Two-photon-excited near-infrared emissive carbon dots as multifunctional agents for fluorescence imaging and photothermal therapy

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1 Electronic Supplementary Material Two-photon-excited near-infrared emissive carbon dots as multifunctional agents for fluorescence imaging and photothermal therapy Minhuan Lan 1,, Shaojing Zhao 1,, Zhenyu Zhang 1, Li Yan 1, Liang Guo 2, Guangle Niu 2, Jinfeng Zhang 1, Junfang Zhao 2, Hongyan Zhang 2, Pengfei Wang 2,3 ( ), Guangyu Zhu 4, Chun-Sing Lee 1, and Wenjun Zhang 1 ( ) 1 Center of Super-Diamond and Advanced Films (COSDAF), Department of Physics and Materials Science, City University of Hong Kong, Hong Kong, China 2 Key Laboratory of Photochemical Conversion and Optoelectronic Materials, Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing , China 3 School of Future Technology, University of Chinese Academy of Sciences, Beijing , China 4 Department of Biology and Chemistry, City University of Hong Kong, Hong Kong, China Minhuan Lan and Shaojing Zhao contributed equally to this work. Supporting information to DOI /s Table S1 Liver function (ALT, AST and ALP) and kidney function (BUN, CRE and UA) of mice after intravenously injected with CDs aqueous solution or PBS Target Normal value CDs PBS ALT ± ± 9.8 AST ± ± 36.5 ALP ± ± 53.8 BUN ± ± 2.0 CRE ± ± 23.3 UA ± ± 13.8 Table S2 Blood analysis of mice after intravenously injected with CDs aqueous solution or PBS. The analyzed targets including red blood cell count (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), red cell distribution width-cell volume (RDW-CV), platelet count (PLT), mean platelet volume (MPV), white blood cell count (WBC), percentage of lymphocyte (LY%), percentage of intermediate cells (Mid%), percentage of granulocyte (GR%) and plateletcrit (PCT) Target Normal value CDs PBS RBC ± ± 0.5 HGB ± ± 0.3 HCT ± ± 0.02 Address correspondence to Wenjun Zhang, apwjzh@cityu.edu.hk; Pengfei Wang, wangpf@mail.ipc.ac.cn

2 (Continued) Target Normal value CDs PBS MCH ± ± 1.6 MCHC ± ± 41.9 RDW-CV ± ± 0.3 PLT 270 1, ± ± MPV ± ± 0.1 WBC ± ± 2.7 LY% ± ± 6.8 Mid% ± ± 0.4 GR% ± ± 7.2 PCT ± ± 0.08 Figure S1 (a) The particle size distribution histograms (n = 40) of CDs. (b) The dynamic light scattering (DLS) analysis of the CDs aqueous solution. DLS analysis suggests an average hydrodynamic diameter of about 23 nm for the CDs in water. Because the hydrodynamic diameter is usually slightly larger than the dry-state diameter, the result of DLS analysis agrees well with the TEM observations. (c) Zeta potential measurement of CDs aqueous solution. Figure S2 Deconvolution of high-resolution (a) N1s, (b) S2p and (c) Se3d XPS spectra of CDs. Figure S3 FT-IR spectrum of the CDs.

3 Figure S4 The time-resolved fluorescence decay of CDs aqueous solution recorded at 731 nm with the excitation 488 nm laser. The luminescence decay process can be fitted by an exponential function with two decay constants of 0.4 ns and 3.9 ns. Figure S5 (a) The FL spectra of CDs in DI water, phosphate-buffered saline (PBS) and fatal bovine serum (FBS) solution. The fluorescence spectrum of CDs in PBS solution is almost the same as that in DI water, and a stronger fluorescence could be observed in FBS solution due to its higher viscosity as compared with PBS and DI water. Effect of (b) ph value, (c) amino acids (20 μm, Mix presents the mixture of all amino acids), and (d) metal ions (20 μm) on the FL intensity ratio of CDs aqueous solution at 731 nm. I and I 0 presents the FL intensity of CDs in the presence and absence of the amino acids or metal ions, respectively. Note that the CDs aqueous solution has its fluorescence intensity at 731 nm almost independent of the ph values in the range of 4 13; and the interference of these amino acids and metal ions only leads to trivial changes in the emission of CDs. (e) Photostability and (f) photobleaching characteristics of CDs in comparison with fluorescein sodium. All samples were continuously irradiated using a 500 W Xenon lamp. The absorbance and FL intensity was normalized. Note that the absorbance of CDs at 526 nm do not present obvious decrease, while the absorbance of fluorescein sodium at 490 nm deceases to ~5% of its original vale. Moreover, under the same irradiation condition, the CDs solution has its fluorescence intensity reduced only by ~40%, much better than the fluorescein sodium (reduced by ~95%). The above results verify the excellent photo and photochemical stabilities of CDs. Nano Research

4 Figure S6 Photodegradation of Na 2 -ADPA in the CDs and Rose bengal aqueous solution, and pure water. A 0 and A t are the absorbance of the sample at 378 nm before and after irradiation, respectively. Figure S7 Temperature variation of (a) CDs solution and (b) water upon the on and off of the laser irradiation (635 nm, 2 W/cm 2 ). (c) and (d) Plot of cooling period (counting after switching off laser) versus negative natural logarithm of driving force temperature (θ). The time constant (τ s ) for heat transfer from the (c) CDs aqueous solution and (d) water to ambient is determined to be s and s, respectively. (e) The absorption spectrum of the CDs aqueous solution used in this experiment. The absorbance at 635 nm is

5 Figure S8 Flow cytometry analysis of apoptosis and necrosis of HeLa cells. (a) Cells incubated with CDs and exposed to laser irradiation, (b) cells incubated with CDs alone, (c) cells exposed to laser irradiation alone, and (d) control sample without CDs and laser irradiation. Annexin V-FITC (fluorescein isothiocyanate) was used as a probe to monitor the apoptotic signals, and PI (propidium iodide) was employed to stain cells in the early stage of necrosis. The quadrants from lower left to upper left (counter clockwise) represent healthy, early apoptotic, late apoptotic, and necrotic cells, respectively. The percentage of cells in each quadrant is shown on the graphs. Figure S9 The viability of (a) A549 and (b) KB cells incubated with CDs of different concentrations for 24 h in the dark or after irradiation by 635 nm laser (2 W/cm 2 ) for 10 min. Figure S10 Confocal bright-field images of HeLa cells in the absence of CDs (a) before and (b) after irradiation by 880 nm laser for 3 min. Nano Research

6 Figure S11 H&E staining images of major organs (heart, liver, spleen, lung, and kidney). Scale bar: 100 μm.