Janus Iron Semiconducting Polymer Nanoparticle Tracer for

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1 Supporting Information Janus Iron Semiconducting Polymer Nanoparticle Tracer for Cell Tracking by Magnetic Particle Imaging Guosheng Song, Min Chen, Yanrong Zhang, Liyang Cui, Haibo Qu, Xianchuang Zheng, Max Wintermark, Zhuang Liu, Jianghong Rao* Experiments 1. Chemicals: All chemicals were obtained from Sigma-Aldrich unless otherwise stated. 2. Materials characterization: TEM images were obtained on a JEM 1230 transmission electron microscope with an accelerating voltage of 200 kv. Dynamic light scattering (DLS) and Zeta potential measurements were conducted on the Malvern ZetaSizer Nano S. UV-vis spectra were recorded on an Agilent spectrophotometer. Fluorescence measurements were carried out on a wavelength-calibrated FluoroMax-3 fluorometer (Horiba Jobin Yvon). The iron concentrations of samples were measured by inductively coupled plasma mass spectrometer (ICP-MS, Thermo). Powder X-ray diffraction (XRD) measurement was conducted with Shimadzu XRD-6000 X-ray diffractometer. 3. Synthesis of iron-oleate complex 1, 2 : 72 mmol of oleic acid was dissolved in a mixture solution composed of 80 ml of methanol, 40 ml of ethanol, 10 ml of H 2 O and 72 mmol of NaOH. The resulting solution was heated to 60 C and kept at that temperature for one hour. Then 40 ml of FeCl 3 (0.6 mol/l) was dropwisely added into the mixture solution, followed by addition of 80 ml of cyclohexane. The resulting solution was heated to 70 C and kept for four hours. When the reaction was completed, the upper organic layer containing the iron- 1

2 oleate complex was washed three times with 30 ml of H 2 O in a separatory funnel. After washing, hexane was evaporated off, resulting in iron oleate complex in a waxy solid form. 4. Synthesis of Fe 3 O 4 nanoparticles: superparamagnetic iron oxide nanocrystals were synthesized following the procedure proposed by Hyeon et al with some modification. 1 In a typical synthesis, iron-oleate (2.7 g, 3 mmol) and oleic acid (1 mmol) was dissolved in 1- octadece (30 ml). The mixture was firstly heated to 120 C for 10 min under a stream of nitrogen to get rid of air and water in the system and then heated to 200 C for 30 min. Subsequently, the reaction mixture was heated to 310 C and kept for different time such as 31 h. The resulting solution was then cooled to room temperature, added with ethanol, and centrifuged to separate the nanocrystals. 5. Preparation of Fe 3 O Janus nanoparticles: 0.25 mg of poly[2,7- (9,9-dioctylfluorene)-alt-4,7-bis(thiophen-2-yl)benzo-2,1,3-thiadiazole] (PFODBT, Mw=10,000-50,000), 1 mg of Fe 3 O 4, 2 mg of poly(styrene-co-maleic anhydride) (PSMA, Mn=1,700) was dissolved in 2 ml of THF under bath sonication. Then, the mixture solution was rapidly injected into 18 ml of H 2 O under bath sonication, followed by addition of 1 ml of Na 2 CO 3 (10 mg/ml). After further sonication 10 min, THF was evaporated by rotary evaporation. The aqueous solution was washed three times using 100 K centrifugal filter units, and stored in dark at 4 C for later use. 6. Preparation of Fe 3 O 4 -COOH: 2 ml of THF containing 1 mg of Fe 3 O 4, 2 mg of PSMA was rapidly injected into 18 ml of H 2 O under bath sonication, followed by addition of 1 ml of Na 2 CO 3 (10 mg/ml). After further sonication 10 min, THF was evaporated by rotary evaporation. The Fe 3 O 4 -COOH solution was washed three times using a 100 K centrifugal filter, and stored at 4 C for later use. 2

3 7. Culture of cell lines: The human cervical cancer HeLa cells were purchased from the American Type Culture Collection (ATCC). HeLa cells were cultured in Dulbecco s modified eagle medium (DMEM) (GIBCO) cultured with 10% fetal bovine serum (FBS) (GIBCO), at 37 C under 5 % CO Cellular labeling: Cells were seeded into 6-well culture plates and allowed to adhere for 24 h in a humidified atmosphere of 5% CO 2 and 95 % air at 37 C. The cells were treated with different concentration of Fe 3 O at 37 C in serum-free Dulbecco s modified eagle medium (DMEM) for 4 h, and then were washed three times with PBS, to remove unbound particles. For confocal imaging, those cells were fixed with 4 % paraformaldehyde for 15 min, stained by DAPI for 10 min, and finally washed three times with 1 PBS. Confocal imaging was conducted on a confocal scope (Zeiss 880). Excitation at 405 nm and emission at 450 nm were used for DAPI; and excitation at 540 nm and emission above 680 nm were used for Fe 3 O For cellular uptake measurement, the iron contents in cells were measured by inductively coupled plasma mass spectrometer (ICP-MS, Thermo). For cytotoxicity test, Fe 3 O of various concentrations were incubated with Hela in 96-well culture plates in serum-free culture medium, followed by repetitive washing with PBS to remove unbound Fe 3 O Labeled cells were further cultured in fresh cell medium for desired periods of time. The cell proliferation was studied by measuring the cell viabilities using the standard MTS assay. 9. Cell tracking in vivo: All animal experiments were performed in compliance with the Guidelines for the Care and Use of Research Animals established by the Stanford University 3

4 Animal Studies Committee. Female mice were subcutaneously injected with different number of cells labelled cells onto abdomen or back. For longitudinal tracking tumor, mice were implanted subcutaneously with Hela cells ( ) pre-labelled with Fe 3 O COOH (31 pg Fe /cell). On day 20, the tumors were dissected, frozen, sliced, fixed and stained with DAPI. Fluorescent imaging was performed on IVIS Spectrum (PerkinElmer) using 1 second scan time, and excitation/emission filters at 540 and 680 nm, respectively. CT imaging was performed on a Micro CT (TriFoil Imaging CT120) with fast scan mode. MR imaging was performed on a MRI scanner (Siemens, 3T) using T 2 sequence. MPI imaging was performed using a MPI scanner (Magnetic Insight Inc, MOMENTUM Imager). The frequency of MPI is 45 khz; The magnetic gradient strenght of MPI is 6T/m. The original dates were processed by VivoQuant software. Supporting Figure 4

5 Figure S1. (a) Picture of the commercial MPI scanner (Magnetic Insight Inc) used for this study. (b) 200 µl of PCR tube was placed on the bed for MPI imaging. Figure S2. Linear scanning MPI spectrum of 200 µl of Feraheme (8 µg of Fe) in a PCR tube. 5

6 Figure S3. Plot of MPI signals of Fe 3 O 4 -COOH in 200 µl H 2 O with a large range of Fe from 62.5 ng to 64 µg. Figure S4. The resolution of MPI phantom images of Fe 3 O nanoparticles. Two microbore tubes were filled with 0.5 µl of Fe 3 O (1.9 mg Fe/mL), and were arranged in a linear array with an edge-to-edge distance of 2.5 mm between these two tubes. (a) Photograph. (b) Two-dimensional projection MPI scanning. (c) Corresponding linear scanning MPI spectra. 6

7 Figure S5. Background subtraction to improve the detection limit of labeled cells. Firstly, two-dimensional projection MPI images of 500 or 250 labeled cells (31 pg Fe /cell) in 20 µl PBS in PCR tube were scaned; Secondly, two-dimensional projection MPI images of PCR tube with no cells was scanned as background; Thirdly, subtraction of the image of No cells in tube from the image of 500 or 250 cells in tube afforded the corrected MPI images of 500 or 250 cells. 7

8 Figure S6. Plot of fluorescent signals as a function of the number of labeled cells ranging from 0 K to 20 K. Figure S7. Different views of three-dimensional MPI and CT imaging of a mouse received local subcutaneous injection of labeled cells. References (1) Park, J.; An, K.; Hwang, Y.; Park, J. G.; Noh, H. J.; Kim, J. Y.; Park, J. H.; Hwang, N. M.; Hyeon, T. Nat. Mater. 2004, 3, 891. (2) Chen, F.; Bu, W.; Chen, Y.; Fan, Y.; He, Q.; Zhu, M.; Liu, X.; Zhou, L.; Zhang, S.; Peng, W.; Shi, J. Chem. Asian. J. 2009, 4,