Supporting Information

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1 Supporting Information For Intracellularly Acid-Switchable Multifunctional Micelles for Combinational Photo/Chemotherapy of the Drug Resistant Tumor Tingting Wang +,#, Dangge Wang +,#, Haijun Yu +, *, Mingwei Wang, Jianping Liu +, Bing Feng +, Fangyuan Zhou +, Qi Yin +, Zhiwen Zhang +, Yongzhuo Huang +, Yaping Li +, * + State key Laboratory of Drug Research & Center of Pharmaceutics, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai , China. Department of Nuclear Medicine, Fudan University Shanghai Cancer Center, Shanghai , China. # Equal contribution to this study. *Corresponding authors: Dr. Haijun Yu (hjyu@simm.ac.cn); Prof. Yaping Li ( ypli@simm.ac.cn), Tel/Fax:

2 Experimental Section Materials. Methoxy polyethylene glycol 5000 (mpeg 114 -OH), copper bromide (CuBr), Pluronic copolymer P85 (PEO 26 -PPO 39 -PEO 26 ), 4-dimethylaminopyridine (DMAP), Triethylamine (TEA), 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), droxybenzotrizole (HOBT), 2-Hydroxyethyl methacrylate (HEMA), anhydrous Dimethylacetamide (DMAc), succine anhydride, Pentamethyldiethylenetriamine (PMDETA), Tert-butyl methacrylate (TBA), 2-(diisopropylamino) ethyl methacrylate (DPA), 2,7 -dichlorfluorescein-diacetate (DCFH-DA) and 9,10-anthracenediyl- bis(methylene) dimalonic acid (ABDA) were all obtained from Sigma-Aldrich (Shanghai, China). LysoTracker green (DND-26) and Hoechst were obtained from Life Technologies (Shanghai, China). Doxorubicin hydrochloride (DOX) was purchased from Shanwei Pharmaceutics Co., Ltd. (Shanghai, China). Photosensitizer chlorin e6 (Ce6) was purchased from Frontier Scientific Inc. (Logan, USA). Reactive Oxygen Species (ROS) Assay Kit was obtained from Beyotime Biotechnology (Shanghai, China). Bafilomycin A1 (Baf-A1) from Gene Operation (Michigan, USA) was dissolved in DMSO at 1.0 mm and used as the stock solution. Dialysis tubing (MWCO 3500 Da) was ordered from Fisher Scientific. Inc. (IL, USA). NH 2 -GFLG-COOH tetrapeptide was synthesized by GL Biochem. Co., Ltd (Shanghai, China). All other reagents were used as received without further purification. Synthesis of Pluronic P85-Conjugated DOX Prodrug (PDOX). Briefly, 15 g of P85 (Mw 4600 Da, 3.3 mmol) was reacted with 1.32 g of succine anhydride (Mw 2

3 100 Da, 13.2 mmol) in 60 ml of anhydrous 1,4-dioxane with the addition of 806 mg of DMAP (Mw Da, 6.6 mmol) and 0.88 ml of TEA (Mw 101 Da, 6.6 mmol). The reaction was continued for 24 h. The solvent was removed under reduced pressure, the reaction mixture was purified by dialyzing against DI water and lyophilized to obtain carboxylated P85. Afterwards, 200 mg of P85-COOH (Mw 4656 Da, mmol) was reacted with 50 mg of NH 2 -GFLG-COOH (Mw 400 Da, mmol) in 10 ml of anhydrous DMAc with the addition of 17.2 mg of EDCI (Mw 191 Da, 0.09 mmol), mg of HOBT (Mw 135 Da, 0.09 mmol) and 72 µl of TEA (Mw 101 Da, 0.52 mmol). The reaction was continued for 24 h. The reaction mixture was then purified by dialyzing against DI water and lyophilized to obtain GFLG conjugated P85. The successful conjugation of GFLG on P85 was confirmed by 1 H NMR spectrum examination. Next, 100 mg of P85-GFLG-COOH (Mw 5038 Da, 0.02 mmol) was reacted with 23 mg of DOX (Mw 580 Da, 0.04 mmol) in 10 ml of anhydrous DMSO with the addition of 7.58 mg of EDCI (0.04 mmol), 5.36 mg of HOBT (0.04 mmol) and 11 L of TEA (0.08 mmol). The reaction was continued for 24 h at RT under Ar protection and then purified by dialyzing against DMSO and water. The final product was obtained by lyophilizing. The successful conjugation of DOX on P85 was confirmed by 1 H-NMR spectrum measurement. The DOX weight percentage in the resulting product was determined to be 8.0 wt.% by fluorescence spectrum measurement. Synthesis of Ce6-Conjugated mpeg 113 -b-p(dpa 45 -r-hema 3 ) and mpeg 114 -b-p(tba 40 -r-hema 3 ) Diblock Copolymers. Two diblock copolymers of 3

4 mpeg 114 -b-p(dpa 50 -r-hema 6 ) (namely PDPA) and mpeg 114 -b-p(tba 50 -r-hema 6 ) (namely PTBA) were synthesized by atom transfer radical polymerization (ATRP) method as we reported previously. [1] Photosensitizer Ce6 was conjugated on the pendant hydroxyl groups of PDPA or PTBA via formation of an ester bond between the carboxyl group of Ce6 and the hydroxyl groups of the diblock copolymer. The reactants were purified by dialyzing against DMSO and high performance liquid chromatography (HPLC) using acetonitrile as the eluent. The chemical structure of the resultant product was examined by 1 H-NMR spectra using d6-dmso as solvent. A Ce6 weight percentage of 8.0 wt% were found in the resulting product as determined using UV-Vis absorption spectra measurement. It was calculated that three molecules of Ce6 were conjugated on each polymer chain. Ce6-conjugated PDPA and PTBA diblock copolymers were referred as PDPC and PTBC, respectively. Cell lines and animals. Parental MCF-7 human breast cancer cell line was obtained from the cell bank of Chinese Academy of Sciences (Shanghai, China). DOX-resistant MCF-7 cell line (MCF-7/ADR) was purchased from Keygen Biotech Co. Ltd (Shanghai, China). MCF-7 cells were cultured in complete RPMI 1640 cell culture medium containing 10% of fetal bovine serum (FBS), 100 U/mL of penicillin G sodium and 100 mg/ml of streptomycin sulfate. MCF-7/ADR cells were maintained in the same culture medium used for MCF-7 cells added with 1.0 µg/ml of DOX. Both kinds of cells were maintained in 37 incubators under a humidified atmosphere with 5% CO 2 supply. All experiments were performed in the logarithmic 4

5 phase of cell growth. The female athymic BALB/c nude mice (4-5 weeks of age, g) and adult Sprague-Dawley (SD) rats (200 ± 20 g) were all purchased from Shanghai Experimental Animal Center (Shanghai, China). All animal procedures were carried out under the guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of the Shanghai Institute of Materia Medica, Chinese Academy of Sciences. Reference [1]. Yu H.; Xu Z.; Wang D.; Chen X.; Zhang Z.; Yin Q.; et al. Polym. Chem. 2013, 4,

6 Supplemental Tables and Figures Table S 1. IC 50 of DOX in MCF-7 and MCF-7/ADR cells determined after 48 h incubation. Table S 2. Pharmacokinetic parameters of DOX, PDOX prodrug and the PDOX/PDPA micelles in rats after i.v. injection. Figure S 1. Synthesis of Ce6-conjugated PDPA diblock copolymer PDPC. Figure S 2. 1 H-NMR spectra of the PDPA diblock copolymer, Ce6 and Ce6-conjugated PDPC diblock copolymer. Figure S 3. Synthesis of Ce6-conjugated PTBA diblock copolymer PTBC. Figure S 4. Particle size distribution of the (A) PTBC and (B) PDPC micelles determined at ph of 7.4 or 6.0; (C-E) Fluorescence spectra of (C) Ce6 and the PDPC micelles at (D) ph 7.4, or (E) ph 6.0; (F) Ce6 loading ratio vs. fluorescence intensity of the PDPC micelles determined at ph 6.0; (G&H) Fluorescence spectra of the (G) PDPC and (H) PTBC micelles. Figure S 5. In vitro stability of the PDPC micelles in Hepes buffer solution (20 mm, ph 7.4 or 6.0) or fresh mouse blood. Figure S 6. (A) T 1 -weighted MR phantom images of a concentration gradient of gadolinium examined at ph 7.4 and 6.0; (B&C) Laser illumination induced significant and comparable temperature elevation of the (B) PDPC and (C) PTBC micelles examined at an identical micelle concentration of 100 g/ml; (D) UV-Vis spectra of the PDPC micelles examined at different ph while an identical micelle concentration of 100 g/ml. 6

7 Figure S 7. Colocalization of the PDPC micelles with the lysosomes as confirmed by CLSM examination. Figure S 8. ICP-MS determined intratumoral Ga 3+ distribution delivered with the PDPC or PTBC micelles. Figure S 9. Influence of P85 concentration on the intracellular uptake and cytotoxicity of free DOX in MCF-7/ADR cells. (A) P85-concentration dependent intracellular accumulation of free DOX in MCF-7/ADR cells; (B) P85 significantly improved the cytotoxicity of DOX to MCF-7/ADR cells in a P85 concentration dependent manner. Figure S 10. Synthesis of the PDOX prodrug. Figure S H-NMR spectra of pluronic P85, P85-COOH, P85-GFLG-COOH and PDOX prodrug. The black arrows in the spectra of PDOX indicated the presence of DOX in the final product. Figure S 12. (A) TEM images of the PDOX and PDOX/PDPC micelles examined at ph 7.4 or 6.0; (B) Hydrodynamic diameter of the PDOX/PDPC micelles; (C) Fluorescence property of the PDOX/PDPC micelles examined at different ph values, the insert shows the fluorescence images of the hybrid micelles examined at ph 6.0, 6.2, 6.4 and 6.6; (D) Acid-triggered ROS generation, and (E) Fluorescence emission of the PDOX/PDPC micelles. Figure S 13. H&E staining of the major organs in the nude mice treated with PDOX/PDPC micelles with or without laser illumination (scale bar 200 m). 7

8 Table S 1. IC 50 of DOX in MCF-7 and MCF-7/ADR cells determined after 48 h incubation. IC 50 ( g/ml) MCF-7 MCF-7/ADR Resistance index (RI) DOX PDOX PDOX/PDPC Table S 2. Pharmacokinetic parameters of DOX, PDOX prodrug and the PDOX/PDPA micelles in rats after i.v. injection. Parameters DOX PDOX PDOX/PDPA C max (µg ml -1 ) 1.1± ± ±3.4 T 1/2α (h) 0.055± ± ±0.02 AUC (0-t) (µg ml -1 h -1 ) 2.8± ± ±2.1 Relative bioavailability* *Determined by normalizing the AUC (0-t) value of DOX. 8

9 Figure S 1. Synthesis of the Ce6-conjugated PDPA diblock copolymer PDPC. Figure S 2. 1 H-NMR spectra of the PDPA diblock copolymer, Ce6 and Ce6-conjugated PDPC diblock copolymer. 9

10 Figure S 3. Synthesis of Ce6-conjugated PTBA diblock copolymer PTBC. 10

11 Figure S 4. Particle size distribution of the (A) PTBC and (B) PDPC micelles determined at ph of 7.4 or 6.0; (C-E) Fluorescence spectra of (C) Ce6 and the PDPC micelles at (D) ph 7.4, or (E) ph 6.0; (F) Ce6 loading ratio vs. fluorescence intensity of the PDPC micelles determined at ph 6.0; (G&H) Fluorescence spectra of the (G) PDPC and (H) PTBC micelles. Figure S 5. In vitro stability of the PDPC micelles in Hepes buffer solution (20 mm, ph 7.4 or 6.0) or fresh mouse blood. 11

12 Figure S 6. (A) T 1 -weighted MR phantom images of a concentration gradient of gadolinium examined at ph 7.4 and 6.0; (B&C) Laser illumination induced significant and comparable temperature elevation of the (B) PDPC and (C) PTBC micelles examined at an identical micelle concentration of 100 g/ml; (D) UV-Vis spectra of the PDPC micelles examined at different ph while an identical micelle concentration of 100 g/ml. 12

13 Figure S 7. Colocalization of the PDPC micelles with the lysosomes as confirmed by CLSM examination. Figure S 8. ICP-MS determined intratumoral Ga 3+ distribution delivered with the PDPC or PTBC micelles. 13

14 Figure S 9. Influence of P85 concentration on the intracellular uptake and cytotoxicity of free DOX in MCF-7/ADR cells. (A) P85-concentration dependent intracellular accumulation of free DOX in MCF-7/ADR cells; (B) P85 significantly improved the cytotoxicity of DOX to MCF-7/ADR cells in a P85 concentration dependent manner. 14

15 Figure S 10. Synthesis of the PDOX prodrug. 15

16 Figure S H-NMR spectra of pluronic P85, P85-COOH, P85-GFLG-COOH and PDOX prodrug. The black arrows in the spectra of PDOX indicated the presence of DOX in the final product. 16

17 Figure S 12. (A) TEM images of the PDOX and PDOX/PDPC micelles examined at ph 7.4 or 6.0; (B) Hydrodynamic diameter of the PDOX/PDPC micelles; (C) Fluorescence property of the PDOX/PDPC micelles examined at different ph values, the insert shows the fluorescence images of the hybrid micelles examined at ph 6.0, 6.2, 6.4 and 6.6; (D) Acid-triggered ROS generation, and (E) Fluorescence emission of the PDOX/PDPC micelles. 17

18 Figure S 13. H&E staining of the major organs in the nude mice treated with PDOX/PDPC micelles with or without laser illumination (scale bar 200 m). 18