Intracellular Adenosine Triphosphate Deprivation Through Lanthanide-doped Nanoparticles

SUPPORTING INFORMATION Intracellular Adenosine Triphosphate Deprivation Through Lanthanide-doped Nanoparticles Jing Tian,, Xiao Zeng, Xiaoji Xie, Sanyang Han, Oi-Wah Liew, Փ Yei-Tsung Chen, Փ Lianhui Wang,*,, and Xiaogang Liu*,,# Laboratory of Advanced Materials, Fudan University, Shanghai 200433, China Department of Chemistry, National University of Singapore, Singapore 117543, Singapore Փ Cardiovascular Research Institute, Department of Medicine, National University of Singapore, National University Health System, Singapore 117599, Singapore Key Laboratory for Organic Electronics & Information Displays, Institute of Advanced Materials, and Jiangsu National Synergistic Innovation Center for Advanced Materials (SICAM), Nanjing University of Posts & Telecommunications, Nanjing 210023, China # Institute of Materials Research and Engineering, 3 Research Link, Singapore 117602, Singapore S1

Supplemented Experimental Section Synthesis of NaYF 4 nanoparticles. NaYF 4 nanoparticles were synthesized according to our previous publication. 1 Typically, to a 50-mL two-neck flask charged with oleic acid (3 ml) and 1-octadecene (7 ml) was added an aqueous solution (2 ml) of Y(CH 3 CO 2 ) 3 (0.4 mmol). The resulting mixture was heated at 150 o C for 1.5 h under stirring and then cooled to 50 o C. Next, a methanol solution (6 ml) of NH 4 F (1.6 mmol) and NaOH (1 mmol) was added and stirred at 50 o C for 30 min, followed by heating to 100 o C for another 30 min to remove the methanol. With the protection under a flow of argon, the reaction solution was heated to 280 o C and kept for 1.5h. After cooling down to room temperature, nanoparticles were precipitated out with ethanol, collected by centrifugation, washed with ethanol three times, and finally dispersed in 4 ml of cyclohexane. Synthesis of NaGdF 4 nanoparticles. The procedure for NaGdF 4 synthesis was similar to that of NaYF 4. 1 A water solution of Gd(CH 3 CO 2 ) 3 (0.4 mmol) was added to a 50-mL twoneck flask containing oleic acid (4 ml). After heating at 150 o C for 1 h, 6 ml of 1-octadecene was added into the resultant mixture and heated for another 30 min at 150 o C before cooling down to 50 o C. After that, a methanol solution (5.4 ml) of NH 4 F (1.36 mmol) and NaOH (1 mmol) was added and stirred at 50 o C for 30 min. To remove the methanol, the reaction mixture was heated at 100 o C for 30 min in vacuo. Subsequently, the temperature was raised to 290 o C and maintained for 1.5 h. The resulting nanoparticles were collected and dispersed in 4 ml of cyclohexane. To increase the size of NaGdF 4 nanoparticles, a shell of identical composition was used. The precursor was prepared in a procedure similar to the core. Subsequently, the core nanoparticles were added to the reaction mixture together with NH 4 F (1.36 mmol) and NaOH (1 mmol), followed by heating at 100 o C for 30 min and then 290 o C for 1.5 h. Finally, the asprepared core-shell NaGdF 4 were collected and redispersed in cyclohexane (4 ml). Preparation of PAA-coated nanoparticles. Ligand-free nanoparticles (10 mg) in water were mixed with 50 mg of PAA (Mw = 1,800) in 4 ml of water. Thereafter, 1 ml of 0.2 M NaOH was added dropwise and stirred for 4 h at room temperature. After the reaction, products were collected by centrifugation at 16,500 rpm for 20 min, washed with water several time, and redispersed in Milli-Q water. The contents of lanthanide ions were determined by ICP-OES. S2

Cell culture. HeLa cells were obtained from American Type Culture Collection (ATCC) and maintained in DMEM supplemented with 10% FBS, 100 U/mL penicillin and 100 g/ml streptomycin at 37 o C in a humidified 5% CO 2 atmosphere. Fluorescence microscopy. Cells were cultured with ibidi 35-mm dishes and treated with nanoparticles. Before staining, cells were fixed with 3.7% formaldehyde in PBS for 15 min at room temperature. Then cell skeleton was stained with 200 L of rhodamine phalloidin (165 nm) in PBS with 1% bovine serum albumin (BSA) for 20 min. After being washed three times with PBS, the cell nuclei were labeled with DAPI (200 L; 1 g/ml) for 10 min. The cells were ready for fluorescence microscopy after washing. The emission/excitation wavelengths for rhodamine and DAPI were 595/613 nm and 359/461 nm. Caspase-3/7 expression assay. HeLa cells were treated with different concentrations of ligand-free NaGdF 4 or NaYF 4 nanoparticles in 96-well or 24-well plates for 3 and 7 d, respectively. In 3-d assay, the cells in 96-well plates were used directly for caspase-3/7 expression measurements with assay kits following manufacturer s instruction. For 7-d assay, the cells in 24-well plates were trypsinized and then transferred to 96-well plates for testing. DNA fragmentation (sub-g1 phase) analysis. HeLa cells were treated with different concentrations of ligand-free NaGdF 4 or NaYF 4 nanoparticles in 6-well plates for 3 and 7 d. After treatment, cells were harvested by trypsinization and suspended with 0.5 ml of PBS. Then, 1.2 ml of absolute ethanol was added to fix cells (final concentration of ethanol is about 70%) on ice for at least 2 h, followed by washing with PBS once. Cell pellets were suspended and stained with PI staining solution (PBS with 0.1% triton X-100, 0.2 mg/ml RNase A, and 20 μg/ml PI) at 37 o C for 15 min. Thereafter, samples were analyzed by a flow cytometry analyser with at least 10,000 cells collected for each sample. ATP binding test. ATP solution of 1000 M was incubated with ligand-free and PAAcoated nanoparticles at a final concentration of 0, 100, 400, and 1600 g/ml, respectively, at 37 o C for 72 h. The ATP content and adenosine concentration of supernatants were measured at every 12-h intervals with ATP test kits and UV-vis absorbance, respectively. ATP hydrolysis product was examined by HPLC. A nanoparticle-atp mixture (200 L) in cell culture medium was withdrawn and diluted by ultra-pure water to 1 ml. Subsequently, the diluted dispersion was centrifuged at 18,500 rpm for 30 min for removal of the nanoparticles. S3

A 5 L of supernatant was withdrawn and injected into an Agilent 1200 series HPLC equipped with a diode array detector. Chromatographic separation was achieved by using a Phenomenex Luna C18 column (250 4.60 mm, 5 mm particle size, 100 Å pore size). Potassium dihydrogen orthophosphate (0.04 M) and dipotassium hydrogen orthophosphate (0.06 M) were used as mobile phase at a flow rate of 1.0 ml/min. Eluent was detected by monitoring the molecular absorption at 254 nm. ATP rescue assay. HeLa cells were seeded at a density of 2000-5000 cells/well (100 μl) in 96-well plates. After 24-h incubation, fresh medium with different concentrations of NaGdF 4 nanoparticles was added to replace the old medium and incubated for another 72 h. Thereafter, 2 μl of ATP solutions (25 mm) were supplemented to the wells to reach a final concentration of 500 M. After culture for 24 h, cell viability was measured with MTS assay and compared to groups without ATP addition. Cellular uptake of nanoparticles. HeLa cells were cultured in T-25 flasks with 100 g/ml of ligand-free NaGdF 4, NaYF 4, NaGdF 4 /PAA or NaYF 4 /PAA nanoparticles for 3 and 7 d. Cells were collected and counted after washed with PBS for three times to eliminate the nanoparticles external of cells. Then, treat cells with HNO 3 : HCl (3:1) in 70 o C oil bath for 24 h until all nanoparticles were digested completely. After that the contents of lanthanide ions were measured with ICP-OES and cellular uptake of nanoparticles per cell was calculated. PAA release experiment. To quantify the released PAA molecules from nanoparticle surface, PAA-coated nanoparticles (NaGdF 4 /PAA) were first labeled with FITC according to procedures described below. To a 4 ml of DMSO solution containing 10 mg of NaGdF 4 /PAA was added with 15.6 mg of N-hydroxysuccinimide and 26 mg of 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide. The mixture was stirred in darkness for 1 h followed by centrifugation at 18,500 rpm for 20 min. A 2 ml of DMSO and a 200 L of amine-modified FITC (1 mm) were added to redisperse the pellet, and the reaction mixture was subsequently stirred overnight at 600 rpm in darkness. After the completion of reaction, the FITC labeled product (NaGdF 4 /PAA-FITC) was washed with Milli-Q water three times and then dispersed into 10 ml Milli-Q water. To study the PAA release behavior of the as-prepared NaGdF 4 /PAA-FITC, a stock solution of 1 ml NaGdF 4 /PAA-FITC was taken each day and subjected to fluorescence measurement S4

to determine the total amount of PAA present in the dispersion. The emission spectrum was recorded from 500 to 600 nm under 493-nm excitation. Subsequently, the dispersion was centrifuged at 18,500 rpm for 30 min to isolate the PAA molecules released in supernatant. The amount of PAA molecules was then determined using the same spectroscopic method as mentioned above. The percentage of released PAA molecules is calculated by dividing FITC fluorescence intensity (at 520 nm) measured in the supernatant by that measured in NaGdF 4 /PAA-FITC dispersion. Lanthanide ion release experiment. PAA-coated NaGdF 4 nanoparticles (10 mg) and ligandfree NaGdF 4 nanoparticles (10 mg) were separately dispersed into 1 ml of ultrapure water. These solutions were dialyzed against 200 ml of ultrapure water for 5 days. The dialysis bag has a molecular-weight-cutoff value of 14,000. During each day 10 ml of aqueous solution was taken for ICP-MS analysis of Gd 3+ ion concentration in water. Meanwhile, the dialysis solution was replenished with 10 ml ultrapure water to keep the total volume at 200 ml. References for supporting information (1) (a) Wang, F.; Deng, R.; Wang, J.; Wang, Q.; Han, Y.; Zhu, H.; Chen, X.; Liu, X. Nat. Mater. 2011, 10, 968. (b) Su, Q.; Han, S.; Xie, X.; Zhu, H.; Chen, H.; Chen, C.-K.; Liu, R.-S.; Chen, X.; Wang, F.; Liu, X. J. Am. Chem. Soc. 2012, 134, 20849. Table S1. Size and zeta potential of the nanoparticles under investigation Nanoparticles Size from TEM (nm) Hydrodynamic size (nm) Zeta potential (mv) in water in medium in water in medium NaGdF 4 28.51±4.06 45.10±10.78 245.6±75.93 27.9±2.62-11.6±0.26 NaGdF 4 /PAA 29.14±6.31 115.3±32.35 124.8±22.28-58.7±2.25-12.8±0.92 NaYF 4 29.32±3.51 56.98±15.88 274.0±97.71 33.0±0.31-10.8±0.36 NaYF 4 /PAA 30.18±2.88 134.6±31.16 139.6±33.56-59.2±1.67-7.91±0.99 S5

Figure S1. TEM images of OA-capped, ligand-free, and PAA-coated NaGdF 4 (a-c) and NaYF 4 :Yb/Tm (d-f). S6

Figure S2. XRD patterns of the as-synthesized NaGdF 4 nanoparticles (a) and NaYF 4 nanoparticles (b). Diffraction patterns of these samples are consistent with those of hexagonal NaGdF 4 crystals (JCPDS file no. 27-0699) and NaYF 4 crystals (JCPDS file no. 28-1192), respectively. S7

Figure S3. (a-c) FTIR spectra of OA-capped, ligand-free, and PAA-coated NaGdF 4 nanoparticles, respectively. (a) Note that the peaks at 3437 and 1637 cm -1 are assigned to the stretching and bending vibrations of O H groups. The peaks at 2925 and 2854 cm -1 result from the stretching of CH 2 groups and the bands at 1558 and 1458 cm -1 are attributed to symmetric and asymmetric stretching vibrations of the carboxylate groups ( COO ) of oleic acid. (b) After HCl treatment, the peaks at 2925, 2854, 1558, and 1458 cm -1 disappeared, indicating a complete removal of oleic acid. (c) The peak at 2951 cm -1 is due to the methylene (CH 2 ) stretching vibrations of the long alkyl chain of PAA. The carbonyl (C=O) stretching peak locates at 1663 cm -1 and the bands at 1409 and 1328 cm -1 result from the C O stretching vibration in the COOH groups of PAA. S8

Figure S4. FTIR spectra of OA-capped (a), ligand-free (b), and PAA-coated (c) NaYF 4 nanoparticles. (a) The peaks at 3443 and 1637 cm -1 are assigned to the stretching and bending vibrations of O H groups. The bands at 2926 and 2856 cm -1 result from the stretching of CH 2 groups and the peaks at 1558 and 1458 cm -1 are attributed to symmetric and asymmetric stretching vibrations of the carboxylate groups ( COO ) of oleic acid. (b) After HCl treatment, the peaks at 2926, 2856, 1558, and 1458 cm -1 disappeared, indicating a complete removal of oleic acid. (c) The peak at 2950 cm -1 results from the methylene (CH 2 ) stretching vibrations of the long alkyl chain of PAA. The carbonyl (C=O) stretching peak locates at 1665 cm -1 and the bands at 1409 and 1329 cm -1 are assigned to the C O stretching vibration in the COOH groups of PAA. S9

Figure S5. LDH release tests of HeLa cells after treatment with ligand-free NaGdF 4 nanoparticles for 24, 48, and 72 h at dosages of 0-1600 g/ml. Figure S6. Cytotoxicity of ligand-free NaYF 4 nanoparticles on HeLa cells. (a) MTS assay, (b) ATP level measurement, and (c) LDH release test after 24, 48, and 72-h exposure at dosages of 0-1600 g/ml. (d) Cytotoxicity tests with MTS assay (blue line) and ATP level test (red line) after treatment with nanoparticles at 0, 50, 100, 200, 400, 800, and 1600 g/ml for 7 d. *P<0.05, **P<0.01. S10

Figure S7. HeLa cell morphologies after treatment with ligand-free NaGdF4 nanoparticles at 0, 100, 400, and 1600 g/ml for 1, 2, 3, and 7 d. Morphological alterations include cytoplasmic vacuolization (marked by arrows) and membrane blebbing (marked by arrowheads). Scale bars are 20 m. S11

Figure S8. HeLa cell morphologies after treatment with ligand-free NaYF 4 nanoparticles at 0, 100, 400, and 1600 g/ml for 1, 2, 3, and 7 d. Morphological alterations show cytoplasmic vacuolization (marked by arrows) and membrane blebbing (marked by arrowheads). Scale bars are 20 m. S12

Figure S9. Effects of ligand-free NaGdF 4 nanoparticles on HeLa cell morphology in 3-d treatment. Cells were exposed to 0, 100, 400, and 1600 g/ml for 3 d and stained with rhodamine palloidin and DAPI for actin and nuclei, respectively. Morphological alterations include cytoplasmic vacuolization (marked by black arrows), cell shrinkage (marked by black thick arrows), and nucleus condensation (marked by arrowheads). All photos were taken under the same settings. Scale bars are 20 m. S13

Figure S10. HeLa cell morphology with ligand-free NaYF 4 nanoparticles for 3 d. Cells were treated with at 0, 100, 400, and 1600 g/ml for 3 d and stained with rhodamine palloidin and DAPI for actin and nuclei, respectively. Morphological alterations show cytoplasmic vacuolization (marked by black arrows), shrinked cells (marked by white and black thick arrows), and condensed and irregular nuclei (marked by arrowheads). All photos were taken under the same settings. Scale bars are 20 m. S14

Figure S11. HeLa cell morphology with ligand-free NaYF 4 nanoparticles in 7-d exposure. Cells were treated with at 0, 100, 400, and 1600 g/ml for 7 d and stained with rhodamine palloidin and DAPI for actin and nuclei, respectively. Morphological alterations include cytoplasmic vacuolization (marked by black arrows), cell shrinkage (marked by white and black thick arrows), and nucleus condensation and fragmentation (marked by arrowheads). All photos were taken under same settings. Scale bars are 20 m. S15

Figure S12. Cell autophagy induced by ligand-free NaYF 4 nanoparticles. (a, b) Fluorescence microscopy of HeLa cells with MDC staining after exposure to 0, 100, 400, and 1600 g/ml of nanoparticles for 3 d (a) and 7 d (b). All photos were taken with the same settings. Scale bars are 20 m. (c, d) Western blotting of LC3 protein. HeLa cells were treated with 0, 100, 200, 400, 800, and 1600 g/ml of nanoparticles for 3 d (c) and 7 d (d). Actin was used as loading control. S16

Figure S13. Effects of ligand-free NaYF 4 nanoparticles on HeLa cell apoptosis. (a, b) Representative profiles of FACS analysis and statistical results of early and late apoptosis at day 3 (a) and day 7 (b) after treatment with 0, 50, 100, 200, 400, 800, and 1600 g/ml of NaYF 4 nanoparticles. (c) Fold changes of caspase-3/7 expression as compared to control groups after exposure to the nanoparticles for 3 and 7 d. (d) DNA fragmentation by measuring cells in sub-g1 phase with flow cytometry after treatment with nanoparticles for 3 and 7 d. *P < 0.05, **P < 0.01. S17

Figure S14. Interaction of NaYF 4 nanoparticles with ATP. (a) ATP and (b) adenosine concentration measurements after binding with ligand-free NaYF 4 nanoparticles at concentrations of 0, 100, 400, and 1600 g/ml for 72 h. (c) Schematic illustration of interaction between the NaYF 4 nanoparticles and ATP. S18

Figure S15. HPLC chromatogram of (a) ATP solution after incubating at 37 C for 24 h, (b) ATP-nanoparticles mixture after incubating at 37 C for 24 h, (c) AMP standard solution, (d) ADP standard solution, (e) and ATP standard solution. S19

Figure S16. Concentration of free Gd 3+ in a NaGdF 4 -containing dispersion. Ligand-free NaGdF 4 and NaGdF 4 /PAA nanoparticles (10 mg each) were dialyzed in 200 ml of ultrapure water. The Gd 3+ concentration in ppb was determined by ICP-MS and further converted into percentage. S20

Figure S17. Rescue effects of ATP addition on cell viability. HeLa cells were exposed to 0, 100, 200, 400, 800, and 1600 g/ml of NaGdF 4 nanoparticles for 72 h, and then supplemented with 500 M of ATP in cell medium followed by 24 h incubation. Figure S18. Inhibition effects of PAA coatings on interaction between NaYF 4 nanoparticles and ATP. (a) Schematic illustration showing how PAA coatings hinder NaYF 4 nanoparticles from binding to ATP. (b) ATP concentration determination after binding with NaYF 4 /PAA nanoparticles at concentrations of 0, 100, 400, and 1600 g/ml for 72 h. S21

Figure S19. Protection effects of PAA coatings on nanoparticle-induced cytotoxicity. (a, b) MTS assays and (c, d) LDH tests of HeLa cells after 24, 48, and 72-h exposure to NaGdF 4 /PAA (a, c) and NaYF 4 /PAA (b, d) nanoparticles at 0-1600 g/ml. *P < 0.05, **p < 0.01. S22

Figure S20. Comparison of cytotoxicity between NaYF 4 and NaYF 4 /PAA. (a) ATP level measurements after treatment with NaYF 4 (left) or NaYF 4 /PAA (right) at 0, 5, 25, 50, 100, 200, 400, 800, and 1600 g/ml for 3 d. (b) MTS assays and (c) ATP tests after 7-d exposure to NaYF 4 or NaYF 4 /PAA nanoparticles at dosages of at 0, 50, 100, 200, 400, 800, and 1600 g/ml. S23

Figure S21. HeLa cell morphologies after NaGdF4/PAA treatment at 0, 100, 400, and 1600 g/ml for 1, 2, 3, and 7 d. Morphological changes reveal cell shrinkage (marked by arrows). Scale bars are 20 m. S24

Figure S22. Cellular uptake of ligand-free and PAA-coated NaGdF 4 /NaYF 4 nanoparticles after treatment at the dosage of 100 g/ml for 3 d and 7 d. S25

Figure S23. Quantification of released PAA in PAA-coated NaGdF 4 nanoparticle dispersion (black line). The red line indicates the PAA releasing behavior when 1 mm ATP is present in the dispersion. S26

Figure S24. Upconversion luminescence of NaGdF 4 :Yb/Tm and NaYF 4 :Yb/Tm nanoparticles in HeLa cells. Cells were incubated with 100 g/ml of nanoparticles for 12 h and then stained with rhodamine palloidin for actin. All photos were taken under the same settings. Scale bars are 20 m. S27