Supplementary Information

Macrophage Cell Tracking PET Imaging Using Mesoporous Silica Nanoparticles via

In Vivo Bioorthogonal F-18 Labeling

Hyeon Jin Jeong†1, Ran Ji Yoo†2, Jin Kwan Kim†1, Min Hwan Kim2, Su Hong Park1, Haebin Kim1, Jae

Wook Lim1, Sun Hee Do3, Kyo Chul Lee2, Yong Jin Lee*2, and Dong Wook Kim*1

1Department of Chemistry and Chemical Engineering, Inha University, 100 Inharo, Namgu, Incheon

22212, Korea, 2Department Molecular Imaging Research Center, Korea Institute of Radiological and

Medical Sciences, 75 Nowon-ro, Nowon-gu, Seoul 139-706, Korea, 3Department of Clinical Pathology,

College of Veterinary Medicine, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 05029,

Seoul, Korea

† These authors contributed equally to this work.

CORRESPONDING AUTHOR E-MAIL ADDRESS: yjlee@kirams.re.kr; kimdw@inha.ac.kr

General. Unless otherwise noted all reagents and solvents were commercially available. Reaction

progress was followed by TLC on 0.25 mm silica gel glass plates containing F-254 indicator.

Visualization on TLC was monitored by UV light or radio-TLC scanner. [18F]Fluoride ion was produced

from a cyclotron (IBA Cyclone 30, Belgium) using the 18O(p,n)18F nuclear reaction with 19 MeV proton

irradiation of an enriched [18O]H2O target. High performance liquid chromatography (HPLC) was

performed with spectra system (Thermo Scientific, Waltham, MA) using semipreparative column (C18

silica gel, 10 µm, 10 × 250 mm) and analytic column (C18 silica gel, 5 µm, 4.6 × 250 mm). The eluant

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was simultaneously monitored by a UV detector (215 nm & 254 nm) and a NaI(Tl) radioactivity detector.

Radioactivity was measured in a dose calibrator.

Characterization. The morphology of the MSNs was observed by high-resolution transmission electron

microscopy (HR-TEM, JEM-2100F, JEOL) operated at accelerating voltage of 200 kV. Fourier transform

infrared (FT-IR) spectra were recorded on a Bruker VERTEX 80V spectrometer in the range between

4000 and 400 cm-1. Zeta potentials were recorded on a zeta potential analyzer (ELS-8000, Photal Inc.).

UV-VIS Spectra were recorded on a HP8453 (Hewlett Packed) spectroscopy. Elemental analysis was

measured using a element analyzer (FLASH2000 series, Thermo Scientific co.).

Figure S1. FT-IR spectra. (a) H2N-MSNs, (b) FmocNH-PEG-MSNs, (c) NH2-PEG-MSNs, and (d)

DBCO-MSNs.

Table S1. Zeta potentials of H2N-MSNs, FmocNH-PEG-MSNs, NH2-PEG-MSNs, and DBCO-MSNs.

Sample H2N-MSNs FmocNH-PEG-MSNs NH2-PEG-MSNs DBCO-MSNs

Zeta Potential (mV) 19.91 ± 1.53 -19.25 ± 2.48 5.46 ± 1.92 -18.59 ± 0.69

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Figure S2. Ninhydrin test for detection of amine groups of (a) MSNs, (b) H2N-MSNs, (c) FmocNH-PEG-

MSNs, (d) NH2-PEG-MSNs, and (e) DBCO-MSNs

The incorporation of functional groups, such as NH2, Fmoc, and DBCO, would result in the change of

surface charges of MSNs after each reaction step. Therefore, zeta-potential assay was employed to further

verify the covalently coupling of various functional groups to MSNs. In contrast to the zeta potential (ζ-

potential) of 21.49 mV for NH2-MSNs, the values of zeta-potential for FmocNH-PEG-MSNs, NH2-PEG-

MSNs, and DBCO-MSNs in 1 mM PBS buffer (pH 7.2) were correspondingly changed from -21.01 mV,

21.38 mV to -18.08 mV, respectively. These results of zeta-potential assay and ninhydrin test (for

detection of amine group on MSN derivatives) suggests that various items/groups were successfully

coupled to MSNs.

As shown in Figure S3, a solution of 3-azidopropylmethanesulfate (200 mg) in 2 mL of EtOH:H2O (1:1)

was added to the suspension of DBCO-MSNs (100 mg) in 4 mL of EtOH/H2O (1:1). The reaction

solution was stirred at 25 oC for 6 h. The methanesulfate-functionalized DBCOT-MSNs were collected by

centrifugation (13200 rpm, 10 min). The nanoparticles were washed several times with EtOH/water (3:1),

and dried overnight under vacuum. After the SPAAC reaction, a sulfur elemental analysis of the

nanoparticle product showed that approximate 0.18 mmole of ADIBO moiety was tethered to per gram of

DBCO-MSN product. Anal.: S 0.58 ± 0.011 (n = 10, ≈0.18 mmol DBCO portion/g).

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Figure S3. SPAAC reaction of DBCO-MSNs with 3-azidopropylmethanesulfonate for measuring the

amount of DBCO group portion in the DBCO-MSNs by sulfur elemental analysis. Anal.: S 0.58 ± 0.011

(n = 10)

Representative procedure for preparation of [18F]fluoropentaethylene glycolic azide ([18F]2):

[18F]Fluoride was produced in a cyclotron (IBA Cyclone 30, Belgium) by the 18O(p,n)18F nuclear reaction

with 19 MeV proton irradiation of an enriched [18O]H2O target. A solution of [18F]fluoride (481 MBq) in

water (200 µL) was added to a vacutainer containing n-Bu4NHCO3 (8% aq, 100 µL, 30 µmol). The

azeotropic distillations were conducted with 0.5 mL aliquots of CH3CN at 90 °C under a stream of

nitrogen. A nucleophilic [18F]radiofluorination reaction of the corresponding mesylate precursor (5.0 mg,

14.6 µmol, prepared according to our previous work [35]) with n-Bu4N[18F]F in t-amyl alcohol (500µL)

was carried out in a reaction vial at 100 °C for 20 min. After cooling to room temperature, the solvent

was removed with a gentle stream of nitrogen. The crude radioactive mixture was injected onto reverse-

phase HPLC with acetonitrile (1 mL) and purified. The [18F]2 was collected from HPLC (tR = 11.1 min;

C18 silica gel, 10 µm, 4.6 × 250 mm; 0.1% TFA in H2O/acetonitrile = 20:80 (v/v); 214 nm; 2 mL/min).

122 MBq of [18F]2 was obtained after the evaporation under a stream of nitrogen in 52% decay-corrected

radiochemical yield with high radiochemical purity (> 99%). The total synthesis time was 55 min.

Preparation of DBCO-MSNs-RAW cells and quantitative analysis of Si. 3.1mg of DBCO-MSN was

incubated with 5 × 106 RAW264.7 cells for 2 h at 37 °C. DBCO-MSNs RAW cells were washed with

PBS. For quantitative analysis of silica (Si) ions, samples were prepared as 1 × 106, 1 × 105, and 1 × 104

cell number of DBCO-MSNs-RAW cells (n = 4 per group). Si from DBCO-MSNs-RAW cells were

quantitatively analyzed using the Inductively Coupled Plasma Mass Spectrometry (ICP-MS) system

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(7900, Agilent, Santa Clara, CA) from the Yonsei Center for Research Facilities (YCRF, Seoul, Korea).

ICP-MS measurements for 1 × 106, 1 × 105, and 1 × 104 DBCO-MSNs-RAW cells showed that the Si

content was 88.67 ± 4.26, 11.56 ± 0.30, and 1.97 ± 0.05 μg, respectively. Thus, the Si content in DBCO-

MSNs-RAW cells showed a positive correlation with an increase in cell number, and approximately 0.1

ng of Si contained per cell in 1 × 106 and 1 × 105 DBCO-MSNs-RAW cells.

Fluorescent intracellular uptake assay. For fluorescent intracellular uptake assay to assess the retention

of DBCO-MSNs in the cells, RAW264.7 cells treated with 30 μg of Cy5.5 labeled DBCOT-MSNs were

incubated in 2-well chamber slide at 5 × 104 per well for 24 h. Cy5.5 labeled DBCOT-MSNs-treated cells

were incubated for 1, 3, 6, and 8 days. After incubation, slides were incubated with 4’,6-diamidino-2

phenylindole dihydrochloride (DAPI, Sigma) for 3 min at room temperature. After rinsing, slides were

mounted with aqueous mounting medium (Dako, Carpinteria CA, USA) and observed using an Olympus

DP74 digital microscopic camera system (Olympus, Tokyo, Japan). Changes in intracellular fluorescence

values by day were quantitatively analyzed using ImageJ software. This fluorescent intracellular uptake

assay show that the MSNs were not observed to be excreted out of the cells up to 8 days (the intracellular

fluorescent value at 8 days was similar with the value at 1 day).

Figure S4. Intracellular fluorescent value in Cy5.5 labeled DBCOT-MSNs-treated RAW264.7 cells at 1,

3, 6, and 8 days.

Tumor xenograft model. In this study, the care, maintenance, and treatment of animals followed

protocols approved by the Institutional Animal Care and Use Committee of the Korean Institute of

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Radiological & Medical Sciences (KIRAMS). Male athymic nude (Nu/Nu) BALB/c mice were purchased

from Nara Biotech Co., Ltd. (Seoul, Korea). Mice 6 weeks of age were injected subcutaneously with 5 ×

106 U87MG cells suspended in 200 μL PBS in the flank of the left thigh. For biodistribution and PET

imaging, the mice were grown for 10 days to reach 0.7 − 0.9 cm3 in tumor diameter.

Figure S5. Control group PET imaging. Three-dimensional reconstruction (upper) and transverse

section (lower) combined PET-CT images of 18F-labeled azide ([18F]2; 11.1 MBq) in U87 MG tumor-

bearing mice treated with only normal RAW 264.7 cells (2 × 106 cells) 1, 3, 6, or 8 days earlier, recorded

1 h after injection of [18F]2. T = tumor.

DBCO-MSNs pretargeting PET imaging. PET/CT imaging was performed with a small animal

PET/CT scanner (NanoScan, Mediso Medical Imaging Systems, Budapest, Hungary). For PET imaging

study, the 100 μg of DBCO-MSNs was administrated to the U87MG tumor-bearing nude mice via the

retro-orbital venous sinus. After 1 and 6 day(s), about 11.1 MBq of [18F]2 in saline (100 L) was

intravenously injected into each mouse under isoflurane anesthesia (2%). For attenuation correction and

anatomical reference, micro-CT imaging was obtained during 2.5 minutes before PET imaging using 50

kVp of X-ray voltage and 0.16 mAs of anode current. Subsequently, PET images were acquired with an

energy window of 400-600 keV during 20 minutes at 1 h after the injection of [18F]2. All obtained images

were reconstructed using four iterations of the 3-dimensional ordered subset expectation maximization

(3D-OSEM) algorithm with 6 subsets. To quantify [18F]2 uptake, standardized uptake value (SUV), which

adjusted voxel value within a region of interest (ROI) was calculated using Inter View Fusion version

2.02.055.2010 (Mediso Ldt., Budapest, Hungary). The SUV (g/mL) of each voxel was calculated as

follow; activity of each voxel (kBq/mL) was multiplied by the mouse body weight (g) and divided by the

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decay-corrected-activity (kBq). The three-dimensional ROI showing high activity was manually drawn on

the tumor region using CT images as a guide. SUVmean value was obtained based on the ROI. Tumor to

muscle ratio in SUVmean at 1 day and 6 days after the pre-treatment of DBCO-MSNs were 1.65 and 1.09,

respectively (Fig. S6B).

The PET images and their tumor to muscle ratio data in SUVmean show that the pre-treated DBCO-MSNs

6 days earlier in tumor mice model could not work to visualize tumor area by bioorthogonal 18F-labeling

(Fig. S6), whereas the pre-treated them 1 day earlier provided good tumor to muscle ratio with tumor PET

images, which were similar results with our previous study [24]. Probably, these results suggested that 6

days may be too long circulation time for the DBCO-MSNs to be accumulate on tumor tissue by the EPR

effect owing to the possibility of excretion and degradation of them.

Figure S6. Free DBCO-MSNs pretargeting PET imaging. (A) Coronal section (upper) and transverse

section (lower) combined PET-CT images of 18F-labeled azide ([18F]2; 11.1 MBq) in U87MG tumor-

bearing mice treated with only DBCO-MSNs 1 or 6 day(s) earlier, recorded 1 h after injection of [18F]2.

White arrows refer to the tumor position. (B) Tumor to muscle ratio in SUVmean.

Biodistribution study. For necropsy data, the DBCO-MSNs-RAW cells (2 × 106 cells; 0.1 ng Si/cell) or

normal RAW264.7 cells (2 × 106 cells) were administrated to the U87MG tumor-bearing nude mice via

the retro-orbital venous sinus. After 3 days, about 11.1 MBq of [18F]2 in saline (100 µL) was

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intravenously injected into each mouse under 2% isoflurane anesthesia (n = 4). The mice were necropsied

at 1 h after injection of [18F]2. Tissues of interest (fat, blood, muscle, heart, lung, liver, spleen, stomach,

intestine, kidney, bone, and tumor) were weighed and counted on WALLAC 1480 WIZARD gamma

counter (Perkin Elmer, Waltham, MA). Uptake in each tissue was expressed as the percentage injected

dose per gram of tissue (% ID/g). All experiments were repeated many times, and the results were

presented as the mean ± standard deviation. A p-value of less than 0.005 was considered statistically

significant.

Immunofluorescence. U87MG tumor–bearing nude mice were sacrificed at 3 and 8 days after injection

of the DBCO-MSNs-RAW cells. Tumor tissues were dissected and fixed with 4% paraformaldehyde for

24-48 h. The tissue was embedded in Tissue-Tek® O.C.T. Compound (Sakura Finetek USA Inc.,

Torrance, CA), and 4µm sections were obtained for staining. Nonspecific background was blocked by 1

hour of incubation in 10% bovine serum albumin in tris-buffered saline (TBS) at room temperature.

Slides were incubated with CD68 (Abcam, Cambridge, UK) for 2 h at RT. After rinsing in TBS,

fluorescein isothiocyanate (FITC)-conjugated secondary antibody (Abcam) was added for 30 min at RT.

After rinsing, slides were incubated with 4’,6-diamidino-2-phenylindole (DAPI) dihydrochloride (Sigma,

St. Louis, MO) for 3 min at RT. After rinsing, slides were mounted with aqueous mounting medium

(Dako, Carpinteria, CA) and observed using an Olympus DP71 digital microscopic camera system

(Olympus, Tokyo, Japan).

Model of atherosclerosis in ApoE-/- mouse. ApoE-/- mice (B6.KOR-Apoeshl) and Balb/C mice (6 to 8

weeks old) were purchased from Central Lab Animal Inc. (Seoul, Korea). At 6 weeks of age, ApoE -/-

animals were placed on a Western diet (21% adjusted caloric diet, 1.5% cholesterol) (Research Diets, Inc.

New Brunswick, NJ) for experiments. Control animals remained on a regular chow diet. All procedures

involving animals were approved by the Institutional Animal Care and Use Committee (IACUC) at the

Korea Institute of Radiological and Medical Sciences (KIRAMS) (IACUC no. KIRAMS 2012-49).

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