF4:15% Eu3+ NPs. (c) and (d) represent tomographic XCT and XLCT images of the nude mouse, respectively. (e) the merged image of XCT and XLCT images with a location error of 1.92 mm. (f) the reconstruction 3D distribution of β-NaGdF4:15% Eu3+ NPs from XLCT result.

4. CONCLUSIONS In summary, the X-ray excited luminescent -NaGdF4:X% Eu3+ NPs with ultra-small size, monodisperse, and highly uniform distribution have been synthesized through the co-precipitation strategy for simultaneously control of the crystal phase and morphology. The experimental results indicate that the doping level of Eu3+ has no effect on the crystal phase and morphology of the NaGdF4 host. However, it has an



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impact on the luminescent intensity of the -NaGdF4:X% Eu3+ NPs upon X-ray excitation and the maximum luminescent intensity was observed in the samples containing 15% Eu3+. Importantly, the PAA-NaGdF4:X% Eu3+ NPs exhibit good water-dispersity and negligible cytotoxicity. The enhanced biocompatibility of the as-obtained NPs, integrated with high X-ray luminescence intensity, makes them good candidates for in vivo XLCT imaging. Both phantom and in vivo experiments performed in this study validate their feasibility for XLCT imaging. What's more, for the first time, we verify that the proposed biocompatible functional NPs could be used as high-quality inner excitation probes for in vivo XLCT, promoting XLCT imaging from a prototype design to preclinical implementation. The current study provides a proof of concept for the designation of new generations of NaGdF4 hosted NPs for in vivo XLCT application. With proper surface modification of these NPs, we believe that functional NPs-based XLCT holds great promise for in vivo target imaging that can detect molecular targets or events specifically. This creates a strong motivation for further modification of these NPs to obtain near-infrared emission and biomarker specificity, enabling deep-seated tumors to be detected accurately by this sensitive imaging technique. In addition, in in vivo studies, the specificity, sensitivity, stability and biosafety of the proposed X-ray excited luminescent NPs should be further explored.

AUTHOR INFORMATION Corresponding Authors *F. Shi: E-mail: [email protected] *H. B. Lu: E-mail: [email protected] Notes: The authors declare no competing financial interest.

ACKNOWLEDGEMENTS This work was financially supported by the National Key Research and Development Program of China (2017YFC0107400, 2017YFC0107401,


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2017YFC0107402, 2017YFC0107403, and 2017YFC0107405), the National Natural Science Foundation of China (Grants Nos. 81230035, 31700865, 61505102 and 21641001), the Natural Science Foundation of Shaanxi Province (2017JQ2020) and the Fundamental Research Funds for the Central Universities

(GK201602004,

GK201703028,

GK201701007

and

GK2017CSY033).

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Table of Contents Graphic


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Sub-10 nm Water-Dispersible Î²-NaGdF4:X% Eu3+ Nanoparticles with

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