Polymer-Caged Nanobins for Synergistic Cisplatin-Doxorubicin Combination Chemotherapy

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1 S1 Polymer-Caged Nanobins for Synergistic Cisplatin-Doxorubicin Combination Chemotherapy Sang-Min Lee, Thomas V. O Halloran,* and SonBinh T. Nguyen* Department of Chemistry and the Center of Cancer Nanotechnology Excellence, Northwestern University, 2145 Sheridan Road, Evanston, Illinois Table of Contents Page S0. Additional supporting figures for the manuscript S1 S1. Spectroscopic study of ligand dissociation of [Pt II (NH 3 ) 2 (N -AcLys)] under acidic conditions S2 S2. Therapeutic efficacy of Pt-PCN DXR against MCF-7 S4 References S5 S0. Additional supporting figures for the manuscript Figure S1. In vitro cytotoxicity profiles of sodium poly(acrylate) against HeLa human cervical cancer cell line. Cells were incubated for 48 h in the cell culture media including sodium poly(acrylate) with the molecular weight of either 2.1 or 5.1 kda. Figure S2. DLS profiles of PCN DXR before and after Pt-conjugation showing similar particle size distributions. DLS profile of bare liposome templates (BL DXR ) is also presented for comparison.

2 S2 S1. Spectroscopic study of ligand dissociation of [Pt II (NH 3 ) 2 (N α -AcLys)] under acidic conditions The acid-sensitive dissociation of N α -AcLys ligand from [Pt II (NH 3 ) 2 (N α -AcLys)] was observed by UV-vis spectroscopy during the incubation of cis-ptlys in ph 4.0-buffered solution (150 mm NaCl) at 37 o C. Because different Pt compounds such as cis-ptlys, cis-[pt II (NH 3 ) 2 (H 2 O) 2 ] 2+, and [Pt II (NH 3 ) 2 Cl 2 ] (cisplatin), present different absorption spectra (Figure S3A) due to the shift of d-d bands, S1 the ligand exchange can be monitored by absorbance changes at 246 nm (Abs 246 ), where the maximum intensity change is observed. S2 Indeed, temporal decrease of Abs 246 was observed during the incubation of cis-ptlys in ph 4.0 conditions (Figure S3B), suggesting the dissociation of N α -AcLys ligand from the Ptprodrug complex (Scheme S1) and the subsequent formation of cisplatin in acidic NaCl solution (150 mm NaCl). Figure S3. (A) UV-vis spectra of [Pt II (NH 3 ) 2 (N α -AcLys)] (cis-ptlys), cis-[(nh 3 ) 2 (H 2 O) 2 Pt II ] 2+ (diamminediaquo Pt II ), and [Pt II (NH 3 ) 2 Cl 2 ] (cisplatin). (B) Time-dependent absorbance (246 nm) change of cis-ptlys incubated in ph 4.0 buffered solution (150 mm NaCl). The inset shows the spectra observed at the corresponding incubation times. Figure S4. ESIMS spectrum of Pt II species released from a sample of Pt-PCN ((Pt/P = 1.29) that has been dialyzed against ph 5.0-bufferred solutions (150 mm NaCl) at 37 o C. Under such high-salt, acidic condition, the release rate of Pt II (NH 3 ) 2 Cl 2 (cisplatin) from acetyl-lysine ligand can be accelerated (Scheme S1a). Alternatively, Pt II (NH 3 ) 2 (H 2 O) 2 can form, S2 which reacts with free Cl - ions to yield Pt II (NH 3 ) 2 Cl 2 (Scheme S1b). Also shown is theoretical isotopic pattern of cisplatin (Inset).

3 S3 Scheme S1. (A) Our proposed mechanism for the release of Pt II (NH 3 ) 2 Cl 2 from the N α -acetylamido ligand in a high Cl - environment. (B) Proposed mechanism for Pt-release from the N α -acetylamido ligand in water, S2 which then reacts with Cl - to form Pt II (NH 3 ) 2 Cl 2. Figure S5. Comparison of cumulative release of Pt and DXR from Pt-PCN DXR (Pt/DXR = 5.9:1) at ph 5.0 and 37 o C. The apparent release rates (k apr ) of each agent are indicated in the legend. Pt/DXR ratio Table S1. Combination index (C.I.) values of Pt-PCN DXR and free drug combination against OCVAR- 3 and MDA-MB-231 human cancer cell lines. OVCAR-3 MDA-MB-231 Pt/DXR Pt-PCN DXR Free Drugs Pt-PCN DXR Free Drugs ratio IC 25 IC 50 IC 75 IC 25 IC 50 IC 75 IC 25 IC 50 IC 75 IC 25 IC 50 IC

4 S4 Figure S6. Fluorescence-activated cell sorter (FACS) plots for MCF-7 cells that were treated for 48 h with drug-free media (Ctrl.), 20 μm solution of cisplatin (CDDP), 20 μm solution of cis-ptlys, and 20 μm solution of Pt-conjugated PCNs (Pt-PCN emp ). S2. Therapeutic efficacy of Pt-PCN DXR against MCF-7 While caspase-3-deficient MCF-7 breast cancer cell line responds to the [Pt + DXR] combination in Pt-PCN DXR, the cellular response level is only boosted to enhanced potency (lower cell viability) at high drug-dose range and without shifting the responsive curve S3 (see red box in Figure S9A). As caspase-3, a member of the cysteine-aspartate protease family, plays a critical role in chemotherapy-responsive cellular apoptosis, S4 MCF-7 cells, which intrinsically lack caspase- 3, do not undergo the DNA fragmentation S5 associated with the apoptosis induced by CDDP S6 or DXR. S7 Instead, MCF-7 was known to undergo senescence as a cell death pathway in response to chemotherapy. S8, 9 This is verified by the negligible apoptosis observed in MCF-7 cells treated with our Pt-PCNs by Annexin-V assay (Figure S10). This data allows the abnormal therapeutic behavior observed for Pt-PCN DXR in Figure S9A to be attributed to the absence of caspase- 3 in MCF-7 cells. Figure S7. (A) Dose-effect profiles and (B) combination index plots for MCF-7 cells that were treated with Pt-conjugated, DXR-loaded PCNs (Pt-PCN DXR ). Cells were exposed to Pt-PCN DXR with various Pt/DXR ratios for 72 h.

5 S5 Figure S8. Comparison of the apoptotic effects of each drug formulation against MCF-7 cells, as evaluated by Guava Nexin assay. (A-E) Fluorescence-activated cell sorter (FACS) plots for MCF-7 cells that were treated for 48 h with: drug-free media (A), 20 μm solution of cisplatin (CDDP) (B), 50 μm solution of CDDP (C), 20 μm solution of Pt-conjugated PCNs (Pt-PCN emp ) (D), and 50 μm solution of Pt-PCN emp (E). In each panel, the lower-left (Annexin-V -, 7-AAD - ), lower-right (Annexin-V +, 7-AAD - ), and upper-right (Annexin-V +, 7-AAD + ) quadrants represent live cells, early apoptotic cells, and late apoptotic/dead cells, respectively. (F) Cumulative bar graphs of apoptotic and dead cell populations as a function of the Pt concentration in Pt-PCNs and CDDP. References: S1. Nishiyama, N.; Yokoyama, M.; Aoyagi, T.; Okano, T.; Sakurai, Y.; Kataoka, K., Langmuir 1998, 15 (2), S2. Hoch, J.; Milburn, R. M., Inorg. Chem. 1979, 18 (3), S3. Lehar, J.; Stockwell, B. R.; Giaever, G.; Nislow, C., Nat. Chem. Biol. 2008, 4 (11), S4. Fesik, S. W., Nat. Rev. Cancer 2005, 5 (11), S5. Jänicke, R. U.; Sprengart, M. L.; Wati, M. R.; Porter, A. G., J. Biol. Chem. 1998, 273 (16), S6. Blanc, C.; Deveraux, Q. L.; Krajewski, S.; Jänicke, R. U.; Porter, A. G.; Reed, J. C.; Jaggi, R.; Marti, A., Cancer Res. 2000, 60 (16), S7. Yang, X.-H.; Sladek, T. L.; Liu, X.; Butler, B. R.; Froelich, C. J.; Thor, A. D., Cancer Res. 2001, 61 (1), S8. Elmore, L. W.; Rehder, C. W.; Di, X.; McChesney, P. A.; Jackson-Cook, C. K.; Gewirtz, D. A.; Holt, S. E., J. Biol. Chem. 2002, 277 (38), S9. Rebbaa, A.; Zheng, X.; Chou, P. M.; Mirkin, B. L., Oncogene 2003, 22 (18),