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Engineering of radioiodine-labeled gold core-shell nanoparticles as efficient nuclear medicine imaging agents for trafficking of dendritic cells Sang Bong Lee, Sang-Woo Lee, Shin Young Jeong, GhilSuk Yoon, Sung Jin Cho, Sang Kyoon Kim, In-Kyu Lee, Byeong-Cheol Ahn, Jaetae Lee, and Yong Hyun Jeon ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b14800 • Publication Date (Web): 20 Feb 2017 Downloaded from http://pubs.acs.org on February 24, 2017

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ACS Applied Materials & Interfaces

Engineering of radioiodine-labeled gold core-shell nanoparticles as efficient nuclear medicine imaging agents for trafficking of dendritic cells Sang Bong Lee

†, ⊥

, Sang-Woo Lee

†,⊥

, Shin Young Jeong†, GhilSuk Yoon‡, Sung Jin Cho§, ††,

Sang Kyoon Kim§§, In-Kyu Lee‡‡, ⊥, Byeong-Cheol Ahn†, Jaetae Lee†,§* & Yong Hyun Jeon†,⊥, §§* †

Department of Nuclear Medicine, Kyungpook National University Hospital, Daegu, Korea

‡

Department of Pathology, School of Medicine, Kyungpook National University, Daegu

§

Daegu-Gyeongbuk Medical Innovation Foundation, Daegu, Korea

⊥

Leading-edge Research Center for Drug Discovery and Development for Diabetes and

Metabolic Disease, Kyungpook National University Hospital, Daegu, Korea ††

New Drug Development Center, Daegu-Gyeongbuk Medical Innovation Foundation, Daegu

41061, South Korea ‡‡

Department of Internal Medicine, School of Medicine, Kyungpook National University, Daegu

41944, South Korea §§

Laboratory Animal Center, Daegu-Gyeongbuk Medical Innovation Foundation, Daegu, Korea

KEYWORDS: Dendritic Cells, Cell Tracking, Tannic Acid, Functionalized Gold Core-Shell Nanoparticles, Detection limit, PET imaging

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ABSTRACT

The development of highly sensitive, stable, and biocompatible imaging agents allowing visualization of dendritic cell (DC) migration is one of the essential factors for effective DCbased immunotherapy. Here, we used a novel and efficient synthesis approach to develop Radioiodine-124-labeled Tannic Acid Gold Core-Shell Nanoparticles (124I-TA-Au@AuNPs) for DC labeling and in vivo tracking of their migration using positron emission tomography (PET). 124

I-TA-Au@AuNPs were produced within 40 min in high yield via straightforward tannic acid-

mediated radiolabeling chemistry and incorporation of Au shell, which resulted in high radiosensitivity and excellent chemical stability of nanoparticles in DCs and living mice.

124

I-TA-

Au@AuNPs demonstrated good DC labeling efficiency and did not affect cell biological functions, including proliferation and phenotype marker expression. Importantly,

124

I-TA-

Au@AuNPs in an extremely low amount (0.1 mg/kg) were successfully applied to track the migration of DCs to lymphoid organs (draining lymph nodes) in mice.


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INTRODUCTION Dendritic cells (DCs) can activate effector T cells via specialized co-stimulatory molecules, including B7, MHC class I/II, and adhesion factors.1-2 DCs can recognize several type of tumor-specific or -associated antigens and induce anti-tumor immune reactions.3 To successfully generate anti-tumor immune response, DCs should migrate to lymphoid organs such as draining lymph nodes to provide tumor antigenic peptides to effector T cells.4-5 Although DCbased immunotherapeutic strategies have been used to treat various cancers, their therapeutic effects are only partially successful because of the lack of appropriate methods to visualize DC migration and vaccination efficiency in living organisms. Therefore, sensitive, quantitative, and biocompatible in vivo imaging tools are urgently required to track the migration of immune cells for the accurate evaluation of immunotherapy. The introduction of direct and indirect labeling methods based on multimodal reporter genes, radioactive probes, MRI agents, and fluorescent probes, allowed developing several imaging approaches including radionuclide imaging, magnetic resonance imaging, and optical imaging to visualize the biological behavior of immunotherapeutic DCs6-8. Among these methods, nuclear imaging and MRI imaging based on direct labeling have been proved effective in tracking the migration of human DCs in clinical situations4-5, 8, indicating the importance of developing clinically relevant straightforward cell tracking imaging agents. Among various imaging approaches based on contrast nanomaterials, nuclear imaging modality has been progressively employed in disease diagnostics and management as well as in visualization of immune cell migration because of their high sensitivity and the absence of depth limitation.9-10 Since the safety of clinically used radioisotopes is well established, radiolabeled nanoparticles have a great potential for wide application in clinical settings.



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Gold nanoparticles (AuNPs) have been extensively examined for their use in molecular imaging owing to their various beneficial characteristics.11-12 To date, extensive research has been conducted to develop sensitive imaging probes, particularly for nuclear medicine imaging, by efficient radiolabeling of AuNPs via direct labeling methods (electrostatic attraction) or chelation.13-16 However, a disadvantage of these labeling approaches is transchelation of radioactive isotopes to biological molecules and their subsequent accumulation in non-target tissues, which increases the risk of image misinterpretation.17-19 This problem characteristic for general radiochemistry approaches would restrict extensive application of AuNPs in nuclear medicine imaging. Therefore, straightforward radiolabeling techniques that can provide high sensitivity and stability without adverse effects are urgently required.16, 20-21 In this study, we attempted to synthesize a highly sensitive, stable, and biocompatible nuclear imaging probe to visualize the migration of DCs in living mice, which is important for the generation of effective anti-tumor immunity. In order to produce highly sensitive and stable imaging probes, we developed a novel type of chemical synthesis that enabled ultrafast (within 40 min) and efficient labeling of tannic acid-functionalized AuNPs (TA-AuNPs) with iodine-124 (124I), a PET imaging radioisotope (Scheme 1). Furthermore, a protective Au shell was constructed around

124

I-labeled TA-AuNPs to form

124

I-TA-Au@AuNPs, which exhibited high

radiochemical stability and biocompatibility. Finally, to investigate the feasibility of

124

I-TA-

Au@AuNPs as a nuclear imaging agent for the in vivo DC tracking, the migration of DCs toward lymphoid organs (draining lymph nodes) was serially monitored with PET/CT after subcutaneous injection of DCs labeled with 124I-TA-Au@AuNPs to footpad (Scheme 2).


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Scheme 1. Procedure for ultrafast and efficient preparation of radioiodine (124I)-labeled tannic acid (TA)-coated gold core gold shell nanoparticles (124I-TA-Au@AuNP).

Scheme 2. Dendritic cell (DC) labeling with

124

I-TA-Au@AuNPs and in vivo tracking of DC

migration by PET imaging.



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MATERIALS AND METHODS Preparation of Radioiodine-labeled Tannic Acid Gold Core-Shell Nanoparticles (124I-TAAu@AuNPs) The synthesis of 124I-TA-Au@AuNPs is described in detail in Supporting information. Sensitivity and stability tests at different pH and in human serum The changes in PET signal intensity depending on

124

I-TA-AuNP and

124

I-TA-

Au@AuNP concentrations (0.1–100 pM) were investigated using a PET scanner. To test the stability of imaging probes, 100 µL of 1.0 nM 124I-TA-AuNPs or 124I-TA-Au@AuNPs was added to human serum at 37°C; the released radionuclide was quantified by TLC using an AR-2000 scanner (Bioscan, Washington, DC, USA). Uptake of 124I-TA-Au@AuNPs by DCs and proliferation assay Detailed protocols for cellular uptake of 124I-TA-Au@AuNPs and cell proliferation assay are described in Supporting information. Apoptosis and phenotypic marker expression analyses Methods to assess apoptosis and phenotypic marker expression are described in Supporting information. In vivo PET imaging Experiment 1: mice (n = 5) were intravenously injected Na124I, 124

I-TA-AuNPs (50 µCi, 1.85 MBq) and analyzed by PET scan.


124

I-TA-Au@AuNPs, or

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Experiment 2: mice were injected with TNF-α subcutaneously into the footpad of the hind leg (n = 5). The next day, 3 × 106

124

I-TA-Au@AuNPs-labeled BMDCs were

subcutaneously administered to the footpad and PET scan was done. During imaging, mice were anesthetized using 1–2% isoflurane. Detail protocols for PET/CT imaging and data interpretation are described in Supporting information.



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RESULTS AND DISCUSSION Synthesis of Radioiodine-124-labeled Tannic Acid Gold Core-Shell Nanoparticles (124I-TAAu@AuNPs) Recently, a facile synthesis of stable spherical AuNPs coated with TA, a plant polyphenol, has been reported,22-23 and currently, TA-AuNPs are a commercially available nanomaterial. Interestingly, an early study24 demonstrated ready TA iodination at a high rate (11:1, iodine : TA), suggesting that TA could provide chemically functional reaction sites for AuNP radiolabeling. Thus, positions 2, 6 of the 3-hydroxy phenyl ring of TA present potential sites for facile radioiodination using Na124I (Figure S1a); therefore, multiple TA molecules on AuNPs offer numerous points of iodination, which should create nanoparticles highly detectable in vivo. Thus, TA-functionalized AuNPs could be a promising approach to improving iodine load on nanoparticles. Furthermore, the formation of an additional Au shell over radiolabeled TAAuNPs can enhance their radiochemical stability in vivo, improving their performance as imaging agents. TA-AuNPs were radiolabeled with 124I, a clinically used radionuclide with an appropriate half-life (4.2 days) for PET.25 The iodination reaction was conducted by mixing Na124I with TAAuNPs (Figure S1a), and the yield of resulting

124

I-labeled TA-AuNPs (124I-TA-AuNPs) was

evaluated by TLC and sedimentation of nanoparticles. The iodination was completed in as short a time as 30 min (Figure S1b), the yield of blue), and the

124

124

I-TA-AuNPs was as high as 99% (Figure 1a, b-

I density per AuNP was 10,575 (Materials and Methods). An additional Au

shell around 124I-TA-AuNPs was created as previously described. 26 The Au shell formation was monitored by the optical density at 520 nm every 5 s after HAuCl4 addition, and was detected as early as 20 s after the start of the reaction with the maximum at 40 s (Figure S2a). Thus, the


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overall time of

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I-TA-Au@AuNPs production was as short as 40 min (Scheme 1),

demonstrating the effectiveness of our novel synthetic approach. The final yield of

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I-TA-

Au@AuNPs was high – about 90% (Figure 1a, b-red). As a result of Au shell modification, the hydrodynamic radius of particles was slightly increased: from 29.8 ± 1.5 nm in

124

I-TA-AuNPs

to 48.9 ± 2.3 nm in 124I-TA-Au@AuNPs (Figure S2b). TA-AuNPs, 124I-TA-AuNPs, and 124I-TAAu@AuNPs had the same UV-visible spectra (Figure S2c) showing no particle aggregation during radiolabeling and Au shell formation. X-ray photoelectron spectroscopy revealed the presence of Au and iodine in

124

I-TA-Au@AuNPs (Figure S2d). High resolution transmission

electron microscopy (HR-TEM) images revealed that

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I-TA-Au@AuNPs exhibited a spherical

shape (Figure 1c), showing the absence of Au-Au interval sizes (Figure S3). The distribution of 124

I in 124I-TA-Au@AuNPs by energy dispersive X-ray spectrometry analysis is shown in Figure

1c. To further characterize each type of obtained particles, we performed zeta potential analysis and Fourier transform infrared (FT-IR) spectroscopy with the aim to detect the introduction of functional groups such as NH2 and COOH, which would change surface charges and spectra of the particles. Figure S4a shows that zeta potential values for TA-AuNPs, 124I-TAAuNPs, and

124

I-TA-Au@AuNPs were -42.2 mV, -53.73 mV, and -36.30 mV, respectively. FT-

IR spectroscopy analysis of the products obtained from chemical reaction of TA-AuNPs detected peaks at 1,213 cm-1 (CH2), 1,651 cm-1 (C=O), 2858 cm-1 (C–H), and 3,408 cm-1 (O–H) (Figure S4b), which were the same as those in

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I-TA-AuNPs and

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I-TA-Au@AuNPs. These results

suggest that various functional groups successfully reacted with gold nanostructures. Although our synthetic approach is in certain aspects similar to previously reported methods (DNA-mediated radiolabeled gold core@shell), there are clear differences conferring



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advantages to the current strategy. First, for DNA-mediated engineering of nuclear imaging agents described before, multiple steps and long time (about 50 h) are required. Thus, specialized oligonucleotides should be synthesized and conjugated with AuNPs (day 1); then, Sulfosuccimidyl-3-[4-hydroxyphenyl]propionate (sulfo-SHPP)-modification of DNA-AuNPs should be conducted (day 2); and finally, radiolabeling of SHPP-DNA-AuNPs and shell formation are required (2 h 10 min). However, in case of TA-mediated production of nuclear imaging agents, commercial TA-functionalized AuNPs already have numerous functional sites for radioiodine labeling, enabling direct isotope labeling of TA-AuNPs without additional modifications. Because of this straightforward approach, it takes only about 40 min to produce 124

I-TA-Au@AuNPs. Second, DNA-modified AuNPs can be labeled with 2,800 iodide atoms,

while TA-AuNPs can be labeled with 10,575. Thus,

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I-TA-Au@AuNPs exhibit higher

sensitivity compared with previously described DNA-mediated synthesis of radiolabeled gold core@shell particles.

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Figure 1. (a) Schematic synthesis of

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I-TA-Au@AuNPs. (b) Radiochemical yield of

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I-TA-

AuNPs and 124I-TA-Au@AuNPs. (c) TEM images of 124I-TA-Au@AuNPs and EDX analysis. Sensitivity of 124I-TA-Au@AuNP Next, we examined the sensitivity of 124I-TA-AuNP and 124I-TA-Au@AuNP detection by PET/CT. In vitro PET/CT imaging of phantom tubes containing

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I-TA-AuNPs and

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I-TA-

Au@AuNPs demonstrated an increase of radioactivity in a particle dose-dependent manner (Figure 2a, b, respectively), and the signal could be detected for as low as 0.1 pM AuNPs and

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124

I-TA-

I-TA-Au@AuNPs. Furthermore, we observed a good linear relationship between



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nanoparticle concentration (0.1–100 pM) and radioactivity (R2 = 0.95 and R2 = 0.87 for 124I-TAAuNPs and 124I-TA-Au@AuNPs, respectively; Figure 2c, d).

Figure 2. Sensitivity and stability of 124I-TA-Au@AuNPs. Radio-detection of 124I-TA-AuNPs at different concentrations (0.1–100 pM). PET/CT imaging of phantom tubes with (a) and

124

124

I-TA-AuNPs

I-TA-Au@AuNPs (b). Correlation between radio-detection and concentration of

TA-AuNPs (c) and 124I-TA-Au@AuNPs (d).

Stability of 124I-TA-Au@AuNPs


124

I-

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Since the detachment of radionuclides from nanoparticles results in a decrease of bioimaging sensitivity and difficulty for long-term tracking, which are important factors in tracking of immunotherapeutic cells, we assessed the radiochemical stability of and

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124

I-TA-AuNPs

I-TA-Au@AuNPs in solutions with different pH values, as well as in human serum and

live mice. Both

124

I-TA-AuNPs and

124

I-TA-Au@AuNPs were highly stable at the broad pH

range of 1–14 at room temperature for 48 h (Figure 3a, b and Figure S5). Human serum stability test indicated that 124I-TA-AuNPs lost about 20% 124I after 24-h incubation in serum (Figure 3cblack and Figure S6a); however, more than 98 % of 124I remained on

124

I-TA-Au@AuNPs after

48 h (Figure 3c-red and Figure S6b).

Figure 3. Time-dependent stability of 124I-TA-AuNPs and 124I-TA-Au@AuNPs in solutions with different pH (a, b) and in human serum (c). Subsequently, the in vivo stability test was conducted by intravenous injection of the nanoparticles into mice. The results of PET/CT imaging revealed that free Na124I was taken up by the thyroid, stomach, and bladder (Figure 4a, d-black) in the process of normal physiological uptake. Mice administered

124

I-TA-AuNPs also showed strong radioactivity in the thyroid,

stomach, and bladder because 124I detached from the nanoparticles (Figure 4b, d-red). In contrast,



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a significant uptake of 124I-TA-Au@AuNPs was observed in the spleen and liver until 24 h postinjection, while low radioactivity was observed in the thyroid of mice administered

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I-TA-

Au@AuNPs (Figure 4c, d-blue), indicating the in vivo radiochemical stability of

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I-TA-

Au@AuNPs due to Au shell protection. These findings indicated that our imaging probe had unique characteristics as an in vivo cell tracker, including high sensitivity, excellent stability, and efficient labeling with a clinically used radionuclide, thus enabling the monitoring of DC migration. Therefore, we next examined the properties of

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I-TA-Au@AuNPs as an imaging

probe for sensitive and quantitative tracking of bone marrow-derived DC (BMDC) migration both in vitro and in vivo.

Figure 4. Time-dependent stability of

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I-TA-AuNPs and

were intravenously injected free Na124I (a),

124

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I-TA-Au@AuNPs in vivo. Mice

I-TA-AuNPs (b), or

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I-TA-Au@AuNPs (c) and

analyzed by PET/CT. Reconstructed 3D PET/CT and axial PET/CT images (focusing on thyroid


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lesions) are shown. T: thyroid; ST: stomach; BL: bladder; LI: liver; SP: spleen. (d) Quantification of radioactivity in the thyroid. Labeling of DCs with 124I-TA-Au@AuNPs To achieve this goal, the labeling efficiency was first evaluated by measuring the radioactivity in DCs after incubation with

124

I-TA-Au@AuNPs. DCs have strong phagocytic

activity, which facilitates the capture of antigens and foreign bodies.3 The particle uptake assay showed that BMDCs efficiently internalized 124I-TA-Au@AuNPs in a dose- and time-dependent manner (Figure 5a, b); they became darker (Figure 5c) and were readily sensed by the PET detector (Figure 5d). As a result, the optimal probe concentration (2.0 nM) and labeling time (3.0 h) were determined. Importantly,

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I-TA-Au@AuNPs should remain inside BMDCs for a long

enough time to track DCs. Although

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I-TA-Au@AuNP radioactivity in BMDCs showed a

gradual decrease with time, the cells retained more than 65% of the initial signal even after 4 days (Figure 5e), indicating the high level of

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I-TA-Au@AuNP intracellular stability essential

for selective, long-term monitoring of BMDC biological activity.27 However, when

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I-

Au@AuNPs were used for BMDC labeling, over 90% radioiodine was released (data not shown), indicating that

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I-Au@AuNPs were not suitable as imaging agents for the in vivo cell

tracking because of poor radiochemical stability.



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Figure 5. (a) Concentration-dependent

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I-TA-Au@AuNP uptake by BMDCs. (b) Time-

dependent 124I-TA-Au@AuNP uptake by BMDCs. (c) Bright-field microscopy of bone marrowderived dendritic cells (BMDCs) after incubation with observed in

124

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I-TA-Au@AuNPs. Dark inclusions

I-TA-Au@AuNP-labeled BMDCs indicate cellular uptake of nanoparticles. (d)

PET/CT imaging of phantom tubes containing unlabeled and

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I-TA-Au@AuNPs-labeled

BMDCs. (e) Time-dependent stability of 2 nM 124I-TA-Au@AuNPs in BMDCs. The effect of 124I-TA-Au@AuNP labeling on biological functions of BMDCs Biocompatibility of imaging agents is another important factor. Therefore, we assessed the cytotoxicity of 124I-TA-Au@AuNPs in BMDCs by analyzing cell proliferation and apoptosis. In the concentration range of 1–4 nM,

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I-TA-Au@AuNPs did not inhibit cell proliferation

(Figure 6a) or induce apoptosis (Figure 6b), indicating good biocompatibility. Next, we further determined the effects of our particles on the phenotype marker expression of BMDC. FACS results revealed that the uptake of

124

I-TA-Au@AuNP by DCs did not affect the expression of

their phenotypic markers (Figure 6c, d). We also compared the levels of Th1-cytokines including TNF-α, IL-12p70, and IL-6 secretion, and the ability to induce T cell proliferation between


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unlabeled and labeled BMDCs. The results indicated that there was no difference in cytokine secretion between these cell groups (Figure S7). Furthermore, unlabeled and labeled BMDCs showed similar potency in the induction of T cell proliferation (Figure S8). These data revealed that 124I-TA-Au@AuNP labeling did not interfere with DC functions.



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Figure 6. (a) BMDC viability 24 h and 48 h post-labeling. (b) Apoptosis of unlabeled and

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I-

TA-Au@AuNP-labeled BMDCs 24 h post-labeling. (c) Levels of phenotypic markers in unlabeled and

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I-TA-Au@AuNP-labeled BMDCs. Black histograms represent cells labeled

with isotype-matched antibodies. (d) Relative expression of the indicated proteins assessed by fluorescence intensity. Sensitive detection of BMDCs labeled with 124I-TA-Au@AuNPs To determine the sensitivity of DC detection in vivo, different numbers of

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I-TA-

Au@AuNP-labeled cells were subcutaneously injected to the dorsal sites of mice (Figure 7a), and the animals were analyzed by PET/CT imaging. The results indicated that the increase in PET signals linearly correlated with the number of injected cells and that as low as 1 × 102 labeled BMDCs could be discovered (Figure 7b, c; R2 = 0.99), suggesting that

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I-TA-

Au@AuNPs were highly detectable by PET/CT imaging in living mice.

Figure 7. (a, b) Detection of 124I-TA-Au@AuNP-labeled BMDCs subcutaneously injected at the indicated concentrations to the dorsal sites of mice; as few as 1 × 102 labeled cells could be detected. (c) Radioactivity directly correlated with BMDC numbers.


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Tracking of

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I-TA-Au@AuNPs-labeled BMDC migration to draining lymph nodes using

PET/CT To induce the antigen-specific immune response in vivo, it is essential that DCs pulsed with tumor antigenic peptides should move towards draining lymph nodes and effectively provide these peptides to effector cells. Therefore, in vivo imaging approaches have recently been tested for monitoring of DC migration in the host. In the next experiment, we attempted to visualize the migration of

124

I-TA-Au@AuNP-labeled BMDCs by PET/CT using the protocol

shown in Figure S9. When BMDCs are subcutaneously injected into the mouse footpad, they mostly migrate to the draining lymph nodes.28 To enhance the migration into lymphoid organs, mice were preconditioned by the injection of 30 ng TNFα into the footpad. The in vivo tracking by PET/CT imaging revealed the migration of

124

I-TA-Au@AuNP-labeled BMDCs to DPLNs,

which was observed at 15 h post-injection and reached a plateau at 24 h (Figure 8a, b); the cells could be detected even at 96 h post-injection. From day 1 post-injection, weak radioactive signal due to some release of radioactive iodide from

124

I-TA-Au@AuNPs was observed in mouse

thyroid glands (Figure S10), but no radioactivity was detected in the stomach, bladder, and spleen. The imaging results obtained in vivo were in agreement with those obtained ex vivo with excised DPLNs (Figure 8c, d), and with the biodistribution of labeled BMDCs (Figure 8e). PET imaging of DPLNs revealed good linearity between the in vivo and ex vivo radioactivity (R2 = 0.97, Figure 8f).



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Figure 8. Application of

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I-TA-Au@AuNPs to in vivo tracking of bone marrow-derived

dendritic cell (BMDC) migration using PET/CT. (a) Reconstructed 3D (top) and axial (bottom) PET/CT images showing time-dependent BMDC migration to draining popliteal lymph nodes (DPLNs). (b) Quantification of radioactivity in DPLNs at different times after injection of

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I-

TA-AuNP-labeled BMDCs. (c, d) Ex vivo PET/CT imaging and radioactivity in dissected PLNs and DPLNs. (e) Radioactivity distribution in indicated mouse organs after subcutaneous injection of 124I-TA-Au@AuNP-labeled BMDCs into the footpad. The right graph presents the magnified area of the left graph showing radioactivity accumulation in DPLNs. (f) Linearity between ex vivo and in vivo radioactivity in DPLNs.


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Immunohistological analysis of DPLNs Immediately after PET/CT imaging at 96 h, DPLNs were excised for histological examination, which revealed that 124I-TA-Au@AuNP-labeled BMDCs were mostly accumulated in CD3-positive T-cell zones (Figure 9c) rather than in CD79a-positive B-cell zones (Figure 9b). Notably, cells expressing a DC-specific marker S-100 were mainly detected within the T-cell zones (Figure 9d), indicating the active movement of DCs to the T-cell area of DPLNs.

Figure 9. Histological analysis of DPLNs. (a) H&E staining, (b) B-cell zone, (c) T-cell zone, (d) S100-positive zone for DCs (arrows indicate 124I-TA-Au@AuNPs). CONCLUSION



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We designed 124I-TA-Au@AuNPs to be used as an advanced nuclear imaging probe. 124ITA-Au@AuNPs were synthesized within 40 min via simple tannic acid-based radiolabeling chemistry and formation of an additional Au shell. The

124

I-TA-Au@AuNPs generated by our

novel ultrafast synthesis demonstrated several advantages over other probes, such as outstanding sensitivity and radiochemical stability in DCs and living organisms provided by the Au shell. The

124

I-TA-Au@AuNPs were easily taken up by DCs without side effects on cell proliferation

or the expression of phenotypic markers, indicating good biocompatibility. Furthermore,

124

I-

TA-Au@AuNPs could be used in an extremely low amount (0.1 mg/kg) for effective tracking of DC migration to lymphoid organs (draining lymph nodes) in living mice. Cumulatively, these results suggest that

124

I-TA-Au@AuNPs are a promising probe for nuclear medicine imaging,

which can be used in labeling and tracking of immune cells used for cancer immunotherapy such as DCs, cytotoxic T cells, and natural killer cells. However, it should be mentioned that although our novel nanoparticles showed a great potential as imaging agents for cell tracking, safety concerns regarding the application of radioisotopes should be addressed. In this study, we used low doses of radioactive iodide; however, long-term exposure to even very low doses of radiation could cause cell and organ damage due to several factors, including apoptosis induction. Therefore, further investigation should be conducted using various in vivo models to determine a safe dose of radiolabeled nanoparticles prior to their clinical application.

ASSOCIATED CONTENT Supporting Information. Supporting Materials and Methods, Figures S1-10 is available in the Supporting Information on the ACS Publications website. ACS Paragon30-22 Plus Environment

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Radio-chemical synthesis of 124I-TA-AuNPs, Time-dependent monitoring of radiolabeling reaction using TLC, Reaction rate of Au shell coverage, Dynamic light scattering analysis, UVvisible spectroscopy, X-ray photoelectron spectroscopy, High resolution transmission electron microscopy (HR-TEM) analysis, Lattice plane of the Au shell, Zeta-potentials and FT-IR spectra analysis, Time-dependent stability of radiolabeled nanoparticles at different pH and human serum, Cytokine secretion analysis, Induction of T cell proliferation by BMDCs, Schematic protocol of in vivo experiments, PET/CT imaging of radioiodine uptake in the mouse thyroid glands. AUTHOR INFORMATION Corresponding Author *Yong Hyun Jeon, Ph.D. Leading-edge Research Center for Drug Discovery and Development for Diabetes and Metabolic Disease, Kyungpook National University Hospital, 807 Hogukro, Bukgu, Daegu, South Korea 702-210 Laboratory Animal Center, Daegu-Gyeongbuk Medical Innovation Foundation (DGMIF) 80 Cheombok-ro, Dong-gu, Daegu, Korea H.P: 82-10-2455-6046 Tel: 82-53-200-3149 E-mail: [email protected] *Jaetae Lee, M.D., Ph.D. Department of Nuclear Medicine Kyungpook National University School of Medicine 50 Samduk-dong 2-ga, Chung Gu, Daegu, South Korea 700-721 Daegu-Gyeongbuk Medical Innovation Foundation (DGMIF) 80 Cheombok-ro, Dong-gu, Daegu, Korea Tel: 82-53-420-5586; Fax: 82-53-422-0864 E-mail: [email protected]

AUTHOR CONTRIBUTIONS



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Y.H.J. and J.T.L. conceived the study and wrote the manuscript. S.B.L prepared and characterized nanoparticles under the supervision of Y.H.J. S.B.L performed all cell and animal experiments and wrote parts of the manuscript. S.G.Y conducted immunohistological analysis. S.W.L., S.Y.J., and B.C.A. performed PET/CT imaging analysis and contributed to the optimization of efficient TA-AuNP radiolabeling. All the authors discussed the results, contributed to data analysis, commented on the manuscript, and approved the final version.

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