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1 Supporting Information Combined Photodynamic and Photothermal Therapy Using Cross-Linked Polyphosphazene Nanospheres Decorated with Gold Nanoparticles Xuan Wei,, Hongzhong Chen, Huijun Phoebe Tham, Nan Zhang,, Pengyao Xing, Guangcheng Zhang,, * Yanli Zhao*, Key Laboratory of Applied Physics and Chemistry in Space (Ministry of Education), Department of Applied Chemistry, School of Natural and Applied Science, Northwestern Polytechnical University, Xi an, China Division of Chemistry and Biological Chemistry, School of Physical and Mathematical Sciences, Nanyang Technological University, Singapore * zhangguc@nwpu.edu.cn * zhaoyanli@ntu.edu.sg S-1
2 Materials. HCCP was purchased from Sigma-Aldrich, and recrystallized from petroleum ether followed by sublimation (60 C and 0.05 mmhg). HAuCl 4, mpeg-cooh, bisphenol S (BPS), branched PEI, N,N -carbonyldiimidazole (CDI), 1-ethyl-3[3-dimethylaminopropyl] carbodiimide hydrochloride, N-hydroxysuccinimide tetra(4-hydroxyphenyl) porphyrin (TPP), and triethylamine (TEA) were purchased from Sigma-Aldrich and used directly without further purification. Reagents for cell culture, including Dulbecco s modified eagle medium, fetal bovine serum, and penicillin/streptomycin, were purchased from Life Technologies Holding Pte Ltd. Characterization. Fourier-transform infrared (FTIR) spectra were carried on a Shimadzu Prestige-21 spectrometer. Powder XRD patterns were collected on a powder diffractometer (XRD-7000/PC, Shimadzu, Japan) using Cu-K irradiation (40 kv, 30 ma). Diffraction patterns were collected from 10 to 90 at a speed of 3 /min. The morphologies of the samples were observed using a JEOL 1400 transmission electron microscopy (TEM). Scanning electron microscope (SEM) images and energy-dispersive spectroscopy (EDS) were recorded on FESEM Dynamic light scattering (DLS) and Zeta potential values were measured by a Mavern Nanosizer. UV-vis spectra were recorded on a Shimadzu UV-3600 UV-vis-NIR spectrophotometer. Fluorescent spectra were obtained on a Shimadzu RF-5301 spectrofluorophotometer. Cell viability assay was recorded on a Tecan Infinte M200. Fluorescence images were observed by a Nikon Eclipse TE2000-E confocal fluorescence microscope. Figure S1. Zeta potential changes in the formation process of CP-TPP/Au/PEG nanospheres. S-2
3 Figure S2. Powder XRD patterns of CP-TPP and CP-TPP/Au/PEG nanospheres. Figure S3. TEM image of CP-TPP nanospheres fully covered by gold nanoparticles. S-3
4 Figure S4. SEM images (left column), elemental mapping (middle two columns), and atomic ratios of elements (right table) for CP-TPP, CP-TPP/AU seeds, and CP-TPP/Au nanospheres. Figure S5. Photothermal properties of CP-TPP/Au/PEG before and after four weeks storage under 808 nm laser (1.5W) irradiation. S-4
5 Figure S6. Confocal microscopy images of carboxy-h 2 DCFDA stained HeLa cells incubated with CP-TPP nanospheres at a concentration of 50 µg/ml (a-c) in the dark or (d-f) under 630 nm LED irradiation (50 mw/cm 2 ) for 15 min. Images from left to right present cell nuclei stained by DAPI (blue), cells treated with carboxy-h 2 DCFDA as a fluorogenic marker for ROS generation (green), and merged images. Scale bar = 50 µm. We characterized the escape ability of the nanospheres from lysosomes to cytosol through the proton sponge effect facilitated by branched PEI. Lysosomes were labeled using green lyso tracker, and the colocalization of the nanospheres (red) with lysosome produced yellow fluorescence in the merged images. The endosome escape efficiency of CP-TPP and CP-TPP/Au/PEG nanospheres was quantified by calculating the colocalization percentage using ImageJ software. The results indicate that both CP-TPP and CP-TPP/Au/PEG have high efficiency to escape from the lysosome to the cytosol. However, the results did not show significant difference between these two kinds of nanospheres, meaning that the branched PEI did not influence the endolysosomal escape ability of the nanospheres. The in vitro PDT studies of these two kinds of the nanospheres showed the same results. On the other hand, the nanospheres with an average diameter of around 200 nm are relatively large and not easy to be trapped in the endosomes. Thus, the colocalization percentage of these two kinds of S-5
6 nanospheres with lysosomes was relatively low and the PDT efficacy would not be influenced. Figure S7. Viability of HUVECs incubated with CP-TPP, CP-TPP/Au, and CP-TPP/Au/PEG nanospheres at different concentrations for 48 h. S-6
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