Noninvasive Multimodal Imaging of Osteosarcoma and Lymph Nodes

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Noninvasive Multimodal Imaging of Osteosarcoma and Lymph Nodes using a 99mTc-Labeled Biomineralization Nanoprobe Zhiming Xu, Yangyun Wang, Jianshan Han, Qingqing Xu, Jiawei Ren, Jiaying Xu, Yong Wang, and Zhifang Chai Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b04925 • Publication Date (Web): 05 Mar 2018 Downloaded from http://pubs.acs.org on March 7, 2018

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Analytical Chemistry

Noninvasive Multimodal Imaging of Osteosarcoma and Lymph Nodes using a 99mTc-Labeled Biomineralization Nanoprobe Zhiming Xu,†,‡ Yangyun Wang,† Jianshan Han,‡ Qingqing Xu,† Jiawei Ren,† Jiaying Xu,† Yong Wang,*,† Zhifang Chai† †

Center for Molecular Imaging and Nuclear Medicine, State Key Laboratory of Radiation Medicine and Protection, School for Radiological and Interdisciplinary Sciences (RAD-X), Collaborative Innovation Center of Radiation Medicine of Jiangsu Higher Education Institutions, Soochow University, 199 Renai Road, Suzhou Industrial Park, 215123, P.R. China ‡ Chinese People's Liberation Army 117 Hospital, 14 Lingyin Road, Hangzhou, 310007, P.R. China ABSTRACT: The accurate imaging of the lymph nodes represents a critical indicator for tumor staging and surgical planning (e.g. osteosarcoma). Clinically, nodal tracing using a radionanocolloid is often limited by the inaccessibility of real-time images and inadequate anatomical information. Herein, we present a 99mTc-labeled biomineralization nanoprobe for the advanced detection of osteosarcoma and lymph nodes with multimodal imaging. By exploiting the complementary strengths of MRI/SPECT/NIR fluorescence, the fabricated nanoprobe exhibited suitable stability and biocompatibility characteristics and was shown to be able to locate in osteosarcoma. The lymphatic drainage and network in healthy mice were imaged in real-time using NIR fluorescence and SPECT/CT. Furthermore, we demonstrated that our 99mTc-biomineralization nanoprobe could be used for the high-resolution and high-sensitivity imaging analysis of lymphatic drainage in an orthotopic osteosarcoma model. Taken in concert, the 99mTc-labeled biomineralization nanoprobe features promising characteristics to be used as an intraoperative visualization tool to aid in precise tumor imaging and nodal resection.

The invasion level of tumor-draining lymph nodes (LNs) is a crucial indicator for tumor phase assessment and treatment planning.1 Particularly, the detection of the sentinel lymph node (SLN), a first-line lymphatic drainage node from the malignant tumor, represents a standard-of-care procedure for the majority of tumors.2 The evaluation of LNs metastases generally relies on the experience level of the surgeon and often requires removal of all anatomically susceptible LNs for ex vivo pathological examination.3 Therefore, the noninvasive tumor-draining LNs imaging represents a promising detection method in real-time for improving treatment outcome and decreasing overall healthcare costs.4 Multimodal imaging has been developed for accurate tumor imaging by exploiting the complementary strengths of each individual modality.5 Obviously, an efficient probe is indispensable for noninvasive multimodal imaging and nanotechnology offers new opportunities to design a multifunctional nanoprobe with different properties.6-8 Gambhir’s group pioneered a unique multimodality MRI–photoacoustic–Raman imaging nanoprobe that could accurately define the boundaries of brain tumor mass for resection in vivo.9 Pre-clinically, some hybrid radionuclide/near-infrared (NIR) fluorescence probes, comprising the non-covalently bound indocyanine green and an albumin-based nanocolloid, were successfully introduced for LNs imaging in patients suffering from prostate cancer or head and neck melanoma.10-13 The previous multimodal nanoprobes, however, were limited by the inaccessible real-time images in SPECT/MRI or PET/MRI,14, 15 the lack of anatomic

information (e.g. NIR fluorescence imaging)16 and poor colloidal stability (e.g. radionanocolloid).17, 18 Herein, we propose a method to gain complementary information from MRI combined with SPECT and NIR fluorescence imaging in an effort to provide more accurate and realtime information of tumor and LNs. Owing to its high resolution, MRI can provide superior anatomical resolution and assessment of tumor dynamics,6 while SPECT/CT imaging has been shown to help identify tumor masses and LNs with highsensitivity.19 Particularly, NIR fluorescence image-navigation during cancer surgery offers the potential to visualize tissue demarcation in real-time, thereby decreasing damages caused to normal tissue structures.20 We produced a multimodal MRI/NIR/SPECT probe based on cypate-grafted biomineralization gadolinium oxide nanoparticles and labeled the material with 99mTc for the enhanced detection of osteosarcoma and LNs (Scheme 1). An ovalbumin-based biomineralization approach was employed to synthesize gadolinium oxide nanoparticles (Gd@OVA), followed by conjugation of a NIR fluorescence dye (cypate, Cy) and 99m Tc with the ovalbumin scaffold. The corresponding MRI/NIR/SPECT probe (99mTc-Gd@OVA-Cy) featured the following properties: (i) high spatial resolution and sensitivity for the accurate visualization of osteosarcoma and LNs in realtime, (ii) appropriate size for rapid uptake and long retention time in osteosarcoma and LNs, (iii) excellent clearance and biocompatibility features.

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Scheme 1. Schematic Illustration of Noninvasive Multimodal Imaging of Osteosarcoma and Lymph Nodes using a 99m Tc-Labeled Biomineralization Nanoprobe.

EXPERIMENTAL SECTION Materials and Chemicals. Gd(NO3)3·6H2O, 1-Ethyl-3-(3dimethylaminopropy) carbodiimide (EDC) and Nhydroxysulfosuccinimide sodium salt (NHS) were obtained from Alfa Aesa (Shanghai, China). 99mTc pertechnetate (99mTcO4-) was purchased from Shanghai GMS Pharmaceutical Co.,Ltd. All other chemicals were obtained from Sinopharm (Shanghai, China). Ultrapure water (18 MΩ*cm) was utilized throughout this work. Instrumentation and Characterization. The morphology of Gd@OVA-Cy was characterized using an FEI Tecnai G2 field emission transmission electron microscope (TEM) with an accelerating voltage of 200 kV. The hydrodynamic size was measured using dynamic light scattering (DLS, Zetasizer ZS90, Malvern). The concentration of Gd element was measured using an inductively coupled plasma mass spectrometer (ICPMS, Elemental-2, ThermoFisher). Fourier transform infrared (FT-IR) spectra were recorded on a VERTEX 70 spectrometer (Bruker). The absorbance and fluorescence spectra were measured using UV-Vis-NIR spectrophotometer (Lambda 35, PerkinElmer) and fluorescence spectrophotometer (FLS 980, Edinburg), respectively. NIR fluorescence, MRI, and SPECT imaging were performed using IVIS Spectrum (PerkinElmer), 1.5 T MR imaging system (Philips, Achieva) and USPECT+/CT (MILABS), respectively. Synthesis of 99mTc-Gd@OVA-Cy Nanoprobe. Gd@OVACy was synthesized according to previously reported methods with modification.21, 22 Ovalbumin (0.25 g) was dissolved in 9.0 mL of de-ionized water, and then 1.0 mL Gd(NO3)3 solution (100 mM) was slowly added under vigorous stirring. Next, NaOH (2.0 M) was used to adjust the pH of the solution to 12, and the mixture was then allowed to react under vigorous stirring at 37 °C for 12 h. The resulting Gd@OVA solution was successively purified by ultrafiltration (Ultrafree-PF filters, 100 kDa, Millipore) and dialysis (hollow fibre dialysis membrance, 50 kDa). Next, the carboxylic acid group on Cy was conjugated with free amine group in ovalbumin through carbodiimide-catalyzed amide formation. Finally, Gd@OVACy was obtained by the dialysis of the solution against Milli-Q water for 24 h. For radiolabeling, 0.5 mL Gd@OVA-Cy (1 mg mL-1 Gd) solution was mixed with 10.0 µL freshly prepared anhydrous stannous chloride solution (1.0 mg mL-1 in 0.012 M HCl), and then 0.5 mL 99mTcO4- (1.2 mCi, half-time of 6.02 h) solution was immediately added, followed by ultrasound for 10 min. The 99mTc-labeled Gd@OVA-Cy solution was purified through ultrafiltration to achieve more than 95% radiochemical purity by using the radioactivity meter. The resulting product was named 99mTc- Gd@OVA-Cy nanoprobe.

Orthotopic Osteosarcoma Model. Animal experiments were approved by Animal Ethics Committee of Soochow University (Suzhou, China). The 4-week-old female BALB/c mice of approximately 18 g were obtained from the Experimental Animals Center of Soochow University (Suzhou, China). All animal procedures were performed under anaesthesia by inhalation of a 2.5% isoflurane/air mixture. The orthotopic osteosarcoma model was established in 5-week-old female BALB/c mice by injection of 1 × 106 of murine osteosarcoma K7M2 cells suspended in 20 µL PBS into the left proximal tibia using an insulin syringe with 29G, 0.5 inch.23 Osteosarcoma progression was visualized and assessed weekly in vivo using an optical and X-ray imaging system (IVIS Lumina XRMS Series III, PerkinElmer). Multimodal Imaging in Orthotopic Osteosarcoma. For MRI, the mice bearing orthotopic osteosarcoma were intravenously administered with 99mTc-Gd@OVA-Cy nanoprobe (200 µL containing 0.01 mM Gd). A series of MRI images were captured at pre-injection (Pre), and 15 min, 1, 2, 6, 12, and 24 h post-injection. For NIR fluorescence imaging, the mice bearing orthotopic osteosarcoma were intravenousy administered with 99mTc-Gd@OVA-Cy nanoprobe (200 µL containing 0.2 mg mL-1 Cy, equal to 0.01 mM Gd). A series of NIR fluorescence images were captured at pre, 15 min, 1, 2, 6, 12, and 24 h post-injection using IVIS Spectrum with ICG filter (excitation: 780 nm; emission: 810 nm). For SPECT imaging, the mice bearing orthotopic osteosarcoma were intravenously administered with 99mTc- Gd@OVA-Cy nanoprobe (200 µL containing 800 µCi 99mTc). A series of SPECT/CT images were captured at pre, 15 min, 1, 2, 6, 12, and 24 h postinjection. Lymphatic Drainage Imaging of Healthy Mice and Orthotopic Osteosarcoma Model. Dynamic NIR fluorescence and SPECT/CT imaging were started immediately after hindpaw and peritumoral subcutaneous injection of 99mTcGd@OVA-Cy nanoprobe (10 µL containing 40 µCi 99mTc, equal to 0.2 mg mL-1 Cy), respectively. For osteosarcomadraining LNs imaging at 2 h post-injection, the mice were executed. Organs were collected for NIR fluorescence imaging and γ counter test, such as heart, liver, spleen, lung, kidney, osteosarcoma, and LNs. The radioactivity of organ was calculated as percentage of the injected dose per gram of tissue (% ID/g). In Vivo Biocompatibility, Biodistribution and Hematoxylin & Eosin (H&E) Staining. To appraise the blood circulation time, an important index of biocompatibility, the health mice (n=3) were intravenously injected with the 99mTcGd@OVA-Cy nanoprobe (200 µL containing 40 µCi 99mTc). Then the blood was obtained from the eye socket and gathered for radioactive tests using γ counter on the fixed time point. After 24 h post-injection, all mice were euthanized and sacrificed for the histopathological analyses. The tissues (e.g. heart, liver, spleen, lung, kidney and bone) were then excised and fixed in 4% paraformaldehyde for 24 h. The shin bone was placed in 10% (wt/wt) EDTA for 7 d to decalcify. The samples were frozen and sectioned at 8 mm using an ultramicrotome (CM1900, Leica). The tissue sections were stained with H&E and their bright-fields were observed using an inverted fluorescence microscope (IX73, Olympus). For the biodistribution, four groups of mice, each group of three mice was intravenously injected with the 99mTcGd@OVA-Cy nanoprobe (200 µL containing 40 µCi 99mTc).


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Analytical Chemistry Four groups of mice were euthanized and sacrificed after 6 h, 12 h, 24 h and 72 h post-injection, respectively. And the following organs were collected for NIR fluorescence imaging analysis: heart, liver, spleen, lung, and kidney. To evaluate whether the 99mTc-Gd@OVA-Cy nanoprobe will cause toxicity or inflammation, hematology were performed on control and nanoprobe-treated mice (intravenous injection, 200 µL containing 0.2 mg mL-1 Cy, equal to 0.01 mM Gd). Blood was collected (days 1, 7, 14) from the mice, followed by centrifugation at 3000 rpm for 10 min. The supernant plasma fraction was stored at -80 ºC for biochemistry analysis such as for alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, albumin, globulin, total protein, and creatinine. In addition, whole blood (100 µL) was collected, and analyzed using a hematology analyzer (Sysmex XN-3000, IL, US). Hematology parameters such as hemoglobin, hematocrit, white blood cell count, red blood cell count, and platelets were measured.

RESULTS AND DISCUSSION

Figure 1. Characterization of physical properties of Gd@OVACy nanoprobe. (A) TEM image of Gd@OVA-Cy nanoparticles. (B) DLS of Gd@OVA-Cy nanoparticles in PBS. (C) FT-IR spectra of OVA, Cy, Gd@OVA and Gd@OVA-Cy. (D) The relaxivity of Gd@OVA-Cy nanoparticles and commercially confined Magnevist (Gd-DTPA) to enhance T1 contrast at 1.5T magnetic fields. (E) Absorbance and fluorescence spectra of Gd@OVA-Cy solution. (F) Radiostability study of 99mTc-chelated Gd@OVA-Cy in PBS (pH = 7.4).

Synthesis and Characterization of 99mTc-Gd@OVA-Cy Nanoprobe. As mentioned before,24 the rich functional groups of the protein ligand offer the possibility to further functionalize the Gd@OVA nanoparticles. For the production of the SPECT/MRI/NIR nanoprobe, an indocyanine green (ICG) analogue Cy was bound directly to the Gd@OVA surface using a succinimide linkage. This effectively solved the problem of clinical ICG lack of reactive groups for further chemical conjugation.25 After conjugation, Gd@OVA binding to Cy formed a Gd@OVA-Cy nano-assembly. TEM images con-

firmed the assembly structure and the average particle size was about 35 nm (Figure 1A). Due the stretch of protein surface, dynamic light scattering (DLS) showed that the hydrodynamic diameter was 65.34 nm (Figure 1B). Compared with the clinical albumin-based nanocolloid,17, 18 the Gd@OVA-Cy nanoparticles exhibited an improved colloidal stability (Figure S2). FT-IR spectra were used to confirm the composition of Gd@OVA-Cy (Figure 1C). The strong band present in the fingerprint region (350 – 1750 cm-1) of Gd@OVA-Cy proved the presence of organic compounds, i.e. OVA and Cy. Next, we determined the in vitro imaging properties of the Gd@OVA-Cy nanoparticles for each modality. First, the MRI ability of Gd@OVA-Cy nanoparticles and the commercially available species Magnevist (Gd-DTPA) were compared to determine the T1 contrast enhancement at a magnetic field of 1.5T. The r1 relaxivity value of the Gd@OVA-Cy nanoparticles was determined to be 11.46 s-1 mM-1 (Figure 1D), roughly threefold compared to the Gd-DTPA chelate. The conjugation was further validated by absorbance and fluorescence spectrum. Gd@OVA-Cy exhibited two absorbance peaks at 725 and 800 nm, respectively, and one fluorescence peak at 820 nm in aqueous solution (Figure 1E). This latter finding indicated the successful conjugation of Cy with Gd@OVA. Due to the strong chelating effect of OVA, radionuclides such as 99m Tc may be used to label the Gd@OVA-Cy nanoparticles for SPECT/CT imaging. Using a radioactivity meter, the labeling yield was determined to be as high as 99%. Furthermore, the obtained 99mTc labeled Gd@OVA-Cy nanoparticles (99mTcGd@OVA-Cy) exhibited an excellent radiostability in phosphate buffered saline (PBS) at 37 °C (Figure 1F). After radiolabelling, 99mTc-Gd@OVA-Cy nanoparticles have a colloidal stability as before (Figure S2).

Figure 2. Multimodal detection of orthotopic osteosarcoma in vivo with 99mTc-Gd@OVA-Cy nanoprobe. The osteosarcomabearing mice were intravenously injected with 99mTc-Gd@OVACy nanoprobe and performed consecutive MRI, NIR fluorescence (FL) imaging on each mouse pre-injection and at 15 min, 1 h, 2 h, 6 h, 12 h and 24 h post-injection, respectively. The SPECT/CT images were executed on each mouse pre-injection and at 15 min, 1 h, 2 h, 6 h, 8 h and 24 h post-injection. The right tibia bone was destruction in the pre CT imaging. The all post-injection images showed clear tumor visualization (red arrow outline the tumor location).

In Vivo Multimodal Orthotopic Osteosarcoma Visualization of 99mTc-Gd@OVA-Cy Nanoprobe. We then explored whether the nanoprobe could be used for orthotopic osteosarcoma visualization in vivo. We hypothesized that the nano-



probe would enter the extravascular space and accumulate in osteosarcoma cells by enhanced permeability and retention (EPR) effect. The latter principle has been reported to be feasible in subcutaneous tumor models.26, 27 The 99mTcGd@OVA-Cy nanoprobe was injected into osteosarcomabearing mice via tail vein, and consecutive MRI, NIR fluorescence imaging and SPECT/CT imaging was performed on each mouse before injection and at 15 min, 1 h, 2 h, 6 h, 8 h, 12 h and 24 h post-injection, respectively (Figure 2). 15 min post-injection, all three modality images showed enhanced signals corresponding to osteosarcoma in the right leg compared to the normal left leg. Therefore, we determined that the 99mTc-Gd@OVA-Cy nanoprobe could be used for the rapid diagnosis and clear visualization of osteosarcoma. The images also showed that the nanoprobe was eliminated through the hepatic and renal pathways. The region of interest intensity of the osteosarcoma region was quantified and exhibited a conspicuous signal enhancement in the MRI, NIR fluorescence and SPECT/CT signals post-injection (Figure S2). The entire signal peak appeared at 6-12 hours post-injection and the osteosarcoma signal gradually declined due to metabolic transformation after 12 hours. The multimodal nanoprobe species described herein enabled us to locate the tumor by exploiting the complementary advantages of various techniques. The nanoprobe was retained in the tumor by ERR effect, allowing for repetitive imaging. The administration of multiple doses was not required. This high resolution image analysis may also be significant for distinguishing tumor margins during surgical tumor mass removal.

Figure 3. Real-time LNs visualization of 99mTc-Gd@OVA-Cy nanoprobe by NIR fluorescence (FL) imaging combined with SPECT/CT. The normal BALB/C nude mice were injected with 99m Tc-Gd@OVA-Cy nanoprobe via intradermally in the right hindpaw. Bimodal imaging was performed on each mouse preinjection and at 5 min, 30 min, 1 h, 2 h and 12 h post-injection, respectively. All two modalities imaging displayed clear LNs visualization (red arrow outline the LNs location: 1, popliteal node; 2, sciatic node; 3, subiliac node; 4, renal node).

Real-time LNs Visualization of 99mTc-Gd@OVA-Cy Nanoprobe. Combining NIR fluorescence with radio-guided sentinel node imaging has proven to be feasible during surgery in many preclinical studies.11-13 This technique describes the direct injection of a probe into the dermis around the tumor and represents a powerful technique to guide surgical resection. To explore the LNs visualization potential of the 99mTcGd@OVA-Cy nanoprobe, we first carried out lymphatic imaging by hindpaw subcutaneous injection into healthy mice. Upon summarizing the representative imaging results from NIR

fluorescence and SPECT/CT, the draining popliteal and sciatic nodes could be clearly visualized 5 min post-injection (Figure 3). After intradermal injection of the nanoprobe, initial lymphatic vessel drainage could be visualized noninvasively by NIR fluorescence imaging with very low tissue autofluorescence.28, 29 An imaging modality based on radioactive tracers such as SPECT offers depth sensitivities up to several centimeters and combined SPECT/CT imaging modalities provide excellent anatomical registration of nanoprobes in the lymph nodes. Therefore, the more extensive lymphatic network (the renal and subiliac LNs) could also be detected by SPECT/CT with minimal background. 30 min post-injection, all two modality images showed that the nanoprobe was eliminated through the renal pathway. Based on these results, we believe that the 99mTc-Gd@OVA-Cy nanoprobe may be used for mapping of sentinel lymph nodes from orthotopic osteosarcoma.

Figure 4. Mapping of SLN from the orthotopic osteosarcoma using 99mTc-Gd@OVA-Cy nanoprobe. The osteosarcoma-bearing mice were intratumorally injected with 99mTc-Gd@OVA-Cy nanoprobe and performed NIR fluorescence (A) and SPECT/CT (C) imaging at 15 min post-injection, respectively (red arrow outline the SLNs location). The biodistribution of heart, liver, spleen, lung, kidneys and lymph nodes was detected with NIR fluorescence imaging (B) and by γ counter (D).

Multimodal Imaging of LNs from the Orthotopic Osteosarcoma using 99mTc-Gd@OVA-Cy Nanoprobe. Clinical LNs imaging is usually performed immediately after intradermal injection of a radiotracer around the primary tumor.10 Following intra-osteosarcoma administration of the 99mTcGd@OVA-Cy nanoprobe into the orthotopic mouse model, NIR fluorescence and SPECT/CT imaging was able to identify the lymphatic location (Figure 4). NIR fluorescence imaging provided a representative whole body image of the nanoprobe


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Analytical Chemistry uptake at two draining nodes 15 min post-injection, including the SLN and sciatic node. The results illustrated that the 99mTcGd@OVA-Cy nanoprobe may represent a critical platform providing complementary observations into lymphatic functions in osteosarcoma models. To study the clearance pathways of 99mTc-Gd@OVA-Cy nanoprobe from the injection site, osteosarcoma-bearing mice were subjected to autopsy to remove various organs 2 h postinjection. The mean uptake in the heart, liver, spleen, lung, kidneys and lymph nodes was detected with NIR fluorescence imaging and using a γ-counter (Figure 4 B and D). The biodistribution results from the radio-fluorescence were consistent. A high level of nanoprobe uptake was detected in the SLN (~6 % ID/g) from the absolute radioactive quantification. Therefore, the SLN localization of the 99mTc-Gd@OVA-Cy nanoprobe may be promising for the use in surgical management.

cells or serum biochemistry could not be detected (Table S1). Taken in concert, the systematic in vivo toxicity evaluation showed that 99mTc-Gd@OVA-Cy was biocompatible and may therefore be potentially used in clinical translation.

CONCLUSIONS In summary, we produced a multimodal nanoprobe, i.e. Tc-Gd@OVA-Cy, for the noninvasive detection of osteosarcoma and LNs with MRI/SPECT/NIR. 99mTc-Gd@OVACy was demonstrated to accurately locate osteosarcoma tumors by exploiting the complementary strengths of each individual modality. Lymphatic drainages in healthy as well as osteosarcoma-bearing mice were also investigated. We demonstrated that 99mTc-Gd@OVA-Cy could be used for highresolution and high-sensitivity imaging analysis of lymphatic drainage in an osteosarcoma model. Potentially, this nanoprobe may be used in a clinical setting to improve preoperative planning for nodal resection and tumor staging. 99m

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]

Author Contributions 99m

Figure 5. (A) Blood clearance profile of Tc-Gd@OVA-Cy nanoprobe in Balb/c mice. (B) Biodistribution of 99mTcGd@OVA-Cy nanoprobe over a period of 72 h in Balb/c mice. (C) Representative histopathology examination of control and nanoprobe-treated mice after 24 h post-injection (scale bar, 200 µm).

In Vivo Biodistribution and Biocompatibility. In order to determine the biodistribution of the 99mTc-Gd@OVA-Cy nanoprobe, the blood clearance level from the body was quantified. Healthy BALB/C mice were intravenously injected with the 99mTc-Gd@OVA-Cy nanoprobe and blood was collected for radioactive testing on fixed time points. The measured blood circulation half-lives (t1/2) indicated that the nanoprobe exhibited a rapid t1/2α of 37.2 min and a t1/2β of 18.34 h (Figure 5A). This two-compartment pharmacokinetic behavior was similar to that of other pre-clinically available contrast agents.30 For in vivo biodistribution, various organs were resected for NIR fluorescence imaging analysis at 6 h, 12 h, 24 h and 72 h post-injection. The most intense uptakes for the nanoprobe were measured in the liver, spleen and kidneys (Figure 5B). At extended time points, the signal of the nanoprobe gradually decreased due to the rapid metabolic removal from the body. To evaluate if the 99mTc-Gd@OVA-Cy causes any toxicity or inflammation characteristics, histopathology and hematology experiments were carried out on control and nanoprobetreated mice. Histopathology examination of major organs (heart, liver, spleen, lung, kidneys and bone) was performed 24 h post-injection. Hematoxylin and eosin (H&E) staining of histological sections revealed no difference in terms of organ abnormalities or lesions in the control or nanoprobe-treated mice (Figure 5C). Furthermore, any indicators related to organ damages or inflammation, abnormalities in red or white blood

The manuscript was written through contributions of all authors. / All authors have given approval to the final version of the manuscript. / Z.M. Xu, Y.Y. Wang and J.S. Han contributed equally.

ACKNOWLEDGMENT This work was supported by National Natural Science Foundation of China (21505096, 51503139), National Key Research Program of China (2016YFC0101200), Postdoctoral Science Foundation of China (2014M560442, 2015M571796, and 2015T80575), Postdoctoral Research Program of Jiangsu Province (1401062C and 1402109C), Natural Science Foundation of the Jiangsu Higher Education Institutions of China (15KJB150025), and The Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

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