In Vivo Tracking of Phagocytic Immune Cells Using a Dual Imaging

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In Vivo Tracking of Phagocytic Immune Cells using a Dual Imaging Probe with Gadolinium-enhanced MRI and Near-infrared Fluorescence Eun-Joong Kim, Sankarprasad Bhuniya, Hyunseung Lee, Hyun Min Kim, Weon Sup Shin, Jong Seung Kim, and Kwan Soo Hong ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b03344 • Publication Date (Web): 08 Apr 2016 Downloaded from http://pubs.acs.org on April 13, 2016

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ACS Applied Materials & Interfaces

In Vivo Tracking of Phagocytic Immune Cells using a Dual Imaging Probe with Gadolinium-enhanced MRI and Near-infrared Fluorescence Eun-Joong Kim,†,¶ Sankarprasad Bhuniya,‡,§,¶ Hyunseung Lee,† Hyun Min Kim,† Weon Sup Shin, ∥

Jong Seung Kim,∥,* and Kwan Soo Hong†,⊥,*

†

Bioimaging Research Team, Korea Basic Science Institute, Cheongju 28119, Korea

‡

Amrita Centre for Industrial Research & Innovation, Amrita University, Ettimadai, Coimbatore

641 112, India. §

Department of Chemical Engineering & Materials Science, Amrita University, Ettimadai,

Coimbatore 641 112, India. ∥ Department

⊥ Graduate

of Chemistry, Korea University, Seoul 02841, Korea.

School of Analytical Science and Technology, Chungnam National University,

Daejeon 34134, Korea.

ACS Paragon Plus Environment

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ABSTRACT: A novel dual imaging probe for in vivo magnetic resonance imaging (MRI) and optical imaging was developed by combining gadolinium (Gd)-chelating MR probe and a nearinfrared (NIR) fluorophore, aza-BODIPY (AB). This aza-BODIPY-based bimodal contrast agent (AB-BCA) showed a significant fluorescence emission around the near infrared range and an enhanced longitudinal relaxivity in MR modality. The probe was easily delivered to phagocytic cells of the innate immune system, together with macrophages and dendritic cells (DCs), and presented high performance fluorescence and MR imaging without obvious cytotoxicity. For in vivo visualization of AB-BCA using MRI and optical imaging, bone marrow-derived DCs were labeled and injected into the footpad of mice and labeled DCs were tracked in vivo. We observed the migration of AB-BCA-labeled DCs into the lymph nodes via lymphatic vessels using NIR fluorescence and T1-weighted MR images. This dual-modality imaging probe was used for noninvasive monitoring of DC migration into lymph nodes and could be useful for investigating advanced cellular immunotherapy.

KEYWORDS: Aza-BODIPY, in vivo tracking, lymph node, magnetic resonance imaging, nearinfrared fluorescence


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INTRODUCTION In vivo cell tracking has become essential for enhancing the efficacy of cell therapy. Improvements in non-invasive imaging strategies have led to the acquisition of detailed information on the transplanted cells for monitoring cellular therapeutics and assessing the immune microenvironment.1 Imaging techniques such as computed tomography (CT), positron emission tomography (PET), ultrasound, and magnetic resonance imaging (MRI) have been commonly used for medical applications.2 MRI is the most attractive modality owing to its safety and high resolution. Recently, cell tracking using MRI has been extensively utilized to assess biological processes by modulating and monitoring immunotherapeutic cells using immunotherapies.3,4 Especially, T1-weighted MR imaging, which can show hyperintense areas and produce bright positive contrast against background tissue, has been known as a suitable method for labeling and tracking of target cells.5,6 Optical imaging techniques using fluorescence for labeling and tracking of immune cells in vitro and in vivo are also appealing because of they are rapid, non-invasive, nontoxic, and relatively sensitive.7 Nevertheless, considerable attention has been paid to fluorescent dyes due to their relatively low photostability and sensitivity at the cellular level in the near-infrared (NIR) region of the spectrum.8 Therefore, various combined imaging modalities have been tested to overcome these problems.9 In addition, several MR contrast agents have been developed for the fluorescence modality that possess high signal-to-noise ratios and allow visualization and analysis of immune cells of interest for immunotherapy.10 Nanoparticle-based imaging contrast agents have been mainly evaluated as carriers of MRI contrast and fluorescent agents for non-invasive monitoring of immune cells in vivo.11 Bimodal probes incorporated with MRI contrast agents and quantum dot (QD) have also been used for immune cell tracking and imaging.12,13 However, the potential toxicity of QD precluded its extensive use in clinical applications.14 Although various bimodal contrast agents using organic dyes have been widely reported, BODIPY-based contrast agents are rarely used for dual imaging modalities. BODIPY derivatives are promising optical probes with high stability, sharp emission bands, high extinction


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coefficients, and high quantum yields.15 Furthermore, aza-BODIPY-based fluorescent probe was successfully used for the detection of nitroxyl in vivo.16 The innate immune response is the first defense of the host against invading pathogens17 and leads to effective adaptive immunity.18 Generally, macrophages and dendritic cells (DCs) are known as the phagocytic cells of innate immune responses and act as professional antigen presenting cells (APCs).19 Macrophages have an innate targeting ability to recognize and accumulate into pathological sites. Moreover, the macrophage-mediated immune response is involved in the clearance of cellular debris by phagocytosis, induction of inflammatory response by cytokines, and modulation of adaptive immunity by the antigen presenting process.20 In particular, because their high phagocytic activity facilitates the detection, quantification, and localization of macrophages labeled with imaging agents, various macrophage-targeted approaches have been widely used as delivery systems for therapeutic and imaging applications.21,22 DCs are known to play essential roles in linking the innate and adaptive immune systems.23 Typically, DCs, differentiated from bone marrow progenitor cells, capture foreign antigens in peripheral tissues and present them to naive T cells after migration to the draining lymph nodes (LNs) via afferent lymphatic vessels for antigen presentation.24 Especially, LNs are crucial organs leading the proper adaptive immunity from innate immune response by providing a space for the interaction of antigen-bearing DCs and naïve T cells through major histocompatibility complex (MHC) class II molecules.25 In recent years, DC-based immunotherapy have increasingly garnered interest for cancer treatments,26–28 suggesting that more improved immunotherapeutic outcomes could be achieved by effective DC tracking during the migration into the LNs using a non-invasive imaging approach such as MRI, PET, and fluorescence imaging.29,30 In this context, we report a new bimodal contrast agent that allows targeted labeling and in vivo tracking of immune cells such as macrophages and DCs. We synthesized an aza-BODIPY-based contrast agent (AB-BCA) possessing two gadolinium chelates for NIR and MR imaging. This dual imaging agent showed substantial NIR emission and reasonable T1 relaxation effect without apparent cytotoxicity.


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Finally, migration of AB-BCA-labeled DCs into the LNs in vivo was non-invasively monitored by optical imaging and MRI.

MATERIALS AND METHODS Synthetic procedures Materials and instrumentations: The aza-BODIPY (b) was synthesized as previously reported.30 4′Hydroxy chalcone (Alfa-aesar, Haverhill, MA, USA), and diethyl amine (Alfa-aesar), nitromethane (Alfaaesar), BF3.Et2O (Sigma-Aldrich, St Louis, MO, USA), ammonium acetate (Sigma-Aldrich), ethanol (Sigma-Aldrich), DIPEA (Sigma-Aldrich), 10% Pd on charcoal (Sigma-Aldrich), diethylene triamine (Sigma-Aldrich), tert-butyl bromoacetate (Sigma-Aldrich); HATU (Carbosynth, San Diego, CA, USA), N-Cbz-1,3-propyldiamine (Sigma-Aldrich), Gadolinium trichloride (Sigma-Aldrich), DMAP (Alfa-aesar), DMF (Sigma-Aldrich), acetonitrile (Sigma-Aldrich), and MeOH (Sigma-Aldrich) were commercially purchased and used without further purification. Column chromatography was achieved using silica gel 60 (70–230 mesh) as the stationary phase. Analytical thin layer chromatography was performed using silica gel 60 (pre-coated sheets with 0.25 mm thickness). Mass spectra were recorded on a mass spectrometer (Ion Spec, Varian, CA, USA). NMR spectra were collected on a 400 MHz spectrometer (Bruker, Germany). Synthesis of 1: tert-butyl bromoacetate (1.17 g, 6.0 mmol) and K2CO3 (1.4 g, 10.0 mmol) were added to a solution of compound b (529 mg, 1.0 mmol) in acetonitrile (20 mL). The reaction mixture was then refluxed for 4 h. Upon reaction completion, the reaction mixture was cooled to room temperature and filtered. The filtrate was then concentrated and passed through silica gel column chromatography using ethyl acetate (20%) in hexane to obtain the pure compound. This compound was then subjected to deprotection in the presence of Trifluoroacetic acid (TFA) in DCM for 12 h at room temperature. The


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reaction mixture was filtered and concentrated to obtain 1 as a brick-red solid. 1H-NMR (400 MHz, DMSO-d6): δ 7.58 (m, 8H); 7.38 (m, 8H); 7.04 (m, 4H); 4.75 (s, 4H). 1H-NMR (100 MHz, DMSO-d6 + CDCl3): 170.11, 160.28, 154.20, 133.42, 129.16, 128.78, 128.39, 115.11, 65.09. ESI-MS m/z (M+Na): calcd 668.198, found 668.239. Synthesis of 2: HATU (740 mg, 2.0 mmol) and a (1,346 g, 2.0 mmol) were added followed by addition of DIPEA (175 µl, 1.0 mmol) to a solution of 1 (645 mg, 1 mmol) in dry THF (20 mL). The reaction mixture was stirred continuously for 6 h at room temperature. After completion of the reaction, the reaction mixture was diluted with water; the crude compound was extracted in EtOAc. The organic layer was dried to obtain the compound. This compound was reacted with TFA (80% in DCM) for 12 h at room temperature. Then, the reaction mixture was evaporated to dryness. The crude product was wash with DCM repeatedly and triturated with ether to obtained 2 (610mg, 40.0%). 1H-NMR (400 MHz, DMSO-d6) δ 8.23 (s, 1H); 8.19 (s, 1H); 8.09 (d, J = 8.02 Hz, 6H); 7.95 (d, J = 8.01 Hz, 2H); 7.45-7.56 (m, 5H); 7.16-7.24 (m 4H); 4.52 (s, 2H); 4.07 (S, 4H); 3.51 (br, 23H); 3.27 (m, 10H); 3,12 (m, 9H), 3.04 (m, 6H); 1.13(q, 4H). 13C-NMR (100 MHz, DMSO-d6): 173.24, 167.82, 165.06, 160.23, 154.67, 133.68, 129.13, 128.97, 128.82, 128.68, 116.64, 67.65, 65.38, 54.74, 54.32, 54.03, 52.58, 49.09, 42.29, 37.02, 36.47, 29.34, 18.49, 17.14, 16,61, 12.91. MALDI-MS m/z (M+K+): calcd 1548.510, found 1,548.522. Synthesis of compound a: N-Cbz-propyl diamine, HATU (380 mg, 1.0 mmol) and DIPEA (1.0 mmol) were added to a solution of compound b (613 mg, 1.0 mmol) in THF (20 mL) and the reaction was continued for 6 h. After completion of the reaction, the reaction mixture was concentrated and dissolved in methanol. The reaction mixture was hydrogenated in presence of Pd/C (50 mg, 10%) for 12 h. After completion of the reaction, the reaction mixture was filtered and concentrated. The crude product was passed through silica column chromatography using methanol (5%) in DCM as eluent to get compound a as oily liquid (500 mg, 74.29%). 1H-NMR (400 MHz, CDCl3): δ 3.57 (t, J = 6.37 Hz, 8H); 2.67 (m, 12H); 2.48 (m, 4H); 1.42 (s, 36H). 13C-NMR (100 MHz, CDCl3): 172.11, 165.37, 84.21, 57.32, 37.83, 27.21. ESI-MS m/z (M+H): calcd 674.463, found 674.47.


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Preparation of Gd3+ complex: The ligand (AB-BCA, 90 µM) was in ultrapure water (10 mL) and the solution was adjusted to pH 7 with sodium bicarbonate. Gadolinium chloride hexahydrate (170 µM) was dissolved in 3.0 mL of ultrapure water and added to the ligand solution (AB-BCA) in three separate aliquots. After the addition of each aliquot, the pH was adjusted back to a pH between 6.5–7.0 using 0.1 M potassium carbonate. The solution was stirred for 30 min to allow the Gd3+ chelation to take place, dialyzed against ultrapure water overnight, and lyophilized to yield respective complex. Characterization of AB-BCA. T1 relaxivities, r1, of aqueous solutions with various concentrations of AB-BCA were measured using 60 MHz and 200 MHz on NMR (mq60, Bruker, Germany), and MRI (Biospec 47/40, Bruker, Germany) systems, respectively, and imaged on a 4.7 T MRI system (Biospec 47/40, Bruker, Germany). The linear fitting of measured relaxation rates (R1 = 1/T1, s-1) vs. the Gd3+ concentration (mM) was acquired for the relaxivity value of r1. Absorption and fluorescence spectra were obtained with a fluorescence spectrometer (FS-2, SCINCO, Korea). The fluorescence spectrum (665−800 nm) was measured with an excitation wavelength at 650 nm. Cell culture. The murine macrophages, RAW 264.7, were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) and 1× Antibiotic-Antimycotic (Invitrogen-Gibco, Carlsbad, CA, USA) in an atmosphere of 5% CO2 and 95% air at 37°C. Bone marrow-derived DCs (BMDCs) were generated from 5–6-week-oldfemale C57BL/6 (H-2b) mice (Orientbio, Seongnam, Korea). The mice were maintained under pathogen-free conditions. After euthanasia, femurs and tibiae were collected. Intact bones were then washed several times with phosphate-buffered saline (PBS). Next, the ends of each bone were carefully cut with scissors and the marrow inside the bones was flushed out by centrifugation with a small amount of RPMI 1640 medium. A red pellet of collected bone marrow was disintegrated by vigorous pipetting, then move to tube filled with 20–30 mL of RPMI 1640 media. After centrifugation (15,000 rpm, 5 min) and pipetting, 5 mL of red blood cell lysis buffer (Sigma Aldrich) were added and incubated at 37°C for 5 min. The suspension was then washed with RPMI 1640 medium and bone marrow cells (> 2 × 106 cells) were collected and cultured in 100 mm Petri dishes containing 10 mL of


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RPMI medium supplemented with 10% heat-inactivated FBS, 50 IU/mL penicillin, 50 mg/mL streptomycin, and 20 ng/mL mouse recombinant granulocyte-macrophage colony-stimulating factor (GMCSF, R&D Systems, Minneapolis, MN, USA). After 3 days, 10 mL of RPMI medium with 20 ng/mL GM-CSF were added. On day 7, non-adherent and loosely adherent cells were harvested just before being used for the experiments. Cell viability assay and cellular imaging. The cytotoxicity of AB-BCA was measured by performing MTT (Roche Diagnostics GmbH, Mannheim, Germany) assay on RAW 264.7 cells and BMDCs. Cells were seeded in a 96-well cell culture plate at 1 × 104 cells per well under a humidified atmosphere containing 5% CO2 at 37°C for 24 h. Samples with different concentrations of AB-BCA were then added to the wells. The cells were subsequently incubated for 24 h and MTT solution was added, followed by an additional incubation for 4 h at 37°C. Next, the medium containing MTT was removed and the solubilization buffer (Roche Diagnostics, Basel, Switzerland) was added to each well to dissolve the MTT formazan crystals. Then, the colorimetric measurements were performed at 570 nm using a microplate reader (VersaMax; Molecular Devices, Sunnyvale, CA, USA). For intracellular imaging of AB-BCA, RAW 264.7 cells or BMDCs, plated in µ-slide 8 well chamber (Ibidi, Munich, Germany) at a density of 1 × 104 cells per well, were treated with various concentrations of AB-BCA. Cells were then incubated at 37°C for 1, 2, 4, and 6 h in culture medium. After washing with PBS, cells were fixed with 4% paraformaldehyde in PBS for 10 min at room temperature (RT). Fluorescence microscopy images were acquired using a laser scanning confocal microscope (LSM 710, Carl Zeiss, Germany). T1-weighted MR images of the cell phantoms with spin-echo pulse sequence were measured from the RAW 264.7 cells and BMDCs labeled with AB-BCA incubated with different concentrations of AB-BCA using a 4.7 T MRI scanner. The following MRI parameters were used: field of view (FOV) = 5 × 3 cm2, matrix size = 256 × 256, slice thickness = 1 mm, echo time (TE) = 10 ms, and repetition time (TR) = 350 ms. In vivo tracking of BMDCs in lymph nodes. For lymph node imaging using fluorescence and MRI, BMDCs were initially labeled with 10 µM AB-BCA for 4 h. BMDCs were then matured by treatment


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with lipopolysaccharide (LPS, Sigma Aldrich, St. Louis, MO, USA) at a concentration of 1 µg/mL for 24 h. After pre-injection of 30 ng of tumor necrosis factor-α (TNF-α) in the footpad, the labeled and unlabeled BMDCs were subcutaneously injected at 5 × 106 cells per hind leg footpad., in vivo NIR and MR images of LNs were obtained under anesthesia and analyzed at 24 h and 48 h using an optical imaging system (Maestro II, CRi Inc., Woburn, MA, USA) and a 4.7 T animal MRI scanner, respectively. After euthanasia at 48 h, ex vivo NIR images of excised LNs were acquired using the optical imaging system. Optical images were normalized and analyzed by using the Maestro 2.10.0 software. T1-weighted MR images were obtained using multi-slice multi-echo (MSME) sequence (TR = 350 ms, TE = 10 ms, slice thickness = 1 mm, FOV = 3 × 3 cm2, NEX = 4, matrix size = 256 × 256).

RESULTS AND DISCUSSION Synthesis and characterization of AB-BCA. As presented in Figure 1A, the aza-BODIPY moiety was utilized as an NIR fluorogenic core (b).31,32 b was reacted with tert-butyl bromoacetate in the presence of K2CO3 in acetonitrile, followed by trifluoroacetic acid treatment. Compound 1 was obtained in moderate yield. Next, 1 was reacted with an in-house prepared compound (a), which was further treated with trifluoroacetic acid to generate ligand 2. Finally, Gd3+ chelated bimodal contrast agent, AB-BCA, was obtained by complexation of GdCl3 with ligand 2 in aqueous medium at pH 7. The detail reaction procedures and supporting spectroscopic evidences are provided in supporting information (Figure S110). We determined the absorption and fluorescence properties of the novel bimodal contrast agent. UV-vis and fluorescence spectra of AB-BCA were recorded in PBS solution. BODIPY chromophores have relatively higher fluorescence maximum emission intensity in favor of the biological and medical aspects, while they are commonly hydrophobic and show an aggregational behavior in aqueous medium, which results in a fluorescence quenching.33,34 However, aza-BODIPY-based AB-BCA showed an increased


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hydrophilicity due to the functionalization with two Gd3+-chelated ligands, and AB-BCA absorption and emission spectra (Figure 1B) exhibited a maximum peak at 580 and 700 nm around the NIR range, respectively. Therefore, NIR fluorescence-based optical imaging using the AB-BCA can be used to facilitate cell detection and tracking.


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Figure 1. (A) Synthesis of AB-BCA. (B) Absorbance spectrum (black line) and emission (red line) spectra of AB-BCA in PBS.

MR properties of AB-BCA. In order to investigate the ability of the contrast agent as an MRI contrast agent, we measured MR longitudinal relaxation rate (1/T1 = R1) in PBS at RT. MR phantoms were imaged using a 200 MHz (4.7 T) MR scanner (Biospec, Bruker) with increasing molecular concentrations of the complex (Figure 2A). The phantom image revealed that the AB-BCA provides brighter images as the concentration increased to 1.0 mM. Before being applied to the biological milieu as a contrast agent, the relaxivity (r1) values at 60 and 200 MHz were measured as 13.3 ± 0.1 and 3.9 ± 0.1 mM-1s-1, respectively (Figure 2B), which is 3.2-fold higher at 60 MHz than the r1 value of the clinically available T1 contrast agent, Gd-DTPA-BMA (Omniscan®, Amersham Biosciences, Piscataway, NJ, USA) (the r1 value of Gd-DTPA-BMA is approximately 4.2 mM-1s-1 at 60 MHz).35


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Figure 2. (A) T1-weighted spin-echo MR image obtained using the following parameters; TE/TR = 7.8/300 ms, FOV = 30 × 60 mm2, slice thickness = 1 mm, number of average = 4, matrix size = 192 × 192 at 200 MHz (4.7 T). (B) T1 relaxivity measurement of AB-BCA in PBS solution with different concentrations at 60 and 200 MHz.

T1-weighted MR imaging has been known as a reliable and promising tool for cell labeling and in vivo tracking alternative to T2 or T2*-weighted MRI.6,36,37 Gd-based MR contrast agents were used for cell labeling in various cell lines such as stem cells and immune cells, and the labeled cells can be easily detected as bright positive signals against the background tissue.38,39 However, these results indicate that AB-BCA is competitive and capable of T1 contrast enhancement and a specific cell labeled with this bimodal contrast agent may be monitored non-invasively by MR imaging. Biocompatibility of AB-BCA. To rationalize the applicability of the bimodal contrast agent in biological milieus, we initially checked the biocompatibility using a murine macrophage cell line, Raw 264.7, and primary dendritic cells derived from the bone marrow. As shown in Figure 3A, the cell viability results indicate that a less apparent cytotoxicity effect was observed in both cell lines at various AB-BCA concentrations lower than 50 µM.


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Figure 3. (A) Effect of AB-BCA on the viability of Raw 264.7 and BMDCs. Cell viability was determined using an MTT assay after 24 h treatment of Raw 264.7 and BMDCs with AB-BCA. (B) Flow cytometry analysis for the expression of the dendritic cell marker CD11c on BMDCs unlabeled or labeled with AB-BCA. BMDCs were treated with 10 µM of AB-BCA for 4 h, followed by incubation with the antibody to CD11c. Flow cytometry was performed at 690/50 nm band-pass filter.

Assessing the cytotoxic effect of imaging probes on a target cell labeled is a very important issue to be considered, since it is quite difficult to non-invasively monitor the damaged cells with migration problem in vivo. In particular, in order to establish an appropriate cell labeling protocol for in vivo cell tracking, it is necessary to determine the optimal and safe dose.40 Furthermore, in this study, because DCs were derived from bone marrow precursors and needed to be activated for their migration to the LNs, the contrast agent should not have any influence on cell differentiation or morphology. In term of differentiation, the expression level of CD11c, a dendritic cell marker, on BMDCs treated with AB-BCA was not significantly different from that of unlabeled BMDCs (Figure 3B). Moreover, AB-BCA labeling did not alter the morphological characteristics of BMDCs treated with LPS such as enlargement and irregular shape with longer protrusion (Figure S11),41 indicating that AB-BCA can be used for cell labeling and prolonged tracking in vivo. Cellular fluorescence and MR imaging of AB-BCA labeling. Cellular uptake and intracellular distribution of AB-BCA, a dual modality imaging probe, in macrophages and DCs was examined by both optical and MR imaging. In the optical imaging study, the fluorescence signal started to be visible in the cytoplasm of Raw 264.7 cells and BMDCs at 1 h after treatment with AB-BCA. NIR fluorescence was increased after 2 h of incubation, and maximized after 4 h in both cell types (Figures S12 and S13). The fluorescence intensities after incubation for 4 h (Figure 4) indicated that the fluorescence in the AB-BCAtreated cells was gradually increased and saturated after treatment with 10 µM of AB-BCA, which means


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the cellular uptake of AB-BCA was concentration-dependent. The fluorescence microscopic analyses revealed that this bimodal imaging probe was more effective in labeling innate immune cells like macrophages and DCs, indicating that the probe can be used to track these cells in vivo.

Figure 4. Cellular fluorescence images of AB-BCA-treated Raw264.7 and BMDCs. Cells were treated with various concentrations of AB-BCA (red; Ex 640/20, Em 685/715) for 4 h. Cells were then fixed in 4% paraformaldehyde after washing in PBS. The nuclei were counterstained using Hoechst (blue; Ex 360/40, Em 455/50). Scale bar: 20 µm

The feasibility of AB-BCA cellular labeling and tracking was also investigated through MR imaging. Based on the fluorescence imaging results, we obtained the T1 contrast enhancement images of both cell types and their signal intensities after incubation for 4 h with various concentrations of AB-BCA. As


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presented in Figure 5, increased signal intensity on MR images was observed with the cell pellet phantom. The enhanced T1-weighted MR image started to appear at 3 µM and reached the maximum level at a concentration of AB-BCA in the range up to 10 µM.

Figure 5. T1-weighted MR images of RAW264.7 and BMDCs labeled with AB-BCA. Both cell types were treated with various concentrations of AB-BCA, followed by incubation for 4 h. (A) MR phantom images of cells (upper panel: Raw264.7, lower panel: BMDCs) were acquired with multi-slice multi-echo (MSME) MRI sequence. (B) Contrast-to-noise ratio (CNR) was calculated within the region of interest (ROI) defined in the MR images.

Various imaging techniques and cell labeling methods have been used for in vivo immune cell imaging.42 The dual-mode imaging approach has been developed in an attempt to achieve a better


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visualization and relevant information of the cellular activities of the immune systems via enhanced sensitivity and specificity. Especially, the integration of MR and optical imaging techniques can be a useful complementary way to overcome the limitation of each technique such as low sensitivity in MRI and restricted penetration depth in optical imaging.10,43 Besides, the direct cell labeling method involving the internalization of some imaging probes into the cells is known as a suitable approach for labeling and in vivo imaging of immune cells such as macrophages and DCs.42 However, the optical and MR imaging results (Figures 4 and 5) demonstrated that AB-BCA labeling efficiency was readily achieved in Raw264.7 cells and BMDCs via their phagocytic ability and both cell types labeled with AB-BCA showed a positive contrast-enhanced optical and MR imaging capability. Furthermore, AB-BCA-treated BMDCs may provide potential benefits for accurate monitoring and tracking of DCs migration to the LNs through lymphatic vessels in vivo. In vivo tracking of AB-BCA-labeled BMDC migration to the lymph nodes. For the in vivo tracking of DC migration by optical and MR imaging, BMDCs labeled and unlabeled with AB-BCA were subcutaneously injected into the footpads of mice and the BMDC migration was monitored from the injection site to the lymph nodes through lymphatic vessels using NIR fluorescence and T1-weighted MR imaging. The fluorescence signals from AB-BCA-labeled BMDCs were obtained and analyzed by the in vivo optical imaging system. While the injection area of unlabeled BMDCs and their migration route to lymph nodes showed less fluorescent, the BMDCs labeled with AB-BCA were clearly detected at the dorsal left footpad, lymphatic vessels, and LNs after 24 h of injection (Figure 6A, B). Strong fluorescent signals were observed in the lymphatic vessel and draining LNs at 48 h later after BMDC injection (Figure 6C), and ex vivo NIR imaging on extracted lymph nodes confirmed the migration of AB-BCAlabeled BMDCs into the LNs (Figure S13). However, we did not observed any noticeable fluorescence enhancement at early time points (< 2 hr) post AB-BCA-labeled BMDCs injection. T1-weighted MR imaging of a lymph node from a mouse injected with the BMDCs was also performed in the same conditions as above (Figure 6D). Although it was difficult to obtain distinct MR images, we observed a


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noticeable difference in the migration into LNs between labeled and unlabeled DCs. In the regional LN of the mouse administered with AB-BCA-labeled DCs, a hyperintense area was detected on axial T1weighted MR images. However, there was no hyperintense signal in the left regional LN on the side of unlabeled DCs-injected footpad. The results of in vivo MR imaging was in concordance with that of NIR optical imaging, indicating that in vivo DCs tracking via T1-weighted MR imaging can be evaluated by AB-BCA labeling.

Figure 6. In vivo NIR and MRI tracking of AB-BCA-treated BMDC migration to LNs in mice. BMDCs treated with or without AB-BCA were subcutaneously injected in both hind footpads at a density of 5 × 106 cells/mouse. NIR fluorescence (A-C) and MR (D) images were then acquired at 24 h (A, B, and D) and 48 h (C) after injection. NIR fluorescence images from whole body (A) and magnified thigh (B, C) were obtained by Maestro In-Vivo Imaging System (emission = 700 nm, exposure time: 1000 ms). In vivo T1-weighted MR images were acquired by a 4.7 T MRI system using multi-slice multi-echo (MSME) sequence. Arrows indicated the LNs.


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There have been many LN imaging studies via in vivo DC tracking, most of which showed that the migration of labeled DCs into the LNs was monitored by MRI and/or fluorescence imaging. Moreover, fluorescence imaging approaches mainly explore the NIR wavelength, which can provide the improved tissue penetration, allowing the detection of in vivo DC migration into the LNs.30 Aza-BODIPY-based NIR-emitting AB-BCA utilized in this study can be indirectly visualized in the lymphatic vessels and LNs through labeling of DCs. Besides, although MRI has been widely applied to non-invasively track specific DCs, most MRI contrast agents are based on superparamagnetic iron oxide (SPIO)-enhanced T2-weighted or 19F/1H imaging.44–46 The Gd-containing bimodal contrast agent, AB-BCA, would be the first attempt to track the migration of DCs into LNs in vivo by using T1-weighted MRI. Furthermore, AB-BCA could be a dual-modality (MRI/NIR fluorescence) imaging probe for non-invasive monitoring and tracking of the DCs by exploiting the complementary strengths of each modality.

CONCLUSIONS We synthesized the NIR-emitting aza-BODIPY with Gd-chelating units at the two arms of the fluorophore. The AB-BCA showed sufficient relaxivity to enable effective signal enhancement in MRI and NIR fluorescence-enhanced optical imaging and low cytotoxicity on innate immune cells, allowing continuous monitoring of labeled cells in vivo. In addition, AB-BCA, as an optical/MR dual imaging agent, can easily be used to label the intracellular regions of macrophages and BMDCs, supporting high performance MR and fluorescence imaging. Finally, in vivo tracking of AB-BCA-labeled DCs to regional LNs was accomplished by NIR fluorescence and T1-weighted MRI. These findings demonstrate that the newly synthesized AB-BCA can serve as a potentially reliable bimodal contrast agent for the noninvasive monitoring of innate immune cells and, consequently, for successful immunotherapy.


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ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website at DOI: 1

H NMR, 13C NMR, and MALDI-MS spectra of all intermediates and final product, microscopic

and flow cytometry analysis of cells, and ex vivo fluorescence image of lymph nodes.

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] (K.S.Hong) and [email protected] (J.S.Kim). Author Contributions ¶

E.J.K and S.B equally contributed to this study.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This study was funded by a grant from Korea Basic Science Institute (D35401, KSH), the Creative Allied Project (CAP) grant (PBC066, KSH) funded by the Korea Research Council of Science and Technology, and the CRI project (No. 2009-0081566, JSK) from the NRF of the Ministry of Science, ICT & Future Planning (MSIP) of Korea.


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Table of Contents Graphic


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In Vivo Tracking of Phagocytic Immune Cells Using a Dual Imaging

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