Electronic Supplementary Information. MMP2-targeting and redox-responsive PEGylated chlorin e6

Transcription

1 Electronic Supplementary Information MMP2-targeting and redox-responsive PEGylated chlorin e6 nanoparticles for cancer near-infrared imaging and photodynamic therapy Wenxiu Hou 1,2, Fangfang Xia 1, Carla S. Alves 3, Xiaoqing Qian 1,2, Yuming Yang 1, Daxiang Cui 1 * 1 Department of Bio-Nano Science and Engineering, Key Laboratory for Thin Film and Microfabrication of Ministry of Education, Research Institute of Micro/Nano Science and Technology, Shanghai Jiao Tong University, Shanghai , P.R. China 2 School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai , P.R. China 3 CQM Centro de Química da Madeira, Universidade da Madeira, Campus da Penteada, Funchal, Portugal * To whom correspondence should be addressed. Tel: ; Fax: addresses: dxcui@sjtu.edu.cn (D. Cui) S-1

2 Experimental Preparation of PEG-SS-Ce6 Nanoparticles: Moreover, the PEGlylation Ce6 (PEG-SS-Ce6) without MMP2-cleavable polypeptide was also synthesized as a control group. In brief, the carboxylic group of Ce6 (10 mg) was activated by NHS (15 mg) and EDC (30 mg) in 5 ml of DMSO for 1 h at room temperature in the dark. Whereafter, the PEG-SS-NH 2 (37.32 mg) dissolved in 5 ml DMSO was added dropwise into the above solution. The reaction mixture was stirred for 24 h at 30 in the dark and then extensively dialyzed against distilled PBS (ph = 8.4) using a dialysis tube (MWCO = 1000 Da) for one day and dialyzed against distilled water for two days and lyophilized to obtain a green powder. Characterization Techniques 1H NMR spectra were acquired using a Bruker Avance-III-HD 600 MHz NMR Spectrometer (Bruker BioSpin Corp., Billerica, MA, USA). Fourier transform infrared (FT-IR) spectra were recorded on a Bio-Rad WinIR instrument using potassium bromide method. UV-Vis spectra were measured with a Varian Cary 50 spectrophotometer (Varian Inc., Palo Alto, CA, USA). The size and morphology were characterized by TEM on a JEM-2100F (JEOL, Japan). DLS (dynamic light scattering) measurements were completed using a NICOMP 380 ZLS Zeta potential/particle sizer (PSS Nicomp, Santa Barbara, CA, USA). PL spectra were recorded on a Hitachi FL-4600 spectrofluorometer. The Redox-responsive Behavior of PEG-SS-Ce6-MMP2 NPs in Vitro The release profile of Ce6 from PEG-SS-Ce6-MMP2 NPs was evaluated using a dialysis method. PEG-SS-Ce6-MMP2 NPs solution (Ce6 concentration 600 µg/ml) were transferred into a S-2

3 dialysis tube (MWCO = 3500 Da). Then, it was immersed in a tube containing 80 ml of phosphate buffered saline (PBS, ph = 7.4) without 10 mm DTT or PBS with 10 mm DTT, and incubated at 37 with continuous shaking of 110 rpm. At desired time intervals, 1 ml of the external buffer was taken out and an equal volume of fresh PBS was returned to the system. The amount of Ce6 released was measured by UV-Vis spectra with a Varian Cary 50 spectrophotometer (Varian Inc., Palo Alto, CA, USA. λ = 405 nm). The release profile of Ce6 from PEG-SS-Ce6 NPs was also measured according to the above method. The singlet oxygen sensor green (SOSG) reagent was employed for detecting the 1 O2 generation of the nanoprobes treated with DTT (excitation = 494 nm). Typically, nanoprobes of PEG-SS-Ce6 NPs and PEG-SS-Ce6-MMP2 NPs (Ce6, 5 µg/ml) treated with 10 mm DTT for 3 h, respectively, and then SOSG under the concentration of 2.5 µm was introduced to measure the 1 O2 generation of the nanoprobes under light irradiation at 633 nm (50 mw/cm2) for 10 min. Tumor-targeting Efficiency in Tumor Bearing Mice The A549 tumor-bearing mice were randomized into five groups of 3 animals per group to estimate the in vivo photodynamic therapy efficiency, when the tumor reach the size of ~100 mm 3. In brief, (1) PBS (150 µl) without laser, (2) PBS (150 µl) with laser, (3) free Ce6 (5 mg/kg) upon laser irradiation, (4) PEG-SS-Ce6 NPs (equivalent Ce6 5 mg/kg), (5) PEG-SS-Ce6-MMP2 NPs (5 mg/kg of Ce6 equivalents) with laser irradiation were injected into the tail vein, respectively. For the irradiated groups, a 633 nm He-Ne laser (50 mw/cm 2, 30 min) was executed after 10 h and 24 h of intravenous injection. The tumor images of A549 tumor-bearing mice were taken at the day before the tumors were exposed to the irradiation (0 day) and at 12 h (1 day), 4 days (4 day), 14 days (14 day) after irradiation, respectively.. The tumor size was measured by a caliper every three days after treatment. S-3

4 Figure S1. Synthetic scheme of the PEG-SS-Ce6-MMP2 conjugates. S-4

5 Figure S2. 1 H NMR spectra of (A) PEG-SS-NH 2 (DMSO-d 6 ), (B) Ce6 (DMSO-d 6 ), (C) PEG-SS-Ce6 (DMSO-d 6 ) and (D) Ce6-MMP2 (DMSO-d 6 ). S-5

6 Figure S3. 1 H NMR spectra of PEG-SS-Ce6-MMP2 in DMSO-d 6 (A) and in D 2 O (B) S-6

7 Figure S4. FTIR spectra of Ce6, Ce6-MMP2, PEG-SS-NH 2, PEG-SS-Ce6 and PEG-SS-Ce6-MMP2, respectively. Figure S5. UV Vis absorption spectra of Ce6, PEG-SS-Ce6 NPs and PEG-SS-Ce6-MMP2 NPs. S-7

8 Figure S6. Characterization of the nanoprobes. (A) TEM images of the PEG-SS-Ce6 NPs and (B) Hydrodynamic diameter of the PEG-SS-Ce6 NPs. Figure S7. Relationship between the fluorescent intensity ratio (I3/I1) and PEG-SS-Ce6 concentration in water. The CAC value is about 26.2 µg/ml of PEG-SS-Ce6. S-8

9 Figure S8. In vitro release profile of PEG-SS-Ce6 NPs and PEG-SS-Ce6-MMP2 NPs in PBS (ph 7.4) in the presence and absence of DTT. S-9

10 Figure S10. Flow cytometry analysis of the median fluorescence of A549 cells after treated with PEG-SS-Ce6 NPs or PEG-SS-Ce6-MMP2 NPs for 4h and 12h, respectively. Figure S11. In vivo therapeutic efficacy of PEG-SS-Ce6-MMP2 NPs. Tumor images after photodynamic therapy with free Ce6 or PEG-SS-Ce6-MMP2 NPs or PEG-SS-Ce6 NPs in A549 tumor -bearing mice S-10

11 Figure S12. H&E stained tissue sections from the heart, liver, spleen, lung, kidney, and brain of the mouse after 30 days post-treatment of PEG-Ce6-MMP2. Scale bar, 100 µm. S-11