Multimodal Nanoprobe Based on Upconversion Nanoparticles for

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Multi-modal nanoprobe based on upconversion nanoparticles for monitoring implanted stem cells in bone defect of big animal Dexin Chen, Daqian Wan, Rongying Wang, Yanyue Liu, Kang Sun, Xiaofeng Tao, Yang Qu, Kerong Dai, Songtao Ai, and Ke Tao ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00763 • Publication Date (Web): 09 Jan 2018 Downloaded from http://pubs.acs.org on January 10, 2018

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ACS Biomaterials Science & Engineering

Multi-modal nanoprobe based on upconversion nanoparticles for monitoring implanted stem cells in bone defect of big animal Dexin Chen,†a Daqian Wan,†bd Rongying Wang, a Yanyue Liu, a Kang Sun,* a Xiaofeng Tao, b Yang Qu, b Kerong Dai, c Songtao Ai,*b Ke Tao* a a. State Key Lab of Metal Matrix Composites, Shanghai Jiao Tong University, Shanghai 200240, P. R. China. b. Department of Radiology, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, P. R. China. c. Department of Orthopaedics, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, P. R. China. d. Department of Orthopedics, Orthopedic Institute of Harbin, The Fifth Hospital in Harbin, Harbin 150040, P. R. China.

KEYWORDS. Stem cells, nanoprobe, bone regeneration, multimodal imaging, big animal. *Email: KS: [email protected]; SA: [email protected]; KT: [email protected]

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ABSTRACT. Monitoring implanted stem cells in bone regeneration and other cell therapies is of great importance to reveal the mechanism of tissue repair and to optimize clinical treatments. However, big challenge still remained in lacking an imaging nanoprobe. Herein, we designed surface modified upconversion nanoparticles (UCNs) with multi-modal imaging capabilities of fluorescence, magnetic resonance imaging (MRI) and dual-energy computed tomography (CT). It was found that the UCNs can label stem cells in an efficient (over 200 pg/cell) and long-term (at least 14 days) manner, with almost no influence on the viability, cell cycle, apoptosis, and multi-lineage differentiation. Thus, clinical dual-energy CT and MRI were successfully applied to observe the migration of labeled cells on a bone-defect model of rabbit for at least 14 days. The results visualized the gathering of stem cells at the defect site of cortical bone, and the in vivo images were well correlated with the in vitro fluorescence observation without extra staining. Therefore, a potentially translatable nanoprobe was developed for non-invasive and real-time tracking of cells, which may be meaningful for understanding the bone regeneration in clinic and shed light on the visualization of cells in other cell therapies.


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1. Introduction Cell therapy, in which stem cells and other mammalian cells are infused or transplanted into tissue, has been pre-clinically applied in diabetes1, myocardial deceases2, liver impairment3, muscle and skeleton deceases4, neurodegenerative diseases5, and tumor6, showing promising prospects in clinical applications. Among these areas, using stem cells to directly treat bone defects or as seed cells to assist tissue engineering scaffold for bone tissue repairing has attracted intensive attentions in recent years7,8. However, so far there have been no definite results to clearly depict the exact mechanism of the role of stem cells in bone regeneration. For example, low concentration of viable stem cells at the defect area was considered as the reason for the failure of autologous bone marrow transplantation9. Some reports, however, showed that successful bone regeneration cases relied only on a very small amount of viable implanted stem cells10,11. In addition, researchers reported inconsistent results concerning whether the implanted stem cells were actively transformed to bone tissue, or only acted as a medium to release related factors to promote endogenous cellular repair12. The unclear mechanism on the role of stem cells severely hampers the development of clinical treatment, and it should be largely ascribed to the lack of a protocol for monitoring the behavior of implant cells in vivo. Optical imaging techniques are of great importance for investigating the behavior of implanted cells. For example, the imaging technique based on the insetion of an optical-reporter-gene is a classical strategy for stem cells monitoring. It generally can offer veracious information on the distribution and proliferation of stem cells via optical imaging in histological analysis

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However, the optical observation in vivo was limited to small animals owing to the high absorption and scattering of visible light in bio-tissues. Besides, this approach is difficult to be translated due to the requirements for DNA modification, transfection of genetic material, and


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the epigenetic gene silencing owing to DNA methylation as well, all of which may lead to the raised concern of adverse cellular effects 15. Positron emission tomography (PET) and magnetic resonance imaging (MRI) are the imaging techniques potentially applicable for tracking cells in big animal or human being. For example, PET possesses extremely high sensitivity to direct observe several tens of cells16-18. However, owing to the limited half-lives of radionuclides, PET techniques are not ideal for long-term imaging studies such as bone regeneration19, Besides, radio-labelling involving chemical conjugation of emitters may be unstable in the body environment. Thus, the PET technique could not correlate with ex vivo studies at a relative long-term endpoint and was limited to the short time in vivo tracking20. An alternative approach is employing superparamagnetic iron oxide nanoparticles to direct label cells21-22. By doing so, tracking of the labeled cells by MRI was widely studied in nervous system disease23-24, myocardial infarction25-26 and angiogenesis in cancer27, etc. In spite of these promising outcome in soft tissues, skeleton system as a biological hard tissue with relatively high density and low hydrogen proton concentration is not suitable for individual MRI imaging techniques28-29. Thus, the main issue for studying the role of stem cells in bone regeneration is lack of an appropriate nanoprobe that can non-invasively track the cells behavior in vivo for a relatively long time in big animals. Upconversion nanoparticles (UCNs), especially doped NaLnF4 (Ln=lanthanides), can convert near infrared (NIR) light to visible and have found wide applications in various biomedical fields owing to their advantages such as low toxicity, resistance to photobleaching and deep penetration of NIR in biotissues30-31. Furthermore, with choosing appropriate element of lanthanides, NaLnF4 can simultaneously possess the imaging contrast of computed tomography (CT) and MRI 32-34. Thus, as shown in Figure 1, we herein designed a nanoprobe based on the surface modification


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of core-shell structured UCNs with fluorescence/MRI/CT multimodal imaging capabilities, resulting in the efficient and stable label of bone marrow mesenchymal stem cells (BMSCs). With such a nanoprobe, BMSCs were tracked by multiple imaging techniques in a bone-defect model of big animal (rabbit), especially by the dual-energy CT that can discriminate the signal from bone and nanoparticles35-38. Meanwhile, the labeled UCNs directly gave corresponding fluorescent images in the in vitro observation without further histological staining. The current work may suggest a non-invasive and real-time route with potential promise in illustrating the mechanism underlying in bone regeneration, and in clinically tracking the implanted cells in cell therapy as well. 2. Results and Discussion 2.1 UCNs and multi-modal imaging capabilities The core-shell upconversion nanoparticles (UCNs in this work) were synthesized with Yb and Tm co-doped NaYF4 as a core and undoped NaGdF4 as a shell, following our previously reported successive hot-injection approach39. Fig. 2A and 2B show the transmission electron microscopy (TEM) images of synthesized NaYF4:Yb, Tm core and NaYF4:Yb, Tm@NaGdF4 core/shell nanocrystals, respectively. Core nanoparticles were spherical with a diameter of ~40nm. After coating with a shell layer, UCNs presented hexagonal-plate shape with a diameter of about 50 nm. High-resolution TEM image in the inset of Figure 2A indicated the highly crystalline nature of the core nanoparticles, whilst Figure 2B inset showed obvious difference in the crystal lattice orientation between the inner part and the outer part, suggesting the core-shell structure. The scanned elemental spectra across a single NaYF4:Yb,Tm@NaGdF4 core-shell nanocrystal with the direction along the line marked in Figure 2C inset demonstrated that Gd mainly distributed at the edges while Y mainly located in the core. Two-color element distribution of the core/shell


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nanocrystals further revealed the obvious Y signal in the cores and Gd signal on the shell (Figure 2D). Then the UCNs were coated by a thin layer of silane with amino group (denoted as UCNsNH2) and the resultant UCNs-NH2 were mixed with a commercially available transfection agent called DNA Transfectin 3000 (TS) for the final nanoparticles (denoted as UCNs-TS). The surface charge of UCNs-NH2 and UCNs-TS was 10.3 and 18.6mV, respectively, which suggests that TS modification further improved hydrophilicity of the UCNs-NH2. The fluorescence spectra of NaYF4:Yb, Tm and NaYF4:Yb, Tm@NaGdF4 nanoparticles under 980 nm continuous-wave excitation were shown in Figure 3A. The emission bands were clearly resolved at ~360 nm, ~450 nm, and ~803 nm, respectively. The intensity of core-shell structured nanoparticles was obviously higher than that of the core, which should be ascribed to the suppressing of surface quenching effect. Under NIR irradiation, the as-prepared UCNs displayed visible purple-blue color on the photograph (Figure 3A inset). Owing to the existence of gadolinium, the core-shell nanoparticles showed obvious MRI and CT imaging contrast. T1-weighted MR images were brightened while T2-weighted images were darkened as the Gd concentration increased over the tested range (Figure 3C and 3D). According to the linear fitting of the experimental data, longitudinal relativity (R1) and transverse relativity (R2) were around 0.86 and 37 mM-1 s-1, respectively. For CT images of the core-shell nanoparticles, the values of the Hounsfield unit (HU) were measured as a function of the lanthanide concentration in the range of 5 to 80 mM, and Iopamiro was used as a control. Both of the samples displayed a well-correlated linear relationship (Figure 3B inset). Importantly, the UCNs-TS possessed a higher HU value than Iopamiro at same molar concentration, indicating the potential of UCNs-TS as a CT contrast agent. We further evaluated the capability of signal separation in the mixture of UCNs-TS and bone tissue together in ethanol by clinical dual-energy


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CT. Owing to that ethanol is a poor-solvent for UCNs-TS, the nanoparticles tend to quickly deposit at the bottom of the container. As shown in Figure 3, dual-energy CT scan acquired the image data simultaneously at two X-ray with the tube voltage of 80 kV and 140 kV, respectively. In each separated dataset, both UCNs-TS and the bone tissue showed enhanced signals. By postprocessing the dual-energy CT data with decomposition method, UCNs-TS and bone tissue could be set apart from each other and presented as individual maps of distribution, as shown in Figure 3. This result provides us the possibility to distinguish the implanted nanoparticleslabeled stem cells and normal bone tissue by dual-energy CT technique. 2.2 The influence of nanoparticles on BMSCs Figure 4a presented that the viability of BMSCs would not decrease after incubating with different concentration of nanoparticles unless it increased to ~500 µg/ml. Even if the concentration was increased to 1000 µg/ml, the cell viability still kept above 80%. The half maximal inhibitory concentration (IC50) of the nanoparticles is about 1500 µg/ml. It should be noted that in our previous work for gold nanoparticles38, the IC50 is only about 500 µg/ml under the same surface modification, indicating that the UCNs show much less cytotoxicity than gold nanoparticles. Therefore, the incubating concentration of 1000 µg/ml for UCNs-TS that may ensure an efficient labeling of cells was used in the following experiments. For further identifying the biocompatibility of UCNs nanoparticles on BMSCs, we detected the apoptosis of BMSCs by Annexin V-FITC/PI apoptosis detection kit after incubation with UCNsTS for 24 hours. The percentage of alive cells of the control group and the UCNs-TS group were 96.9% and 96.7% respectively (Figure 4B), which demonstrated that the nanoparticles did not induce obvious apoptosis. In order to determine the effect of UCNs-TS on the proliferation of BMSCs, CCK-8 assay was conducted at the 1st day, 3rd day, 5th day and 7th day after labeling


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with the UCNs, the observation at 450 nm had no difference between the control group and UCNs-TS group (Figure 4C). After transfected with UCNs nanoparticles for 24 hours, the cell cycle of BMSCs was detected. It was found that there was almost no difference between each group at G0/G1 phase, G2/M phase and S phase, demonstrating that the nanoparticles did not cause cell cycle arrest (Figure 4D). The effects of UCNs-TS on the differentiation capability of BMSCs to osteoblasts, chondrocytes and adipocytes was respectively investigated. As shown in Figure 5a inset, in osteogenic differentiation assay, alizarin red was used to label the formed osteoblast. The results showed that UCNs-transfected experimental group and positive control group were almost same in color, while the negative control group did not show the color of alizarin red. At the same time, as an important signal that presents the osteogenic differentiation, ALP did not differ significantly in the expression between the UCNs-transfected experimental group and the positive control group, while the expression level in the negative control group was significantly lowered. These results demonstrated that the UCNs-TS nanoparticles hardly affect the osteogenic differentiation of stem cells. Similarly, in the study of chondrogenic differentiation, alcian blue staining on cartilage cells and the expression of cartilage cell gene marker, SOX9, showed that the UCNs-TS nanoparticles had no effect on the chondrogenic differentiation of stem cells. In adipogenic differentiation assay, oil red O staining on adipose cells and the expression of adipose cells marker gene, PPARγ, also showed that the UCNs-TS nanoparticles had no effect on the adipogenic differentiation of stem cells, as shown in Figure 5c. We further investigated the effect of UCNs-TS on the differentiation of BMSCs into osteoblasts at different time points. The expression of key osteoblast differentiation genes, OSX, OCN and Collagen I, were detected. As shown in Figure 5d-f, after being co-cultivated with UCNs-TS


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nanoparticles for 1 day, 7 days, 14 days and 21 days, the expression of OSX, OCN and mRNA of Collagen I were almost identical with that in the control group. The above results showed that UCN-TS nanoparticles have excellent biocompatibility and no effect on the differentiation of stem cells in different stages of osteoblast differentiation. 2.3 Efficient labeling of BMSCs The results of inductively coupled plasma mass spectrometry (ICP-MS) showed that the number of nanoparticles uptaken by BMSCs were 244 pg/cell for UCNs-TS after incubated for one day. In contrast, for UCNs-NH2 as a control, the uptaken dose at the 1st day was 134 pg/cell. After 24 h of incubation, the labelled cells were transferred to fresh culture media without nanoparticles to measure the uptaken stability of UCNs-TS. It was found that the uptaken dose of UCNs-TS nanoparticles per cell had been around 200 pg without perceptible change within 14 days observation. On the contrary, without modification of the transfection agent TS, UCNs-NH2 nanoparticles were obviously excreted by the BMSCs in 14 days to be a remained dose of ~20 pg/cell. These results implied that the modification of TS rendered the retention of UCNs in BMSCs. For further identifying the long-term labeling of nanoparticles, CT images were obtained for the labeled cells in culture solution at different time, as shown in Figure 6b. It should be noted that before the imaging of the solution at each time-point, extracellular nanoparticles were completely washed out, thus the signal of UCNs-TS directly represents those labeled stem cells. The corresponding volumes of deposited labeled cells were 22.8 mm3, 24.7 mm3 25.2 mm3, 24.6 mm3, and 24.1 mm3, and the HU values were 1620, 1310, 1344, 1295, 1277 for 1, 4, 7, 10, 14 days, respectively. Owing to that the HU value is directly related to the local concentration of contrast agent, the slightly increase of the volume of the labelled cells, resulting in the decrease


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of HU value, indicate that the fluctuation of the number of nanoparticles were imperceptible. Meanwhile the signal from MRI T1 weight also kept the same with time extending, as shown in Figure 6c, indicating that the local concentration of labelled cells would not change. Therefore, the results further evidenced that with the help of transfection agent, the UCNs-TS nanoparticles can be retained in BMSCs for at least 14 days. For the techniques based on nanoparticulate contrast agent, the capability of labeling cells with long-term, high-dose internalization and non-cytotoxicity is of great importance. Higher intracellular concentration of nanoparticles generally results in a better imaging contrast. In last two decades, researchers reported various approaches to tailor the internalization of nanoparticles. For example, positively charged nanoparticles are more favored for being uptaken than their negatively charged counterparts in different cancer cell types, such as MCF-7 cells, endothelial cells, and HeLa cells40-41. Several kinds of cell penetrating peptides (CPPs), including trans-activating transcriptional activator (TAT)

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model amphipathic peptide (MAP)47, were modified to guide the nanoparticles traversing through the cellular membranes in a so-called "protein transduction" process. Alternatively, modification with targeting ligands, such as folic acid48, albumin49-50 and cholesterol51, also results in the uptaken of nanoparticles by caveolin-mediated endocytosis. Despite of these significant achievements, the intracellular dose of contrast agent for cellular imaging still has plenty room for the improvement. For example, the cellular uptake of gold nanoparticles recently was increased from ~30 pg/cell52 to ~88.98 pg/cell53-54, and thus can give an observable contrast on a microCT. However, it is still insufficient for the imaging on clinical CT so that the challenge still remains for the cellular monitoring in big animals. In our work, a commercially available transfection agent TS and thin layer of SiO2 significantly improved the


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biocompatibility of the nanoparticles. Therefore, the labeling dose was tremendously increased to above 200 pg/cell. This result shows promising potentials in sensitive and precise imaging of labeled cells, especially for investigation of big animals and the applications of clinical imaging techniques. Besides, high-dose of cellular uptake also benefits for the area of drug delivery, as the cargo such as drugs, proteins, peptides or nucleic acids could be released to directly exert therapeutic effects on cytoplasmic and nuclear targets55. On the other hand, exocytosis of the contrast agent is expected to be avoided as it may give artifacts, or gradually lower the contrast for long-term observation. However, high-dose of cellular uptake did not ensure the retention of nanoparticles in cells for a relatively long term. For example, about 84%, 66% and 49% of mesoporous silica nanoparticles modified with phosphonate, folate and polyethylenimine, respectively, were found to be excreted from cells after 6h56. In contrast, our experiments showed that UCNs-TS almost would not be excreted in 14 days. The excretion or retention of nanoparticles in cells may relate to their surface modifications and cell lines, and the mechanism should be further studied in the future. Nevertheless, our results suggest that long-term and high-efficient labeling of BMSCs without side-effect was achieved by UCNs based on the surface modification, leading to the potential of UCNs as the multi-modal imaging nanoprobe. 2.4 In vivo clinical imaging and the histological analysis Based on the above results, with the injection of UCNs-TS labeled BMSCs in a bone defect model of rabbit, we further explored the capability of tracking these cells by multiple imaging methods including dual-energy CT and MRI. As shown in Figure 7a-d and g-j, the signals of UCNs-TS labeled BMSCs were extracted by dual-energy CT scanning from the bone of rabbit as


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a big animal model. At the first day after the implantation, some of the labeled cells were kept at the injected place, while contrast signal was also observed far from the bone defect. Over 14 days following up the implantation, the contrast signals of UCNs-TS labeled BMSCs near the bone defect maintained and the distribution of these cells has the obvious tendency to migrate to defect site, as observed from the difference between Figure 7c and i. In the meantime, the signal far from the bone defect gradually vanished. This result might be attributed to two possible reasons: first, the nanoparticles-label cells at the other end of bone may enter the body cycle and therefore were invisible on CT owing to the low concentration, and second, they may be homed to the defect site and the signal was combined to that of the cells originally at there. By using clinical MRI, axial and coronal scanning of T1 weight contrast can also image the UCNs-TS labeled BMSCs in a bone defect model of rabbit at the 1st day (Figure 7e and f) and the 14th day (Figure 7k and l), respectively. The contrasts of UCNs-TS labeled BMSCs on MRI images were marked in red dashed lines, while the defect of the bone was marked by red arrow. It can be observed that at the first day after implantation, the contrast area was larger than that on the CT image. After 14 days, the movement of UCNs-TS labeled BMSCs towards bone defect areas was also observed as same as the result of CT imaging. Moreover, it was found that the contrast areas of UCNs-TS labeled BMSCs are somewhat larger than those of CT imaging for both the first day and after 14 days. This may be owing to the higher sensitivity of MRI than CT so that the circumjacent area of UCNs-TS labeled BMSCs with lower concentration was detected by MRI. Furthermore, the bright area increased (as shown in Figure 7k and l, pointed by the arrow), which reveals the initiation of regenerated callus. On the 14th day after the implantation, the rabbit was sacrificed and the slices at the site of bone defect were obtained. As shown in Figure 8, for the control group without UCNs-TS


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labeled BMSCs, tissue with the morphology of directional alignment to some extent can be observed around the defect area, which should be attributed to the new bone in terms of trabecula. For the experiment group, the blue-purple fluorescence of the UCNs-TS labeled BMSCs can be observed without any further staining. Interestingly, the UCNs-TS labeled BMSCs only appears along the interface between normal tissues and bone defect area and tended to an orientation array that would be related to the initiation of regenerated trabecula. This result strongly suggested that a part of the nanoparticle-labeled cells might integrate into or transform to new tissue, and therefore the nanoparticles were integrated into bone tissue. The fluorescence observation provided further evidence for the results of clinical imaging that the contrast signal indicated the migration of BMSCs, and the signal around defect area could last for at least 14 days after implantation in contrast to the disappearance on the other end. Thus, the histological analysis identified the reliability of the clinical imaging, and demonstrated UCNs-TS as a cellular nanoprobe for long term multi-modal imaging. In fact, taking advantages of the NIR excited fluorescence, upconversion nanoparticles have found wide applications in the tracking of cells, including BMSCs57, human amniotic fluid stem cells58, and dendritic cells59. However, the observation of cells by fluorescence is limited to in vitro studies or small animals so far. Besides fluorescence imaging, Liu et. al.60 designed NaYbF4:Er nanoparticles to combine upconversion fluorescence and CT contrast capabilities together and act as the contrast agent for angiography and lymph node mapping. On the other hand, the clinical imaging capability such as MRI was also integrated into the lanthanides-based nanoparticles with MRI contrast agents or by lanthanide itself. Shi et. al.61 conjugated gold and upconversion nanoparticles together to form MRI and CT imageable nano-balls, which increased signal-to-noise ratio of tumors after the local-injection in vivo. However, CT/MRI imaging of


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UCNs was limited in tissue-scale, such as tumors and blood pool. Thus, this paper for the first time developed a cellular nanoprobe based on the appropriate design of upconversion nanoparticles for the non-invasive monitoring implanted cells by using the clinical CT/MRI imaging techniques. More importantly, the fluorescence signal in vitro can be well correlated with the in vivo CT/MRI imaging results especially for the model of big animals. 3. Conclusion In summary, we developed a cellular nanoprobe that not only visualized the integration of the implanted UCNs-TS labeled BMSCs in rabbit in vivo with clinical instruments of dual-energy CT and MRI, but also directly observed the histological graphs without further staining. The protocol is based on a facile modification of UCNs with commercially available transfection agents to tailor the internalization. The resultant UCNs-TS nanoparticles as the cellular nanoprobe can label BMSCs with a dose of over 200 pg/cell without affecting the cell cycle, apoptosis, cell proliferation and multi-lineage differentiation, and were almost not excreted for at least 14 days, which ensures the possibility and veracity of cellular monitoring in big animal model. By the correlation between both clinical imaging results of dual-energy CT & MRI and the histological observation, we found that the implanted BMSCs tended to migrate to the bone defect border and were related to the initiation of regenerated callus tissue. Thus, the current study provides a translatable proof-of-concept for tracking implanted cells based on the clinical CT and MRI instruments, and allows the longitude research on big animal without sacrificing at certain endpoints.

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Supporting Information. The Supporting Information is available free of charge on the ACS Publications website, including the experimental section. AUTHOR INFORMATION Corresponding Author * Email: [email protected] * Email: [email protected] * Email: [email protected]; Tel: 86-21-34202956; Fax: 86-21-34202745 Author Contributions † The first two authors contributed equally to this work.

Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work was financially supported by National Natural Science Foundation of China (Project No. 81301260, No. 31470958, No. 31671027 and No. 31671004), Shanghai Jiao Tong University Medical Engineering Cross Grant (YG2013MS55), and a Global Innovation Initiative (GII) project funded by the British Council and the Department of Business, Innovation and Skills in the United Kingdom. S. A. thanks the support from Shanghai Pujiang Program (15PJD025) and Shanghai Municipal Education Commission-Gaofeng Clinical Medicine Grant (20152221). We thank Instrumental Analysis Center of SJTU for assistance with the instrumentation.


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Figure 1. Schematic illustration of the design for the non-invasive imaging of implanted stem cells with MRI/dual energy CT and the “in situ” observation of the fluorescence of UCNs by a confocal laser scanning microscopy (CLSM).


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Figure 2. TEM images of a) NaYF4:Yb, Tm core nanoparticles, b) NaYF4: Yb, Tm @NaGdF4 core/shell nanoparticles. Insets show the magnified high-resolution images of the corresponding nanoparticles. c) The chemical mapping of core/shell structure with marking Gd in green and Y in red, respectively, and d) the distribution of Gd and Y along the line marked in c).


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Figure 3. a) The fluorescence spectra of the NaYF4:Yb, Tm core nanoparticles and NaYF4: Yb, Tm@NaGdF4 core/shell nanoparticles (UCNs), respectively. Inset shows the photograph of UCNs under the irradiation of a 980nm excitation laser. b) CT contrast of UCNs and commercial Iopamiro in terms of Hounsfield Unit and (inset) contrast images. The T1 weight (c) and T2 weight (d) of the UCNs in the form of relaxation time and contrast images. e) Dual energy CT imaging and signal separation of UCNs and bone scaffold in ethanol.


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Figure 4. a) Concentration-dependence of cell viability after co-cultivate with UCNs-TS, and the influence of 1000 µg/mL UCNs-TS on the b) apoptosis, c) cell proliferation, and d) cell cycle of BMSCs.


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Figure 5. The influence of 1000 µg/mL UCNs-TS on the multi-differentiation capability: Expression of key mRNA in (a) osteogenic differentiation, (b) chondrogenic differentiation, (c) adipogenic differentiation. Insets show the immunostaining of BMSCs of corresponding sample. (d-f) the expression of mRNA in osteogenic differentiation when incubated for different time.


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Figure 6. The maintenance of UCNs-TS nanoparticles in cells within 14 days after incubation with 1000 µg/mL UCNs for 24 h: a) The dose of nanoparticles retained in cells at various time points, with a control group of UCNs-NH2; b) CT and c) MRI T1 weight images at 1st, 4th, 7th, 10th and 14th after 24-hour cultivation, respectively.


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Figure 7. CT (a, b, g, h), decomposed dual energy CT images (c, d, i, j), and MRI T1 weight images (axial: e, k, and coronal: f, l) of UCNs-TS nanoparticles-labelled stem cells in rabbit bone-defect model.


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Figure 8. Observation of the slices at bone defect at 14th day after implanting cells with (lower panel) and without (upper panel) UCNs under a two-photon fluorescent confocal microscopy.


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REFERENCES 1.

Voltarelli, J. C.; Couri, C. E. B.; Stracieri, A. B. P. L.; Oliveira, M. C.; Moraes, D. A.;

Pieroni, F.; Coutinho, M.; Malmegrim, K. C. R.; Foss-Freitas, M. C.; Simões, B. P.; Foss, M. C.; Squiers, E.; Burt, R. K., Autologous nonmyeloablative hematopoietic stem cell transplantation in newly diagnosed type 1 diabetes mellitus. Journal of the American Medical Association 2007, 297 (14), 1568-1576. DOI: 10.1001/jama.297.14.1568. 2.

Beltrami, A. P.; Barlucchi, L.; Torella, D.; Baker, M.; Limana, F.; Chimenti, S.;

Kasahara, H.; Rota, M.; Musso, E.; Urbanek, K.; Leri, A.; Kajstura, J.; Nadal-Ginard, B.; Anversa, P., Adult cardiac stem cells are multipotent and support myocardial regeneration. Cell 2003, 114 (6), 763-776. DOI: 10.1016/S0092-8674(03)00687-1 3.

Vessey, C. J.; Hall, P. D. L. M., Hepatic stem cells: A review. Pathology 2001, 33 (2),

130-141. DOI: 10.1080/00313020124028. 4.

Jackson, K. A.; Majka, S. M.; Wang, H.; Pocius, J.; Hartley, C. J.; Majesky, M. W.;

Entman, M. L.; Michael, L. H.; Hirschi, K. K.; Goodell, M. A., Regeneration of ischemic cardiac muscle and vascular endothelium by adult stem cells. Journal of Clinical Investigation 2001, 107 (11), 1395-1402. DOI: 10.1172/JCI12150. 5.

Lindvall, O.; Kokaia, Z.; Martinez-Serrano, A., Stem cell therapy for human

neurodegenerative disorders-how to make it work. Nature Medicine 2004, 10 (7), S42-S50. DOI: 10.1038/nm1064. 6.

Stuckey, D. W.; Shah, K., Stem cell-based therapies for cancer treatment: Separating

hope from hype. Nature Reviews Cancer 2014, 14 (10), 683-691. DOI: 10.1038/nrc3798. 7.

Bianco, P.; Robey, P. G., Stem cells in tissue engineering. Nature 2001, 414 (6859), 118-

121. DOI: 10.1038/35102181. 8.

Schachter, B., Therapies of the state. Nature Biotechnology 2014, 32 (8), 736-741. DOI:

10.1038/nbt.2984.


24

Page 25 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


9.

Hernigou, P.; Poignard, A.; Manicom, O.; Mathieu, G.; Rourd, H., The use of

percutaneous autologous bone marrow transplantation in nonunion and avascular necrosis of bone. Journal of Bone and Joint Surgery - Series B 2005, 87 (7), 896-902. DOI: 10.1302/0301620X.87B7.16289 10.

Grayson, W. L.; Bunnell, B. A.; Martin, E.; Frazier, T.; Hung, B. P.; Gimble, J. M.,

Stromal cells and stem cells in clinical bone regeneration. Nat Rev Endocrinol 2015, 11 (3), 140150. DOI: 10.1038/nrendo.2014.234. 11.

Dupont, K. M.; Sharma, K.; Stevens, H. Y.; Boerckel, J. D.; García, A. J.; Guldberg, R.

E., Human stem cell delivery for treatment of large segmental bone defects. Proceedings of the National Academy of Sciences 2010, 107 (8), 3305-3310. DOI: 10.1073/pnas.0905444107. 12.

Rubio, R.; Abarrategi, A.; Garcia-Castro, J.; Martinez-Cruzado, L.; Suarez, C.; Tornin, J.;

Santos, L.; Astudillo, A.; Colmenero, I.; Mulero, F.; Rosu-Myles, M.; Menendez, P.; Rodriguez, R., Bone environment is essential for osteosarcoma development from transformed mesenchymal stem cells. Stem Cells 2014, 32 (5), 1136-1148. DOI: 10.1002/stem.1647. 13.

Frangioni, J. V.; Hajjar, R. J., In vivo tracking of stem cells for clinical trials in

cardiovascular disease. Circulation 2004, 110 (21), 3378-3383. DOI: 10.1161/01.CIR.0000149840.46523.FC. 14.

Crigler, L.; Robey, R. C.; Asawachaicharn, A.; Gaupp, D.; Phinney, D. G., Human

mesenchymal stem cell subpopulations express a variety of neuro-regulatory molecules and promote neuronal cell survival and neuritogenesis. Experimental Neurology 2006, 198 (1), 5464. DOI: 10.1016/j.expneurol.2005.10.029. 15.

Kircher, M. F.; Gambhir, S. S.; Grimm, J., Noninvasive cell-tracking methods. Nature

Reviews Clinical Oncology 2011, 8 (11), 677-688. DOI: 10.1038/nrclinonc.2011.141. 16.

McCracken, M. N.; Gschweng, E. H.; Nair-Gill, E.; McLaughlin, J.; Cooper, A. R.;

Riedinger, M.; Cheng, D.; Nosala, C.; Kohn, D. B.; Witte, O. N., Long-term in vivo monitoring of mouse and human hematopoietic stem cell engraftment with a human positron emission


25


Page 26 of 32

tomography reporter gene. Proceedings of the National Academy of Sciences of the United States of America 2013, 110 (5), 1857-1862. DOI: 10.1073/pnas.1221840110. 17.

Bhaumik, S.; Gambhir, S. S., Optical imaging of Renilla luciferase reporter gene

expression in living mice. Proceedings of the National Academy of Sciences of the United States of America 2002, 99 (1), 377-382. DOI: 10.1073/pnas.012611099. 18.

Grimm, J.; Swirski, F.; Pittet, M.; Josephson, L.; Weissleder, R., A nanoparticle-based

cell labeling agent for cell tracking with SPECT/CT. Mol Imaging 2006, 5 (Suppl 3), 364. 19.

Xu, C.; Mu, L.; Roes, I.; Miranda-Nieves, D.; Nahrendorf, M.; Ankrum, J. A.; Zhao, W.;

Karp, J. M., Nanoparticle-based monitoring of cell therapy. Nanotechnology 2011, 22 (49), 494001. DOI: 10.1088/0957-4484/22/49/494001. 20.

Naumova, A. V.; Modo, M.; Moore, A.; Murry, C. E.; Frank, J. A., Clinical imaging in

regenerative medicine. Nature Biotechnology 2014, 32 (8), 804-818. DOI: 10.1038/nbt.2993. 21.

Lewin, M.; Carlesso, N.; Tung, C. H.; Tang, X. W.; Cory, D.; Scadden, D. T.;

Weissleder, R., Tat peptide-derivatized magnetic nanoparticles allow in vivo tracking and recovery of progenitor cells. Nature Biotechnology 2000, 18 (4), 410-414. DOI: 10.1038/74464. 22.

McCarthy, J. R.; Weissleder, R., Multifunctional magnetic nanoparticles for targeted

imaging and therapy. Advanced Drug Delivery Reviews 2008, 60 (11), 1241-1251. DOI: http://doi.org/10.1016/j.addr.2008.03.014. 23.

Guzman, R.; Uchida, N.; Bliss, T. M.; He, D.; Christopherson, K. K.; Stellwagen, D.;

Capela, A.; Greve, J.; Malenka, R. C.; Moseley, M. E.; Palmer, T. D.; Steinberg, G. K., Longterm monitoring of transplanted human neural stem cells in developmental and pathological contexts with MRI. Proceedings of the National Academy of Sciences of the United States of America 2007, 104 (24), 10211-10216. DOI: 10.1073/pnas.0608519104. 24.

Liu, Y.; He, Z. J.; Xu, B.; Wu, Q. Z.; Liu, G.; Zhu, H.; Zhong, Q.; Deng, D. Y.; Ai, H.;

Yue, Q.; Wei, Y.; Jun, S.; Zhou, G.; Gong, Q. Y., Evaluation of cell tracking effects for transplanted mesenchymal stem cells with jetPEI/Gd-DTPA complexes in animal models of


26

Page 27 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


hemorrhagic spinal cord injury. Brain Research 2011, 1391, 24-35. DOI: 10.1016/j.brainres.2011.03.032. 25.

Arai, T.; Kofidis, T.; Bulte, J. W. M.; De Bruin, J.; Venook, R. D.; Berry, G. J.;

McConnell, M. V.; Quertermous, T.; Robbins, R. C.; Yang, P. C., Dual in vivo magnetic resonance evaluation of magnetically labeled mouse embryonic stem cells and cardiac function at 1.5 T. Magnetic Resonance in Medicine 2006, 55 (1), 203-209. DOI: 10.1002/mrm.20702. 26.

Kraitchman, D. L.; Tatsumi, M.; Gilson, W. D.; Ishimori, T.; Kedziorek, D.; Walczak, P.;

Segars, W. P.; Chen, H. H.; Fritzges, D.; Izbudak, I.; Young, R. G.; Marcelino, M.; Pittenger, M. F.; Solaiyappan, M.; Boston, R. C.; Tsui, B. M. W.; Wahl, R. L.; Bulte, J. W. M., Dynamic imaging of allogeneic mesenchymal stem cells trafficking to myocardial infarction. Circulation 2005, 112 (10), 1451-1461. DOI: 10.1161/CIRCULATIONAHA.105.537480. 27.

Arbab, A. S.; Frenkel, V.; Pandit, S. D.; Anderson, S. A.; Yocum, G. T.; Bur, M.; Khuu,

H. M.; Read, E. J.; Frank, J. A., Magnetic resonance imaging and confocal microscopy studies of magnetically labeled endothelial progenitor cells trafficking to sites of tumor angiogenesis. Stem Cells 2006, 24 (3), 671-678. DOI: 10.1634/stemcells.2005-0017. 28.

Tautzenberger, A.; Kovtun, A.; Ignatius, A., Nanoparticles and their potential for

application in bone. International Journal of Nanomedicine 2012, 7, 4545-4557. DOI: 10.2147/IJN.S34127. 29.

Accomasso, L.; Gallina, C.; Turinetto, V.; Giachino, C., Stem cell tracking with

nanoparticles for regenerative medicine purposes: An overview. Stem Cells International 2016, 2016, 7920358. DOI: 10.1155/2016/7920358. 30.

Dong, H.; Du, S. R.; Zheng, X. Y.; Lyu, G. M.; Sun, L. D.; Li, L. D.; Zhang, P. Z.;

Zhang, C.; Yan, C. H., Lanthanide Nanoparticles: From Design toward Bioimaging and Therapy. Chemical Reviews 2015, 115 (19), 10725-10815. DOI: 10.1021/acs.chemrev.5b00091. 31.

Gu, Z.; Yan, L.; Tian, G.; Li, S.; Chai, Z.; Zhao, Y., Recent Advances in Design and

Fabrication of Upconversion Nanoparticles and Their Safe Theranostic Applications. Advanced Materials 2013, 25 (28), 3758-3779. DOI: 10.1002/adma.201301197.


27


32.

Page 28 of 32

Xu, C. T.; Zhan, Q.; Liu, H.; Somesfalean, G.; Qian, J.; He, S.; Andersson-Engels, S.,

Upconverting nanoparticles for pre-clinical diffuse optical imaging, microscopy and sensing: Current trends and future challenges. Laser & Photonics Reviews 2013, 7 (5), 663-697. DOI: 10.1002/lpor.201200052. 33.

Xia, A.; Chen, M.; Gao, Y.; Wu, D.; Feng, W.; Li, F., Gd3+ complex-modified NaLuF4-

based upconversion nanophosphors for trimodality imaging of NIR-to-NIR upconversion luminescence, X-Ray computed tomography and magnetic resonance. Biomaterials 2012, 33 (21), 5394-5405. DOI: 10.1016/j.biomaterials.2012.04.025 34.

Shen, J. W.; Yang, C. X.; Dong, L. X.; Sun, H. R.; Gao, K.; Yan, X. P., Incorporation of

computed tomography and magnetic resonance imaging function into NaYF4:Yb/Tm upconversion nanoparticles for in vivo trimodal bioimaging. Analytical Chemistry 2013, 85 (24), 12166-12172. DOI: 10.1021/ac403486r. 35.

Bhavane, R.; Badea, C.; Ghaghada, K. B.; Clark, D.; Vela, D.; Moturu, A.; Annapragada,

A.; Johnson, G. A.; Willerson, J. T.; Annapragada, A., Dual-energy computed tomography imaging of atherosclerotic plaques in a mouse model using a liposomal-iodine nanoparticle contrast agent. Circulation Cardiovascular Imaging 2013, 6 (2), 285-294. DOI: 10.1161/CIRCIMAGING.112.000119. 36.

Ashton, J. R.; Clark, D. P.; Moding, E. J.; Ghaghada, K.; Kirsch, D. G.; West, J. L.; Badea,

C. T., Dual-energy micro-CT functional imaging of primary lung cancer in mice using gold and iodine nanoparticle contrast agents: A validation study. PLoS ONE 2014, 9 (2), e88129. DOI: 10.1371/journal.pone.0088129. 37.

Zamora, C. A.; Lin, D. D., Utility of dual-energy CT in differentiating contrast

extravasation from intracranial hematoma response. Journal of Neurosurgery 2016, 124 (1), 280280. DOI: 10.3171/2015.5.JNS15953. 38.

Wan, D.; Chen, D.; Li, K.; Qu, Y.; Sun, K.; Tao, K.; Dai, K.; Ai, S., Gold Nanoparticles

as a Potential Cellular Probe for Tracking of Stem Cells in Bone Regeneration Using DualEnergy Computed Tomography. ACS Applied Materials and Interfaces 2016, 8 (47), 3224132249. DOI: 10.1021/acsami.6b11856.


28

Page 29 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


39.

Tian, Q.; Tao, K.; Li, W.; Sun, K., Hot-injection approach for two-stage formed

hexagonal NaYF4: Yb, Er nanocrystals. The Journal of Physical Chemistry C 2011, 115 (46), 22886-22892. DOI: 10.1021/jp208569q. 40.

Torchilin, V. P., Recent approaches to intracellular delivery of drugs and DNA and

organelle targeting. Annual Review of Biomedical Engineering, 2006, 8, 343-375. DOI: 10.1146/annurev.bioeng.8.061505.095735. 41.

Stewart, M. P.; Sharei, A.; Ding, X.; Sahay, G.; Langer, R.; Jensen, K. F., In vitro and ex

vivo strategies for intracellular delivery. Nature 2016, 538 (7624), 183-192. DOI: 10.1038/nature19764. 42.

Al-Jamal, W. T.; Al-Jamal, K. T.; Tian, B.; Lacerda, L.; Bomans, P. H.; Frederik, P. M.;

Kostarelos, K., Lipid - Quantum dot bilayer vesicles enhance tumor cell uptake and retention in vitro and in vivo. ACS Nano 2008, 2 (3), 408-418. DOI: 10.1021/nn700176a. 43.

Al-Jamal, W. T.; Al-Jamal, K. T.; Bomans, P. H.; Frederik, P. M.; Kostarelos, K.,

Functionalized-quantum-dot–liposome hybrids as multimodal nanoparticles for cancer. Small 2008, 4 (9), 1406-1415. DOI: 10.1002/smll.200701043. 44.

Leroueil, P. R.; Hong, S.; Mecke, A.; Baker Jr, J. R.; Orr, B. G.; Holl, M. M. B.,

Nanoparticle interaction with biological membranes: Does nanotechnology present a janus face? Accounts of Chemical Research 2007, 40 (5), 335-342. DOI: 10.1021/ar600012y. 45.

Kumar, S.; Harrison, N.; Richards-Kortum, R.; Sokolov, K., Plasmonic nanosensors for

imaging intracellular biomarkers in live cells. Nano Letters 2007, 7 (5), 1338-1343. DOI: 10.1021/nl070365i. 46.

Kumar, S.; Aaron, J.; Sokolov, K., Directional conjugation of antibodies to nanoparticles

for synthesis of multiplexed optical contrast agents with both delivery and targeting moieties. Nature Protocols 2008, 3 (2), 314-320. DOI: 10.1038/nprot.2008.1. 47.

Heitz, F.; Morris, M. C.; Divita, G., Twenty years of cell-penetrating peptides: From

molecular mechanisms to therapeutics. British Journal of Pharmacology 2009, 157 (2), 195-206. DOI: 10.1111/j.1476-5381.2009.00057.x.


29


48.

Page 30 of 32

Oehlke, J.; Scheller, A.; Wiesner, B.; Krause, E.; Beyermann, M.; Klauschenz, E.;

Melzig, M.; Bienert, M., Cellular uptake of an α-helical amphipathic model peptide with the potential to deliver polar compounds into the cell interior non-endocytically. Biochimica et Biophysica Acta - Biomembranes 1998, 1414 (1-2), 127-139. DOI: 10.1016/S00052736(98)00161-8. 49.

Shi, X.; Wang, S.; Meshinchi, S.; Van Antwerp, M. E.; Bi, X.; Lee, I.; Baker Jr, J. R.,

Dendrimer-entrapped gold nanoparticles as a platform for cancer-cell targeting and imaging. Small 2007, 3 (7), 1245-1252. DOI: 10.1002/smll.200700054. 50.

Dixit, V.; Van Den Bossche, J.; Sherman, D. M.; Thompson, D. H.; Andres, R. P.,

Synthesis and grafting of thioctic acid-PEG-folate conjugates onto Au nanoparticles for selective targeting of folate receptor-positive tumor cells. Bioconjugate Chemistry 2006, 17 (3), 603-609. DOI: 10.1021/bc050335b. 51.

Cui, B.; Wu, C.; Chen, L.; Ramirez, A.; Bearer, E. L.; Li, W. P.; Mobley, W. C.; Chu, S.,

One at a time, live tracking of NGF axonal transport using quantum dots. Proceedings of the National Academy of Sciences of the United States of America 2007, 104 (34), 13666-13671. DOI: 10.1073/pnas.0706192104. 52.

Tekle, C.; Van Deurs, B.; Sandvig, K.; Iversen, T. G., Cellular trafficking of quantum

dot-ligand bioconjugates and their induction of changes in normal routing of unconjugated ligands. Nano Letters 2008, 8 (7), 1858-1865. DOI: 10.1021/nl0803848. 53.

Meir, R.; Motiei, M.; Popovtzer, R., Gold nanoparticles for in vivo cell tracking.

Nanomedicine 2014, 9 (13), 2059-2069. DOI: 10.2217/nnm.14.129. 54.

Nam, S. Y.; Ricles, L. M.; Suggs, L. J.; Emelianov, S. Y., In vivo ultrasound and

photoacoustic monitoring of mesenchymal stem cells labeled with gold nanotracers. PLoS ONE 2012, 7 (5), e37267. DOI: 10.1371/journal.pone.0037267. 55.

Betzer, O.; Shwartz, A.; Motiei, M.; Kazimirsky, G.; Gispan, I.; Damti, E.; Brodie, C.;

Yadid, G.; Popovtzer, R., Nanoparticle-Based CT Imaging Technique for Longitudinal and


30

Page 31 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Quantitative Stem Cell Tracking within the Brain: Application in Neuropsychiatric Disorders. ACS Nano 2014, 8 (9), 9274-9285. DOI: 10.1021/nn503131h. 56.

Yanes, R. E.; Tarn, D.; Hwang, A. A.; Ferris, D. P.; Sherman, S. P.; Thomas, C. R.; Lu,

J.; Pyle, A. D.; Zink, J. I.; Tamanoi, F., Involvement of lysosomal exocytosis in the excretion of mesoporous silica nanoparticles and enhancement of the drug delivery effect by exocytosis inhibition. Small 2013, 9 (5), 697-704. DOI: 10.1002/smll.201201811. 57.

Wang, C.; Cheng, L.; Xu, H.; Liu, Z., Towards whole-body imaging at the single cell

level using ultra-sensitive stem cell labeling with oligo-arginine modified upconversion nanoparticles. Biomaterials 2012, 33 (19), 4872-4881. DOI: 10.1016/j.biomaterials.2012.03.047. 58.

Xu, Y.; Xiang, J.; Zhao, H.; Liang, H.; Huang, J.; Li, Y.; Pan, J.; Zhou, H.; Zhang, X.;

Wang, J. H.; Liu, Z.; Wang, J., Human amniotic fluid stem cells labeled with up-conversion nanoparticles for imaging-monitored repairing of acute lung injury. Biomaterials 2016, 100, 91100. DOI: 10.1016/j.biomaterials.2016.05.034. 59.

Xiang, J.; Xu, L.; Gong, H.; Zhu, W.; Wang, C.; Xu, J.; Feng, L.; Cheng, L.; Peng, R.;

Liu, Z., Antigen-Loaded Upconversion Nanoparticles for Dendritic Cell Stimulation, Tracking, and Vaccination in Dendritic Cell-Based Immunotherapy. ACS Nano 2015, 9 (6), 6401-6411. DOI: 10.1021/acsnano.5b02014. 60.

Liu, Y.; Ai, K.; Liu, J.; Yuan, Q.; He, Y.; Lu, L., A High‐Performance Ytterbium‐

Based Nanoparticulate Contrast Agent for In Vivo X‐Ray Computed Tomography Imaging. Angewandte Chemie International Edition 2012, 51 (6), 1437-1442. DOI: 10.1002/anie.201106686. 61.

Xing, H.; Bu, W.; Zhang, S.; Zheng, X.; Li, M.; Chen, F.; He, Q.; Zhou, L.; Peng, W.;

Hua, Y.; Shi, J., Multifunctional nanoprobes for upconversion fluorescence, MR and CT trimodal imaging. Biomaterials 2012, 33 (4), 1079-1089. DOI: 10.1016/j.biomaterials.2011.10.039. For Table of Contents Use Only


31


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Multi-modal nanoprobe based on upconversion nanoparticles for monitoring implanted stem cells in bone defect of big animal Dexin Chen,†a Daqian Wan, † bd Rongying Wang, a Yanyue Liu, a Kang Sun,* a Xiaofeng Tao, b Yang Qu, b Kerong Dai, c Songtao Ai,*b Ke Tao* a

For Table of Contents Use Only


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