Integrating Enzymatic Self-Assembly and Mitochondria Targeting for Selectively Killing

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1 Supporting Information for Integrating Enzymatic Self-Assembly and Mitochondria Targeting for Selectively Killing Cancer Cells without Acquired Drug Resistance Huaimin Wang,, Zhaoqianqi Feng, Youzhi Wang, Rong Zhou, Zhimou Yang *,, and Bing Xu *, Department of Chemistry, Brandeis University, 415 South Street, Waltham, MA 02453, USA State Key Laboratory of Medicinal Chemical Biology, Key Laboratory of Bioactive Materials, Ministry of Education, College of Life Sciences, Collaborative Innovation Center of Chemical Science, Nankai University, Tianjin , P.R. China Content S1. Experiment materials and instruments... 3 S2. Synthesis and characterization of the precursors... 3 S3. TEM sample preparation... 4 S4. Cell culture and MTT assay S5. CLSM images of Saos2 cell... 5 S6. Actin staining... 5 S7. Apoptotic signaling assay... 6 S8. Time-dependent Western blot... 6 S9. Supplemental figures... 7 Fig. S1. TEM images of L-1P and D-1P (50 µm) in PBS buffer (ph 7.4) without or with ALP (1 U/mL) after 24 h. Fig. S2. TEM images of L-1P and D-1P (100 µm) in PBS buffer (ph 7.4) without or with ALP (1 U/mL) after 24 h. Fig. S3. TEM images of L-1P and D-1P (50 µm) in PBS buffer (ph 7.4). Scale bar is 50 nm. S1

2 Fig. S4. Time-dependent dephosphorylation process of the precursors L-1P and D-1P at the concentration of 0.1 wt% treated with ALP (0.1 U/mL) at 37 C. Fig. S5. Size distribution of L-1P (D-1P) at PBS buffer (ph = 7.4) without or with ALP (1 U/mL) obtained by dynamic light scattering. Fig. S6. TEM images of L-2P and D-2P (50 µm) in PBS buffer (ph 7.4) without or with ALP (1 U/mL) after 24 h. Fig. S7. Cell viability of Saos2 cells treated with L-1P, D-1P, L-2P, D-2P and L-2P plus 3, D-2P plus 3 (the concentration of 3 is the same as L-2P or D-2P, that is start from 20 μm to 500 μm) at 24, 48 and 72 h Fig. S8. Time dependent intracellular concentration of L-1P/L-1 (D-1P/D-1) inside/outside of Saos2 cell lines. The incubating concentration of L-1P or D-1P is 50 µm. Fig. S9. Cell viability of HS-5 cells or HeLa cells treated with L-1P and D-1P at 24, 48 and 72 h and 48 h cell viability of different cell lines treated with 200 µm of L-1P or D-1P. Fig. S10. Cell viability of HepG2, T98G and MCF-7 cells treated with L-1P and D-1P at 24, 48 and 72 h. Fig. S11. Confocal laser scanning microscopy (CLSM) images of Saos2 cells treated with L-1P or D-1P (the concentration is from 10 µm to 50 µm) lasted two months and washed the compounds, the trypsin-digested-then-reattached cells as the first passages. Fig. S12. Confocal laser scanning microscopy (CLSM) images of Saos2 cells treated with L-1P or D-1P (50 µm) for 4 h at the temperature of 4. Fig. S13. CLSM images (Gray scale) of Saos2 cells treated with L-1P or D-1P (50 µm) for 1 h in the absence (control) or presence of the inhibitors EIPA (100 µm, ethyl-isopropyl-amiloride), CPZ (30 µm, chlorpromazine), Filipin Ш (5 µg/ml) and M-βCD (5 mm). Fig. S14. Cell viability of Saos2 cell line treated by L-1P or D-1P in the presence of phosphatase inhibitors or external ALP for 24, 48 or 72h ([L-Phe] = [levamisole] =1 mm, GinnGEL 2Me=2 µm, [ALP] = 10 U/mL) S2

3 Fig. S15. Confocal laser scanning microscopy (CLSM) images of Saos2 cells treated with L-1P (50 µm) for 4 h in the absence or with ALP or levamisole (TNAP inhibitor, 1 mm) and L-Phe (PLAP inhibitor, 1 mm). Fig. S16. Confocal laser scanning microscopy (CLSM) images of Saos2 cells treated with D-1P (50 µm) for 4 h in the absence or with ALP or levamisole (TNAP inhibitor, 1 mm) and L-Phe (PLAP inhibitor, 1 mm). Fig. S17. Cell viability of Saos2 cell line treated by L-1P or D-1P in the presence of cell death signaling inhibitors at 24, 48 and 72 h ([zvad-fmk] = 45 μm, [Nec-1] = 50 μm). Fig. S18. Time-dependent western blot analysis of cytochrome c (cyt c) from the whole cell fraction (contains both fraction of cytosol and mitochondria) of Saos2 cell treated with L-1P or D-1P (50 µm). Fig. S19. The resistant test of Saos2 cells treated with L-1P or D-1P for five weeks, and use MTT to test the cell viability of corresponding compounds at 24 h, 48 h and 72 h. Fig. S20. 1 H NMR of L-1P in DMSO-d6. Fig. S21. 1 H NMR of D-1P in DMSO-d6. Fig. S22. 1 H NMR of L-2P in DMSO-d6. Fig. S23. 1 H NMR of D-2P in DMSO-d6. Fig. S24. HPLC trace of L-1P, D-1P, L-2P and D-2P. Fig. S25. MS spectrum of L-1P, D-1P, L-2P and D-2P. S1. Experiment materials and instruments 2-Cl-trityl chloride resin (0.6 mmol/g), Fmoc protected amino acid, HBTU and Fmoc-OSu were obtained from GL Biochem (Shanghai, China). N, N-Diisopropylethylamine (DIPEA), TPP and other chemical reagents and solvents were obtained from Fisher Scientific; all chemical reagents and solvents were used as received from commercial sources without further purification; alkaline phosphatase was purchased from Biomatik. Dulbecco s modified Eagle s medium (DMEM), McCoy's 5a Medium and 1640 Medium were purchased from ATCC and fetal bovine serum (FBS) and penicillin/streptomycin were purchased from Gibco by life technologies. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from ACROS Organics. All precursors were purified with Water Delta600 HPLC system, equipped with S3

4 an XTerra C18 RP column. LC-MS spectrum was obtained on Waters Acquity Ultra Performance LC with Waters MICROMASS detector, and 1 H-NMR spectra on Varian Unity Inova 400, and TEM images on Morgagni 268 transmission electron microscope. MTT assay for cell viability test on DTX880 Multimode Detector. S2. Synthesis and characterization of the precursors Solid phase peptide synthesis All the peptides were prepared by standard solid-phase peptide synthesis (SPPS) using 2-chlorotrityl chloride resin and the corresponding Fmoc-protected amino acids with side chains properly protected. The first amino acid was loaded onto the resin at about 0.6 mmol/g of resin. After loading the first amino acid to the resin, the capping regent (DCM: MeOH: DIPEA = 17: 2: 1) was used to ensure all the active sites of the resin were protected. The solution of 20% piperidine in DMF was used to remove the Fmoc group, the next Fmoc-protected amino acid was coupled to the free amino group using HBTU as the coupling reagent. The growth of the peptide chain followed the established Fmoc SPPS protocol. The last capping group of NBD-β-Alanine was synthesized according to the reported method. 1 The crude peptides were collected using TFA-mediated cleavage method: The peptide derivative was cleaved using 95% of trifluoroacetic acid with 2.5% of TIS and 2.5% of H2O for 1 h. 20 ml per gram of resin of ice-cold diethyl ether was then added to cleavage reagent. The resulting precipitate was centrifuged for 10 min at room temperature at 10,000 rpm. Afterward the supernatant was decanted and the resulting solid was use for the next step for synthesis. Synthesis of L-1P (or D-1P) 100 mg of 3-carboxypropyl triphenylphosphonium bromide (TPP) was dissolved in 10 ml of dichloromethane (DCM), followed by 1.1 equiv. (29 mg) of N-Hydroxysuccinimide (NHS) and 57.6 mg of N,N'-dicyclohexylcarbodiimide (DCC) with catalytic amount of 4-dimethylamiopryidine were added. After being stirred at room temperature for 2hrs, the solution was filtered by a filter paper to remove precipitations. The filtrate was evaporated under reduced pressure to yield a white power, which was used directly for the next step. After the white powder obtained above being dissolved in 5 ml of N,N-dimethylformamide, 1.3 equiv. of corresponding peptide was then added with 3 equiv. N-diisopropylethylamine (DIPEA). The resulting reaction mixture was stirred overnight and the title products were purified by reverse phase HPLC. For L-2P (or D-2P), acetic anhydride was used directly to react with peptide at the solution of DMF with DIPEA (adjusting ph to 8), after stirred at room temperature for 1h, remove DMF by air compressor, and add methanol to dissolve the product. Finally, we purify the product by reverse phase HPLC. S3. TEM sample preparation 1. First place sample solution by pipettor on the carbon coated grid (5 µl, sufficient to cover the grid surface). 2. After 30 seconds, put the grid with the face of sample to a large drop of the ddh2o on parafilm and let the grid touch the water drop for 5 seconds, tilt the grid and gently absorb S4

5 water from the edge of the grid using a filter paper sliver. This process repeated for 3 times. 3. Staining (immediately after rinsing): place a large drop of the UA (uranyl acetate, 2% v/v) stain solution on parafilm and let the grid touch the stain solution drop, with the sample-loaded surface facing the parafilm. Tilt the grid and gently absorb the stain solution from the edge of the grid using a filter paper sliver. 4. Allow the grid to dry in air and examine the grid as soon as possible. S4. Cell culture and MTT assay Cell culture: Saos2, HeLa, HepG2, T98G, MCF-7 and HS-5 cells were purchased from American-type Culture Collection (ATCC, USA). Saos2 cells were cultured in Macyo s 5A medium supplemented with 15% v/v fetal bovine serum, 100 U/mL penicillin and 100 μg/ml streptomycin. HeLa, HepG2, T98G, MCF-7 cells were cultured in MEM Medium supplemented with 10% v/v fetal bovine serum, HS-5 cells were cultured in Dulbecco s Modified Eagle s medium (DMEM) supplemented with 10% v fetal bovine serum (FBS), 100 U/mL penicillin and 100 μg/ml streptomycin. All cells were cultured at 37 C in a humidified atmosphere of 5% CO2. MTT assay: All cells were seeded in a 96-well plate with the density of 1*10 4 cells per-well (total medium volume of 100 μl). 24 hours post seeding, the solutions with a serial of concentrations (5 concentrations) of different precursors were added to each well. Cells without the treatment of the precursors were used as the control. At designated time (24/48/72 hours), 10 μl MTT solution (5 mg/ml) was added to each well and incubated at 37 C for another 4 h, and then 100 μl of SDS-HCl solution was added to stop the reduction reaction and dissolve the purple formazan. The absorbance of each well at 595 nm was measured by a multimode microplate reader. The cytotoxicity assay was performed three times and the average value of the three measurements was taken. S5. CLSM images of Saos2 cell General image process: Saos2 cells at the density of 1.5*10 5 is seeded onto 3.5 cm confocal dish. After the growth of Saos2 cells in cell incubator for 24h, L-1P (or D-1P) at the designed concentration is added to the above solution with completed medium. After 1h or 4h, use PBS to wash Saos2 cells for 3 times per minute. We use Hoechst 3342 to stain cell nucleus for 10 minutes and wash Saos2 cells by live cell imaging solution for 3 times per minute, keep the Saos2 cells in live cell imaging solution for CLSM immediately. For stain lysosome (or mitochondria), after Saos2 cells treated with L-1P (or D-1P) for 1h or 4h, remove the medium from the confocal dish and washed by PBS for 3 times, and then add the pre-warmed (at 37 C) LysoTracker (or Mito-Tracker) containing medium. Incubate the Saos cells for 1h (for Mito-Tracker 30 to 45 minutes is fine) under growth conditions. Then remove the staining solution and washed by live cell imaging solution 3 times, after stain the cell nucleus by Hoechst Then the cells were washed by live cell imaging solution and imaged immediately by confocal laser scanning microscope. S5

6 For endocytosis mechanism experiment, we first pre-incubate different endocytosis inhibitors of EIPA (100 µm, ethyl-isopropyl-amiloride), CPZ (30 µm, chlorpromazine), Filipin Ш (5 µg/ml) and M-βCD (5 mm) with Saos2 cells for 30 minutes, and then add 50 μm of L-1P (or D-1P) to the above confocal dish. After co-incubate the inhibitor with L-1P (or D-1P) for another 1h, remove the culture medium, wash the Saos2 cells by live cell imaging solution for 3 times. After statin with Hoechst 3342 for 10 minutes, wash the Saos2 cells by live cell imaging solution for another 3 times and image by CLSM immediately. S6. Actin Staining We used the procedure recommended by Molecular Probes (Thermo Fisher Scientific) for actin staining, briefly: 1. The cells were seeded in 3.5 cm confocal dish at cells per dish; 2. After incubate for 24 h, we removed culture medium, and added fresh medium containing L-1P or D-1P for 4 h; 3. After 4 h, we removed the medium and use PBS to wash the cells for three times. After fixing by 4% paraformaldehyde for 15 minutes, we added 1 ml of 0.1% Triton X-100 in PBS buffer for 30 minutes. 4. After washing the cells three times by PBS, we added 1 ml of 0.1% BSA in PBS for 30 minutes, and then washed the cells by PBS for three times ml of PBS containing 5 unit of Alexa 633 was added to the cells for 1 h. 6. After removing the staining solution and washing the cells three times by PBS, we added 1 ml of Hoechst (1 µg/ml) for 10 minutes. Then, the cells were washed three times with PBS buffer before imaging. S7. Apoptotic signaling assay According to the procedure provided by Cell Signaling Technology, Inc. We perform this assay, briefly: 1. After the Saos2 cells grows to 80 90% confluence in 10 cm culture dish, treated cells with L-1P (or D-1P) at desired time, untreated cells as control 2. Remove the culture medium and wash the cells by pre-cold PBS for 3 times, then add 0.5 ml ice-cold 1* cell lysis buffer plus protease inhibitors to each plate and incubate the plate on ice for 5 min. 3. Scrape cells off the plate and transfer to a 1.5 ml tube. 4. Freeze-thaw 3 cycles of collected cell fraction. Use a microcentrifuge for 20 min (12,000 rpm) at 4 C and transfer the supernatant to a new tube. The supernatant is the cell lysate. 5. Following the detail protocol as provided by cell signaling technology for EISA test. S8. Time-dependent western blot Mitochondria fraction purify: 1. After the Saos2 cells reach to about 90% confluence in 10 cm culture dish, remove the culture medium, add the fresh culture medium contains 50 µm L-1P (or D-1P) at different times. 2. At desired time, collect cells (each time contains two 10 cm culture dish) by trypsin and centrifuge at 600g for 5 minutes at 4 C. S6

7 3. Wash the Saos2 cells with 5 ml of pre-cold PBS for two times, each time centrifuge at 600g for 5 minutes and remove supernatant. 4. Re-suspend cell with 0.3 ml of cytosol extraction buffer containing DTT and protease inhibitors. 5. Incubate above suspension on ice for 10 minutes. 6. Homogenize cells on ice and transfer homogenate to a 1.5 ml tube, and centrifuge at 700g for 10 minutes at 4 C. Collect the supernatant carefully and discard the pellet. 7. Transfer the supernatant to a fresh 1.5 ml tube and centrifuge for 30 minutes at 4 C. 8. Carefully transfer the supernatant from 7 to a new 1.5 ml tube, this is the cytosolic fraction of Saos2 cells. 9. Use bradford protein assay to quantify the concentration of protein. 10. Perform standard Western blot. S9. Supplemental figures Figure S1. TEM images of L-1P and D-1P (50 µm) in PBS buffer (ph 7.4) without or with ALP (1 U/mL) after 24 h; Scale bar is 100 nm. S7

8 Figure S2. TEM images of L-1P and D-1P (100 µm) in PBS buffer (ph 7.4) without or with ALP (1 U/mL) after 24 h; Scale bar is 100 nm. Figure S3. TEM images of L-1P and D-1P (50 µm) in PBS buffer (ph 7.4). Scale bar is 50 nm. Figure S4. Time-dependent dephosphorylation process of the precursors L-1P and D-1P at the concentration of 0.1 wt% treated with ALP (0.1 U/mL) at 37 C. The precursors dissolve in PBS (ph 7.4) buffer. S8

9 Figure S5. Size distribution of L-1P (D-1P) at PBS buffer (ph = 7.4) without or with ALP (1U/mL) obtained by dynamic light scattering. Figure S6. TEM images of L-2P and D-2P (50 µm) in PBS buffer (ph 7.4) without or with ALP (1 U/mL) after 24 h; Scale bar is 100 nm. S9

10 Figure S7. Cell viability of Saos2 cells treated with L-1P, D-1P, L-2P, D-2P and L-2P plus 3, D-2P plus 3 (the concentration of 3 is the same as L-2P or D-2P, that is start from 20 μm to 500 μm) at 24, 48 and 72 h. Figure S8. Time dependent intracellular concentration of L-1P/L-1 (D-1P/D-1) inside/outside of Saos2 cell lines. The incubating concentration of L-1P or D-1P is 50 µm. This experiment performs twice. S10

11 Figure S9. Cell viability of HS-5 cells or HeLa cells treated with L-1P and D-1P at 24, 48 and 72 h and 48 h cell viability of different cell lines treated with 200 µm of L-1P or D-1P. S11

12 Figure S10. Cell viability of HepG2, T98G and MCF-7 cells treated with L-1P and D-1P at 24, 48 and 72 h. Figure S11. Confocal laser scanning microscopy (CLSM) images of Saos2 cells treated with L-1P or D-1P (the concentration is from 10 µm to 50 µm) lasted two months and washed the compounds, the trypsin-digested-then-reattached cells as the first passages. Scale bar is 25 m. S12

13 Figure S12. Confocal laser scanning microscopy (CLSM) images of Saos2 cells treated with L-1P or D-1P (50 µm) for 4 h at the temperature of 4 C. Scale bar is 20 m. Figure S13. CLSM images (Gray scale) of Saos-2 cells treated with L-1P or D-1P (50 µm) for 1 h in the absence (control) or presence of the inhibitors EIPA (100 µm, ethyl-isopropyl-amiloride), CPZ (30 µm, chlorpromazine), Filipin Ш (5 µg/ml) and M-βCD (5 mm). Scale bar is 15 µm. S13

14 Figure S14. Cell viability of Saos2 cell line treated by L-1P or D-1P in the presence of phosphatase inhibitors or external ALP for 24, 48 or 72 h ([L-Phe] = [levamisole] = 1 mm, GinnGEL 2Me = 2 µm, [ALP] = 10 U/mL) Figure S15. Confocal laser scanning microscopy (CLSM) images of Saos2 cells treated with L-1P (50 µm) for 4 h in the absence or with ALP or levamisole (TNAP inhibitor, 1 mm) and L-Phe (PLAP inhibitor, 1 mm). Scale bar is 10 m. S14

15 Figure S16. Confocal laser scanning microscopy (CLSM) images of Saos2 cells treated with D-1P (50 µm) for 4 h in the absence or with ALP or levamisole (TNAP inhibitor, 1 mm) and L-Phe (PLAP inhibitor, 1 mm). Scale bar is 10 m. Figure S17. Cell viability of Saos2 cell line treated by L-1P or D-1P in the presence of cell death signaling inhibitors at 24, 48 and 72 h ([zvad-fmk] = 45 μm, [Nec-1] = 50 μm). Figure S18. Time-dependent western blot analysis of cytochrome c (cyt c) from the whole cell fraction (contains both fraction of cytosol and mitochondria) of Saos2 cell treated with L-1P or D-1P (50 µm). S15

16 Figure S19. The resistant test of Saos2 cells treated with L-1P or D-1P for five weeks, and use MTT to test the cell viability of corresponding compounds at 24h, 48h and 72h. Figure S H NMR of L-1P in DMSO-d6. S16

17 Figure S21. 1 H NMR of D-1P in DMSO-d6. Figure S22. 1 H NMR of L-2P in DMSO-d6. S17

18 Figure S23. 1 H NMR of D-2P in DMSO-d6. Figure S24. HPLC trace of L-1P, D-1P, L-2P and D-2P. S18

19 Figure S25. MS spectrum of L-1P, D-1P, L-2P and D-2P (the first peak in L-2P or D-2P is the mass of (M-H) - /2, for L-2P and for D-2P; the other peak is the (M-H) -, for both L-2P and D-2P). LC-MS (ESI) L-1P: calc. M = , obsvd. (M-H) - = D-1P: calc. M = , obsvd. (M-H) - = L-2P: calc. M = , obsvd. (M-H) - = D-2P: calc. M = , obsvd. (M-H) - = (1) Cai, Y.; Shi, Y.; Wang, H.; Wang, J.; Ding, D.; Wang, L.; Yang, Z. Anal. Chem. 2014, 86, S19