in Î²-Ca - ACS Publications - American Chemical Society

Article Cite This: ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

Cosubstitution of Lanthanides (Gd3+/Dy3+/Yb3+) in β‑Ca3(PO4)2 for Upconversion Luminescence, CT/MRI Multimodal Imaging Rugmani Meenambal and S. Kannan* Centre for Nanoscience and Technology, Pondicherry University, R. V. Nagar, Kalapet, Puducherry 605 014, India S Supporting Information *

ABSTRACT: Multifunctional Gd3+, Dy3+ and Yb3+ cosubstitutions in β-Ca3(PO4)2 were achieved through a facile synthetic technique. Gd3+ and Dy3+ prefer to occupy the 7-fold coordinated Ca2+(1), 6- or 8-fold coordinated Ca2+(2), and 8-fold coordinated Ca2+(3) sites of the β-Ca3(PO4)2 structure. The shortest Ca(5)O bond length of te Ca 2+ (5) site with 6-fold co-ordination favors Yb 3+ accommodation. The substitution limit of each dopant to attain β-Ca3(PO4)2 solid solution is determined as ∼1.25 mol %. The combined substitutions displayed upconversion emission in blue, yellow and red regions on excitation at 980 nm via the energy transfer mechanism from Yb3+ to Dy3+. Yb3+ and Dy3+ combination induces a high X-ray absorption ability for computed tomography (CT). The materials also displayed paramagnetic behavior and exhibited high longitudinal (r1 = 48.71 mM−1 s−1) and transverse relaxivity (r2 = 61.79 mM−1 s−1) indicating their potential as T1 and T2 contrast agents in magnetic resonance imaging (MRI). The negligible toxicity of Gd3+/Dy3+/Yb3+ cosubstitutions in β-Ca3(PO4)2 is also demonstrated. The overall results justify the potential of the developed material as possible contrast agent for T1 and T2 MRI/ CT/upconversion luminescence. KEYWORDS: β-Ca3(PO4)2, cosubstitutions, structure, MRI, CT, luminescence

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INTRODUCTION

Several carrier host systems are reported for utility in imaging-guided therapy and there is a thrust to develop multimodal imaging contrast agents. Some of the investigated host systems are organically modified silica, hydrophobic and hydrophilic organic polymers, quantum dots, inorganic fluorophores, upconversion materials, magnetic nanoparticles, graphene oxide, and other metal oxides.9−12 A recent report emphasize the design of magnetite/dextran-functionalized graphene oxide nanosheets for in vivo positive contrast MRI.10 Biocompatible core/shell (α-NaYbF4:Tm3+)/CaF2 nanoparticles with effective NIRin−NIRout upconversion (UC) features have also been developed for high contrast and deep bioimaging.11 Nonetheless, the functional utility of these systems is restricted on the grounds of toxicity, leakage of encapsulated agent, lack of colloidal and photochemical stability, susceptibility to photobleaching and decomposition under repeated excitation.12 In lieu of the limitations from the existing systems, the authors have recently demonstrated single and dual Ln3+ substitutions in β-tricalcium phosphate [βCa3(PO4)2, β-TCP] β-Ca3(PO4)2 for optical, CT, and MR imaging applications.13−16 β-Ca3(PO4)2 is promising as a host for bioimaging since the Ln3+ incorporation in calcium phosphates improve the optical and magnetic features of the encapsulated contrast agents. Besides, β-Ca3(PO4)2 offers

Multimodal imaging attracts a great interest in diagnosis and therapy to attain high-resolution, three-dimensional images and molecular features of tissues. Magnetic resonance imaging (MRI) and X-ray computed tomography (CT) provides information in terms of anatomical reconstruction while, fluorescence imaging is more sensitive and serves as a tool for visualization at the cellular level and in vivo animal models.1,2 In view of better spatial resolution, penetration depth in tissues and application in target site, a blend of CT, MRI, and luminescence imaging using multifunctional bioprobes are in high demand. Lanthanide (Ln3+)-based materials are considered ideal multimodal contrast agents because of their unique 4f electronic configuration, which present excellent optical, electronic, and magnetic features.3 Additionally, Ln3+-doped systems exhibits upconversion mechanism under an excitation at 980 nm, which results in the conversion of lower energy photons in near-infrared region (NIR) to higher energy photons in the visible region that facilitates high signal-to-noise ratio and deep light penetration for in vivo imaging.4 In the context of upconversion materials, NaYF4:Yb/Er upconversion nanophosphors have been developed for bioimaging and real time tracking.5 Currently reported upconversion materials includes NaYF4:Yb:Er nanoparticles doped with Gd3+ and core−shell structures of NaGdF4:Yb/Tm.6,7 There are also few illustrations on Ln3+-substituted apatite systems for upconversion luminescence and bioimaging.8 © XXXX American Chemical Society

Received: October 6, 2017 Accepted: December 12, 2017

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DOI: 10.1021/acsbiomaterials.7b00742 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

Article

ACS Biomaterials Science & Engineering

Table 1. Elemental Composition and Phase Fractions of Gd3+/Dy3+/Yb3+ Cosubstitutions in β-Ca3(PO4)2 Determined from ICP-AES and Rietveld Refinement, Respectively elemental analysis from ICP-AES (mol %) sample code β-TCP 1GDY 2GDY 3GDY 4GDY

Ca

2+

(mol %)

50.01 47.82 45.64 43.46 41.30

Dy

3+

(mol %) 0.73 1.45 2.17 2.90

3+

Gd

(mol %) 0.73 1.46 2.17 2.87

Yb

phase fractions from refinement (wt %) 3+

(mol %) 0.73 1.43 2.20 2.89

DyPO4 (wt %)

GdPO4 (wt %)

YbPO4 (wt %)

100 100 100 94.90 90.30

1.60 3.40

1.80 3.30

1.70 3.00

aqueous (NH4)2HPO4 solution was added dropwise to the mixture of cationic nitrate solution containing Ca2+, Gd3+, Dy3+, and Yb3+ under constant stirring conditions at 90 °C. Following the dissolution of solution mixture, NH4OH solution was added in excess to attain a pH of ∼8, thereby forming a white precipitate. To this above mixture was added 5% of cationic surfactant cetyltrimethylammonium bromide (CTAB) respective of overall cationic concentrations to enhance the dispersion ability of the system. Further, the suspension was kept under constant stirring conditions at 90 °C for 3 h. The obtained precipitate was filtered, dried at 120 °C overnight, and followed by the heat treatment at selective temperatures for further studies. Powder Characterization. Crystallinity and phase behavior of the synthesized powders after heat treatment at 900 °C were determined with X-ray powder diffractometer (RIGAKU, ULTIMA IV, Japan) using CuKα radiation (λ= 1.5406 Å). The powder samples were scanned in 2θ range of 5−90° at a scanning rate of 0.01°/s at 40 kV and 30 mA. The phase matches were made using standard ICDD (International Centre for Diffraction Data) card Nos. 00−009−0169 for β-Ca3(PO4)2, 01−083−0657 for GdPO4, 00−045−0530 for YbPO4, and 00−026−0593 for DyPO4. The quantitative analysis of the powder XRD patterns were also performed using Rietveld refinement through GSAS-EXPGUI software package.15 The standard crystallographic data for the refinement of β-Ca3(PO4)2, GdPO4, YbPO4 ,and DyPO4 were respectively obtained from Yashima et al.,23 Ni et al.,24 Herrmann et al.,25 and Milligan et al.26 Raman spectra of the all the powders were recorded in the scan range of 100 to 1100 cm−1 at an excitation wavelength of 785 nm with data acquisition time of 30s and 0.5% of power using Raman Spectrometer (Renishaw, United Kingdom). Determination of the elemental contents was performed by inductively coupled plasma-atomic emission spectroscopy (ICP-AES) Analysis (ARCOS, SPECTRO, Germany). The absorption spectra of the samples were recorded with UV−vis−NIR spectrometer (SHIMADZU 3600, Japan) in the range of 300−1400 nm. Upconversion luminescence emission measurements were carried out using UV−vis−NIR fluorescence spectrophotometer (FLS-980, Edinburgh, United Kingdom) using a 980 nm semiconductor laser. The magnetization curves were recorded on a Physical Properties Measurement System-Vibrating Sample Magnetometer (SystemQuantum Design, United States) in the field range of −1 to 1 T. Cytotoxicity and Live−Dead Cell Imaging in Vitro. A characteristic MTT [3-(4, 5-dimethylthiazol-2-yl) - 2, 5 diphenyltetrazolium bromide] assay was carried out to appraise the cytotoxicity of Gd3+/Dy3+/Yb3+ cosubstitutions in β-Ca3(PO4)2 on human hepatocellular carcinoma, Hep G2 cell lines procured from the National Centre for Cells Science (NCCS), Pune, India. Cells were cultured in Dulbecco’s modified Eagle’s medium with standard culture conditions at 37 °C in 5% CO2, supplemented with 10% fetal calf serum. The procedure is briefed as follows: 1 × 104 cells were seeded in 96-well plates as triplicate at the different concentrations of 10, 50,100 and 200 μg and then incubated for 96 h at 37 °C. All in vitro experiments were compared with positive control, doxorubicin hydrochloride drug, at a concentration of 0.48 μg. The assay was performed in accordance with EZcount MTT Cell Assay Kit. (Himedia, Cat. No. CCK003) and the absorbance were measured on ELISA reader. Cell viability was deliberated by live/dead cell assay (Live/Dead_ Viability/Cytotoxicity Kit, Invitrogen, Eugene, OR, USA). Briefly, Hep G2 cells were seeded on 96-well tissue culture plates with 1 ×

salient features of biocompatibility, osteoinductivity and nontoxicity that aids in bone and dental repair allied by the attractive possibility for bioimaging and therapeutic delivery applications. Sufficient reports are available on single and coupled ionic substitutions in β-Ca3(PO4)2 that targets specific applications. This is due to the flexibility of five different Ca2+ sites of β-Ca3(PO4)2 with wide range of sizes and coordination spheres that favors ionic substitutions at its lattice.17−19 Moreover, lanthanides functionally mimic with Ca2+ because of (i) similar ionic size of Ln3+ (ranging from 0.84−1.06 Å) with Ca2+ (0.99 Å) (ii) Ln3+ ions are spherical and hard and (iii) the bonding behavior of Ln3+ is essentially ionic with an exception of slight covalent contribution from s-orbitals.20 The biological properties of Ln3+ are profound as they unveil physiological effects by blocking the Ca2+ synaptic plasma membrane exchange and thereby inhibiting skeletal, smooth, and cardiac muscle contraction. The Ln3+ substitution either activates or inhibits calcium dependent enzymes and proteins while, they inhibit calcium-mediated processes associated with immune cell function.21 Among the available trivalent lanthanides, Dy3+, Gd3+, and Yb3+ are chosen as dopants because of their characteristic properties. Dy3+ and Yb3+ possess comparatively large K-edge value and high X-ray mass absorption coefficient suitable for CT contrast agents. Additionally, Dy3+ display high magnetic moment and short electronic relaxation time specific for T2 MRI contrast agents. On the contrary, Gd3+ with seven unpaired 4f electrons has been already commercialized as MRI contrast agents. Yb3+ gains substantial interest as it acts as sensitizer that absorbs NIR excitation over a range of 700−1000 nm and transfer the energy to the activators (Ho3+, Er3+, Tm3+, etc.), exhibiting short-wavelength upconversion emission.22 Herein, we designed biocompatible β-Ca3(PO4)2 substituted with Gd3+, Dy3+, and Yb3+ for upconversion luminescence/ MRI/CT multimodal imaging. A thorough investigation on the substitutions of Gd3+, Dy3+, and Yb3+ in β-Ca3(PO4)2 were illustrated through physiochemical characterization techniques. The energy-transfer upconversion emission has been demonstrated under NIR excitation. The cytotoxic effects along with multimodal imaging efficiencies for MRI, CT, and upconversion luminescence were established.

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β-Ca3(PO4)2 (wt %)

EXPERIMENTAL METHODS

Preparation of Gd3+/Dy3+/Yb3+ Cosubstitutions in βCa3(PO4)2. The lanthanide Gd3+, Dy3+, and Yb3+ cosubstitutions in β-Ca3(PO4)2 were prepared by facile aqueous precipitation technique. In the typical experiment, a series of four different concentrations of cosubstitutions in β-Ca3(PO4)2 were synthesized with varying levels of Ln3+. A standard stoichiometric β-Ca3(PO4)2 was also synthesized for effectual comparison. The (Ca2++Gd3++Dy3++Yb3+)/P ratio was maintained at 1.5 with equimolar concentrations of Ln3+. The sample codes along with the elemental compositions are listed in Table 1, which is used throughout the article. In brief, a freshly prepared B


Article


Figure 1. Refined diffraction patterns of (a) 1GDY, (b) 2GDY, (c) 3GDY, and (d) 4GDY. Red lines, yellow lines, pink lines, and green ticks, respectively, indicate calculated, background, difference, Bragg of β-Ca3(PO4)2 in 1GDY and 2GDY. Blue ticks, orange ticks, purple ticks, and maroon ticks indicate Bragg of β-Ca3(PO4)2, Bragg of DyPO4, Bragg of GdPO4, and Bragg of YbPO4, respectively, in 3GDY and 4GDY. 104 cells in 100 μL of media per well. After 24 h of stabilization of the cells, they were treated with different concentrations of cosubstituted β-Ca3(PO4)2 for 24 h. At the end of the exposure, the cells were washed with PBS followed by the addition of 1 μL of acridine orange and 1 μL of ethidium bromide. After a brief incubation of 30 min at 37 °C, the stained cells were examined under Fluorescence microscope (OLYMPUS) at 400× magnification. Computed Tomography (CT) Phantom Imaging and X-ray Attenuation. CT phantom images of Ln3+-cosubstituted βCa3(PO4)2 samples were acquired from a multi slice spiral CT system (GE HISPEED CT/e). The samples were prepared by dispersing in deionized water by varying dopant concentrations (0, 1.25, 2.5, 5, 10 mg/mL). Along with the samples, phantom image of water with zero HU were obtained as reference. Similarly, the analysis has been carried out for different concentrations of Omnipaque for effective comparison of the results. . The following specifications were used for the measurements 120 kVp and 160 mA: field of view (FOV) = 54.07 mm × 146.00 mm, thickness = 0.9 mm, exposure time = 800 ms/rotation. The images were resolved using the Kodak molecular imaging software embedded in the CT scanner. The attenuation values in terms of Hounsfield units (HU) were determined by the following equation:27

HU =

μ − μwater μwater

Relaxivity maps based on the intensity of MR images were obtained using Matlab software, The Mathworks Inc., MA. T1 and T2 maps are acquired with variable flip angle method and fast spin echo (FSE) sequence, correspondingly. The relaxivity values were calculated in accordance with the earlier literature reports by the authors.28 The sequence specifications for T1 weighted images are flip angles = 6−32°, slice thickness = 4 mm, matrix size = 256 × 256, echo time (TE) = 4.9 ms, repetition time (TR) = 20 ms. The sequence specifications for T2 weighted images are slice = 1, TR = 3000 ms, matrix size = 256 × 128, slice thickness = 5 mm, varied TE = 22−352 ms (difference 22 ms).

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RESULTS Structural Analysis. The previous report by the authors indicate the formation of unique β-Ca3(PO4)2 structure by hosting a selective amount Ln3+ at its lattice.13−16 The ability of β-Ca3(PO4)2 to retain a discrete solid solution depends on the quantity of Ln3+ concentrations either based on single and cosubstitutions. β-Ca3(PO4)2 attains a distinct solid solution by hosting respectively a maximum of ∼4.35 mol % and an equal share of ∼2.20 mol % in case of single and coupled Ln3+ substitutions. Based on the outcome of our previous investigations, the present study aims to combine the influence of three different Ln 3+ namely Gd 3+ , Dy 3+ , and Yb 3+ substitutions in β-Ca3(PO4)2. Akin with our previous reports, the qualitative X-ray analysis ensured the formation βCa3(PO4)2 at 900 °C with the combined substitutions of Gd3+, Dy3+ and Yb3+. The elemental analysis data of the powders at 900 °C (Table 1) exhibit good corroboration with

1000

Where μ and μwater are the X-ray attenuation coefficient values of the material and water, respectively. MR Imaging and Relaxivity in Vitro. The longitudinal proton relaxation time T1 and T2 relaxivity values of cosubstituted βCa3(PO4)2 were measured as a function of trivalent lanthanide ion concentration in a 1.5 T MRI scanner (Siemens Magnetom Avanto). C


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ACS Biomaterials Science & Engineering Table 2. Lattice Parameters Determined from Rietveld Refinement β-Ca3(PO4)2 (Å) sample code crystal system space group β-TCP 1GDY 2GDY 3GDY 4GDY

a = b axis

c axis

GdPO4 (Å) a axis

Dy(PO4) (Å)

b axis

c axis

a = b axis

Yb(PO4) (Å)

c axis

a = b axis

c axis

rhombohedral

monoclinic

tetragonal

tetragonal

R3c (167) 10.4420(4) 37.4110(3) 10.4427(3) 37.4108(1) 10.4438(4) 37.4095(1) 10.4445(2) 37.3999(1) 10.4473(5) 37.3896(2)

P121/c1

I41/amd

I41/amd

6.3236(4) 6.2467 (3)

6.8153(4) 6.8288(3)

8.1689(5) 7.9198(4)

6.8929(7) 6.9014(2)

6.0428(1) 6.1775(4)

6.8997(1) 6.8946(7)

6.1034(2) 6.0438(9)

refinement agreement factors χ2

RBragg

1.75 1.22 1.87 1.05 1.02

6.62 7.88 6.77 7.29 8.35

spectra of 1GDY and 2GDY unveil the bands characteristic of β-Ca3(PO4)2. Nonetheless, 3GDY and 4GDY exhibited bands typical of GdPO4 and DyPO4 at 995 cm−1 (AIIG internal mode) and YbPO4 at 1054 cm−1 [Γ4 (B2g) vibrational modes] respectively].31−33 It is noteworthy that the 945 and 969 cm−1 modes of β-Ca3(PO4)2 displayed a contrasting shift with progressive dopant concentrations, wherein the former and the latter indicated prominent Raman shift toward respective lower and high frequency regions. UV−Vis−NIR Absorption and Upconversion Emission. The room-temperature absorption spectra of all the synthesized powders recorded in the range of 300−1400 nm (Figure 3a) exhibits good agreement with the literature reports.34 The 2F5/2 → 2F7/2 transition typical of Yb3+ is centered at ∼980 nm and the absorption peak at 273 nm is attributed to the 8S7/2 → 6I7/2 transitions of Gd3+. All other distinct peaks are typical of Dy3+ transition from ground state 6 H15/2 to various excited states. In addition, the absorption intensities demonstrate a progressive trend as a function of dopant concentrations. Figure 3b represents the upconversion luminescence spectra of all the powders that were excited with 980 nm laser diode at room temperature. Interestingly, three major up-converted visible light emission peaks at 480 nm (blue), 571 nm (yellow), and 656 nm (red) are respectively assigned to 6H15/2, 6H13/2, and 6H11/2 transitions from ground state of 2F5/2 due to the energy transfer process from Yb3+ to Dy3+.35 A schematic representation of the partial energy levels of Yb3+ and Dy3+ and the possible energy transfer mechanisms is shown in Figure 3c. It is noteworthy to mention that the emission intensities exhibited a gradual upsurge with simultaneous increase in the dopant concentrations. The altering luminescence intensities under 980 nm excitation wavelength of Gd3+, Dy3+, and Yb3+ cosubstitutions in βCa3(PO4)2 are also represented by the CIE chromaticity diagram as illustrated in Figure 3d. The Commission International de I’Eclairage (CIE) 193136 has been used to determine the (x, y) chromaticity coordinates. The results

the predetermined concentrations considered during the synthesis. The XRD patterns of the powders recorded after heat treatment at 900 °C (Figure S1) specify the presence of βCa3(PO4)2 in all the compositions and additional DyPO4, GdPO4, and YbPO4 phases in 3GDY and 4GDY compositions. The powder XRD patterns were refined to gain quantitative information on the influence of Gd3+, Dy3+, and Yb3+ in βCa3(PO4)2 crystal structure. Figure 1 graphically represents the quality of the refined diffraction patterns and the corresponding data (Table 2) unveils the formation of unique βCa3(PO4)2 in 1GDY and 2GDY. Although DyPO4, GdPO4, and YbPO4 were detected as additional components along with β-Ca3(PO4)2 in case of 3GDY and 4GDY. The refined data of rhombohedral β-Ca3(PO4)2 with R3c(167) space setting, monoclinic GdPO4 with P121(c1) space setting, and tetragonal DyPO4 and YbPO4 with I41(amd) space setting are presented in Table 2. As a result of increased Ln3+ dopant additions during the synthesis, a gradual upsurge in the phase fractions of corresponding phosphates is witnessed in 3GDY and 4GDY. Besides, Table 2 illustrates the contrasting variations in lattice parameters with increasing dopant concentrations, wherein a = b-axis exhibits lattice expansion while c-axis displays a contraction of the unit cell. The refined occupancy values are attributed to the preferential accommodation of Gd3+ and Dy3+ at Ca2+(1), Ca2+(2), and Ca2+(3) lattice sites, whereas Yb3+ prefers to occupy at the Ca2+(5) lattice site of βCa3(PO4)2 (Table 3). Raman spectroscopy was recorded to examine the active vibrational modes of β-Ca3(PO4)2 due to the three different Ln3+ cosubstitutions. The resultant spectra (Figure 2) ensures the presence of Raman active bands typical of β-Ca3(PO4)2 at 945 and 969 cm−1 (υ1 symmetric P−O stretching), 406, 441, and 481 cm−1 (υ2 double degenerate O−P−O bending), 1046 cm−1 (υ3 triple degenerate asymmetric P−O stretching) and 548, 612, and 627 cm−1 (υ4 triply degenerate O−P−O bending modes) for all the investigated compositions.29,30 Raman

Table 3. Preferential Occupancy of Gd3+, Dy3+ and Yb3+ at Five Different Ca2+ Sites of β-Ca3(PO4)2 Crystal Structure occupancy factors Dy

3+

Gd3+

Yb3+

sample code

2+

Ca (1)

2+

Ca (2)

2+

Ca (3)

2+

Ca (1)

2+

Ca (2)

2+

Ca (3)

Ca2+(5)

1GDY 2GDY 3GDY 4GDY

0.049 0.052 0.074 0.093

0.018 0.038 0.055 0.119

0.031 0.040 0.061 0.102

0.050 0.069 0.078 0.098

0.039 0.057 0.084 0.121

0.026 0.037 0.083 0.104

0.039 0.054 0.067 0.121

D


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Figure 2. Raman spectra recorded for β-Ca3(PO4)2 and four different Ln3+ cosubstituted β-Ca3(PO4)2 after heat treatment at 900 °C.

Figure 3. (a) UV−vis−NIR absorption spectra. (b) Upconversion luminescence spectra of Ln3+ cosubstitutions in β-Ca3(PO4)2. (c) Energy level diagram of Yb3+ → Dy3+ corresponding to an excitation at 980 nm. (d) CIE diagram along with chromatic coordinates as a function of dopant concentrations.

Figure 4. (a) Cell viability of Ln3+ substitutions in β-Ca3(PO4)2 in HepG2 human liver hepatocellular carcinoma cells. Live/dead cell staining with (b) PBS (control) and (c) 4GDY after 24 h incubation.

E


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displayed an apparent signal enhancement with the increasing dopant concentrations. Simultaneously, varying concentrations of the GDY samples were monitored using X-ray CT to verify the Hounsfield Unit. The results ensure a linear correlation between the HU values and the concentration of GDY samples (Figure 5b). At equimolar concentrations, the calculated slopes for the developed samples exhibit higher HU values than Iopromide, a clinically used commercial contrast agent.39 The outcome signpost a promising feature to tailor Ln 3+ cosubstituted β-Ca3(PO4)2 as probes for X-ray CT imaging in vivo. Magnetic Measurements and MRI in Vitro. Apart from upconversion luminescence and CT contrast effectiveness, Ln3+-cosubstituted β-Ca3(PO4)2 as a prospective MRI contrast agent were evaluated. The paramagnetic behavior of all the compositions at room temperature magnetization is illustrated in Figure 6a. A magnetic moment of 0.79 emu/g exhibited by 4GDY is appropriate for MRI contrast agents, which is comparable with the literature reports.40 The developed Ln3+ cosubstitutions in β-Ca3(PO4)2 were evaluated for their usefulness as T1 and T2 MR imaging contrast agents using a 1.5T clinical MRI scanner at assorted concentrations in the range of 0.2−1.0 mM. With a steady increase in the dopant concentration, the T1 MRI signal intensity gradually increases with the resultant hypointense images (Figure 6b) whereas, the T2 MRI signal intensity displays a constant reduction with the resultant hyperintense images (Figure 6d). The witnessed trend is a clear reflection of the characteristic relaxivity measurements. From the slopes of 1/T1 and 1/T2 versus GDY concentration plot (Figure 6c), the r1 and r2 relaxivity values are respectively determined as 48.71 and 61.79 mM−1 s−1, which exhibits good coincidence with the literature data.41,42 These observations signify the feasibility of Ln3+ cosubstituted β-Ca3(PO4)2 for MR contrast imaging applications.

comprehend the multicolour luminescence of the developed materials in the visible region on excitation at 980 nm.37 Cytotoxicity Tests and Live/Dead Cell Imaging in Vitro. Prior to the in vitro imaging experiments, the cytotoxicity tests of Ln3+-cosubstituted β-Ca3(PO4)2 powders were performed using HepG2 human liver hepatocellular carcinoma cells as demonstrated in Figure 4a. The MTT assay reveals negligible level of toxicity for the tested concentration range up to 200 μg/mL. The cytologically compatible nature could be justified by the chemical nature of β-Ca3(PO4)2 that biologically mimics the inorganic component of the hard tissues.38 Figures 4b, c shows live (green)/dead (red) stain of (b) phosphate buffer saline (PBS) control and (b) 4GDY induced Hep G2 cell lines for 18 h incubation at a concentration of 100 μg/mL. PBS was used as a control to evaluate the relative cytotoxic effect of Ln3+ cosubstitutions in β-Ca3(PO4)2 on human liver cells. In all the cell images, the dominant green fluorescence emission signal demonstrates the absence of toxic effects in pure β-Ca3(PO4)2, cosubstituted βCa3(PO4)2 powders and PBS control. These results infer the stability of cells to remain alive under the tested conditions. X-ray Attenuation and CT Imaging in Vitro. Considering the high X-ray absorption efficiency of Ln3+, the CT contrast efficiencies of the Ln3+ cosubstituted β-Ca3(PO4)2 were evaluated. The developed contrast agent (Figure 5a)

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DISCUSSION The results ensures the flexibility of β-Ca3(PO4)2 crystal structure to host Gd3+, Dy3+, and Yb3+. The experimental observations are apparent in which the individual entry of each Ln3+ in the β-Ca3(PO4)2 lattice to a retain solid solution is limited to ∼1.25 mol %. Beyond this saturation limit, the lasting ions crystallize in the form of their corresponding phosphate components namely GdPO4, DyPO4, and YbPO4. It is worth mentioning that single or coupled Ln3+ substitutions exhibit a similar trend of additional lanthanide phosphate or lanthanide oxide phase formation beyond the substitution limit of ∼4.35 and ∼2.2 mol %, respectively.14−16 In continuation

Figure 5. (a) CT images of Ln3+ cosubstitutions in β-Ca3(PO4)2 in the concentration range of 0−10 mg/mL and (b) CT value (HU) as a function of Gd3+/Dy3+/Yb3+ concentrations.

Figure 6. (a) Room-temperature magnetic hysteresis plot of Ln3+ cosubstitutions in β-Ca3(PO4)2; (b) T1 MR images; (c) r1 and r2 relaxivity curves; and (d) T2 MR images of different concentrations of Gd3+/Dy3+/Yb3+ cosubstitutions in β-Ca3(PO4)2. F


Article

ACS Biomaterials Science & Engineering with our previous studies, the following reaction mechanisms have been proposed for the collective occupancy of Gd3+, Dy3+, and Yb3+ in β-Ca3(PO4)2:

Upconversion materials based on lanthanide-doped system gains interest for its application of multiplexing in optical and magnetic imaging.49−51 Gd3+, Dy3+, and Yb3+ cosubstitutions in β-Ca3(PO4)2 inferred upconversion emission in blue, yellow and red regions on 980 nm excitation via energy transfer process from Yb3+ to Dy3+ ions. The simultaneous presence of Yb3+ and Dy3+ stimulates energy transfer from Yb3+ (2F5/2 level) to Dy3+ (6H5/2, 6F7/2, and 6H7/2 levels) due to similar energy levels among Yb3+ and Dy3+. The absorption occurs via the process of either excited state absorption (ESA) or energy transfer (ET) or the combination of both the processes.35 The energy transfer process between the Gd3+ and Dy3+ in βCa3(PO4)2 system detailed in our previous study establishes the typical emissions of Dy3+ under 273 nm excitation.16 Despite the coexistence of Gd3+, Dy3+, and Yb3+ in the βCa3(PO4)2 system, Gd3+ imparts rather a negligible influence in case of near-infrared to visible upconversion. The CIE coordinates were used to determine the color tuning ability of Ln 3+ cosubstitutions in β-Ca 3 (PO 4 ) 2 . The CIE color coordinates (x, y) of all the powders with assorted dopant concentrations were determined with reference to the upconversion emission spectra. The resultant calculated values are (0.28, 0.29), (0.29, 0.31), (0.31, 0.33), and (0.33, 0.36) for 1GDY, 2GDY, 3GDY, and 4GDY, respectively. It is thus determined (Figure 3d) that the CIE coordinates of 1GDY are located in the blue region, 2GDY and 3GDY are positioned in the white region, and 4GDY are slightly found in the yellow region. These results indicate that cosubstitutions of Ln3+ realize multicolour luminescence in the visible region when excited at 980 nm. This result warrants great attraction to tailor a novel upconversion luminescent probe for multicolor imaging of biological tissues. As the study has been focused for biomedical applications, it is essential to evaluate the toxicity of developed material. The results from MTT assay and live (green)/dead (red) staining reveal the nontoxic nature of β-Ca3(PO4)2 due to Ln3+ cosubstitutions. The nontoxic nature of stoichiometric βCa3(PO4)2 is well-emphasized from the previous reports.52,53 The negligible toxic effect witnessed in β-Ca3(PO4)2 due to Ln3+ cosubstitutions is mainly attributed to the minimum concentration of the dopant level subjected for investigation. The CT efficiency of β-Ca3(PO4)2 due to Ln3+ cosubstitutions were determined as 332 Hounsfield units (HU) for 10 mg/mL, which originates from the collective effect of Gd3+, Dy3+, and Yb3+. Ln3+ exhibits higher instrument sensitivity and X-ray attenuation coefficient, which is mainly due to their high atomic number and higher K absorption edge values (50 keV for Gd, 54 keV for Dy, and 61 keV for Yb) than iodine (33 keV), which is a major component of the existing commercial contrast agents. It is necessary to emphasize that low molecular weight of iodinated contrast agents lead to their rapid excretion from kidney and consequently results in short circulation times in vivo. Hence, a large dose of contrast agents is required for better visualization of the tissues and moreover their rapid renal elimination is expected to cause adverse health effects in patients.54,39 Herein, the combined Ln3+ substitutions in βCa3(PO4)2 unveils enhanced signal intensity at a lower dose thus recommending reduced contrast administration to the patients. As shown in Figure 6a, Ln3+ cosubstitutions in β-Ca3(PO4)2 demonstrates paramagnetic hysteresis at room temperature, which arises from their inherent characteristics.55 Gd3+ with seven unpaired 4f electrons enjoys the largest pure electron

Ca 9GdDyYb(HPO4 )(PO4 )6 (OH) 900° C

⎯⎯⎯⎯⎯→ Ca 9GdDyYb(PO4 )7 + H 2O

The refined occupancy factors of the multiple Ln 3+ substitutions at different Ca2+ sites of β-Ca3(PO4)2 are wellresolved as reported in Table 3. The substitution model for trivalent element in β-Ca3(PO4)2 due to the charge imbalance between Ca2+ and M3+ is established by Yoshida et al.43 as follows: 3Ca2+ → 2M3+ + VCa(4), where M3+ is a trivalent element and VCa(4) is the vacancy. It is postulated that the defective and half-filled Ca2+(4) site compensates for the charge imbalance induced by a trivalent cation substitution. The average Ca2+O bond length and the ionic size of five different Ca2+ sites are considered the prime factors to determine the accommodation of any cation in β-Ca3(PO4)2 structure. The comparable ionic sizes of both Gd3+ (0.94 Å for 6-fold coordination, 1.05 Å for 8-fold coordination) and Dy3+ (0.91 Å for 6-fold coordination, 1.03 Å for 8-fold coordination) with that of Ca 2+ (1.00 Å for 6-fold coordination, 1.12 Å for 8-fold coordination) favors their occupancy at Ca2+(1), Ca2+(2) and Ca2+(3) sites of βCa3(PO4)2. It is also necessary to emphasize that the average bond length of Ca2+(1)O (2.32−2.51 Å), Ca2+(2)O (2.35−2.74 Å), and Ca2+(3)O (2.35−2.68 Å) contribute to rather negligible differences among each other.44 The Ca2+(5)O distance (2.21−2.31 Å) with 6-fold coordination is the least among the five different Ca2+ sites that facilitates to accommodate small sized cations like Yb3+ and Lu3+.45 In agreement with the aforementioned facts, the Yb3+ occupancy occurs at Ca2+(5) site of β-Ca3(PO4)2. A close examination of the refined lattice data (Table 2) unveils the significant impact induced by the multiple Ln3+ substitutions in β-Ca3(PO4)2 unit cell. In case of the investigated compositions, the refined lattice data confirm the rhombohedral β-Ca3(PO4)2 unit cell with R3c space setting that exhibits good corroboration with the data of Dickens et al.46 (a = b axis = 10.439 Å, c axis = 37.375 Å, α = β = 90°, γ = 120°) and Yashima et al.23 The lattice parameters displayed a constant expansion along a = b-axis along with the simultaneous contraction along c-axis due to Gd3+, Dy3+, and Yb3+ cosubstitutions. The contraction of c-axis is justified by considering the substitution of smaller-sized Gd3+, Dy3+, and Yb3+ for the higher sized Ca2+, whereas the high cationic mass of Dy3+ (162.50), Gd3+ (157.25), and Yb3+ (173.04) compared to the lower cationic mass of Ca2+ (40.08) contributes to the expansion along a = b-axis. Similar to the observations in refined lattice parameters, Raman spectra also displays a contrasting trend wherein, the respective shift of 969 and 945 cm−1 band toward higher and lower frequency regions are clearly justified. The same anion with a similar site symmetry in different compounds show evidence of differences in the frequency of internal vibrational modes that displays close proximity with various cationic parameters such as cationic mass, ionic size, coordination number, polarization power, cation−anion bonding, and electronic configuration.47 Similar shifts in the spectral vibrations are explained by Weir et al. as being attributed to the effect of varying cationic mass and ionic radius.48 G


Article


(CIF), Pondicherry University, for instrumentation facilities. The first author Rugmani Meenambal acknowledge Lady Tata Memorial Trust (year of award- 2016-17) for providing Junior Research Scholarship. The authors also acknowledge Mr. Biji M. Cheriyan, Radiographer, Tellicherry Co-operative Hospital.

spin magnetic moment and induces longitudinal water proton spin relaxation. On the contrary, Dy3+ with its asymmetric ground state (6H15/2) and 4f9 orbital possess the largest magnetic moment (10.65 μB) among all the Ln3+ and induces transverse relaxation,56,57 whereas Yb3+ contributes only to feeble magnetic moment that originate from its 4f13 orbital. Thus, the Gd3+ and Dy3+ combinations in β-Ca3(PO4)2 favors their potential as both T1 and T2 MRI contrast agents. The r1 = 61.79 mM−1 s−1 and r2 = 48.71 mM−1 s−1 values (Figure 6c) obtained for Ln3+-cosubstituted β-Ca3(PO4)2 are appreciably higher than the relaxivity values of the reported contrast agents.58,59 Considering the multifunctional upconversion luminescence, magnetic and X-ray attenuation properties, the proposed material could be a prospective candidate for multimodal imaging.

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CONCLUSION In summary, the cosubstitutions of Gd3+, Dy3+, and Yb3+ in βCa3(PO4)2 were successfully accomplished as contrast agent for multimodal imaging application. The critical limit to retain β-Ca3(PO4)2 solid solution considering the individual contribution of each dopant is determined as 1.25 mol %. The occupancy of Gd3+, Dy3+, and Yb3+ were determined and their incorporation induced expansion of a = b axis and the simultaneous contraction along c-axis of β-Ca3(PO4)2. The upconversion luminescence in blue, yellow and red regions on NIR (980 nm) excitations is realized by the energy transfer process from Yb3+ ions to Dy3+ ions. Meanwhile, Ln3+ cosubstitutions in β-Ca3(PO4)2 with high X-ray absorption coefficient exhibits higher CT contrast efficiency. In addition to the excellent paramagnetic characteristics, the toxicity results demonstrate the biocompatibililty and negligible cytotoxicity of Ln3+ cosubstitutions in β-Ca3(PO4)2 to Hep G2 human liver cell lines. Further, the positive and negative contrast enhancements were illustrated in T1 and T2 MR images with r1 = 61.79 mM−1 s−1 and r2 = 48.71 mM−1 s−1. Thus, the overall study validates the prospective nature of Gd3+, Dy3+ and Yb3+ cosubstitutions in β-Ca3(PO4)2 for upconversion luminescence, CT, and MRI multimodal imaging applications.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomaterials.7b00742. Figure S1: XRD patterns of all the powders heat treated at 900 °C (PDF)

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

* E-mail: [email protected]. Phone: 0091-413-2654973. ORCID

S. Kannan: 0000-0003-2285-4907 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The financial assistance received from DST-SERB [Reference: EMR/2015/002200 dated 20.01.2016], India, is acknowledged. We acknowledge Central Instrumentation Facility H


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