Supporting Information

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1 Supporting Information Dual-Modal Probe based on Polythiophene Derivative for Pre- and Intraoperative Mapping of Lymph Nodes by SPECT/Optical Imaging Bing Jia,,, Xin Zhang,, Bing Wang, #, Muhua Chen, Fengting Lv, # Shu Wang, *,# and Fan Wang *,, Medical Isotopes Research Center and Department of Radiation Medicine, School of Basic Medical Sciences, Peking University, Beijing , P. R. China Medical and Healthy Analytical Center, Peking University Health Science Center, Beijing , P. R. China # Beijing National Laboratory for Molecular Sciences, Key Laboratory of Organic Solids Institute of Chemistry, Chinese Academy of Sciences, Beijing , P. R. China Key Laboratory of Protein and Peptide Pharmaceuticals, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing , P. R. China * wangfan@bjmu.edu.cn * wangshu@iccas.ac.cn General materials and methods S-1

2 Chemicals, such as succinic acid, trisodium triphenylphosphine-3, 3, 3 -trisulfonate (TPPTS) and tricine were purchased from Sigma Aldrich (St. Louis, MO, USA). Indocyanine green (ICG) was purchased from J&K Scientific Company (Beijing, China) and stored at 4 C. Na 99m TcO 4 was obtained from a commercial 99 Mo/ 99m Tc generator (Beijing Atom High Tech Co., Ltd.). 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) and N-Hydroxysulfosuccinimide sodium salt (NHS-SO - 3 ) was dissolved with distilled water before use. All organic solvents were purchased from Beijing Chemical Works and used as received. Water with the resistivity of 18.2 mω cm from Milli-Qreference Water Purification System (Millipore) was used for reactions and solution preparation. The high performance liquid chromatography (HPLC) method used a LabAlliance HPLC system equipped with a UV/Vis detector (λ= 460 nm) and Superose 12 HR 10/30 column (Code No ; Amersham Pharmacia Biotech AB, Uppsala, Sweden). The flow rate was 1.0 ml/min. The mobile phase was 0.01 M of phosphate buffered saline. The radio-hplc method used the LabAlliance HPLC system (State College, PA) equipped with a β-ram IN/US detector (IN/US System, Tampa, FL) and Superose 12 HR 10/30 column. The flow rate and mobile phase were the same as that described above. The hydrodynamic diameter of the synthesized HY-PTP was analyzed by using dynamic light scattering (DLS, Zetasizer Nano, Malvern Instruments, UK). UV-vis spectra were obtained using a Varian Cary 300 UV-visible spectrometer and fluorescence spectra were obtained using Cary Eclipse spectrometer. SPECT/CT images were performed using the NanoSPECT/CT camera (Mediso Lnc., Budapest, Hungary). The SPECT and CT fusion images were obtained using the automatic fusion feature of the InVivoScope program (Bioscan Inc.). The 1 H NMR spectra were recorded on a Bruker Avance 300 MHz spectrometer. Water soluble polythiophene (PTP) was synthesized as literature. 1 The sizes of particles were measured on a Nano ZS (ZEN3600) system. Animal Preparation Female BALB/c nude mice (4 weeks of age) and female Kunming (KM) mice (4 weeks of age) were purchased from the Department of Experimental Animal, Peking University Health Science Center. All animal experiments were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee at Peking University. References (1) Xing, C.; Xu, Q.; Tang, H.; Liu, L.; Wang, S. Conjugated Polymer/Porphyrin Complexes for Efficient Energy Transfer and Improving Light-Activated Antibacterial Activity. J. Am. Chem. Soc. 2009, 131, S-2

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4 Figure S1. (A) 1 H-NMR spectrum of PTP-NHBOC, 1 H NMR (300 MHz, D 2 O, δ): (br), (br), (br), (br). (B) 1 H-NMR spectrum of PTP-NH 2, 1 H NMR (300 MHz, D 2 O, δ): (br), (br), (br). (C) 1 H-NMR spectrum of HY-PTP, 1 H NMR (300 MHz, DMSO,δ): 8.86(s, 1H), 8.54(s, 1H),8.02(d, 2H),7.74(d, 2H), (m), 7.15 (d, 1H), (br), 2.73 (s), (br). The chemical shifts observed at ppm can be attributed to the protons of the conjugated HYNIC-NHS. S-4

5 Figure S2. In vivo (A and B) and in vitro (C and D) fluorescence image (pseudocolor) of 99m Tc-PTP at different concentrations. S-5

6 Figure S3. Radio-HPLC analysis of 99m Tc-PTP after purification (A). The solution stability of 99m Tc-PTP in saline and in the presence of excess cysteine (B and C). Radio-HPLC chromatogram of urine samples collected at 4 h p.i.(d) S-6

7 Figure S4. SPECT/CT imaging of lymph nodes in a nude mouse from 0.5 to 24 h post intradermal injection. The standard (Std.) accounted for 1% of the injected dose. Figure S5. Complete blood count of toxicity test on nude mice at imaging dose. A) WBC (white blood cell),b) RBC (red blood cell), C) HGB (hemoglobin), and D) PLT (platelet). S-7

8 Figure S6. Complete blood count of acute toxicity test on Kunming mice at a high chemometry. A) WBC (white blood cell), B) RBC (red blood cell), C) HGB (hemoglobin), and D) PLT (platelet). Data are expressed as the mean ± SD (n=7), and analyzed by Two-tailed t test. The level of significance was set at p=0.05. Figure S7. Body weight evolution of nude mice (A) and Kunming mice (B) in biosafety studies (n=7). S-8