Ho3+ Co-Doped Apatite Upconversion Nanoparticles to

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Yb /Ho Co-Doped Apatite Upconversion Nanoparticles to Distinguish Implanted Material from Bone Tissue Xiyu Li, and Haifeng Chen ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b05514 • Publication Date (Web): 27 Sep 2016 Downloaded from http://pubs.acs.org on September 28, 2016

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ACS Applied Materials & Interfaces

Yb3+/Ho3+ Co-Doped Apatite Upconversion Nanoparticles to Distinguish Implanted Material from Bone Tissue Xiyu Li and Haifeng Chen* Department of Biomedical Engineering, College of Engineering, Peking University, Beijing, 100871, China KEYWORDS: Rare earth, apatite, upconversion fluorescence, lattice model, imaging and tracking

ABSTRACT: The exploration of bone reconstruction with time requires the combination of a biological method and a chemical technique. Lanthanide Yb3+ and Ho3+ co-doped fluorapatite (FA:Yb3+/Ho3+)

and

hydroxyapatite

(HA:Yb3+/Ho3+)

particles

with

varying

dopant

concentrations were prepared by hydrothermal synthesis and thermal activation. Controllable green and red upconversion emissions was generated under 980 nm near-infrared excitation; the FA:Yb3+/Ho3+ particles resulted in superior green luminescence, while HA:Yb3+/Ho3+ dominated in red emission. The difference in the green and red emission behavior was dependent on the lattice structure and composition. Two possible lattice models were proposed for Yb3+/Ho3+

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co-doped HA and FA along the hydroxyl channel and fluorine channel of the apatite crystal structure. We first reported the use of the upconversion apatite particles to clearly distinguish implanted material from bone tissue on stained histological sections of harvested in vivo samples. The superposition of the tissue image and material image is a creative method to show the material-tissue distribution and interrelation. The upconversion apatite particles and image superposition method provide a novel strategy for long-term discriminable fluorescence tracking of implanted material or scaffold during bone regeneration.

Introduction Bone reconstruction is both a biological process and a chemical process. The formation of bone apatite mineral is attributed to the chemical reaction or pile-up process of Ca2+, PO43-, and OH- ions from body fluid, enzymes and cells.1-2 Synthetic apatite usually acts as a bioactive material or scaffold component to bond with bone tissue in bone repair and reconstruction, mainly due to its affinity and similarity to bone mineral.3-4 The light images of histological sections with hematoxylin/eosin (H.E.) staining or Masson’s staining are adopted to show the newly formed bone tissue and biomaterial-bone interface.5 However, the implanted biomaterial often cannot be precisely imaged or discriminated on the stained histological section, especially for the biomaterial composed of synthetic apatite, which is too similar to the bone apatite to identify. A novel method is required to realize the imaging and tracking and to distinguish the implanted material from the newly formed bone tissue. We postulate that incorporating stable and long-lasting fluorescence into the apatite material is a promising strategy to achieve this purpose, on the premise of not losing the bone-bonding ability of synthetic apatite.


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Fluorescent probes play a key role in bioimaging to label the target and to amplify the signal. As microscopes have become more advanced, traditional organic fluorophores, such as green fluorescent protein (GFP), have demonstrated insufficient probe brightness and stability.6 The newly developed semiconductor quantum dots (QDs) may have also shortcomings in potential toxicity, photo-blinking, and short circulation half-times.7 Recently, lanthanide (Ln)-doped downconversion (DC) and upconversion (UC) fluorescent particles have drawn much attention due to their photostability, high contrast and low toxicity.8 Besides, the UC process using lower energy excitation such as near-infrared (NIR) light, shows advantages like deep penetration in tissues, negligible photodamage to cells and low auto-fluorescence.9 Table 1 briefly summarizes the limitations and merits of some fluorescent probes. Although some studies have shown upconversion applications in long-term real-time cell imaging10 and in small-animal imaging,11 currently most reported upconversion materials are based on matrixes of NaYF4,12 NaYbF4,13 and LaPO4.14 There have been few reports on lanthanide co-doped apatite matrixes for upconversion luminescence, and there are no reports on the potential of upconversion material for labelling implanted material and precisely tracking material-tissue interrelation.


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Table 1. Limitations and merits of some fluorescent probes. Rare-earth doping particles Organic Quantum dots fluorophores

Downconversion Upconversion by NIR by UV

easy to use, photobleaching,

high

high brightness,

high brightness,

brightness,

high contrast,

high contrast,

high contrast,

photo-stability,

photo-stability,

potential

low toxicity,

low or no toxicity,

conflict with

deep penetration in

photoblinking,

tissue

tissues,

short

auto-fluorescence,

circulation

photodamage to

auto-fluorescence and

half-time

cells

photodamage to cells

toxicity, poor stability, short lifetime avoidance of tissue

The apatite hexagonal structure was used as a stable matrix to host lanthanides in our previous studies to label and track the stem cell differentiation both in vitro and in vivo.15-17 Herein, we hypothesize that lanthanide co-doped apatites with stable upconversion luminescence and bone-bonding capacity will provide a new method to label the implanted material and precisely track the material-tissue interrelation, to overcome the insufficiency of H.E. or Masson’s stained histological sections. The method is an effective combination of a chemically synthesized


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fluorescent material and biological histological analysis. We also demonstrated that NIR upconversion excitation can avoid the auto-fluorescence of natural bone tissue (Figure S1), i.e., natural bone tissue could present blue, green, and red auto-fluorescence (Figure S1B-D) under excitation of UV, blue, and green light, respectively, but no luminescence or auto-fluorescence could be observed under 980 nm near-infrared light (Figure S1E). Therefore, the following research is focused on upconversion apatite materials.

Experimental section Materials:

Analytical

grade

Ca(NO3)2·4H2O,

Ho(NO3)3·6H2O,

Yb(NO3)3·6H2O,

Na3PO4·12H2O, NaF, and ethanol were obtained from Beijing Chemical Reagents Company, China. Preparation of Yb3+/Ho3+ co-doped apatite materials: Yb3+/Ho3+ co-doped apatite precipitates were hydrothermally synthesized based on a previous method.15 Briefly, an aqueous solution of Na3PO4 (for HA) or Na3PO4 plus NaF (for FA) was slowly dripped into the mixture solution of Ca(NO3)2, Ho(NO3)3, and Yb(NO3)3. Surfactants of octadecylamine, oleic acid or ricinoleic acid were selectively added during synthesis. The molar concentrations of Yb3+ and Ho3+ ions relative to Ca2+ ions were 10:0.5, 10:1, 10:2 and 10:3. After agitated for 10 min the solution mixture was treated hydrothermally at 120-160 °C for 12-16 h. The powder was obtained by centrifugation at 2700×g for 10 min and was then rinsed in deionized water and ethanol. Samples were freeze-dried and activated at approximately 700 °C for 2 h. Characterization: X-ray diffraction (XRD) data were obtained by a Philips X’ Pert Pro MPD (Netherland) with Cu Kα radiation (λ = 1.5406 Å). Transmission electron microscopy (TEM) imaging and elemental analyses were carried out on FEI TecnaiG2 T20 instrument (USA). The


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Fourier-transformed infrared (FTIR) spectra were obtained on a Perkin-Elmer 6000 spectrometer (USA).

The

photoluminescence

(PL)

spectra

were

detected

using

a

fluorescence

spectrophotometer - Hitachi F-7000 (Japan) equipped with a 2 W diode laser (Beijing Hi-Tech Optoelectronic Co., China). In vitro cell culture: The cell viability of MG63 cells cultured with various concentration of FA:10Yb3+/0.5Ho3+ particles for 1, 4, 7 and 11 days was evaluated via MTT assay (Promega, USA) with a microplate spectrophotometer (PerkinElmer Wallac 1420, USA) at 570 nm, using a method similar to our previous report.18 The MTT experimental results were presented in terms of the cell survival rate. For cell imaging, the FA:Yb3+/Ho3+ nanoparticles were modified with

dextran by immersing the particles (0.5 g) in a dextran solution (1 g, 7 mL). Then BMSCs were treated with 100 µg mL-1 dextran-modified FA:Yb3+/Ho3+ nanoparticles, as in our previous method.15 In vivo experiments: Six adult New Zealand white rabbits were randomly separated into 2-month group, 4-month group and 6-month group (n=2 in one group). Anesthesia was induced by ear vein injection. After shaving and disinfection of the hind limbs, a cylindrical bone defect (diameter 6 mm, depth 5 mm) was drilled into the left distal femoral condyle. The FA:Yb3+/Ho3+ nanoparticles were implanted into the defects (0.2 g in each defect, Figure S2) and were harvested with the surrounding tissue at 2, 4, and 6 months after implantation. The harvested samples were fixed in 10% formalin, dehydrated through an ethanol gradient, then embedded in PMMA and cut into 3 µm thick sections. After Masson staining, the stained sections were observed under Nikon TE2000-U light microscope (Japan). The fluorescent images were obtained using Nikon Ti-U inverted fluorescence microscope (Japan) and ZEISS LSM 780 NLO two-photo confocal microscope (Germany).


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Statistical analysis: Data are presented as the mean ± standard deviation (SD). A statistical comparison between two groups was analyzed by one-way ANOVA. A difference was considered to be statistically significant when P < 0.05.

Results and discussions The morphologies of hydrothermally synthesized Yb3+/Ho3+ co-doped FA and HA particles are shown in Figure 1A, B, and both particles show rod-like nanoscale morphology. After thermal activation at approximately 700 °C, the morphologies of FA:Yb3+/Ho3+ and HA:Yb3+/Ho3+ nanoparticles were not as uniform as before (inset in Figure 1A, B). The morphological change of HA:Yb3+/Ho3+ nanoparticles was more obvious due to its structure stability; the FA crystal structure is more stable than the HA crystal structure.2, 15 The phase composition of the activated particles

was examined using the XRD patterns. After thermal activation, the Yb3+/Ho3+ co-doped FA and HA particles still presented a hexagonal crystal structure, and their crystallinity increased distinctly

(Figure 1E, F). The XRD patterns match the classical hexagonal phase of FA (Ca10(PO4)6F2, ICDD 77-0120) and HA (Ca10(PO4)6OH2, ICDD 79-1572). The sharp characteristic peaks at approximately 26°, 32°, 33°, 40°, 47°, and 50° correspond to the (002), (211), (300), (212), (222), and (213) lattice planes, respectively. High-resolution TEM (HRTEM) was applied to reveal the crystallographic features of the nanoparticle (Figure 1C); the interplanar spacing is 0.347 nm corresponding to the (002) lattice plane. The selected area electron diffraction (SAED) pattern (inset) reveals (002) and (211) planes, and the EDS element mapping (Figure 1D) demonstrates the successful incorporation of Yb3+ and Ho3+ ions into the apatite crystal structure.


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Figure 1. The morphologies of FA:Yb3+/Ho3+ nanorods (A) and HA:Yb3+/Ho3+ nanorods (B) after hydrothermal synthesis and after thermal activation (inset). The HRTEM image (C) with the SAED pattern (inset) and EDS element mapping (D) of FA:Yb3+/Ho3+. The XRD patterns of FA:Yb3+/Ho3+ (E) and HA:Yb3+/Ho3+ (F) before and after thermal activation. The upconversion emissions were investigated under 980 nm excitation and 400 mW pump power (the powder density was approximately 22.6 W/cm2), with a fixed Yb3+ concentration of


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10 mol% and Ho3+ concentrations varying from 0.5 to 3 mol%. We observed different upconversion emission behaviors when altering the doping content of Ho3+ ions. For the Yb3+/Ho3+ co-doped FA matrix, the green emission had a much higher intensity than the red emission, and the peak width of the green emission was relatively narrow (Figure 2A). The FA:10Yb3+/0.5Ho3+ sample had the strongest emission intensity, with both the green and red emissions decreasing with increasing Ho3+ ion concentration from 0.5 to 3 mol, which may be a result of the concentration-dependent quenching. Others have reported a similar phenomenon on different doping matrixes.19-20 Figure 2B shows the upconversion emission spectra of HA:Yb3+/Ho3+, in which the peak intensity of the red emission was dominant and the HA:10Yb3+/1Ho3+ sample had the highest emission intensity. Under excitation of 980 nm near-infrared laser, the Yb3+ ions (sensitizer) absorbed energy from the 980 nm NIR light and transferred it to the adjacent Ho3+ ions (activator), which emitted two-photon upconversion luminescence.15, 21 The green and red emissions centered at 543 nm and 654 nm originated from the transitions of Ho3+: (5F4, 5S2) - 5I8 and 5F5 - 5I8, respectively (Figure 2C). Figure 2D illustrates the dependence of the photoluminescence (PL) intensity of FA:10Yb3+/0.5Ho3+ and HA:10Yb3+/1Ho3+ on the excitation power density. The intensities of the green and red emissions were enhanced by rising the excitation power density, and the enhancement was more rapid and evident during the initial period. A saturation effect might be present with a further increase in the power density, as shown in the curve trend.


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Figure 2. Upconversion luminescence spectra and fluorescent images (insets) of FA:Yb3+/Ho3+ (A) and HA:Yb3+/Ho3+ (B) with various dopant concentrations. The upconversion energy transfer diagram of Yb3+ and Ho3+ ions under 980 nm NIR excitation (C) and the dependence (D) of the PL intensity of FA:10Yb3+/0.5Ho3+ and HA:10Yb3+/1Ho3+ on the excitation power density.

The difference in the green and red emission behavior of FA:Yb3+/Ho3+ and HA:Yb3+/Ho3+ is dependent on their lattice structure and composition,15-18 as well as the distance-related efficiency of the energy transfer from the Yb3+ to the Ho3+ ions.21 Because FA and HA have the same hexagonal crystal structure belonging to the P63/m (176) space group, the difference in dominant


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emissions between the FA (green) matrix and HA (red) matrix may have been caused by the difference in the F- ions and OH- ions. When the F- ion substitutes for the OH- ion, which has a smaller ionic radius and stronger electronegativity, the hexagonal apatite crystal structure becomes more compact and the binding energy increases.18 These small changes in the crystal structure affect the efficiency of Yb3+ to Ho3+ energy transfer and alter the green and red emissions. In addition, F- ions have a much lower phonon energy than OH- ions.22-23 Stronger red upconversion is often reported by other researchers with a relatively high concentration of Ho3+ ions, and it is attributed to cross-relaxation among different levels of Ho3+ ions, which may inhibit green upconversion.24-25 Figure 3A-C shows the crystal structure of the unit cell of HA (A) and the OH channel in the center (B) surrounded by two calcium triangles rotated away from each other by 60º (C), which is from our calculated simulation using Material Studio 8.0 software (BIOVIA, USA). Figure 3D, E shows two possible lattice models proposed for Yb3+/Ho3+ co-doped HA and FA unit cells along the hydroxyl channel and fluorine channel. In the structure, there are two calcium triangles surrounding the two OH- ions or two F- ions. The hydroxyl or fluorine channel has enough lattice space for the substitution of lanthanide ions. To insure charge balance, the three Ca2+ ions in one Ca-triangle should be preferentially substituted by two Ln3+ ions, leaving a Ca2+ vacancy. When one Yb3+ ion and one Ho3+ ion substitute for two Ca2+ ions in a calcium triangle, a Ca2+ vacancy occurs for the charge balance, and the ion substitutions slightly change the lattice parameters. Compared to the lattice parameters of the pure FA structure (a = b = 9.45 Å, c = 6.88 Å), the FA:10Yb3+/0.5Ho3+ sample had smaller lattice parameters of a = b = 9.38 Å and c = 6.88 Å. After activation at approximately 700 °C, the parameters further decreased to 9.36 Å and 6.87 Å. Compared to the pure HA structure (a = b = 9.42 Å, c = 6.88 Å), the HA:10Yb3+/1Ho3+ sample had also smaller lattice parameters of a = b =


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9.39 Å and c = 6.88 Å. After thermal activation, the parameters further decreased to 9.38 Å and 6.84 Å. The decrease in the unit cell dimensions suggests a more stable crystal structure and a stronger ion interaction in the co-doped apatite lattice, which could enhance the distance-related efficiency of the energy transfer from Yb3+ to Ho3+.

Figure 3. The crystal structure of the unit cell of HA (A), the OH channel in the center (B) surrounded by two calcium triangles rotated away from each other by 60º (C). Lattice models for the Yb3+/Ho3+ co-doped HA (D) and FA (E) unit cells along the hydroxyl channel and fluorine channel of the apatite crystal structure; the broken circle represents the Ca2+ vacancy.

Although the brightness of both the green and red emissions was strong (fluorescent insets in Figure 2A, B), the green fluorescence has an advantage in distinguishing itself from the blue and red of the stained bone tissue on the histological sections. Thus, we selected and concentrated on the FA:10Yb3+/0.5Ho3+ sample with superior green luminescence for further cell and animal experiments. To evaluate the cytocompatibility of FA:10Yb3+/0.5Ho3+, we used the MTT assay


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to investigate the cell viability of MG63 cells cultured with various concentrations of FA:10Yb3+/0.5Ho3+ for 1, 4, 7, and 11 days. As shown in Figure 4A, the Yb3+/Ho3+ co-doped FA sample had no negative effect on the viability of MG63 cells at dosages of 100, 200, and 400 µg/mL. The cells proliferated with culture time; on day 1 and day 4, all experimental groups and the control showed similar cell survival rates. On days 7 and 11, although the experimental groups showed slightly lower survival rates than the control (*p

Ho3+ Co-Doped Apatite Upconversion Nanoparticles to

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