Supporting Information. sensitive bioimaging in vivo

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1 Supporting Information Sub-10 nm hexagonal lanthanide-doped NaLuF 4 upconversion nanocyrstals for sensitive bioimaging in vivo Qian Liu, Yun Sun, Tianshe Yang, Wei Feng, Chenguang Li, Fuyou Li * Department of Chemistry & Institute of Biomedical Science, Fudan University, Shanghai, , P. R. China fyli@fudan.edu.cn Materials. All starting materials were obtained from commercial supplies and used as received. Rare earth oxides Lu 2 O 3 (99.999%), Y 2 O 3 (99.999%), Yb 2 O 3 (99.999%), Er 2 O 3 (99.999%), Tm 2 O 3 (99.999%), Gd 2 O 3 (99.999%), were purchased from Shanghai Yuelong New Materials Co. Ltd. Oleyl amine (OM) (>90%) was purchased from Alfa. Aesar Ltd. Citric acid and trifluoroacetic acid (99%) were supplied from Sinopharm Chemical Reagent Co., Ltd. (Shanghai). All other chemical reagents with analytical grade were used directly without further purification. Deionized water was used throughout. RE(CF 3 COO) 3 were prepared with the literature method. 1 Characterization. Powder X-ray diffraction (XRD) measurements were performed on a Bruker D4 diffractometer at a scanning rate of 1 /min in the 2θ range from 10 to 80 (Cu Kα radiation, λ = Å). The size and morphology of UCNPs were determined at 200 kv at a JEOL JEM-2010 low to high resolution transmission electron microscope (HR-TEM). These as prepared samples were dispersed in cyclohexane and dropped on the surface of a copper grid for TEM test. Dynamic light scattering (DLS) experiments were carried out on an ALV-5000 spectrometer-goniometer S1

2 equipped with an ALV/LSE-5004 light scattering electronic and multiple tau digital correlator and a JDS Uniphase He-Ne laser (632.8 nm) with an output power of 22 mw. The size distribution was measured at 25 C with a detection angle of 90. The upconversion luminescence emission spectra were recorded on Edinburgh LFS-920 instrument, but the excitation source using an external 0-1 W adjustable 980 nm semiconductor laser (Beijing Hi-Tech Optoelectronic Co., China) with an optic fiber accessory, instead of the Xeon source in the spectrophotometer. Upconversion luminescence lifetimes was measured with phosphorescence lifetime spectrometer (FSP920-C, Edinburgh) equipped with a tunable mid-band OPO pulse laser as excitation source ( nm, 10 Hz, pulse width 5 ns, Vibrant 355II, OPOTEK). Upconversion luminescence quantum yield was measured with fluorescence spectroscope (Edinburgh LFS920) equipped with a tunable CW 980 nm laser (0 10 W). All the photoluminescence studies were carried out at room temperature. The images of upconversion luminescence were obtained digitally on a Nikon multiple CCD Camera. Synthesis of Lu1. Thermal decomposition procedure is as follow: 2 to a three-necked flask of 20 ml OM at room temperature were added given amounts of Na(CF 3 COO) (2mmol), RE(CF 3 COO) 3 (78%mol Lu, 20%mol Yb, 2%mol Er, total amount: 1 mmol). The resulting mixture was heated to 110 C with constant stirring to remove water and oxygen. After 30 min, the solution was then heated to 330 C and the temperature was maintained for 2.5 hour under an Ar atmosphere. When the reaction was completed, an excess amount of ethanol was poured into the solution at room temperature. The resultant mixture was centrifugally separated, and the products were collected. The as-precipitated nanocrystals were washed several times with ethanol and dried in vacuum overnight. All of these as-prepared nanocrystals could be easily redispersed in various nonpolar organic solvents such as cyclohexane, toluene, and chloroform. S2

3 Synthesis of Lu2, Lu3, Lu4, Lu5, and Lu6. The synthetic procedures of the Lu2, Lu3, Lu4, Lu5, and Lu6 nanocrystals were similar as that used to synthesize Lu1, except some change in reaction condition. The details in reaction condition were listed in the Table S1. Synthesis of Lu6-Tm. The synthetic procedure was the same as that used to synthesize Lu6, except RE(CF 3 COO) 3 (54%mol Lu, 24%mol Gd, 20%mol Yb, 2%mol Er, total amount: 1 mmol) was replaced by RE(CF 3 COO) 3 (55%mol Lu, 24%mol Gd, 20%mol Yb, 1%mol Tm, total amount: 1 mmol). Synthesis of Y1 and Y1-Tm. To a three-necked flask of 20 ml OM at room temperature were added given amounts of Na(CF 3 COO) (2mmol), RE(CF 3 COO) 3 (Y1: 78%mol Y, 20%mol Yb, 2%mol Er, or Y1-Tm: 79%mol Y, 20%mol Yb, 1%mol Tm, total amount: 1 mmol). The resulting mixture was heated to 110 C with constant stirring to remove water and oxygen. After 30 min, the solution was then heated to 320 C at a rate of 20 K min -1 and the temperature was maintained for 1 hour under an Ar atmosphere. When the reaction was completed, an excess amount of ethanol was poured into the solution at room temperature. The resultant mixture was centrifugally separated, and the products were collected. The as-precipitated nanocrystals were washed several times with ethanol and dried in vacuum overnight. All of these as-prepared nanocrystals could be easily redispersed in various nonpolar organic solvents such as cyclohexane, toluene, and chloroform. Synthesis of cit-lu6-tm. The mixed solution of diethylene glycol (DEG) (15.0 ml) and sodium citrate (2 mmol) was heated to 110 ºC for 30 min under argon. Then, 10 mg Lu6-Tm dispersed in chloroform and toluene (v/v = 3:2) solution (5 ml) were injected into the above mixed solution and the system was heated to 160 ºC. After the chloroform and toluene had evaporated, the system retained the temperature for 3 h until the solution became clear. The resulting solution was cooled S3

4 down to room temperature and treated with 0.1 M HCl aqueous solution, and the products were subsequently deposited. The precipitates were collected by centrifugation (14,000 rpm, 15 min) and washed three times with ethanol and deionized water, then the final citrate capped nanocrystals (abbreviated as cit-lu6-tm) were dispersed in water. Synthesis of cit-y1-tm. The synthetic procedure was the same as that used to synthesize cit-lu6-tm, except that Lu6-Tm was replaced by Y1-Tm. Measurement of upconversion quantum yield. According to the method reported by van Veggel et al., 3 fluorescence spectroscopy (Edinburgh LFS920) was modified by using NIR PMT(HAMAMATSU, C , No. CA0142) as detector for detection the excitation light from continuous-wave 980 nm laser and upconversion luminescence (800 nm) from Tm 3+. An integrating sphere was also used for measure the efficiency data. Because the molar extinction coefficient of lanthanide ions is very low, 4 meaning that the absorbance of excitation light at CW 980 nm is very low compared with the total excitation light of CW 980 nm, a attenuation slice (from Giai Photonics Co., Ltd.) for 980 nm CW laser is necessary. Using the attenuation slice, excitation intensity at 980 nm decreased to 1/68396 of the original value. In addition, pure NaYF 4 nanocrystals without doped lanthanide ions were used as reference for measurement of 980 nm absorption. The power density of excitation light is 17.5 W cm 2. Then according to the equation (1), the quantum yield of upconversion luminescence emission of the nanocrystals was calculated. QY = Photons emitted = L[sample ] photons absobed E[reference ] E[sample ] (1) where QY is the quantum yield, L[sample] is the emission intensity, E[reference] and E[sample] are the intensities of the excitation light in the presence of the pure NaYF 4 nanocrystals (reference) and the upconversion nanocrystals sample, respectively, S4

5 Cell Culture. A human nasopharyngeal epidermal carcinoma cell line (KB cell) was provided by the Institute of Biochemistry and Cell Biology, SIBS, CAS (China). Cells were grown in RPMI 1640 (Roswell Park Memorial Institute s medium) supplemented with 10% FBS (fetal bovine serum) at 37 C and 5% CO 2. Cells ( /L) were plated on 14 mm glass cover-slips under 100% humidity condition and allowed to adhere for 24 h. Cytotoxicity of cit-lu6-tm. In vitro cytotoxicity was measured by performing methyl thiazolyl tetrazolium (MTT) assays on the KB cells. Cells were seeded into a 96-well cell culture plate at /well, under 100 % humidity, and were cultured at 37 C and 5% CO 2 for 24 h; different concentrations of cit-lu6-tm (0, 200, 400, and 800 μg ml -1, diluted in RPMI 1640) were then added to the wells. The cells were subsequently incubated for 5 h and 24 h at 37 C under 5% CO 2. Thereafter, MTT (10 μl; 5 μg ml -1 ) was added to each well and the plate was incubated for an additional 4 h at 37 C under 5% CO 2. After the addition of 100 μl DMSO, the assay plate was allowed to stand at room temperature for 2 h. The OD570 value (Abs.) of each well, with background subtraction at 690 nm, was measured by means of a Tecan Infinite M200 monochromator-based multifunction microplate reader. The following formula was used to calculate the inhibition of cell growth: Cell viability (%) = (mean of Abs. value of treatment group/mean of Abs. value of control) 100%. Cellular Staining. To ensure complete dispersion of the cit-lu6-tm in PBS, their solutions (200 μg/ml) obtained from an ultrasonic to get homogeneous colloidal solution. For single-label imaging, KB cells were stained with 200μg ml -1 cit-lu6-tm in a 5% CO 2 incubator at 37 C for 2 h, cell imaging was then carried out after washing the cells with PBS three times to remove the excess cit-lu6-tm. S5

6 Confocal UCL Imaging of Living Cells Incubated with cit-lu6-tm. Confocal imaging of cells was performed with a modified Olympus FV1000 laser scanning upconversion luminescence microscope (LSUCLM) 5 equipped with a continuous-wave (CW) laser at 980 nm (Connet Fiber Optics, China). A 60 oil-immersion objective lens was used. For the cit-lu6-tm, the CW laser at 980 nm provided the excitation, and UCL emission was collected at 470 ± 20 nm. Animal: All animal procedures were performed according to institutional and national guidelines. Male athymic mice (athymic nu/nu - ) weighing g and black mouse were purchased from Shanghai SLAC Laboratory Animal CO. LTD (Shanghai, China). Animals were maintained in our animal care facility and housed eight per cage at room temperature (22±2 ºC ) with food and water ad libidum. In vivo imaging of cit-lu6-tm and cit-y1-tm. 200 µl cit-lu6-tm or cit-y1-tm with the concentration of 1 mg ml -1 were subcutaneously injected into black mouse, then the injected mouse was imaged by UCL in vivo imaging system designed by our group 6. In vivo imaging of cit-lu6-tm-labeled cells. 50 KB cells, which were labeled with cit-lu6-tm, were subcutaneously injected into athymic nude mice, then cells-injected athymic nude mice was imaged by UCL in vivo imaging system designed by our group KB cells, which were labeled with cit-lu6-tm, were intravenously injected into athymic nude mice, then cells-injected athymic nude mice was imaged by UCL in vivo imaging system designed by our group 6. S6

7 Table S1. The crystal structure and diameter of the Gd 3+ -doped NaLuF 4 nanocrystals under different conditions, from the co-thermolysis of Na(CF 3 COO) and RE(CF 3 COO) 3 in oleylamine. Table S2. The analysis of signal to noise ratio (SNR) of Figure 4a. SNR = [(mean luminescence intensity of 1) - (mean luminescence intensity of 3)] / [(mean luminescence intensity of the 2) - (mean luminescence intensity of 3)]. S7

8 Figure S1. Crystal structures of α-naref 4 and β-naref 4 built by CERIUS2 software (Ref.: cited from J. Am. Chem. Soc. 2006, 128, Figure S2. TEM of samples Lu3 (a, b) and Lu4 (c, d). Figure S3. TEM of the Lu5 nanocrystals. S8

9 Number distribution (a.u.) F NaLuF 4 :Gd,Yb,Er(24:18:2 mol %) Gd Cu Gd Lu Lu Lu Gd Gd Gd Lu Lu Gd LuLu Lu kev Figure S4. Energy-dispersive X-ray analysis (EDXA) patterns of nanocrystals Lu6. c b a Diameter (nm) Figure S5. DLS of samples Lu5 (a), Lu6 (b) and cit-lu6-tm (c). S9

10 Figure S6. Proposed energy transfer mechanisms under CW 980 nm excitation in NaLuF 4 :Yb, Er/Tm codoped with Gd 3+. S10

11 UCL Intensity(a.u.) Lu1 Lu2 Lu3 Lu4 Lu5 Lu6 Y Wavelength(nm) Figure S7. Upconverision luminescent spectra of the Lu1 Lu6 and Y1 nanocrystals, which were dispersed in cyclohexane with the concentration of 3.1 mm, under excited by CW 980 nm laser. Figure S8. TEM (a) and XRD (b) of the Y1 nanocrystals. S11

12 Figure S9. The bright-field imaging (a-b) and upconversion luminescence imaging (c) of Lu6 and Y1 powders measured on upconversion luminescence imaging in vivo system designed by our group 6. (d) Diagram depicts of experimental setup for the upconversion luminescence imaging system. The excitation laser beam pathway and the emission pathway are shown in pink and green, respectively. S12

13 Figure S10. UCL emission decays of the 4 H 11/2 (a) and 4 F 9/2 (b) levels of Er 3+ ions in the Y1 and Lu6 nanocrystals, and the 1 G 4 (c) and 3 H 4 (d) of Tm 3+ in Y1-Tm and Lu6-Tm, under the excitation of 976 nm pulsed laser. S13

14 Cell Viability (%) Figure S11. TEM (a) and upconversion luminescence spectrum (b) under CW excitation at 980 nm of cit-lu6-tm h 24 h Concentration ( g/ml) 800 Figure S12. In vitro cell viability of KB cells incubated with cit-lu6-tm at different concentration for 5 and 24 hours. S14

15 Figure S13. UCL intensity along the line shown in UCL image (insert) of KB cell incubated with 200 μg ml -1 cit-lu6-tm at 37 C for 2 h. λ ex = 980 nm, λ em = nm. S15

16 Figure S14. Z-scan confocal imaging and three-dimensional luminescence images of live KB cells stained with 200 µg ml -1 cit-lu6-tm nanocrystals for 2 h at 37 ºC. λ ex = 980 nm, λ em = nm. S16

17 References: 1. Roberts, J. E. J. Am. Chem. Soc. 1961, 83, (a) Mai, H. X.; Zhang, Y. W.; Si, R.; Yan, Z. G.; Sun, L. D.; You, L. P.; Yan, C. H.; J. Am. Chem. Soc. 2006, 128, (b) Boyer, J. C.; Vetrone, F.; Cuccia, L. A.; Capobianco, J. A. J. Am. Chem. Soc. 2006, 128, Boyer, J. C.; van Veggel, F. C. J. M. Nanoscale 2010, 2, Stouwdam, J. W.; Hebbink, G. A.; Huskens, J.; van Veggel, F. C. J. M. Chem. Mater. 2003, 15, Yu, M. X.; Li, F. Y.; Chen, Z. G.; Hu, H.; Zhan, C.; Huang, C. H. Anal. Chem. 2009, 81, Xiong, L. Q.; Chen, Z. G.; Tian, Q. W.; Cao, T. Y.; Xu, C. J.; Li, F. Y. Anal. Chem. 2009, 81, S17