shell nanophosphors for

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PEGylated #-NaGdF:Tb@CaF core/shell nanophosphors for enhanced radioluminescence and folate receptor targeting Yufu Ren, Hayden Winter, Justin Rosch, Kyungoh Jung, Allison Duross, Madeleine Landry, Guillem Pratx, and Conroy Sun ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.9b00629 • Publication Date (Web): 10 May 2019 Downloaded from http://pubs.acs.org on May 11, 2019

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ACS Applied Nano Materials

PEGylated β-NaGdF4:Tb@CaF2 Core/Shell Nanophosphors for Enhanced Radioluminescence and Folate Receptor Targeting Yufu Ren,a Hayden Winter,b Justin G. Rosch,a Kyungoh Jung,c Allison N. DuRoss,a Madeleine R. Landry,a Guillem Pratxc and Conroy Sun*,a,d

a

Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State

University, 2730 SW Moody Ave, Portland, OR 97201, USA b

Department of Chemistry, College of Liberal Arts & Sciences, Portland State

University, 1719 SW 10th Ave, Portland, OR 97201, USA c

Department of Radiation Oncology, School of Medicine, Stanford University, 300

Pasteur Drive, Stanford, CA 94305, USA d

Department of Radiation Medicine, School of Medicine, Oregon Health & Science

University, 3181 S.W. Sam Jackson Park Road, Portland, OR 97239, USA

*Corresponding

authors.

E-mail addresses: [email protected] (C. Sun).

Keywords: Nanophosphors, core-shell nanoparticles, lanthanide, radioluminescence, optical imaging, X-ray

ACS Paragon Plus Environment

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Abstract Lanthanide-doped nanocrystals have been examined extensively as contrast agents for various optical molecular imaging techniques. One of greatest strengthens of these nanomaterials is their ability to enable novel imaging modalities, such as X-ray excited radioluminescence imaging, which leverages the exceptional tissue depth penetration of X-rays and reduced tissue autofluorescence. Here, we report a uniquely engineered NaGdF4:Tb@CaF2 nanoscintillator with substantial lattice mismatch through integration of coprecipitation and thermal decomposition synthetic routes. We observed greatly enhanced radioluminescence by the NaGdF4:15%Tb@CaF2 core/shell nanocrystals, which results from the minimized surface quenching and localized structure

transformation.

Polyethylene

glycol

coated

NaGdF4:15%Tb@CaF2

nanocrystals demonstrated robust aqueous colloidal stability and were well tolerated by a panel of cell lines. The core/shell NaGdF4:15%Tb@CaF2 nanophosphors were subsequently decorated with targeting folate ligands and investigated as an X-ray luminescence imaging probe in vitro. Overall, the results suggest that these optimized radioluminescent nanophosphors have the potential to enable X-ray excited optical emission for biological imaging and serve as energy mediators in theranostic applications.


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Introduction Optical bioimaging approaches that utilize nanoparticle-based contrast agents often offer superior sensitivity and specificity compared to conventional medical imaging technologies. Recent advances in these novel imaging modalities have shown great potential for biomedical applications including early tumor detection, in vivo molecular diagnosis, and monitoring of treatment response.1-3 To yield high normal-to-diseased tissue contrast efficiency, various optical probes have been investigated, such as semiconductor quantum dots (QDs),4-5 organic fluorescent dyes6-7 and lanthanidedoped nanophosphors.8-10 As compared to the conventional fluorescent dyes, rare earth fluoride nanoparticles doped with trivalent lanthanide ions NaREF4:Ln3+ (RE = rare earth, Ln = lanthanide) possess significant advantages, including narrow emission bands, excellent photochemical and colloidal stability, long luminescence lifetimes, low toxicity and minimal autofluorescence.11 Due to advancement in nanomaterials chemistry, rare earth-doped nanoparticles with tunable emission and desirable surface functionalities have emerged as one of the most promising platforms for multifunctional

bio-applications.12-13

For

example,

image-guided

photothermal/photodynamic synergistic therapy has been developed using lanthanidedoped nanophosphors through activation of semiconductor shell or surface-bound photosensitizer/photothermal agents via a fluorescence resonance energy transfer process (FRET).14-15 It is worth noting that the lanthanide activated nanophosphors can be categorized as up-conversion and down-conversion (quantum cutting) nanoparticles according to the selection of Ln3+ dopants and the involved transition pathway.16 Due



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to the absence of multiphoton processes, down-conversion NPs are expected to achieve the higher quantum yield of photoluminescence at low excitation power than that of upconversion NPs.17 To further advance the spatial resolution and deep tissue detection capability of the optical bioimaging systems, we have previously developed combined X-rays/optical imaging techniques, such as X-ray luminescence computed tomography (XLCT), that has recently drawn increasing interests.18 Notably, the narrowly collimated X-ray beam could substantially eliminate the autofluorescence generated by scattered high energy photons within the tissue, exciting the luminescent probes located in deep tissue and enable the reconstruction of an optical tomographic image. The feasibility of this approach had been demonstrated using inorganic scintillators, including rare earth oxides

Gd2O3:Eu3+,

oxysulfides

Gd2O2S:Eu3+,

and persistent

luminescence

nanoparticles ZnGa2O4:Cr.18-21 However, the lack of solution-based synthetic routes and the extremely high reaction temperatures necessary for solid state synthesis limited their widespread applications in the biomedical field. Recently, lanthanide-doped nanophosphors, especially those in the class of down-conversion nanophosphors had been investigated as X-rays excitable bioimaging probes.3,

22-23

Generally, these

radioluminescent nanophosphors efficiently down convert the high energy X-ray photons to luminescence emission over the visible to short-wave infrared wavelength.22 Of particular interest has been β-NaGdF4 nanocrystals, especially in combination with Eu3+ or Tb3+ to tune the radioluminescence emission wavelength.23-25 As a high-Z component, Gd3+ not only exhibits favorable X-ray mass absorption properties, but also


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serves as the intermediate to activate the down-conversion light-emitting lanthanide ions in a Gd3+-containing host matrix.25-28 Moreover, these phosphors benefit from the intrinsic low phonon energy of the NaGdF4 host nanocrystals, avoiding the undesired nonradiative bridging that can compromise luminescent efficiency.23,

29-30

A recent

study has also shown that the NaGdF4 nanocrystals can serve as a contrast agent for magnetic resonance (MR) angiography, which exhibit significant r1 longitudinal relaxivity and negligible leakage of gadolinium ions in physiological environment.31 Thus, the NaGdF4 nanocrystals could be further developed as a multimodal optical-MR bioimaging nanoprobe. Although great progress has been achieved in the past decade leading to enhanced luminescence of lanthanide-doped nanophosphors, the discrepancy of luminescence efficiency between nanophosphors and their bulk counterparts remains significant.32 The low luminescence efficiency of lanthanide doped nanocrystals is often attributed to excited-state quenching by surface defects, impurities, ligands and solvent molecules.9, 16, 33 To suppress the surface quenching losses and subsequently increase quantum yield, the growth of inert shells comprised of undoped NaREF4 encapsulating the active luminescent cores was developed.22, 32-33 For example, with the protection of a 13 nm thick β-NaLuF4 shell, the up-conversion quantum yield of Yb3+, Er3+-doped nanocrystals increased over 50-fold.32 In addition to the undoped matching shells, CaF2 has been identified as another appealing candidate for the epitaxial protection of lanthanide-doped nanophosphors, due to its optical transparency, high crystallinity, and good chemical stability.34 Also importantly, the inherent biocompatibility of CaF2 make



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it particularly advantageous over the nonluminescent NaREF4, due to the long-term toxicity concern towards the presence of rare earth elements.35-36 Recently, the selective cation exchange strategy was proposed to construct core/shell nanoparticles with dissimilar crystal structures leading to greatly intensified up-conversion emission.37 In this work, β-NaGdF4:Tb@CaF2 core/shell X-ray excitable nanophosphors were synthesized through the combination of coprecipitation and thermal decomposition methods. The phase evolution and the microstructure of the as-prepared core and core/shell nanoparticles were examined by X-ray diffraction (XRD) and transmission electron microscopy (TEM). In comparison to the parent core nanoparticles, the βNaGdF4:Tb cores encapsulated by the heterostructured CaF2 shell exhibit significantly greater

luminescence

intensity

upon

X-rays

excitation.

This

enhanced

radioluminescence observed from β-NaGdF4:Tb@CaF2 core/shell nanophosphors can be mainly attributed to the surface passivation effects. Furthermore, the β-NaGdF4: Tb@CaF2 nanoparticles decorated with PEGylated phospholipid layer exhibit excellent aqueous colloidal dispersion stability and negligible in vitro cytotoxicity. In addition, the DSPE-PEG-folate functionalized β-NaGdF4:Tb@CaF2 core/shell nanophosphors were prepared and assessed as a X-ray excitable luminescence imaging probe in vitro. With these favorable materials properties, which include intense radioluminescence yield

and

biocompatible

response,

the

as-synthesized

β-NaGdF4:Tb@CaF2

nanoparticles with modular surface functionalization provide a promising X-ray excitable imaging probe platform that may soon enable novel radioluminescent imaging technologies.


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Results and discussion Physiochemical characterization. Scheme 1 illustrates the stepwise synthesis process for the β-NaGdF4:Tb@CaF2 core/shell nanoparticles and subsequent surface modification. In this process, β-NaGdF4:15%Tb nanoparticles were synthesized by a previously reported co-precipitation method and employed as particle cores.9 The Scheme 1. Schematic illustration of stepwise NaGdF4@CaF2 core/shell nanoparticle synthesis and surface modification procedure.

atomic percentage of 15% was consistent with recent studies, which demonstrates the addition of 15% Tb3+ correlates with the maximized radioluminescence due to the relatively weak cross-relaxation quenching between luminescence activators.26 The representative TEM images showed that the as-synthesized NaGdF4:Tb core only NPs have a quasi-spherical morphology with highly monodispersity and were approximately 8.2 nm in diameter (Figure 1a-c) as measured by dynamic light scattering (DLS). The corresponding the high-resolution TEM (HRTEM) image revealed continuous lattice fringes with d-spacing of 0.31 nm throughout the core nanoparticle, which can be assigned to the (110) plane of the hexagonal-phase NaGdF4. The formation of hexagonal (β-) phase NaGdF4 was further confirmed by the X-ray



diffraction (XRD) pattern of the as-synthesized core nanoparticles (Figure 1d), displaying all the characteristic diffraction peaks of pure β-NaGdF4 (PDF# 97-0415868) and no other phase or impurities were identified. Notably, the intensity of the of the β-NaGdF4 cores decreased substantially as the CaF2 shell was deposited. The newly emerged diffraction peaks agree well with the standard pattern of the cubic phase CaF2 (PDF# 98-000-0218), which is indicative of the CaF2 layer heteroepitaxially grown on NaGdF4 cores. It is can be seen that the sphere like nanoparticles transformed to nanocubes with the hydrodynamic size increased to 11.4 nm after grafting the CaF2 shells, as shown in Figure 1e-h. Thus, the average thickness of the as-prepared CaF2 shell could be estimated to be ca. 1.6 nm accordingly. Moreover, the oleate-capped nanoparticles were rendered hydrophobic, which are readily dispersible in the nonpolar solvents (i.e. cyclohexane) resulting in a transparent colloidal solution (inset of Figure 1e). The HRTEM image (Figure 1f) of the individual β-NaGdF4:Tb@CaF2 core/shell nanophosphor exhibited clear lattice fringes of the (220) crystal plane of cubic phase CaF2 with the observed d-spacing of 0.20 nm in the CaF2 layer. The core/shell geometry of the β-NaGdF4:Tb@CaF2 was clearly illustrated by the discernible contrast of STEM image (Figure 1g) obtained in high angular annular dark-field (HAADF) mode, wherein the high contrast arises from the distinction in atomic number and mass thickness of the elements present in the core (i.e. Gd and Tb) and shell (i.e. Ca). Epitaxial growth is usually performed on the materials with closely matched lattice structure to construct the core/shell geometry as the slight lattice mismatch could result in the anisotropic shell growth and unexpected shape deformation.36, 38-40 In particular, due to the negative


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Figure 1. (a) TEM and (b) HRTEM images of the as-prepared β-NaGdF4:15%Tb core only NPs. (c) Size distribution of the as-prepared β-NaGdF4:15%Tb NPs measured by DLS. (d) XRD patterns of the as-prepared β-NaGdF4:15%Tb core only and β-NaGdF4:15%Tb@CaF2 core/shell NPs. (e) TEM, (f) HRTEM and (g) HADDF-STEM images of the β-NaGdF4:15%Tb@CaF2 core/shell NPs (insets represent the photograph of as-synthesized NaGdF4:15%Tb@CaF2 core/shell NPs dispersed in cyclohexane and the core/shell geometry). (h) Size distribution of the as-prepared βNaGdF4:15%Tb@CaF2 core/shell NPs measured by DLS.

mismatch, the compressively strained shell growth leads to the increasing structural anisotropy.41 Conversely, here the hexagonal β-NaGdF4:Tb parent cores were purposely encapsulated in cubic CaF2 shell to primarily achieve the conformal heteroepitaxial core/shell structure with large magnitude of lattice mismatch (> 5%). It was found that the cation exchange between the Ca and Na could trigger the formation of a

(Na/Ca)REF4 buffer layer to remediate the mismatched the core/shell interface

and facilitate the heteroepitaxial growth of CaF2 shell.37

Moreover, the relatively

small sized nanocrystals are more favorable for the lattice-mismatched epitaxial shell growth, as the misfit stress could be appropriately distributed to the large amount of constituent atoms over their high surface area and greatly curved surfaces.42 Surface Modification. For biological applications, the nanophosphors capped with hydrophobic oleate ligands necessitate subsequent surface modifications to achieve



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Figure 2. (a) Representative TEM image of the β-NaGdF4:15%Tb@CaF2@DSPE-PEG-NH2 NPs in water (inset shows the corresponding photograph of the β-NaGdF4:15%Tb@CaF2@DSPEPEG-NH2 NPs dispersed in water). (b) DSL profile of the β-NaGdF4:15%Tb@CaF2@DSPE-PEGNH2 NPs. (c) FTIR spectra of the oleate capped β-NaGdF4:15%Tb@CaF2 NPs and PEGylated βNaGdF4:15%Tb@CaF2 NPs.

aqueous colloidal stability. Here we utilized polyethylene glycol (PEG) for its hydrophilicity and steric repulsion/non-fouling properties as a biocompatible coating. It is well established that PEG-modified nanoparticles could minimize the nonspecific interactions with protein and increase the in vivo circulation time. Thus, the as-prepared nanophosphors were functionalized with DSPE-PEG-NH2 to render them usable in biologically relevant media and provide a chemical functionality for potential bioconjugation of targeting ligands, such as antibodies. The representative TEM image (Figure 2a) of the DSPE-PEG-NH2 decorated β-NaGdF4:Tb@CaF2 core/shell nanophosphor showed that the nanophosphors preserved monodispersity without noticeable

aggregation

and

the

NaGdF4:Tb@CaF2@DSPE-PEG-NH2

identical

cubic

nanoparticles

shape. possess

In

addition,

excellent

β-

aqueous

dispersion stability leading to the formation of an optically clear suspension in water (inset of Figure 2a). The mean hydrodynamic diameter of the DPSE-PEG-NH2 coated NPs was determined to be 26.16 nm by DLS, which increased about 14 nm after the surface modifications. However, due to the low electron density of the polymer, the


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monolayer of the PEGylated phospholipids was only faintly observed in the TEM micrograph. The β-NaGdF4:Tb@CaF2 nanophosphors coated with DSPE-PEG-NH2 was further characterized by FTIR to detect the surface functional groups. As seen in Figure 2c, the oleate capped nanoparticles exhibited the typical asymmetric and symmetric stretching vibrations of methylene (-CH2) with the peaks centered at 2920 and 2850 cm-1 respectively. The intensive peaks at approximately 1450 and 1560 cm-1 could be ascribed to the COO- vibrations of oleic acid.43 However, peaks related to the vibrations of above-mentioned bands were significantly suppressed after the surface treatment of DSPE-PEG-NH2. In contrast, the new characteristic bands at 1340 (bending of C-H) and 1110 cm-1 (C-O stretching vibrations) emerged, which demonstrates the NPs surface were successfully engineered with phospholipids. The peak at 1640 cm-1 was attributed to the N-H bending vibration of the amine group, while the small bump located near the 3220 cm-1 (N-H stretching) was greatly masked by the broad band of hydroxyl (OH) stretching vibrations.44 More importantly, the DSPEPEG-NH2 was introduced to the surface of β-NaGdF4:Tb@CaF2 nanophosphors by the hydrophobic van der Waals interactions between the hydrophobic tail of the phospholipids and the oleate ligands, which results in the amine group to face toward the aqueous environment. This favorable orientation of the PEG lipid confirms the potential for further conjugated with diverse biomolecules for cell specific targeting applications.45 The core/shell nanophosphors were further endowed with tumor targeting capability by introducing the folate (FA) ligand on the surface of the nanophosphors, which can facilitate the specific binding to the folate receptor positive



tumor cells.46 As seen in Figure S1, yellow tinted solid was obtained after freeze drying of the DSPE-PEG-FA functionalized core/shell nanophosphors. Radioluminescence Characterization. The radioluminescence of the as-prepared βNaGdF4:Tb core and β-NaGdF4:Tb@CaF2 core/shell nanoparticles were measured using a custom fabricated spectrometer (Stellarnet Inc) system under X-ray (130 kV and 5 mA) irradiation. The radioluminescence spectra were initially acquired on the nanoparticle powder samples to gain insight into the luminescence mechanisms. As shown in Figure 3a, the powdered β-NaGdF4 nanoparticle doped with 15% mol of Tb exhibited the distinct Tb3+ emissions at 489, 548, 584 and 629 nm, which are believed to arise from the 5D4→ 7Fj (j=6-3) transitions of Tb3+.26, 47 The Gd component (K-edge at 50.2 eV) present in the rare earth fluoride nanocrystal not only serves as the highly efficient X-ray absorber but also contributes to energy transfer from the host lattice to the luminescent center.23, 48 Therefore, to demonstrate the versatility of the β-NaGdF4

Figure 3. (a) X-ray excited luminescence spectra of the β-NaGdF4:15%Tb and β-NaGdF4:15%Eu powder samples. (b) X-ray excited luminescence spectra of the colloidal β-NaGdF4:15%Tb and βNaGdF4:15%Tb@CaF2 NPs (cyclohexane suspension) along with reference organic liquid scintillator p-Terphenyl in toluene.

as the X-ray excitable host material, the β-NaGdF4:Eu (15 mol%) nanophosphor was also synthesized and its X-ray excited luminescence properties were evaluated


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correspondingly. The radioluminescence spectrum of β-NaGdF4:15%Eu powder sample showed three characteristic red emissions bands of Eu3+ ranging from 590 to 700 nm, which are attributed to the 5D0→ 7Fj (j=1, 2 and 4) transitions of Eu3+ respectively.47 It is known that radioluminescence output of the nanophosphors is greatly influenced by the activators (Tb3+ and Eu3+), as the photon emission in the Xray luminescence relies on the relaxation of the excited luminescent centers.25 In the excited state absorption (ESA) process, the emitting ions consecutively absorb multiple photons with suitable energy to reach the excited state which the emission occurs. In contrast, the X-ray excited luminescence process involves the generation of highly excited electron-hole (e-h) pairs in the host lattice due to the interaction between the Xray photons and the extranuclear electrons of Gd3+ and Tb3+. Subsequently, the migration of e-h pairs through the host lattice and the eventual radiative recombination at the luminescent center result in radioluminescence emission.25-26,

49

Thus, the

scintillation efficiency is considerably influenced by the crystal structure and surface state of the nanophosphors. The radioluminescence spectra of the colloidally dispersed nanoparticles and the reference organic liquid scintillator p-Terphenyl are displayed in Figure 3b. The p-Terphenyl presented an intense and broad peak centered at 395 nm, which is in agreement with previous reports.50 As expected, the radioluminescence intensity of the colloidal β-NaGdF4:15%Tb core nanoparticles decreased dramatically mainly due to the nonradiative surface quenching, while appreciable enhancement in the radioluminescence intensity was observed on the β-NaGdF4:15%Tb@CaF2 core/shell nanoparticles. It was demonstrated that the luminescence output of the



lanthanide doped nanophosphors was significantly lowered by excited state quenching due to the surface defects, vibrations of solvent and ligand molecules.33 For instance, the dipole-dipole coupling of lanthanide dopants to the vibrational mode of environment result in undesired excitation energy migration to the nanophosphor surfaces and irreversible energy loss.51-52 However, the solvent quenching rate could be significantly suppressed by the growth of a non-luminescent shell (i.e. CaF2), as the dipole-dipole coupling is highly dependent on distance and the associated energy migration could be restrained.52 Moreover, the presence of heterostructured CaF2 and the distinct oxide state between the Gd3+, Tb3+ and Ca2+ resulted in the largely diminished interfacial diffusion and leakage of the lanthanide ions, which are beneficial for the radioluminescence enhancement and long-term photostability.37 In addition to the surface passivation arising from the CaF2 shell encapsulation, the local asymmetry resulted by the cation exchange between Ca2+ and Na+ could promote the transition of luminescent ions and the enlarged nanoparticle size was found to increase the radiative recombination rate of e-h pairs.37, 53 X-ray Excited Imaging. To compare the performance of these two types of nanoparticles for X-ray luminescence imaging, we imaged various dilutions with Gd3+ concentration ranging from 0.5 to 10 mg/ml. The nanoparticles were suspended in water and the solution was dispensed in a 96-well microplate. The plate was then irradiated using a conformal small-animal irradiation system equipped with a bioluminescence imaging camera (X-RAD SmART, Precision X-Ray). In this system, the X-ray tube and optical camera are mounted on a rotating gantry, perpendicular to each other


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(Figure S2a). The gantry was set at a 90° angle to irradiate the microplate from the side and the beam was left uncollimated (open). In this configuration, the EMCCD is located above the microplate for imaging (Figure S2b). To assess the luminescence of the nanoparticles, optical images were acquired with the X-ray tube set to maximum power (225 kVp, 13 mA). However, in this open-field configuration, significant X-ray noise was detected by the camera. Since the noise appears randomly in the field of view, the camera was set to acquire 100 frames, each with an exposure of 100 ms, resulting in a total exposure time of 10 sec. The radiation dose received by the samples during the radiation exposure is estimated to be approximately 0.5 Gy. The goal here was to acquire high-quality images for the purpose of characterizing the nanoparticles; it is possible to acquire images with much shorter exposure resulting in radiation dose below 5 cGy. X-ray luminescence images were then displayed after processing to remove Xray noise and compensate for background signal (Figure 4a and 4b). Luminescence was visible mostly at higher concentration of nanoparticles and it matched the outlines of the wells. To better quantify the intensity of the signal and compare the two nanoparticle systems, we performed region-of-interest (ROI) analysis and found that the measured

Figure 4. X-ray excited optical phantom imaging of the aqueous solution of the (a) βNaGdF4:15%Tb@CaF2 and (b) β-NaGdF4:15%Tb NPs with various concentration of Gd3+. (c) Camera counts of the β-NaGdF4:15%Tb and the β-NaGdF4:15%Tb@CaF2 NPs water suspensions as a function of the Gd3+ weight concentration in the solution.



scintillation light increases in a concentration-dependent manner (Figure 4c). Furthermore, at the same concentration, the core-shell nanoparticles emitted about 7× more light than the core-only nanoparticles, confirming our initial hypothesis. Cytotoxicity. Prior to the in vivo bioimaging applications, the cytotoxicity assay is an important assay to evaluate the biological response of the nanophosphors. Here, the cytotoxicity of the PEGylated β-NaGdF4:Tb@CaF2 core/shell nanophosphors were measured on three cell lines including NIH/3T3 (murine fibroblast), HepG2 (human hepatocellular carcinoma) and 4T1-tdTomato-luc (murine breast cancer) using MTS assay (CellTiter 96® aqueous kit). As shown in Figure 5, after being incubated with the β-NaGdF4:15%Tb@CaF2@DSPE-PEG-NH2 nanoparticles for 24h, the cellular viability of the 3T3, HepG2 and 4T1 cells were all above 80% at the highest concentration of 400 µg/ml, which is indicative of the outstanding biocompatibility of the β-NaGdF4:Tb@CaF2@DSPE-PEG-NH2 nanoparticles. Surprisingly, about 10% decrease in viability of 4T1 cells cultured with PEGylated nanophosphors at a low concentration of 6.25 µg/ml was observed, which could be result of multiple random variables including the inconsistent initial cell seeding density. However, with the increased concentration of NPs up to 400 µg/ml, no further significant cell death was detected. This reveals that the as-prepared β-NaGdF4:15%Tb@CaF2@DSPE-PEGNH2 nanoparticles did not induce any appreciable toxicity at a concentration below 400 µg/ml. In addition, the live/dead fluorescence kit was employed to visually assess the viability and morphology of the 3T3, HepG2 and 4T1 cells cultured with the nanoparticles at the concentration of 200 µg/ml for 24 hrs. No significant inhibition of


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cell proliferation (blue) or increase in cell death (green) was observed in the presence of 200 µg/ml β-NaGdF4:15%Tb@CaF2@DSPE-PEG- NH2 nanoparticles, as compared to

those

of

the

untreated

cells.

When

incubated

with

the

β-

NaGdF4:15%Tb@CaF2@DSPE-PEG- NH2 nanoparticles, all three cell lines presented normal morphologies. Therefore, the PEGylated β-NaGdF4:Tb@CaF2 nanophosphors present the promising potential to serve as the bioimaging probe owing to their excellent aqueous dispersion stability and biocompatibility. The Cellular Uptake. To quantitatively assess the DSPE-PEG-NH2 and DSPE-PEGFA functionalized core/shell nanophosphors internalized in CT26 cells, the ICP-MS

Figure 5. Cell viability of the NIH/3T3, HepG2 and 4T1 cells incubated with βNaGdF4:15%Tb@CaF2@DSPE-PEG-NH2 NPs for 24 hrs and the corresponding the live/dead (Bluelive, Green-dead) fluorescence viability assay of the NIH/3T3, HepG2 and 4T1 cells cultured with NaGdF4:15%Tb@CaF2@DSPE-PEG- NH2 NPs at 24 hrs.

ACS Paragon Plus Environment Figure 6. Cellular uptake of the core/shell nanophosphors coated with DSPE-PEG-NH2 and DSPEPEG-FA in CT26 cells for 2h, as analyzed by ICP-MS.


was employed to measure the total Gd3+ content in CT 26 cells upon treatment with 2 concentrations (i.e. 100 and 400 μg/mL) for 2h. As shown in Figure 6, the mass of gadolinium appeared to be markedly higher in the CT26 cells cultured with DSPEPEG-FA functionalized β-NaGdF4:Tb@CaF2 core/shell nanophosphors, compared to the those treated with DSPE-PEG-NH2 functionalized nanophosphors. After 2h incubation, the presence of targeting folate ligand resulted in 5.3 and 4.6-fold higher intracellular Gd3+ concentration for the cells cultured with DSPE-PEG-FA functionalized β-NaGdF4:Tb@CaF2 core/shell nanophosphors at 100 µg/ml and 400 µg/ml respectively than those cultured with nontargeted nanophosphors. This demonstrates the FA targeted nanophosphors can be efficiently taken up by the folate receptor-overexpressing CT26 cancer cells through a receptor-mediated endocytosis process. Further, the cellular uptake of the FA modified nanophosphors was visualized using a confocal laser scanning microscopy (CLSM). It can be seen that green fluorescence emitted from Tb3+ and red emission from Eu3+ distributed throughout the cell cytoplasm (Figure S3), thus suggesting DSPE-PEG-FA treated nanophosphors can bind to and be internalized by the CT26 cells. In Vitro X-ray Luminescence Cell Imaging. To assess the efficacy of the DSPE-PEGFA functionalized β-NaGdF4:Tb@CaF2 core/shell nanophosphors serving as a

Figure 7. In vitro imaging of CT26 cell pellets with various treatments under ambient light and XACS Paragon Plus Environment ray excitation; (a) blank cells and cells labeled with β-NaGdF4:15%Tb@CaF2@DSPE-PEG-FA for 4h (b) and 2h (c), and (d) β-NaGdF4:15%Tb@CaF2@DSPE-PEG-NH2 for 2h.

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radioluminescent cell labeling probe, an in vitro X-ray excited optical luminescence experiment was carried out with the CT26 cell pellets.

Figure 7 shows the

photographs of the CT26 cell pellets under the ambient light and irradiated with X-ray. The relatively high X-ray scintillation light outputs were detected from cell pellets in tube b and c, corresponding to the cells incubated with FA targeted nanophosphors for 4h and 2h respectively. The more intense X-ray luminescence achieved by cell pellet b can be attributed to the robust intracellular uptake of the nanophosphors in CT26 cells with the prolonged incubation time. The cells cultured with PEG-amine functionalized nanophosphors displayed comparable X-ray luminescence intensity with reference to the untreated cells (cell pellet a), which is considered to be the result of the non-specific cellular uptake and the low intracellular concentration of the nanophosphors. It is noted that background optical signals were unexpectedly high when the X-ray beam was on, preventing lower concentrations of nanoparticle from being detected. Here, background luminescence in control samples may be an artifact of the experimental setup where the tube containing the cells serves to reflect or pipe light from the nearby samples. Further optimization of this imaging/therapeutic instrument will be required to detect of lower concentrations of nanoprobe, in an in vivo setting.

Conclusion In summary, we have designed and synthesized the β-NaGdF4:15%Tb@CaF2 core/shell nanophosphors with high magnitude of lattice mismatch between core and shell by combining the coprecipitation and thermal decomposition methods. The CaF2 shells



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were heteroepitaxially grown on the β-NaGdF4:15%Tb core, which resulted in the quasi-spherical

β-NaGdF4:15%Tb

cores

transformed

to

the

cube

like

β-

NaGdF4:15%Tb@CaF2 core/shell nanoparticles with the narrow size dispersion. The radioluminescence efficiency of the Tb3+ doped β-NaGdF4 nanophosphor was significantly improved by the heteroepitaxial shelling of CaF2. The enhancement in the X-ray excited luminescence could be the result of the effective surface passivation and the unique microstructure evolution occurred in the centrosymmetric shell growth process. Moreover, the NaGdF4:15%Tb@CaF2 core/shell nanoparticles decorated with DSPE-PEG-NH2 exhibit high dispersion stability in aqueous media and excellent biocompatibility. NaGdF4:15%Tb@CaF2@DSPE-PEG-NH2 core/shell nanoparticles surface engineered with targeting folate ligand exhibit efficient intracellular uptake by folate receptor overexpressing CT26 cells and in vitro cell labeling capability. Given the great potential of lanthanide-doped nanophosphors as the bioimaging probe, the NaGdF4:Tb@CaF2 core/shell nanoparticles are believed to be very attractive for X-ray excited deep tissue imaging and X-ray activated photodynamic therapy with precise imaging guidance.

Experimental Materials. Gadolinium acetate hydrate Gd(CH3CO2)3∙xH2O (99.9%), terbium acetate hydrate Tb(CH3CO2)3∙xH2O (99.9%), europium acetate hydrate Eu(CH3CO2)3∙xH2O (99.9%), chloroform (99.8%) were purchased from Alfa Aesar. Ammonium fluoride NH4F (99.9%), calcium carbonate CaCO3 (99%), 1-octadecene (ODE, 90%), and oleic


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acid (OA, 90%) were obtained from Sigma-Aldrich. Trifluoroacetic acid (CF3-COOH, 99%), hydrochloric acid solution (HCl, 1.0 N) and dimethyl sulfoxide (DMSO) were purchased from Fisher Scientific. 1,2-distearoyl-sn-glycero-3-phosphoethanolamineN-[amino(polyethylene glycol)-2000] (Ammonium salt) (DSPE-PEG(2000)-Amine) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[folate(polyethylene glycol)-2000] (ammonium salt) (DSPE-PEG(2000)-Folate) were obtained from Avanti Polar Lipids (Alabaster, AL, USA) and Nanocs Inc, respectively. All the chemicals were used without further purification. Synthesis of β-NaGdF4:Tb Core Nanoparticles. Briefly, the β-NaGdF4 core nanoparticles doped with Tb3+ were synthesized following the protocol reported in the literature with slight modifications.9 In a typical synthesis route, Gd(CH3CO2)3∙xH2O (0.85 mmol) and Tb(CH3CO2)3∙xH2O (99.9%) (0.15 mmol) were dissolved in a combination of oleic acid (6 ml) and octadecene (15 ml). The resultant mixture was heated to 130 °C under vacuum for 60 min and then cooled down to room temperature naturally under a nitrogen atmosphere. Next, 2.5 mmol of NaOH and 4 mmol of NH4F were dissolved in 2.5 ml and 7.5 ml of methanol respectively. After adding the methanol solution, the reaction mixture was further stirred overnight at room temperature and then heated to 110 °C for 30 min under vacuum to remove residual methanol and moisture. Following that, the reaction mixture was purged with nitrogen for three times and heated to 300 °C for 120 min under a nitrogen atmosphere. The obtained nanoparticles were precipitated and washed three times with ethanol, and lastly dispersed in cyclohexane (10 ml). β-NaGdF4:Eu nanoparticles were prepared using the



same manner, but replacing the terbium acetate hydrate Tb(CH3CO2)3∙xH2O with europium acetate hydrate Eu(CH3CO2)3∙xH2O. Preparation of Calcium Trifluoroacetate Precursor. 300mg of calcium carbonate CaCO3 (99%) was added to the flask containing 10 ml of deionized water (DI water) and 10 ml of trifluoroacetic acid (CF3-COOH, 99%). The mixture was then heated to 100 °C with vigorous stirring to form a clear solution. The residual water and excessive acid were evaporated at 100 °C in an oven. Synthesis of β-NaGdF4:Tb@CaF2 Core/Shell Nanoparticles. The CaF2 shell was synthesized via the thermal decomposition of calcium trifluoroacetate precursor. In brief, 2 mmol of Ca(CF3COO)2 and 5 ml of the colloidal solution of β-NaGdF4: Tb were added to a 100 ml three-neck bottom-round flask containing 10 ml of oleic acid and 10 ml of octadecene at room temperature. Next, the cyclohexane was removed by evaporation at 110 °C under vacuum for 30 min. After purging with nitrogen for three times, the reaction mixture was quickly heated to 300 °C for 60 min. The final product was collected by centrifuge and washed with ethanol for three times, and re-dispersed in ~5 ml of cyclohexane. Preparation of Ligand-free Nanoparticles. The ligand-free nanoparticles (LF-NPs) were achieved by removing the surface ligands of NPs via the HCl treatment. 54-55 In a typical procedure, 20 mg of oleate-capped NPs were dissolved in 10 ml of ethanol, and then a certain amount of 1M HCl solution was added to the system to maintain the pH value at 1. Next, the mixture was ultra-sonicated for 30 min to strip off the oleate. Subsequently, the NPs were isolated by centrifuge and dispersed in acidic ethanol


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solution (pH=4) for further purification. After the acidic treatment, the LF-NPs were washed with water and ethanol for three times and re-dispersed in DI water. Surface modification of β-NaGdF4:Tb@CaF2 Core/Shell Nanoparticles. 10 mg of oleate-capped NPs and 10 mg of DSPE-PEG(2000)-NH2 were added to two scintillation vials containing 10 ml of chloroform separately and sonicated for 30 min. Then the two kinds of chloroform solution with NPs and DSPE-PEG(2000)-NH2 were mixed in a glass vial and stirred for 1 hour at room temperature. The solvent was evaporated using a rotary evaporator (BUCHI, Germany) with a water bath of 30 °C for 1 h under vacuum. Following that, the film was hydrated with DI water (5 ml) and resuspended by ultrasonication (30 min). The resulting suspension was centrifuged and filtered through a 0.22 µm PTFE membrane filter to remove large aggregates and excess lipids prior to further use. To prepare the folate decorated NPs, the abovementioned procedure was followed but the 10 mg of DSPE-PEG(2000)-NH2 was replaced by the mixture of 8 mg of DSPE-PEG(2000)-NH2 and 2 mg of DSPE-PEG(2000)-Folate. Characterization. Hydrodynamic size distributions of the nanoparticle suspensions were measured using dynamic light scattering (DLS, Malvern Zetasizer NanoDS, Westborough, MA, USA). Transmission electron microscopy (TEM) images were obtained using a Tecnai F-20 TEM (FEI, Hillsboro, OR, USA) at the accelerating voltage of 200 kV. The TEM Samples were prepared by drop casting of NPs colloidal solution onto the copper surface of the formvar/carbon backed TEM grids (Ted Pella, Redding, CA, USA), and allowed to air dry overnight. The XRD patterns were acquired in focused beam (Bragg−Brentano) geometry on a Rigaku Ultima IV X-ray diffraction



system (Woodlands, TX, USA) using graphite monochromatized Cu Kα radiation and were recorded over the 2θ range of 20−70◦. The functional bands present on the NPs were characterized by a Fourier transform Infrared Spectrometer (Nicolet iS5, Thermo Fisher Scientific, US) using an ATR diamond crystal at the range between 4000 and 500 cm−1. The radioluminescence spectra were collected using a compact cabinet X-ray system (CellRad, Faxitron) coupled with the StellarNet Silver Nova 200 spectrometer (StellarNet Inc) and an optic fiber cable (Thorlabs) under ambient conditions. The samples in quartz cuvettes were placed 13 cm away from the X-ray source, which was operated at the maximum power without beam broadening filter (130 kV, 5 mA). The integration time was set at 60 s. The concentration of the nanophosphors in suspension was held constant in all cases. An organic liquid scintillator comprising p-terphenyl in toluene (5 mg/ml) was employed as a reference standard. Phantom X-ray Luminescence Experiments. Various concentrations of ligand free nanoparticles (core and core-shell; ranging from 0.5 to 10 mg/ml) were prepared in black-bottom 96-well plates (200 ul per well). The plate was placed on the treatment bed of a small-animal conformal irradiator (X-RAD SmART, Precision X-ray Irradiation). The gantry was rotated 90° to allow for the sample to be imaged optically from the top. In that configuration, the X-ray tube is on the side. An X-ray CT scan was performed to ensure that the plate is within the irradiated field. For X-ray luminescence imaging, the X-ray tube was turned to full power (225 kVp, 13 mA); at this power, the dose rate is approximately 3 Gy/min for open field (uncollimated). After ≈10 seconds, a sequence of optical images (n=100) were acquired with 0.1 s exposure, 2×2 pixel


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binning, and 50× electron-multiplication gain. For background correction, the process was then repeated, but with the samples removed from the imaging chamber. We also acquired dark images with the X-ray turned off. The images were processed to remove hot pixels due to X-ray photons hitting the CCD sensor. The basic procedure relies on the fact that X-ray noise is randomly distributed in the image, therefore there always exist at least one image (out of 100) for which a given pixel is free of X-ray noise. Images taken for the same experimental conditions were averaged (while excluding hot pixels), then filtered with a 2D Gaussian filter (σ=0.5 pixels). Background images were subtracted. ROI analysis was performed by placing circular regions of interest (diameter = 10 pixels) on the wells and on the background to calculate the mean signal within the ROI. Error bars are ± 1 standard error of the mean. For cell imaging, the cell pellets were placed in 15 ml conical tubes, placed on the treatment bed, and irradiated using the same procedure. Cell Culture and Cytotoxicity Assay. To evaluate the cytotoxicity of the as-prepared β-NaGdF4:15%Tb@CaF2@DSPE-PEG-NH2 NPs, 4T1 (mouse breast cancer), HepG2 (human liver cancer) and NIH-3T3 (mouse embryo fibroblast cell) were employed in the study. The cells were firstly cultured in media (RMPI 1640 Medium for 4T1; DMEM for HepG2 and NIH/3T3; all media was supplemented with 10% fetal bovine serum or bovine calf serum and 1% penicillin-streptomycin) under a humidified atmosphere of 5% CO2 at 37 °C. For cytotoxicity tests, the cells were seeded in 96-well microtiter plates at a density of 10,000/well for overnight to allow attachment. Next, the culture medium in each well was aspirated off cells and replaced with 100 µL of



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fresh medium containing the various concentration of PEG-NH2 functionalized coreshell NPs (400, 200, 100, 50, 25, 12.5, 6.25 µg/ml) or 10% DMSO. Then, the cells were incubated at 37 °C and 5% CO2 for another 24 h. After that, the cell viabilities were determined using CellTiter 96 aqueous one solution cell proliferation assay (Promega, USA), as per the manufacturer’s protocol. Moreover, fluorescence-based LIVE/DEAD assay (Thermo Fisher Scientific) was conducted to assess the cytotoxicity of the βNaGdF4:15%Tb@CaF2@DSPE-PEG-NH2 NPs. Cellular uptake of the PEGylated NaGdF4:15%Tb@CaF2 nanoparticles. To evaluate the cellular uptake of the nanophosphors with different surface functionalization, the CT26 (murine colon carcinoma, ATCC) cells were seeded into a 6-well plate at a cell density of 2×105 cells/well and cultured with RMPI 1640 in a humidified incubator (at 37 °C, 5% CO2) for 48 h. Then, the culture medium was replaced with fresh medium containing NaGdF4:15%Tb@CaF2@DSPE-PEG-NH2 or NaGdF4:15%Tb@CaF2@DSPE-PEG-FA

nanoparticles

with

two

kinds

of

concentrations (100 and 400 µg/ml). After 2h incubation, the cells were washed with PBS buffer for three times, detached with trypsin and resuspended in PBS buffer. The content of Gd3+ per cell was quantified by an inductively-coupled plasma mass spectroscopy (ICP-MS). In addition, the cellular uptake and in vitro cell imaging were visualized using an Olympus FluoView 1000 confocal microscope.

Author information Corresponding authors C.S. Email: [email protected] Notes The authors declare no competing financial interest.

Acknowledgments ACS Paragon Plus Environment

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This work was supported by the NIH NIGMS as a Maximizing Investigators’ Research Award 1R35GM119839-01 (C.S.) and Oregon State University College of Pharmacy Start-up Funds. We gratefully acknowledge the laboratory facilities and equipment support provided by the Portland State University (PSU) Center for Electron Microscopy & Nanofabrication.

Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: XX.XXXX/XXXXXXXXX. FTIR spectra and picture of β-NaGdF4:Tb@CaF2@DSPE-PEG-FA nanoparticles; photographs of X-ray luminescence imaging system; confocal images of CT26 cells incubated with nanophoshors (PDF)

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Figures Legends Scheme 1. Schematic illustration of stepwise NaGdF4@CaF2 core/shell nanoparticle synthesis and surface modification procedure. Figure 1. (a) TEM and (b) HRTEM images of the as-prepared β-NaGdF4:15%Tb core only nanoparticles. (c) Size distribution of the as-prepared β-NaGdF4:15%Tb NPs measured by DLS. (d) XRD patterns of the as-prepared β-NaGdF4:15%Tb core only and β-NaGdF4:15%Tb@CaF2 core/shell NPs. (e) TEM, (f) HRTEM and (g) HADDFSTEM images of the β-NaGdF4:15%Tb@CaF2 core/shell NPs (insets represent the photograph of as-synthesized NaGdF4:15%Tb@CaF2 core/shell NPs dispersed in cyclohexane and the core/shell geometry). (h) Size distribution of the as-prepared βNaGdF4:15%Tb@CaF2 core/shell NPs measured by DLS. Figure 2. (a) Representative TEM image of the β-NaGdF4:15%Tb@CaF2@DSPEPEG-Amine NPs in water (inset shows the corresponding photograph of the βNaGdF4:15%Tb@CaF2@DSPE-PEG-Amine NPs dispersed in water). (b) DSL profile of the β-NaGdF4:15%Tb@CaF2@DSPE-PEG-Amine NPs. (c) FTIR spectra of the oleate capped β-NaGdF4:15%Tb@CaF2 NPs and PEGylated β-NaGdF4:15%Tb@CaF2 NPs. Figure 3. (a) X-ray excited luminescence spectra of the β-NaGdF4:15%Tb and βNaGdF4:15%Eu powder samples. (b) X-ray excited luminescence spectra of the colloidal β-NaGdF4:15%Tb and β-NaGdF4:15%Tb@CaF2 NPs (cyclohexane suspension) along with reference organic liquid scintillator p-Terphenyl in toluene. Figure 4. X-ray excited optical phantom imaging of the aqueous solution of the (a) βNaGdF4:15%Tb@CaF2 and (b) β-NaGdF4:15%Tb NPs with various concentration of Gd3+. (c) Camera counts of the β-NaGdF4:15%Tb and the β-NaGdF4:15%Tb@CaF2 NPs water suspensions as a function of the Gd3+ weight concentration in the solution. Figure 5. Cell viability of the NIH/3T3, HepG2 and 4T1 cells incubated with βNaGdF4:15%Tb@CaF2@DSPE-PEG-NH2 NPs for 24 hrs and the corresponding the live/dead (Blue-live, Green-dead) fluorescence viability assay of the NIH/3T3, HepG2 and 4T1 cells cultured with NaGdF4:15%Tb@CaF2@DSPE-PEG- NH2 NPs at 24 hrs. Figure 6. Cellular uptake of the core/shell nanophosphors coated with DSPE-PEG-NH2 and DSPE-PEG-FA in CT26 cells for 2h, as analyzed by ICP-MS. Figure 7. In vitro imaging of CT26 cell pellets with various treatments under ambient light and X-ray excitation; (a) blank cells and cells labeled with βNaGdF4:15%Tb@CaF2@DSPE-PEG-FA for 4h (b) and 2h (c), and (d) βNaGdF4:15%Tb@CaF2@DSPE-PEG-NH2 for 2h.



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shell nanophosphors for

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