Supporting Information

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1 Supporting Information Functional Near Infrared Emitting Cr 3+ /Pr 3+ Co doped Zinc Gallogermanate Persistent Luminescent Nanoparticles with Super Long Afterglow for in vivo Targeted Bioimaging Abdukader Abdukayum,, Jia Tong Chen, Qiang Zhao, and Xiu Ping Yan, State Key Laboratory of Medicinal Chemical Biology (Nankai University), Synergetic Innovation Center of Chemical Science and Engineering (Tianjin), and Research Center for Analytical Sciences, College of Chemistry, Nankai University, Tianjin, , China College of Life Sciences, Nankai University, Tianjin, , China S1

2 Materials and Reagents. All reagents were used as received without further purification. Zn(NO 3 ) 2 6H 2 O (99.99%), Ga 2 O 3 (99.99%), GeO 2 (99.999%), Cr(NO 3 ) 3 9H 2 O (99.99%), Pr(NO 3 ) 3 6H 2 O (99.9%), C 6 H 8 O 7 H 2 O (99.5%), APTES (99%), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC HCl) and N-hydroxysuccinimide (NHS) were purchased from Aladdin (Shanghai, China). SC-PEG-COOH (average MW=3400) was obtained from Laysan Bio Inc. (AL, USA). c(rgdyk) peptide (MW=619.7) was obtained from ChinaPeptides Co. Ltd. (Shanghai, China). Ultrapure water (Hangzhou Wahaha Group Co. Ltd., Hangzhou, China) was used throughout. Measurement of Absolute Quantum Yield. The absolute photoluminescence quantum yield (QY) of aqueous dispersion of the LPLNPs (excitation at 254 nm) at room temperature was determined on an FLS920 spectrometer (Edinburgh, UK) with an integration sphere attachment. A 10 mm path length quartz cuvette for the solution sample is set in the integrating sphere. The absolute photoluminescence QY is given by: 1 QY PN( em) PN( abs) E L reference sample E sample where PN (abs) is the number of photons absorbed by the sample and PN (em) is the number of photons emitted from the sample, L sample is the photoluminescence intensity of the sample (aqueous dispersion of the LPLNPs), E reference and E sample are the excitation light intensities of the reference and the sample, respectively. The pure solvent (ultrapure water) was employed as reference. S2

3 Cytotoxicity Assay. In vitro cytotoxicity of the LPLNPs was assessed using a cell counting assay. 2 Cell numbers were counted with a Coulter Particle Size Analyzer (Beckman Coulter, High Wycombe, UK). Mouse embryo fibroblast cell lines (Balb/3T3) obtained from China Center for Type Culture Collection (Wuhan, China) were cultured in Dulbecco s modified Eagle s high glucose medium (DMEM) supplemented with 10% fetal bovine serum (FBS). U87MG cell lines obtained from the Cell Culture Center (IBMS, CAMS/PUMC) ( Peking, China) were cultured in MEM Eagles with Earle s Balanced Salts (MEM-EBSS). 3T3 cell lines and U87MG cell lines were plated at a density of cells per well in 24-well plates, and grown for 24 h at 37 C in 5% CO 2. The PEG-LPLNPs dispersed in 10 mm PBS solution with a wide concentration range from 50 to 1000 μg ml -1 were subsequently added into the cell and incubated for another 24 h under the same conditions as above. The cells were then washed with 10 mm PBS (ph 7.4), trypsinized with 200 μl trypsin/edta solution, and resuspended to a final volume of 1 ml cell medium for further measurement of cell viability by a cell counting chamber. Procedures for Cell Imaging. The U87MG cells were cultured for 24 h, and then incubated with PEG-LPLNPs or RGD-LPLNPs (200 μg ml -1 ) in the 6-well plate for 24 h. The resulting cells were washed with PBS (10 mm, ph 7.4) three times before imaging. The cell images were observed on an IX81 motorized inverted microscope (Olympus, Japan) (ocular, WHN 10 ; objective, UPLFLN 10 /0.30; excitation filter, BP ; dichroic beamsplitter, DM400; S3

4 barrier filter, BA420) with X-cite series 120 illuminator (Lumen Dynamics Co., Ontario, Canada) and Retiga 2000R cooled CCD. Animal Model. The adult athymic BALB/c (BALB/c-nu) mice (15~20 g) were obtained from Beijing HFK bioscience Co., LTD. (Beijing, China). Nude mice (15~20 g) harboring U87MG tumors (7 mm) were obtained from the Tianjin Medical University Cancer Institute and Hospital. All animal experiments were carried out in accordance with guidelines of the Tianjin Medical University Cancer Institute and Hospital. Histopathology. The histological changes in the main organs of mice injected with PEG-LPLNPs (0.4 ml, 1.0 mg ml 1 ) were observed after 7 days of injection. The selected organs (heart, liver, spleen, lung and kidney) were fixed in 10% neutral buffered formalin,embedded in paraffin, sectioned (5 μm thick), and stained with hematoxylin and eosin (H&E). The histological sections were observed under an optical microscope (Figure S14b). Ex Vivo Biodistribution Analysis. The PEG-LPLNPs (0.4 ml, 1 mg ml -1 ) and RGD-LPLNPs (0.4 ml, 1 mg ml -1 ) were injected through the tail vein into normal nude mice (n = 3) and U87MG tumor-bearing mice (n = 3), respectively. The mice were sacrificed and major organs were collected for ex vivo luminescence imaging. All organs were excited 5 min under UV light before imaging. Other conditions for ex vivo organ imaging are similar to those for in vivo whole-body imaging. The mean luminescence intensity in each individual organs was calculated by using the region of interest functions of indigo software. S4

5 Table S1. The parameters of photoluminescence decay curve fitting [a] τ 1 (s) A 1 τ 2 (s) A 2 τ 3 (s) A 3 τ av (s) [b] LPLNPs powder ± ± ± ± ± ± ± 1.65 aqueous dispersion of LPLNPs ± ± ± ± ± ± ± 0.05 [a] I(t)= I 0 +A 1 e -t/τ1 + A 2 e -t/τ2 + A 3 e -t/τ3 [b] τ av = (A 1 τ 1 + A 2 τ 2 + A 3 τ 3 )/( A 1 + A 2 + A 3 ) S5

6 Figure S1. Phosphorescence emission spectra (excitation at 254 nm) of LPLNPs powder prepared at different ph values of starting solution (sinter at 900 C). Figure S2. Phosphorescence emission spectra (excitation at 254 nm) of LPLNPs powder prepared at different temperatures. S6

7 Figure S3. NIR afterglow decay curve of LPLNPs with different sinter times after 5 min irradiation with a 254 nm UV lamp. Persistent luminescence intensity was monitored at 695 nm as a function of time (logarithmic scale). Figure S4. Phosphorescence emission spectra (excitation at 254 nm) of Zn z Ga 1.96 Ge 2 O 10 :Cr 0.01 Pr 0.03 with various contents of Zn (z). Insert: digital photo of LPLNPs powder (z=2.95) under 254 nm UV light excitation. S7

8 Figure S5. TEM size distribution of the LPLNPs. Figure S6. Dynamic light scattering spectra of LPLNPs (a), PEG-LPLNPs (b) and RGD-LPLNPs (c). S8

9 Figure S7. Zeta potential of unmodified, APTES-, PEG- and RGD-LPLNPs at neutral ph. Figure S8. FT-IR spectra of c(rgdyk) peptide. S9

10 Figure S9. Thermogravimetric analysis of SC-PEG-COOH Figure S10. Phosphorescence excitation spectra of LPLNPs powder sinter at 600 C. S10

11 Figure S11. The photoluminescence decay curve of LPLNPs powder Figure S12. The photoluminescence decay curve of aqueous LPLNPs suspension. S11

12 Figure S13. NIR afterglow decay of LPLNPs powder at 700 nm after 5 min irradiation with a 254 nm UV lamp as a function of time recorded by CCD camera. Figure S14. NIR afterglow decay of LPLNPs aqueous solution at 700 nm after 5 min irradiation with a 254 nm UV lamp as a function of time recorded by CCD camera. S12

13 Figure S15. Afterglow decay curve of undoped ZGGO powder after 5 min irradiation with a 254 nm UV lamp. Persistent luminescence intensity monitored at 515 nm as a function of time (logarithmic scale). Figure S16. (a) In vitro cell viability of 3T3 cells and U87MG cells incubated with PEG-LPLNPs at different concentrations for 24 h. (b) Representative H&E stained images of major organs including heart, liver, spleen, lung, and kidney collected from PEG-LPLNPs (0.4 ml, 1.0 mg ml 1 ) injected mice (n=4) and the control mice (n=3, injected with PBS) at 7 day after administration. The scale bars is 50 μm for all images. (c) Body weight changes of the mice (n=3) injected with PEG-LPLNPs (0.4 ml, 1.0 mg ml 1 ) and the control mice (n=3) injected with PBS. The error bars represent standard deviation. S13

14 Figure S17. In vivo NIR luminescence images of a normal mouse after subcutaneous injection of PEG-LPLNPs (0.4 mg, 10 min irradiation with a 254 nm UV lamp before injection). (a) Images taken without illumination source after injection. (b) Images taken at 1 min after the stimulation with a NIR light. A continuous-wave 980 nm laser (the power density of ~ 40 mw cm -2 ) to illuminate the whole-body mouse for 10 s. S14

15 Figure S18. Calculation of signal-to-noise ratio from the in vivo NIR luminescence image of a normal mouse at 450 min post-intravenous injection of LPLNPs-PEG (0.6 mg, 10 min irradiation with a 254 nm UV lamp before injection). Calculation of signal-to-noise ratio: 3 Signal-to-noise ratio = [(mean intensity of the specific uptake, 1) - (mean intensity of background, 3)] / [(mean intensity of the nonspecific uptake, 2) - (mean intensity of background, 3)]. region mean intensity of 1 mean intensity of 2 mean intensity of 3 signal-to-noise ratio counts S15

16 Figure S19. Fluorescence images of U87MG cells after incubation with the PEG-LPLNPs and RGD-LPLNPs for 24 h, respectively. The scale bar is 100 μm. References (1) de Mello, J. C.; Wittmann, H. F.; Friend, R. H. Adv. Mater. 1997, 9, 230. (2) Wang, Y.; Chen, J. T.; Yan, X. P. Anal. Chem. 2013, 85, (3) Liu, Q.; Sun, Y.; Yang, T.; Feng, W.; Li, C.; Li, F. J. Am. Chem. Soc. 2011, 133, S16