Targeted Imaging of Damaged Bone in Vivo with Gemstone Spectral

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Targeted Imaging of Damaged Bone in Vivo with Gemstone Spectral Computed Tomography Ying Wang, Chunhuan Jiang, Wenhui He, Kelong Ai, Xiaoyan Ren, Lin Liu, Mengchao Zhang, and Lehui Lu ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.5b07401 • Publication Date (Web): 04 Apr 2016 Downloaded from http://pubs.acs.org on April 5, 2016

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Targeted Imaging of Damaged Bone in Vivo with Gemstone Spectral Computed Tomography Ying Wang,†, ∥, ‡ Chunhuan Jiang,†, ‡ Wenhui He,†, ∥ Kelong Ai,† Xiaoyan Ren,† Lin Liu,§ Mengchao Zhang,*, § and Lehui Lu*, †, ∥ †

State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied

Chemistry, Chinese Academy of Sciences, 5625 Renmin Street, Changchun 130022, China ∥

University of Chinese Academy of Sciences, Beijing 100049, China

§

Department of Radiology, China-Japan Union Hospital of Jilin University, 126 Xiantai Street,

Changchun 130033, China KEYWORDS: gemstone spectral imaging, damaged bone, detection, ytterbium, nanoparticles.

ABSTRACT Achieving high-resolution imaging of bone-cracks and even monitoring them in live organisms are of great significance for understanding their extreme biological effects but remain quite challenging, especially only adopting commercial imaging systems. Herein, we explore the use of clinical gemstone spectral computed tomography (GSCT) technique as a powerful tool for targeted imaging of bone-cracks in rats via intramuscularly administrating crack-targeted ytterbium-based contrast agents (CAs). Material density images of GSCT reveal that bone-crack targeted with CAs can be successfully differentiated from healthy bone based on their different X-ray attenuation characteristics, giving GSCT a distinct advantage over

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conventional CT. More importantly, the superior imaging capability of GSCT allows us to realtime monitor the targeting and accumulation of CAs towards bone-crack in vivo. These results highlight that clinical GSCT, combined with ytterbium-based CAs, will provide a promising opportunity for understanding bone-related diseases in the future.

Bone as the major structural material in body supports a variety of organs, keeps mineral balance and, more importantly, enables the body mobility. During performing these functions, bone is subjected to continuous stresses and strains that may cause damage, such as diffuse damage and linear cracks. If the damage is not timely detected and repaired, accumulation of bone-cracks would lead to fractures in a relatively short time and is also likely linked to osteoporosis and osteoarthritis.1-6 Thus, the development of effective imaging systems that can help to detect and even monitor bone-cracks in live organisms would be greatly valuable for clinical research of their extreme biological effects. Histological study has been proposed to monitor the amount and type of bone damage, but it is inherently invasive and only suitable for in vitro study.7-11 To noninvasively detect damaged bone in vivo, positron emission tomography (PET) that is capable of imaging bone metabolic activity has sparked great research interest in imaging bone-cracks in vivo. Nevertheless, its resolution is relatively low and contrast agents (CAs) are not specific toward bone-cracks but rather cellular activity in the vicinity of bone-cracks.12,13 Considering the high-resolution and nondestructive features, conventional computed tomography (e.g., micro-CT) is anticipated to be a promising candidate for imaging bone-cracks. However, high background signal from strong X-rays absorption of electron-dense bone masks the efficient signal from CAs. Despite enormous research efforts in rational design of targeted CAs, imaging bone-cracks with conventional contrast-enhanced CT remains extremely challenging.14-16 One underlying problem is thought to


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be the inherent limitation of conventional CT technique with regard to its polychromatic images that can only reflect the integrated Hounsfield values and lose specific X-ray attenuation characteristics of healthy bone and CAs.17,18 Clinical gemstone spectral CT (GSCT) has made a great breakthrough in differentiating different matters owing to its technological innovations in detector and tube. With the development of the latest gemstone detector, GSCT is able to perform fast kilovoltage switching between 80 kVp and 140 kVp with one tube (gemstone spectral imaging (GSI) mode), which is challenging for conventional CT.19,20 The GSI mode enables acquisition of energy-independent basis material density that can specify X-ray attenuation characteristics of the scanned matter, such as the amounts of photoelectric effect and Compton scattering contributing to the X-ray attenuation, thus achieving material differentiation.21 We wonder whether the same concept can be applied to bone-cracks detection. However, a key issue in exploiting GSCT for imaging bonecracks in live organisms has been that bone-cracks and healthy bone share the same composition and almost the same Hounsfield value, hence making it difficult to distinguish bone-cracks from healthy bone. To overcome these challenges, especially, to achieve targeted imaging and enhance the CT signal contrast, we turn our attention to the recently reported ytterbium-based nanoparticulate CAs. We and others have demonstrated that ytterbium-based nanoparticle can provide high contrast efficacy for conventional or spectral CT 22,31 and, more importantly, ytterbium (Yb) can present different X-ray attenuation characteristics from calcium (Ca, main metal element in cortical bone),36 all of which are highly desirable in exploring the use of GSCT technique for the targeted imaging of bone-cracks in vivo. With this inspiration, here we demonstrate that GSCT technique can efficiently differentiate bone-crack targeted with CAs from healthy bone based on


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their different X-ray attenuation characteristics. More importantly, the superior imaging capability of GSCT allows us to real-time monitor the targeted accumulation of CAs towards bone-crack in vivo. To the best of our knowledge, our findings constitute the valuable experimental evidence for the use of GSCT technique as a powerful tool to image bone-crack in vivo.

Figure 1. Schematic illustration of the synthesis of YbNP@SiO2-NTA; TEM images of (A) OAYbNPs, (B) YbNP@SiO2-NH2; (C) Zeta potentials of corresponding samples. RESULTS AND DISCUSSION Synthesis and characterization of YbNP@SiO2-NTA. The synthesis of NaYbF4: Gd3+/Er3+@SiO2-NTA nanoparticles (NPs) is illustrated in Figure 1. NaYbF4:Gd3+/Er3+ NPs were firstly prepared through a modified solvothermal route and then coated by a biocompatible amorphous silica shell (YbNP@SiO2) via a simple water-in-oil reverse microemulsion method.2325

The obtained YbNP@SiO2 possessed a distinct core-shell structure with a diameter of around

46 nm (Figure 1B and Figure S4-5 in Supporting Information). Subsequently, YbNP@SiO2 were further modified by 3-aminopropyl triethoxysilane (APTES), endowing the YbNP@SiO2 with


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plenty of exposed amino groups that could be easier to covalently link with carboxyl groups of N-nitrilotriacetic acid (NTA), a critical calcium chelator.26-28 The surface modification process was monitored with Fourier transform infrared spectra (Figure S6), X-ray photoelectron spectrometry (XPS) (Figure S1-3) and zeta potential evaluations. The decline of zeta potentials from +27.0 mV to -38.4 mV proved the successful conjugation of NTA (Figure 1C). This large negative zeta potential improved the stability of YbNP@SiO2-NTA in various solutions, owing

Figure 2. (a) XPS survey spectra of YbNP@SiO2-NTA after immersed in CaCl2 aqueous solution for 24 h. The inset showed the high-resolution Ca2p XPS spectra; (b) Backscattered SEM micrographs showed the surface density of YbNP@SiO2-NTA inside the scratch (top) and outside the scratch (bottom); (c) T1-weighted MR images and relaxation rate r1 (1/T1) versus various Gd3+ concentrations. (d) T1-weighted MRI of control bone and the second labeled damaged bone model.


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to the intense electronic repulsion between the nanoparticles (Figure S7). Moreover, biotransmission electron microscopy (bio-TEM) images of muscle injected with YbNP@SiO2-NTA indicated that most of nanoparticles also maintained monodisperse in vivo (Figure S19). Damaged bone targeted by YbNP@SiO2-NTA. To specifically target bone-cracks, iminodiacetate functionalities were applied to effectively chelate exposed calcium ion. First, XPS spectra evidenced the ability of YbNP@SiO2-NTA as calcium ion binder in calcium chloride aqueous solution (Figure 2a and Figure S18a-b).29,30 Then, we proceeded to confirm the targeting specificity of YbNP@SiO2-NTA for damaged bone tissue which exposed calcium ion on the surface of damaged hydroxyapatite crystal and underwent hydrolysis to release free calcium ion.1,10,11 The pig tibiae bone specimens were sectioned, polished, scratched and finally soaked in YbNP@SiO2-NTA solution. High difference in concentration of YbNP@SiO2-NTA between the crack and healthy bone was detected by backscattered scan electron microscopy images (Figure 2b and Figure S18c-d). In addition, much greater T1-weighted enhancement was observed in labeled damaged murine bone compared with the control group in T1-weighted magnetic resonance images owing to the paramagnetism of gadolinium (r1=1.14 mM-1S-1) (Figure 2c,d). These results provided sufficient evidences for the targeting property of YbNP@SiO2-NTA for damaged bone. Imaging of damaged bone in vitro. Considering the complex physiological environment of damaged bones in vivo, we first established the ability of the clinical GSI mode of GSCT to differentiate YbNP@SiO2-NTA, as CAs, from calcium chloride, simulating the bone tissue with both experimental and theoretical analysis. The GSI mode enabled acquisition of synthesized monochromatic images derived from dual-energy CT data (Figure S15). As shown in Figure 3a, the CT signal of YbNP@SiO2-NTA enhanced linearly with the increased concentration of Yb


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element on GSI monochromatic energy of 70 KeV, which endowed the GSCT technique with an excellent quantitative imaging property.31,32 On the other hand, the X-ray attenuation of YbNP@SiO2-NTA declined slowly with the increasing monochromatic energy of GSI in the whole energy range (Figure 3b). In stark contrast, the X-ray attenuation of calcium chloride decreased very swiftly, hence leading to a remarkable difference from that of YbNP@SiO2-NTA, especially at a relatively higher monochromatic energy. These results revealed that YbNP@SiO2-NTA and calcium chloride possessed different X-ray attenuation characteristics.

Figure 3. (a) Gemstone spectral CT values (HU) and spectral CT images (inset) of YbNP@SiO2NTA with varied concentrations in water at 70 keV; Spectral CT HU curve (b) and synthesized monochromatic images (c) of YbNP@SiO2-NTA (16 mg/ml) and calcium chloride powder. We hypothesized that this difference in attenuation characteristics between calcium chloride and YbNP@SiO2-NTA was related to their large difference in atomic number (Z) of Ca and Yb elements according to the formula of X-ray absorption coefficient (µ):

µ=

ρZ 4

(1)

AE 3


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where ρ is the density, A is the atomic mass and E is the X-ray energy.33 It is apparent that the Xray absorption coefficient is proportional to the fourth power of Z. To gain more insight into the mechanisms responsible for different attenuation characteristics between Yb and Ca, we employed the interaction of X-rays with matter to investigate the role of atomic number in X-ray attenuation.34-36 It has been demonstrated that X-ray attenuation in CT imaging mainly derives from coherent scattering (ω)，the photoelectric effect (τ), and Compton scattering (σ) between X-ray photons and the matter.37 The X-ray attenuation coefficient (µ) of the matter is the sum of these three interactions: (2)

µ = ω +τ + σ

In this case, coherent scattering is so minor that it can be excluded from further consideration. Oppositely, the photoelectric effect and Compton scattering play leading roles in the attenuation. The photoelectric effect arising from the interaction between the X-ray photons and the inner shell electrons, takes place when the X-ray photon energy is greater or the same as the electronic binding energy. In this context, the atomic number (Z) and photon energy (E) significantly influence the occurrence of this process showed as follows:

µ∝

1 E3

(3)

τ ∝ Z3

(4)

Clearly, the matters with higher atomic number are more likely to be involved in the photoelectric effect. Additionally, the greater the photon energy, the less X-ray absorption will occur in this interaction. For Compton scattering, it occurs when an X-ray photon collides with an outer-shell electron and it gradually diminishes as the X-ray photon energy increases. It has been reported that the contribution of Compton scattering is high for the matter with low Z (e.g.,


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ZCa, 20), but its contribution is greatly diminished for the matter with high Z (e.g., ZYb, 70) as the photoelectric effect predominates. Accordingly, it is reasonable to conclude that Compton scattering is responsible for the X-ray attenuation in calcium chloride, while most of X-ray absorption in YbNP@SiO2-NTA mainly originates from photoelectric effect. Therefore, the atomic number indeed affects contribution of photoelectric effect and Compton scattering for X-

Figure 4. (a) Spectral HU curve of the healthy bone (L1) and the crack (L2); (b-d) GSI monochromatic images of the in vitro damaged bone at 40, 70, 140 keV; 2D coronal CT crosssections of iodine (HAP) base image (e) and its real-time pseudocolor image (f); (g) volumerendered image of the model at 70 keV; (h) pseudocolor image of iodine (HAP) base image at oblique section.


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ray attenuation of Yb and Ca referring to aforementioned attenuation mechanism. Furthermore, photoelectric effect and Compton scattering show different energy-dependent responses. Based on this, YbNP@SiO2-NTA and calcium chloride could be differentiated by their attenuation trend along with the increase of X-ray photon energy under the GSI mode. Impressively, GSCT could differentiate YbNP@SiO2-NTA from calcium chloride by virtue of their different attenuation characteristics. Encouraged by this success, we then developed an in vitro damaged bone model (see in Supporting Information) and proceeded to distinguish the

bone-crack, where YbNP@SiO2-NTA were concentrated, from healthy bone (hydroxyapatite, Ca10(PO4)6(OH)2, HAP) under GSI mode in vitro. As shown in Figure 4, the attenuation difference with increased monochromatic energy between YbNP@SiO2-NTA and healthy bone was confirmed. With increasing monochromatic energy, the signal intensities of the first and the second cracks labeled with YbNP@SiO2-NTA varied a little while a marked decrease in signal intensities for healthy bone could be identified (Figure 4 and Figure S9), well consistent with the trend of CaCl2. This difference should be attributed to the different contribution of photoelectric effect and Compton scattering for their X-ray attenuation, i.e. Compton scattering for most attenuation of healthy bone and photoelectric effect for YbNP@SiO2-NTA. Furthermore, based on the difference in X-ray attenuation characteristics of healthy bone and YbNP@SiO2-NTA, GSCT was capable of differentiating them through a few constants (e.g., basis material density) which were independent of energy. This ability of GSCT was usually defined as material decomposition, which is challenging for conventional CT. The basic principle of material decomposition relies on the assumption that the energy-dependent attenuation coefficient (µ) of all materials could be expressed as a combination of the Compton


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and the photoelectric coefficients7 and consequently also be represented by the weighted sum of the attenuation coefficient of two so-called basis materials:17, 18

µ ( x, y , E ) = m ( x, y ) µ ( E ) + m ( x, y ) µ ( E ) 1

1

2

2

(5)

Where, µ, µ1, and µ2 are the attenuation coefficients of the scanned matter, the first and the second basis materials respectively. m1(x,y) and m2(x,y) are the equivalent density of the scanned matter represented by the first and second basis material at coordinate (x,y). The two basis materials should be sufficiently different in their atomic number and thereby also in their Compton and photoelectric attenuation characteristics.17,18 At this point, material density images quantitatively reflect the amount of each basis material (i.e. the amount of photoelectric effect and Compton scattering) that contributes to the observed X-ray attenuation in different scanned matters and achieve materials separation in some degree. Thus, we used material decomposition to differentiate Yb-based CAs from healthy bone based on the different contribution of photoelectric effect and Compton scattering for their X-ray attenuation. In our case, iodine/calcium and iodine/HAP were chosen as basis materials to acquire four material density images, namely, iodine (calcium) base image, calcium (iodine) base image, HAP (iodine) base image (Figure S10), and iodine (HAP) base image (Figure 4e). As shown in Figure 4f, the contribution of iodine to total X-ray attenuation in healthy bone was much lower than that in cracks labeled with YbNP@SiO2-NTA in iodine (HAP) base image. This phenomenon revealed that the contribution of photoelectric effect to X-ray attenuation for YbNP@SiO2-NTA was higher than that for healthy bone, which was consistent with their attenuation characteristics. Great differences in basis material contents between the labeled cracks and healthy bone also existed in other three base images (Figure S10). Therefore, material decomposition of GSCT could separate bone-cracks labeled with YbNP@SiO2-NTA from healthy bone. Moreover, at


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oblique section, the labeled cracks (I, II) could be displayed much more really (Figure 4g), which was in good agreement with the volume-rendered image of this model at 70 keV (Figure 4h). However, in these images, no signal was achieved in the control bone-crack (III) (Figure 4e-h).

Imaging of damaged bone in vivo. Linear crack in long bones where they experience shear due to their orientation is regarded as typical bone damage and, if not detected and thus repaired in a timely manner, would lead to fractures in a relatively short time. Thus, in vivo real-time imaging of bone-cracks is of great importance. To this end, we developed an in vivo damaged bone model by scratching a crack on the right tibia (a kind of long bone) of a rat (Figure S11). Then, the rat was administered with intramuscular injection of YbNP@SiO2-NTA into the right hind leg and scanned with GSI mode (fastly switching between 80 kVp and 140 kVp) of GSCT at different time points after injection. To make a parallel comparison between GSCT and conventional CT, the same treated rat was also scanned with a single kVp (120 kVp) that possessed one polychromatic spectrum (Figure S15) at each selected time point. As expected, GSCT significantly improved signal-to-background ratio and successfully imaged the bone-crack, while the control group that was imaged with conventional CT did not (Figure 5a and Figure S14). In iodine (HAP) base images, GSCT offered excellent bone-crack targeted with YbNP@SiO2-NTA to healthy bone contrast by virtue of their large difference in the contribution of photoelectric effect and Compton scattering for X-ray attenuation. In addition, the superior imaging capability of GSCT can be further utilized to real-time monitor the targeting and accumulation of YbNP@SiO2-NTA towards bone-crack in vivo. As shown in Figure 5b, the iodine density measured in the bone-crack region was very low at 4h post injection and reached a maximum level at 24 h. The density was then followed by a gradual decrease from 24 h post injection, indicating that the diffusion of YbNP@SiO2-NTA towards bone-crack reduced. This


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Figure 5. (a) The conventional CT image at 120 kVp, iodine (HAP) base image and 3D image of the bone-cracks in the rat after intramuscular injection of YbNP@SiO2-NTA and YbNP@SiO2NH2 solution at 4 h, 24 h, 3 d, 15 d; Iodine denstiy (b) and CT value (c) changes of the bonecrack in rats after intramuscular treatment with YbNP@SiO2-NTA (line 1, 2, 3) and YbNP@SiO2-NH2 (line 4) solution at all time points (the region of interest: 1.39 mm2). (d) The conventional CT image at 120 kVp and iodine (HAP) base image of the healthy but SHAM operated leg after intramuscular injection of YbNP@SiO2-NTA at 24 h, 3 d, 7 d.


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reduced signal might be as a result of the decreased amount of exposed calcium ion in bonecrack, deriving from the formation of soft callus around the bon-crack during bone-crack healing phases.39,40 This process was also confirmed by the gradual change of CT signal at 70 keV in Figure 5c. These results proved that GSCT was indeed much sensitive in bone-crack detection and could achieve in vivo real-time imaging of bone-crack. However, in conventional CT, the healthy bone itself could absorb lots of X-ray photons and thus generated strong background noise, which concealed the effective signal from targeted YbNP@SiO2-NTA. On the other hand, conventional CT image was only capable of reflecting their integrated HU values which lose their according inherent X-ray attenuation characteristics, leading to the failure in imaging bonecracks. To further investigate the in vivo targeting ability of YbNP@SiO2-NTA, additional control groups were conducted, including i) healthy but sham-operated legs with YbNP@SiO2-NTA and ii) crack-bearing legs with unlabelled contrast agents (YbNP@SiO2-NH2, NH2Control). For the healthy but sham-operated group, YbNP@SiO2-NTA distributed in the near of the tibia but did not reach the tibia (Figure 5d). This phenomenon indirectly indicated that YbNP@SiO2-NTA targeted the bone-crack through chelating with the exposed calcium ion in the crack. For the NH2Control groups, bone-cracks could not be detected by using conventional CT and even GSCT. This result was reasonable because YbNP@SiO2-NH2 could not chelate with the exposed calcium ion. On the other hand, Sen et al. has demonstrated that the bone-crack could generate ion-gradient-driven electric fields oriented away from the crack, due to the large difference in diffusion coefficients (D) between the cation (Ca2+) and the faster anion (OH-),11 resulting in that the positively-charged YbNP@SiO2-NH2 could not reach the bone-crack. Moreover, most of NH2Control retained and aggregated inside the muscle where it was injected and only a small


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percentage of NH2Control was metabolized at 15 d post injection (Figure 5 and Figure S13), which was also confirmed by conventional CT (Figure S12). In contrast, the GSCT signal of YbNP@SiO2-NTA diffused throughout the muscle over time and decreased significantly after 15 d post injection (Figure 5a). All these results demonstrated that the iminodiacetate functionalities of YbNP@SiO2-NTA indeed played a vital role in targeting the bone-crack.

Biocompatibility of YbNP@SiO2-NTA. Biocompatibility assay of YbNP@SiO2-NTA was performed. Long-term cell viability was not affected by YbNP@SiO2-NTA within the tested concentration range for 24 h in vitro (Figure S8). To further investigate whether YbNP@SiO2NTA caused any harmful effect or disease in vivo, a single dose of YbNP@SiO2-NTA were

Figure 6. (a) Biodistribution of YbNP@SiO2-NTA in mouse at 4 h, 24 h, 7 d, 15 d, 30 d after intramuscular injection. (b) Body weight changes of the mouse with and without intramuscular injection of YbNP@SiO2-NTA solution versus time. (c) Histological changes in the heart, liver, spleen, lung, kidney and the injected muscle of the rats 30 days after intramuscular injection. These tissues are stained with H&E and observed under a light microscope.


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injected intramuscularly into healthy mice. The YbNP@SiO2-NTA accumulated in the liver and the spleen, but the concentration was much lower than that in the injected muscle after 24 h injection (Figure 6a and Figure S16), in agreement with its GSCT signal (Figure 5). One month later, most of the initial dose had been cleared from the body (Figure 6a). The mechanism of clearance may be attributed to the faecal excretion through liver or other organs (Table S1).38 As the metabolism of highly localized the YbNP@SiO2-NTA may induce subsequent damage in muscle and other organs related to NPs clearance, the in vivo toxicity of YbNP@SiO2-NTA was examined. After 30 days post injection, histopathology examination showed that the main organs including heart, liver, spleen, lung, kidney and muscle retained the similar morphologies in control and treated group, which indicated YbNP@SiO2-NTA had not remarkable adverse effect to tissues (Figure 6c). The key hepatic indicators including alkaline phosphatase, alanine transaminase, aspartate transaminase, total protein and albumin were tested and the liver did not exhibit noticeable lesions compared with those of the control group. Clinical routine blood test revealed no obvious interference with the physiological regulation of haem or immune response (Figure S17). In addition, over one-month period the treated rats did not show abnormalities in eating, drinking, grooming, activity, exploratory behaviour, urination or neurological status and the body weight of experimental group kept growing which was comparable to the control group (Figure 6b). These preliminary results suggested that YbNP@SiO2-NTA may be suitable for biological application, although detailed studies would be necessary to further explore the potential toxicity.

CONCLUSION In summary, we have demonstrated the use of crack-targeted ytterbium-based nanoparticles (YbNP@SiO2-NTA) as GSCT contrast agents to image bone-cracks under GSI mode in vitro and


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in vivo. Owing to the vital ability of GSCT to extract the difference between the intrinsic X-ray attenuation characteristics of YbNP@SiO2-NTA and healthy bone, GSCT can not only achieve a higher signal-to-background ratio in the iodine (HAP) base images than that of conventional CT, but also achieve in vivo real-time imaging of bone-crack. Moreover, measurements of iodine density and CT values at 70 keV in the bone-crack can afford further information to quantitatively identify the targeted accumulation of YbNP@SiO2-NTA on bone-crack in real time. Our results shed light on the role of crack-targeted contrast agents in accelerating the application of clinical gemstone spectral CT for imaging damaged bone in vivo.

METHODS Synthesis of OA-NaYbF4:Gd3+(20%)/Er3+(2%) nanoparticles. The OA-YbNPs were synthesized using a modified method as previously reported. In a typical procedure, YbCl3 aqueous solution (1.17 mL, 1 M), GdCl3 aqueous solution (0.3 mL, 1 M) and ErCl3 aqueous solution (0.3 mL, 0.1 M) were injected into a 50 mL flask and dried by heating. Then, OA (15 mL) and ODE (23 mL) were added into the flask. The mixture was heated to 160 ℃ under Ar protection to obtain a clear solution and then cooled down to room temperature. Subsequently, methanol solution (15 mL) containing NaOH (3.75 mmol) and NH4F (6 mmol) was added dropwise into the flask and the solution was stirred for 30 min. Under Ar protection, the solution was slowly heated to 220 ℃ to remove the residual water and impurities with low boiling point. Afterwards, the solution was heated to 300 ℃ and remained at this temperature for 1.5 h. Cooled down to ambient temperature, these nanoparticles were obtained through centrifugation at 10000 rpm for 20 min and washed two times with ethanol. Finally, the nanoparticles were dispersed in cyclohexane.


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Synthesis of NaYbF4:Gd3+/Er3+@SiO2-NH2 nanoparticles. In a typical procedure, CO-520 (0.5 mL) was dispersed in cyclohexane (10 mL) and stirred for 30min. Subsequently, OANaYbF4:Gd3+/ Er3+ (0.04 mmol) cyclohexane solution was added into the mixture. After stirring for 3 h, NH3·H2O (80 µL) was added and the system was stirred for another 2 h. Then, TEOS (0.04 mL) was added and the mixture kept stirring for 24 h. The final nanoparticles were precipitated by ethanol and washed with ethanol three times. Primary amine was grafted onto the silica shell via the hydrolysis of APTES (50 µL) in the 2-propanol (18 mL) at 80 ℃ for 12 h. Then, the product was collected by centrifugation at 9900 rpm for 15 min and washed with ethanol three times. Finally, the YbNP@SiO2-NH2 was dried under vacuum.

Synthesis of NaYbF4:Gd3+/Er3+@SiO2-NTA nanoparticles. Typically, NTA (24 mg) and CDI (20 mg) were dispersed in 5 mL anhydrous N, N-Dimethylformamide (DMF) and stirred at 30 ℃ for 1h. Then, YbNP@SiO2-NH2 anhydrous DMF solution (0.04 mmol, 5 mL) was added. After continuous stirring for 36 h, products were collected by centrifugation at 9900 rpm for 15 min and washed by water three times to remove the unreacted NTA and CDI. The collected products were re-dispersed in water.

Damaged bone model in vivo. The three-month-old female Wistar rats with average weight of 300 g were used to develop an in vivo model of bone-cracks in tibia (n=2/group). All animal procedures were in accord with the guidelines of the Institutional Animal Care and Use Committee. All efforts were made to minimize suffering of the rats. Firstly, the fur surrounding the hind legs of anesthetized rats was removed for surgery by applying a small drop of depilatory cream. After 30 seconds, aseptic cotton buds and warm water were used to remove the depilatory cream and clean the skin. The iodophor was used to disinfect the surgery site. Afterwards, a longitudinal incision (~0.8 cm) was made in the inner thigh to expose the adductor longus


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muscle and gracilis muscle. Then, we found the tibia through intermuscular space between the adductor longus muscle and gracilis muscle. A bone-crack (~0.6 cm) was manually created on the tibia with a surgical scalpel. After that, the wound was cleaned with saline and stitched up. The iodophor was used to disinfect the surgery site again. Then, a single dose of YbNP@SiO2NTA solution (40 mg/ml, 200 µL) was immediately injected into the muscle surrounding the right tibia. Finally, we dressed the wound. The wound was healing after 1 week. During the operation, we avoided the major arteries, veins and nerve tract. In addition, we did not destruct the muscle tissue and there was not massive bleeding in the muscle. There was only minimal bleeding with spontaneous hemostasis when the skin was incised. For the healthy but sham-operated leg group (n=2/group), the operation was the same with the experimental group, except that the tibia was healthy.

Gemstone spectral CT imaging in vivo. Twenty-four hours, four hours, seven days or fifteen days after injecting YbNP@SiO2-NTA solution into the muscle, anesthetized rats were imaged in vivo by 64-row MDCT scanner (GE Discovery HD 750) with GSI mode. X-ray attenuation and material density were measured within a volume of interest surrounding the bone-crack and healthy bone. Imaging parameters were given as follows: Gemstone spectral imaging (GSI) mode with fast tube voltage switching between 80 kVp and 140 kVp on adjacent views during a single rotation; tube current, 600 mA; thickness, 0.625 mm; pitch, 0.18; field of view, 250 mm; gantry rotation time, 0.355 s; All acquired images were transferred to a dedicated workstation (AW 4.6; GE Healthcare) for quantitative and qualitative analyses.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge via the Internet at http://pubs.acs.org.


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AUTHOR INFORMATION Corresponding Author *[email protected] *[email protected].

Author Contributions ‡These authors contributed equally.

ACKNOWLEDGMENT Financial support by NSFC (No.21125521, 21303178) is gratefully acknowledged.

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Targeted imaging of bone-crack in vivo was achieved by using clinical gemstone spectral CT (GSCT) technique. The superior imaging capability of GSCT allows us to real-time monitor the targeting and accumulation of contrast agents towards bone-crack in vivo, hence opening a path to understand the extreme biological effects of damaged bone.


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