Supplementary Information

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1 Supplementary Information Redox-Hypersensitive Organic Nanoparticles for Selective Treatment of Cancer Cells Wei Zhang,, Wenhai Lin,, Qing Pei,, Xiuli Hu, Zhigang Xie, *, and Xiabin Jing State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, 5625 Renmin Street, Changchun, Jilin , P. R. China University of Chinese Academy of Sciences, Beijing , P. R. China * corresponding authors: xiez@ciac.ac.cn

2 Content 1. Experiment section 2. Figure S1. (A) The synthesis routes and (B) 1 H NMR spectrum and (C) ESI-MS spectra of 6,6'-diselanediyldihexanoic acid. 3. Figure S2. (A) 1 H NMR spectrum and (B) MALDI-TOF-MS spectrum of SeBDP. 4. Figure S3. The changes of the diameter and PDI of SeBDP NPs over different time measured by DLS in PBS with FBS (10%) at 37 o C. 5. Figure S4. Zeta potential changes over time of SeBDP NPs after being incubated with 10 mm GSH. 6. Figure S5. (A) The size changes of SeBDP NPs after being incubated with serum containing 10 mm GSH for 24 h. (B) Photos of the nanoparticles in the serum with or without 10 mm GSH after 24 h. The arrows refers to the flocculent suspension appeared in the solution after treatment by GSH. 7. Figure S6. In vitro cytotoxicities of SeBDP NPs toward HeLa cells for 48 h. 8. Figure S7. Flow cytometry analysis of HeLa cells incubated with SeBDP NPs (SeBDP: 2µg/mL) for 2h. 9. Figure S8. (A) The synthesis route and (B) 1 H NMR spectrum of SePTX. 10. Figure S9. (A) TEM image and (B) DLS of SePTX NPs. (C) The changes of the diameter and PDI of SePTX NPs in water and (D) in PBS with FBS (10%) measured by DLS. 11. Figure S10. (A) Size changes of SePTX NPs over time and (B) Size distribution changes of SePTX NPs after 7 h determined by DLS in the presence of different concentrations of GSH

3 12. Figure S11. (A) SEM image and (B) corresponding EDS mapping images of the selected region of the co-nps. 13. Figure S12. TEM image of co-nps after the treatment of 10 mm GSH for 0.5 h. 14. Figure S13. The representative CLSM images of (A) MCF-7 and (B) L929 cells treated with co-nps for 0.5 and 2 h. 15. Figure S14. In vitro cytotoxicities of different concentrations of PTX and co-nps toward L929 cells incubated for 48h. 16. Figure S15. DCF fluorescence in a cell which are marked in the red boxes: (A) HeLa cells, (B) MCF-7 cells, (C) BEAS-2B cells and (D) L929 cells treated with SePTX (PTX: 5 µg ml -1 ) for 4 h (the left one) and without any treatment as the controls (the middle one) and the average green fluorescence intensity of the boxes marked cells (the right one). 17. Figure S16. DCF fluorescence of the tumor cells (HeLa and MCF-7 cells) and normal cells (BEAS-2B cells) of different regions with or without treatment of NPs. 18. Figure S17. Quantitative analysis of DCF fluorescence via flow cytometry of (a)-(c) HeLa cells, (d)-(e) MCF-7 cells and (g)-(i) BEAS-2B cells after the treatment with SePTX NPs (PTX: 5 µg ml -1 ) for 4 h.

4 Experiment section Materials. Selenium powder and sodium borohydride were purchased from Tianjin Fu Chen Chemical Reagents Factory. 6-bromohexanoic acid was purchased from Tianjin Heowns Biochem LLC. Glutathione (GSH) was purchased from Shanghai Yuanye Biological Technology Co., Ltd. N-ethylmaleimide (NEM) was purchased from Shanghai Macklin Biochemical Co., Ltd. 2,7 -dichlorofluorescence diacetate (DCFH-DA) and Lyso Tracker Red were purchased from Shanghai Beyotime Biotechnology Co., Ltd.. O-nitrobenzaldehyde were purchased from Shanghai Sun Chemical Technology Co., Ltd. 4,4-difluoro- 8-(4-isocyanophenyl) -3,5-dimethyl-4- bora-3a,4a-diaza-s-indacene (NC-BDP) was synthesized according to literature methods. 49 All reagents were purchased from commercial sources and used without further treatment, unless indicated otherwise. Characterizations. 1 H NMR spectra were measured in CDCl 3 and DMSO-d 6 at room temperature by an AV-400 NMR spectrometer from Bruker. Size, size distribution and the zeta potential of the NCs were determined by Malvern Zeta-sizer Nano for dynamic light scattering (DLS). The measurement was carried out at 25 o C and the scattering angle was fixed at 90 o. The morphology of the NPs was measured by transmission electron microscopy (TEM) performed on a JEOL JEM-1011 electron microscope operating at an acceleration voltage of 100 kv. To prepare specimens for TEM, a drop of NPs solution (0.1 mg ml -1 ) was deposited onto a copper grid with a carbon coating. The specimens were air-dried and measured at room temperature. SEM micrographs were performed on JEOL JXA-840 under an accelerating voltage

5 of 15 kvx and the elemental mapping was also characterized on it under an accelerating voltage of 20 kvx. For SEM imaging, a drop of freshly prepared sample solution was cast onto a silicon slice, and then Au (1 2 nm) was sputtered onto the grids to prevent charging effects and to improve the image clarity. Synthesis of 6,6'-diselanediyldihexanoic acid. The synthesis of 6,6'-diselanediyldihexanoic acid was according to the literature methods. S1 Disodium diselenium was prepared through a reaction between Se powder and sodium borohydride in water. Se powder (0.32 g, 2 mmol) was firstly diffused in deionized water (5 ml) and sodium borohydride (0.15 g, 4.0 mmol) was dissolved in deioized water (5 ml). Then the sodium borohydride solution was added dropwise into the Se dispersion solution. The Se powder was dissolved with the generation of H 2 and a colorless solution was obtained within 5 min. After the flask was sealed with a rubber plug, the solution was heated to 70 o C. 20 min later the Se powder completely disappeared and the solution turned dark wine. A solution of 6-bromohexanoic acid (0.78g, 4 mmol) in 5 ml refined THF was injected into it under Ar 2 flow. The reaction was performed at 50 o C for 12 h, and then the product was extracted with CH 2 Cl 2 and dried with anhydrous MgSO 4. After evaporation and vacuum drying, the crude product was purified by a silica gel column (CH 2 Cl 2 : MeOH = 5:1). Synthesis of the SeBDP. A mixture of NC-BDP (34.9 mg, 0.1 mmol), 6,6'- diselanediyldihexanoic acid (19.5 mg, 0.05 mmol) and o-nitrobenzaldehyde (22.7 mg, 0.15 mmol) in CH 2 Cl 2 (0.5 ml) was stirred at room temperature for 4 days. Finally,

6 the reaction mixture was chromatographed on a silica gel column (CH 2 Cl 2 : EtOAc = 10:1). Synthesis of the SePTX. Paclitaxel (PTX) (85 mg, 0.1 mmol) was dissolved in refined CH 2 Cl 2 (10 ml), and then 6,6'-diselanediyldihexanoic acid (20 mg, 0.05 mmol), EDC HCl (38 mg, 0.2 mmol) and DMAP (1.2 mg, 0.01 mmol) were added sequentially at room temperature. After stirring for 1h at room temperature, additional EDC HCl (19 mg, 0.1 mmol) and DMAP (1.2 mg, 0.01 mmol) were added and the solution was stirred overnight. The reaction mixture was quenched with a saturated aqueous solution of NH 4 Cl and washed with water and brine. The mixture was then dried over MgSO 4, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified using silica gel column chromatography (EtOAc: hexane =5:1). Preparation of SeBDP NPs. The nano-precipitation method was used to prepare NPs. The SeBDP (2 mg) was dissolved in THF (5 ml). After stirred about 5 min, the solution was added dropwise to deionized water (10 ml) and stirred to evaporate the organic solvent followed by dialysis for 24 h. The concentration of the SeBDP was determined by the UV absorption at 500 nm for the SeBDP, according to the standard calibration curve of SeBDP in DMF: H 2 O=1:1 (v/v). The solution of SeBDP NPs was diluted 10 times with DMF: H 2 O=1: 1 (v/v) and then was measured by UV-vis spectrophotometer performed on Shimadzu UV-2450 PC.

7 Preparation of the SePTX NPs and co-nps. The experiment was similar to the preparation of SeBDP NPs, just the solution was changed to the SePTX or the mixture of SeBDP and SePTX. The concentration of the SePTX NPs was measured by high-performance liquid chromatography (HPLC, Shimadzu, CBM-20A) with an UV-vis detector. The NPs solution was diluted 10 times with mobile phase (methanol: acetonitrile: water = 42.5: 42.5: 15 (v/v/v)) and then was injected through a 20 µl sample loop. The elution rate was 1.0 ml min -1. SePTX was detected at 239 nm. The retention time of SePTX was 6.5 min under these conditions. The concentrations of SePTX and SeBDP of co-nps were determined by HPLC and UV-vis spectrophotometer mentioned above. Reductive behaviors of the SeBDP NPs. For reductive degradation, the SeBDP NPs (2 ml) were added with 10 mm GSH under shake at 37 o C for 0.5 h, and then were measured by DLS. For the reduction sensitive behaviors, the SeBDP NPs (2 ml) was added with 10 mm and 1 mm GSH, respectively, and then the size, PDI and the zeta potential over different time were measured. All the experiments above were also done with the SePTX NPs and co-nps. Cell culture. The human cervical cancer cell line HeLa, human mammary tumor cell line MCF-7 and the mice fibroblast cell L929 were routinely grown in Dulbecco's Modified Eagle Medium (DMEM) (Gibco) containing 10 % fetal bovine serum (FBS) (Hyclone), 100 U ml -1 penicillin, and 100 U ml -1 streptomycin. The human bronchial epithelial cell line BEAS-2B were routinely grown in RPM culture medium (Gibco, USA) containg 10 % FBS, 100 U ml -1 penicillin, and 100 U ml -1

8 streptomycin. All the cells were cultured in a humidified incubator at 37 o C with 5 % CO 2. Confocal laser scanning microscopy (CLSM). The intracellular GSH reduction-responsive disassembly behaviors of SeBDP NPs were determined by confocal laser scanning microscopy (CLSM) toward HeLa cells. The cells were seeded in 6-well plates at about 500,000 cells per well in 2 ml complete DMEM containing 10% FBS, supplemented with 100 U ml -1 penicillin and 100 U ml -1 streptomycin, and incubated at 37 o C in 5 % CO 2 atmosphere for 24 h. After removing culture medium, the cells were then treated with 10 mm GSH for 2 h and NEM for 15 min. Then the cells were washed with PBS and incubated at 37 o C for additional 2 h with SeBDP NPs at a final concentration of 2 µg ml -1 in complete DMEM. Cells without any additional treatment were used as the control. Then the culture medium was removed and cells were washed with PBS three times. Thereafter, the cells were fixed with 4% formaldehyde for 10 min at room temperature, and the cell nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI, blue). CLSM images of cells were obtained with an Olympus FluoView 1000 CLSM. Cellular uptake was quantified by flow cytometry, which collected 5,000 cells per each sample. Cells were seeded in 6-well plates at the density of 500,000 cells per well in 1.5 ml medium and cultured for 24 h. The medium was replaced with SeBDP NPs at the concentration of 2 µg ml -1. After incubation at 37 o C for 2 h, the cells were washed with PBS and treated with trypsin. The harvested cells were suspended in PBS and centrifuged at 1000 rpm for 5 min. The supernatants were discarded and the cells were washed again with PBS

9 to remove the medium. After washing, the cells were re-suspended in 1 ml PBS before analysis by FCM (Berkman, California, USA). We also studied the cellular uptake of co-nps (SeBDP: 2 µg ml -1 ) by the human mammary tumor cell line MCF-7, the mice fibroblast cell L929 and the human bronchial epithelial cell line BEAS-2B with CLSM. The similar experiment was done according to the experimental method above. For the colocalization of lysosomes, the Lyso tracker red (Beyotime Biotechnology Co., Ltd.) was also used to stain lysosomes and the similar experiment was done as metioned above. Cell viability assays. The biocompatibility of the SeBDP NPs was assessed with methyl tetrazolium (MTT) viability assay against HeLa. The cells were seeded in 96-well plates at about cells per well in 100 µl complete DMEM, and incubated at 37 o C in 5 % CO 2 atmosphere for 24 h. After removing culture medium, SeBDP NPs diluted in complete DMEM (100 µl) were added to cell wells with various concentrations from 0.5 to 25 µg ml -1. The cells were incubated for another 48 h. After the culture medium was removed, 200 µl fresh DMEM was added and then 20 µl of MTT was added to cell wells and cells were incubated for another 4 h. The absorbance of the solution was measured on a Bio-Rad 680 microplate reader at 490 nm. Cell viability (%) was calculated based on the following equation: (A sample /A control ) 100 %, where A sample and A control denote as absorbancies of the sample well and control well, respectively.

10 The cytotoxicities of SePTX NPs and co-nps toward the human cervical cancer cell line HeLa, human mammary tumor cell line MCF-7, the mice fibroblast cell L929 and the human bronchial epithelial cell line BEAS-2B were also assessed with MTT. The similar experiment was done according to the experimental method above. Intracellular ROS assays. The intracellular generation of ROS was determined by the fluorescence by CLSM and flow cytomerty. HeLa, MCF-7, L929 and BEAS-2B cells were treated with SePTX NPs (PTX: 5 µg ml -1 ) for 4 h. Then, the medium was replaced. DCFH-DA (final concentration 10 µm) was added and the cells were incubated for 20 min. Later, the DCFH-DA solution was removed and then repeatedly washed 3 times with PBS. Subsequently, another 1 ml PBS was added and the cells were incubated for additional 20 min. Finally, the cells were observed as soon as possible via CLSM (ex=488 nm). For the quantitative analysis of the DCF fluorescence of the different cells (HeLa, MCF-7 and BEAS-2B cells), we used the flow cytometry and similar experiment was done according to the experimental method above.

11 Figure S1. (A) The synthesis routes and (B) 1 H NMR spectrum and (C) ESI-MS spectrum of 6,6'-diselanediyldihexanoic acid.

12 Figure S2. (A) 1 H NMR spectrum and (B) MALDI-TOF-MS spectrum of SeBDP.

13 Figure S3. The changes of the diameter and PDI of SeBDP NPs over different time measured by DLS in PBS with FBS (10%) at 37 o C. Figure S4. Zeta potential changes over time of SeBDP NPs after being incubated with 10 mm GSH.

14 Figure S5. (A) The size changes of SeBDP NPs after being incubated with serum containing 10 mm GSH for 24 h. (B) Photos of the nanoparticles in the serum with or without 10 mm GSH after 24 h. The arrows refers to the flocculent suspension appeared in the solution after treatment by GSH. Figure S6. In vitro cytotoxicities of SeBDP NPs toward HeLa cells for 48 h.

15 Figure S7. Flow cytometry analysis of HeLa cells incubated with SeBDP NPs (SeBDP: 2µg/mL) for 2 h. Figure S8. (A) The synthesis route and (B) 1 H NMR spectrum of SePTX.

16 Figure S9. (A) TEM image and (B) DLS of SePTX NPs. (C) The changes of the diameter and PDI of SePTX NPs in water and (D) in PBS with FBS (10%) measured by DLS. Scale bar: 500 nm. Figure S10. (A) Size changes of SePTX NPs over time and (B) Size distribution changes of SePTX NPs after 7 h determined by DLS in the presence of different

17 concentrations of GSH (The size distribution of 10 mm GSH was out of the range of the equipment, thus the results were submitted). Figure S11. (A) SEM images and (B) corresponding EDS mapping images of the selected region of the co-nps. Figure S12. TEM image of co-nps after the treatment of 10 mm GSH for 0.5 h.

18 Figure S13. The representative CLSM images of (A)MCF-7 and (B) L929 cells treated with co-nps for 0.5 and 2 h. For each panel, the images from left to right show cell nuclei stained by DAPI (blue), BDP fluorescence in cells (green) and overlays of both images. Scale bar: 20 µm.

19 Figure S14. In vitro cytotoxicities of different concentrations of PTX and co-nps toward L929 cells incubated for 48 h.

20 Figure S15. DCF fluorescence in a cell which are marked in the red boxes: (A) HeLa cells, (B) MCF-7 cells, (C) BEAS-2B cells and (D) L929 cells treated with SePTX (PTX: 5 µg ml -1 ) for 4 h (the left one) and without any treatment as the controls (the middle one) and the average green fluorescence intensity of the boxes marked cells (the right one). Scale bar: 20 µm.

21 Figure S16. DCF fluorescence of the tumor cells (HeLa and MCF-7 cells) and normal cells (BEAS-2B cells) of different regions with or without treatment of NPs. Figure S17. Quantitative analysis of DCF fluorescence via flow cytometry of (a)-(c) HeLa cells, (d)-(e) MCF-7 cells and (g)-(i) BEAS-2B cells after the treatment with SePTX NPs (PTX: 5 µg ml -1 ) for 4 h.

22 Table S1. The zeta potential of the nanoparticles SeBDP NPs SePTX NPs co-nps Zeta potential (mv) Table S2. The IC50 value of different cells after treatment of co-nps for 48h HeLa MCF-7 BEAS-2B L929 IC50 (µg/ml) >5 >5 Reference: (S1) Klayman, D. L.; Griffin, T. S., Reaction of Selenium with Sodium Borohydride in Protic Solvents. A Facile Method for the Introduction of Selenium into Organic Molecules. J. Am. Chem. Soc. 1973, 95,