Department of Chemical and Biomolecular Engineering, 4 Engineering Drive 4, National University of Singapore, Singapore,

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1 Supporting Information Targeted Theranostic Platinum(IV) Prodrug with a Built-in Aggregation-Induced Emission Light-up Apoptosis Sensor for Noninvasive Early Evaluation of Its Therapeutic Responses In-situ Youyong Yuan, Ryan T. K. Kwok, # Ben Zhong Tang *,#, and Bin Liu *,, Department of Chemical and Biomolecular Engineering, 4 Engineering Drive 4, National University of Singapore, Singapore, # Department of Chemistry, Institute for Advanced Study, State Key Laboratory of Molecular Neuroscience, Institute of Molecular Functional Materials, Division of Biomedical Engineering, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China SCUT HKUST Joint Research Laboratory, Guangdong Innovative Research Team, State Key Laboratory of Luminescent Materials and Devices, South China University of Technology, Guangzhou, China, Institute of Materials Research and Engineering, 3 Research Link, Singapore, Table of Contents Scheme S1. Synthetic route to TPS-CH 2 N 3. Figure S1. 1 H (A) and 13 C NMR (B) spectra of TPS-CH 2 N 3 in CDCl 3. Figure S2. High resolution mass spectrum (MALDI-TOF) of TPS-CH 2 N 3. Scheme S2. Synthetic route to amine terminated TPS-DEVD. (S4) (S4) (S4) (S5) Figure S3. HPLC spectra of amine terminated TPS-DEVD monitored using S1

2 absorbance at 214 nm (A) or 365 nm (B). Figure S4. 1 H NMR spectrum of amine terminated TPS-DEVD in DMSO-d 6. Figure S5. Mass spectrum of amine terminated TPS-DEVD. Scheme S3. Synthetic route to NHS activated platinum(iv) complex. Figure S6. 1 H NMR spectrum of Pt-2COOH in DMF-d 7. Figure S7. Mass spectrum of Pt-2COOH. (S5) (S5) (S6) (S6) (S6) (S7) Figure S8. HPLC spectrum of Pt-2NHS monitored using absorbance of 214 nm. (S7) Figure S9. 1 H NMR spectrum of Pt-2NHS in DMF-d 7. Figure S C NMR spectrum of Pt-2NHS in DMF-d 7. Figure S11. Mass spectrum of Pt-2NHS. (S7) (S7) (S8) Figure S12. HPLC spectra of TPS-DEVD-Pt-cRGD monitored using absorbance at 214 nm (A) or 365 nm (B). (S8) Figure S13. 1 H NMR spectrum of TPS-DEVD-Pt-cRGD in DMSO-d 6. (S8)Figure S14. Mass spectrum (ESI) of TPS-DEVD-Pt-cRGD (A), the calculated isotope pattern (B), and the observed pattern (C). (S9) Figure S15. Mass spectrum of Pt(DDTC) 2 formed from the adduction of DDTC with the reduced prodrug determined by IT TOF-MS spectrometry. (S9) Figure S16. The chemical structure and mass spectrum of the released apoptois sensor of TPS-DEVD determined by IT TOF-MS spectrometry. (S9) Figure S17. (A) UV-vis absorption spectra of TPS-CH 2 N 3 in THF and TPS-DEVD-Pt-cRGD in DMSO/PIPES (v/v = 1/199); (B) Hydrodynamic diameter of TPS-CH 2 N 3 in DMSO/PIPES (v/v = 1/199) obtained from LLS. (S10) Figure S18. (A) PL spectra of TPS-DEVD-Pt-cRGD and the apoptosis sensor TPS-DEVD in DMSO/PIPES (v/v = 1/199) with NaCl concentrations varying from 0, 240, 480 to 960 mm and in cell culture medium (DMEM). The PL spectrum of TPS-CH 2 N 3 in DMSO/PIPES (v/v = 1/199) is shown for comparison. (B) Fluorescence spectra of TPS-DEVD-Pt-cRGD (10 μm) in DMSO/PBS (v/v = 1/199, ph 7.4) or in DMSO/acetate buffer (v/v = 1/199, ph 5.5). (S10) Scheme S4. The caspase-catalyzed hydrolysis of the apoptosis sensor TPS-DEVD. (S10) S2

3 Figure S19. HPLC spectrum of caspase-catalyzed hydrolysis of TPS-DEVD-Pt-cRGD in the presence of ascorbic acid monitored at absorbance at 365 nm. Figure S20. Mass spectrum of TPS residue. (S11) (S11) Figure S21. Hydrodynamic diameters obtained from LLS for the residue of TPS-DEVD-Pt-cRGD upon ascorbic acid treatment and after caspase-3 cleavage in DMSO/PIPES (v/v = 1/199). (S11) Figure S22. Plot of (I I 0 )/I 0 versus different caspase enzymes, where I and I 0 are the PL intensities at caspase concentrations of 100 and 0 pm, respectively. (S12) Figure S23. CLSM images of TPS-DEVD (10 μm) pre-incubated U87-MG cells in the absence (A) or in the presence ofcisplatin (B) or staurosporine (STS, C). Nuclei were living stained with DRAQ5. D, E, F are the corresponding fluorescence/nucleus overlay images of A, B, C, respectively. All images share the same scale bar (20 μm). (S12) Figure S24. PL intensities of TPS-DEVD-pre-incubated U87-MG cells upon treatment with different amounts of cisplatin. λ ex = 365 nm; λ em = 480 nm. (S12) Figure S25. CLSM images of U87-MG cells upon treatment with TPS-DEVD-Pt-cRGD (5 μm) for 6 h. The cells were pretreated with inhibitor (5 μm) or/and free RGD (25 μm). Nuclei were living stained with DRAQ5. D, E, F are the corresponding fluorescence/nucleus overlay images of A, B, C, respectively. All images share the same scale bar of 20 μm. (S13) Figure S26. Cell viability of U87-MG (A) and MCF-7 (B) cells upon treatment with TPS-DEVD-NH 2 at different concentrations for 72 h. (S13) S3

4 Scheme S1. Synthetic route to TPS-CH 2 N 3. A a, 6H B a Si N 3 b a a' ph, 19H b, 2H b * * Chemical Shift (ppm) Chemical Shift (ppm) Figure S1. 1 H (A) and 13 C NMR (B) spectra of TPS-CH 2 N 3 in CDCl M % m/z Figure S2. High resolution mass spectrum (MALDI-TOF) of TPS-CH 2 N 3. S4

5 Scheme S2. Synthetic route to amine terminated TPS-DEVD. Figure S3. HPLC spectra of amine terminated TPS-DEVD monitored using absorbance at 214 nm (A) or 365 nm (B). Figure S4. 1 H NMR spectrum of amine terminated TPS-DEVD in DMSO-d 6. S5

6 Figure S5. Mass spectrum of amine terminated TPS-DEVD. Scheme S3. Synthetic route to NHS activated platinum(iv) complex. (a) Hydrogen peroxide; (b) succinic anhydride, DMSO; (c) 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, DMF. Figure S6. 1 H NMR spectrum of Pt-2COOH in DMF-d 7. An expanded view is inserted in the figure. S6

7 Figure S7. Mass spectrum of Pt-2COOH. Figure S8. HPLC spectrum of Pt-2NHS monitored using absorbance of 214 nm. Figure S9. 1 H NMR spectrum of Pt-2NHS in DMF-d 7. An expanded view is inserted in the figure. Figure S C NMR spectrum of Pt-2NHS in DMF-d 7. S7

8 Figure S11. Mass spectrum of Pt-2NHS. Figure S12. HPLC spectra of TPS-DEVD-Pt-cRGD monitored using absorbance at 214 nm (A) or 365 nm (B). Figure S13. 1 H NMR spectrum of TPS-DEVD-Pt-cRGD in DMSO-d 6. S8

9 Figure S14. Mass spectrum (ESI) of TPS-DEVD-Pt-cRGD (A) and the calculated isotope pattern (B), the corresponding observed pattern (C). Figure S15. Mass spectrum of Pt(DDTC) 2 formed from the adduction of DDTC with the reduced prodrug determined by IT TOF-MS spectrometry. Figure S16. The chemical structure and mass spectrum of the released apoptois sensor of TPS-DEVD determined by IT TOF-MS spectrometry. S9

10 Figure S17. (A) UV-vis absorption spectra of TPS-CH 2 N 3 in THF and TPS-DEVD-Pt-cRGD in DMSO/PIPES (v/v = 1/199); (B) Hydrodynamic diameter of TPS-CH 2 N 3 in DMSO/PIPES (v/v = 1/199) obtained from LLS. Figure S18. (A) PL spectra of TPS-DEVD-Pt-cRGD and the apoptosis sensor TPS-DEVD in DMSO/PIPES (v/v = 1/199) with NaCl concentrations varying from 0, 240, 480 to 960 mm and in cell culture medium (DMEM). The PL spectrum of TPS-CH 2 N 3 in DMSO/PIPES (v/v = 1/199) is shown for comparison. (B) Fluorescence spectra of TPS-DEVD-Pt-cRGD (10 μm) in DMSO/PBS (v/v = 1/199, ph 7.4) or in DMSO/acetate buffer (v/v = 1/199, ph 5.5). Scheme 4. The caspase-catalyzed hydrolysis of the apoptosis sensor TPS-DEVD. S10

11 Figure S19. HPLC spectrum of caspase-catalyzed hydrolysis of TPS-DEVD-Pt-cRGD in the presence of ascorbic acid monitored at absorbance of 365 nm. Figure S20. Mass spectrum (IT-TOF) of TPS residue. Figure S21. Laser light scattering data (A) and atomic force image (AFM, B) for the residue of TPS-DEVD-Pt-cRGD upon ascorbic acid treatment and after caspase-3 cleavage in DMSO/PIPES (v/v = 1/199). S11

12 Figure S22. Plot of (I I 0 )/I 0 versus different caspase enzymes, where I and I 0 are the PL intensities at caspase concentrations of 100 and 0 pm, respectively. Figure S23. CLSM images of TPS-DEVD (10 μm) pre-incubated U87-MG cells in the absence (A) or in the presence of cisplatin (B) or staurosporine (STS, C). Nuclei were living stained with DRAQ5. D, E, F are the corresponding fluorescence/nucleus overlay images of A, B, C, respectively. All images share the same scale bar (20 μm). Figure S24. PL intensities of TPS-DEVD-pre-incubated U87-MG cells upon treatment with different amounts of cisplatin for 2 h. λ ex = 365 nm; λ em = 480 nm. S12

13 Figure S25. CLSM images of U87-MG cells upon treatment with TPS-DEVD-Pt-cRGD (5 μm) for 6 h. The cells were pretreated with inhibitor (5 μm) or/and free crgd (25 μm). Nuclei were living stained with DRAQ5. D, E, F are the corresponding fluorescence/nucleus overlay images of A, B, C, respectively. All images share the same scale bar of 20 μm. Figure S26. Cell viability of U87-MG (A) and MCF-7 (B) cells upon treatment with the apoptosis sensor TPS-DEVD at different concentrations for 72 h. S13