Smart Nanoreactors for ph-responsive Tumor Homing, Mitochondria-Targeting, and Enhanced Photodynamic-Immunotherapy of Cancer

Transcription

1 Smart Nanoreactors for ph-responsive Tumor Homing, Mitochondria-Targeting, and Enhanced Photodynamic-Immunotherapy of Cancer Guangbao Yang, Ligeng Xu, Jun Xu, Rui Zhang, Guosheng Song, Yu Chao, Liangzhu Feng, Fengxuan Han, Ziliang Dong, Bin Li,*, and Zhuang Liu*, Institute of Functional Nano & Soft Materials (FUNSOM), Jiangsu Key Laboratory for Carbon-Based Functional Materials & Devices, Soochow University, 199 Ren ai Road, Suzhou, , Jiangsu, China. Orthopaedic Institute, Medical College, Soochow University, Suzhou, Jiangsu, China. * Experimental section 1. Materials (3-aminopropyl)triethoxysilane (APTES), tetraethyl orthosilicate (TEOS), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC-HCl), N-hydroxysuccinimide (NHS), poly(allylamine hydrochloride) (PAH, MW 15000), 2,3-dimethylmaleic anhydride (DMMA), succinic anhydride (SA) were purchased from Sigma Aldrich. PEG polymers were purchased from JiaXingBoMei, China. Catalase (CAT), hydrogen peroxide (H 2 O 2 ) 30 wt % solution, (3-carboxypropyl)triphenylphosphonium bromide (CTPP), chlorin e6 (Ce6) were obtained from J&K chemical CO. 2. Synthesis of CAT@S/Ce6-CTPP/SPEG and CAT@S/Ce6-CTPP/DPEG PAH-DMMA-PEG and PAH-SA-PEG were obtained following the reported method. 1, 2 For 1

2 synthesis of hollow silica nanoparticles, 2.2 mg Ce6, 4 mg NHS, 6 mg EDC and 12 L APTES mixed in 200 L of DMSO for 2 h was then mixed with 100 L TEOS and rapidly added into 10 ml water containing 200 L NaHCO 3 (1 M) and 100 L catalase (10 mg/ml) under magnetic stirring. The solution was stirred for 12 h at room temperature before the prepared CAT@S/Ce6 was collected by centrifugation. (3-Carboxypropyl)triphenylphosphonium bromide (CTPP, 8 mg) pre-dissolved in 200 L DMSO was mixed with EDC (9 mg), NHS (6 mg) and APTES (11 L) for 2 h, and then added into 10 ml of water containing 200 L NaHCO 3 (1 M) and 20 mg CAT@S/Ce6. The solution was stirred for 12 h at room temperature. The prepared CAT@S/Ce6-CTPP was washed and centrifuged three times. Lastly, to prepare CAT@S/Ce6-CTPP/SPEG or CAT@S/Ce6-CTPP/DPEG, 20 mg CAT@S/Ce6-CTPP was dropwisely added into 10 ml of PAH-DMMA-PEG or PAH-SA-PEG (10 mg/ml) aqueous solution, respectively and then stirred for 12 h. The final nanoparticles were washed with water for three times and then stored at 4 o C. Before those nanoparticles were used for further experiments, the samples were centrifuged at 3000 rpm for 5 min to remove possible large aggregates (small amounts of precipitates were observed for certain batches of samples). 3. Synthesis of CAT@S/Ce6/DPEG and BSA@S/Ce6-CTPP/DPEG For the synthesis of CAT@S/Ce6/DPEG, 20 mg of CAT@S/Ce6 was added into 10 ml of PAH-DMMA-PEG aqueous solution under ultrasonication and then stirred for 12 h. The precipitate was obtained by centrifugation at rpm. To prepare BSA@S/Ce6-CTPP/DPEG, 100 L of BSA solution (10 mg/ml) was used to replace catalase. The other steps were identical to the synthesis of CAT@S/Ce6-CTPP/DPEG. 2

3 4. Characterizations TEM images of the nanoparticles were obtained by Transmission electron microscopy (TEM, FEI Tecnai F20). The absorbance spectra of nanoparticles were recorded using a UV-vis-NIR spectrophotometer (Thermo Fisher). The hydrodynamic diameters and zeta potentials of different samples were measured by a Zetasizer Nano-ZS (Malvern Instruments, UK). The dissolved O 2 was measured by a portable dissolved oxygen meter (Rex, JPBJ-608, China). 5. Detection of singlet oxygen The SO generation was tested by previously reported procedure. 3 Free Ce6, BSA@S/Ce6-CTPP/DPEG and CAT@S/Ce6-CTPP/DPEG with or without H 2 O 2 were incubated with SOSG (2.5 M) to test SO generation by irradiated under 660-nm light (5 mw cm -2 ) with various periods of time. The generated SO was determined by measuring recovered SOSG fluorescence under 494 nm excitation. 6. Cellular experiments Murine breast cancer 4T1 cells were cultured under standard medium and conditions. The standard thiazolyl tetrazolium (MTT, Sigma-Aldrich) test was used to measure the cell viabilities. For PDT within hypoxic environments, 4T1 cells were incubated with various nanoparticle formulations in 96-well plates under the hypoxic atmosphere (1 % O 2 and 5 % CO 2 balanced with N 2 ) for 2 h and then exposed to the 660-nm light at a power density of 5 mw cm -2 for 1 h. The 96-well plates by multiple layers of transparent sealing membrane were sealed during the light irradiation. Cells were then replaced with fresh medium and further incubated for another 24 h with the normal 3

4 atmosphere before the MTT assay. For ph-responsive PDT, 4T1 cells were incubated with different concentrations of or under ph 6.8 or 7.4 for 4 h. After removal of nanoparticles, cells were transferred into fresh medium and further irradiated by the 660-nm light at the power density of 5 mw cm -2 for 1 h. The cells were incubated for another 24 h before the MTT assay. For mitochondria-targeted PDT, 4T1 cells were incubated with various concentrations of CAT@S/Ce6/DPEG or CAT@S/Ce6-CTPP/DPEG for 12 h. After removal of nanoparticles, the cells were added into fresh medium and irradiated with 660-nm light (5 mw cm -2, 1 h). The cells were incubated for another 24 h before the MTT assay. For confocal fluorescence imaging, 4T1 cells were cultured with CAT@S/Ce6-CTPP/SPEG or CAT@S/Ce6-CTPP/DPEG (Ce6 = 8 M) under ph 6.8 or 7.4 for 4 h. 4, 6-diamidino-2-phenylindole (DAPI) was used to label cells, which was imaged by a laser scanning confocal fluorescence microscope. Alternatively, 4T1 cells were cultured with CAT@S/Ce6/DPEG or CAT@S/Ce6-CTPP/DPEG (Ce6 = 8 M) for different time points and stained by DAPI and MitoTracker to visualize cell nuclei and mitochondria, respectively, under confocal fluorescence microscope. Mitochondria were stained by MitoTracker (Life Technologies, Catalog number: M7512), strictly following the standard protocol Animal models Female Balb/c mice (18-20 g, 6-8 weeks) were obtained from Nanjing Peng Sheng Biological Technology Co.Ltd, and used under protocols approved by Soochow University Laboratory Animal 4

5 Center. To generate the 4T1 tumor model, cells in 50 L phosphate buffered saline (PBS) were subcutaneously injected into the back of Balb/c mouse. The mice were anesthetized by intraperitoneal injection of pentobarbital sodium (Beijing Zhongcheng Jinnian limited Co.). 8. In vivo pharmacokinetics study To test blood circulation, L of blood were obtained from the tail vein in Balb/c mice at different time intervals after i.v. injection of CAT@S/Ce6-CTPP/DPEG (Ce6 = 5 mg kg -1 ). The Si concentration of blood samples were measured by ICP-AES after being solubilized with aqua regia. For biodistribution study, three 4T1 tumor bearing Balb/c mice were sacrificed at 24 h after i.v. injection of CAT@S/Ce6-CTPP/DPEG (Ce6 = 5 mg kg -1 ). The various organs from mice were then collected and solubilized by aqua regia. The concentrations of Si in those organs was also measured by ICP-AES. 9. In vivo imaging For in vivo imaging, different samples were i.v. injected into mice. In vivo fluorescence imaging was recorded using a Maestro in vivo optical imaging system (Lumina Ⅲ, PerkinElmer, Inc.). For PA imaging, 4T1-tumor bearing mice were accomplished by a Visualsonic Vevo 2100 LAZER system utilizing the Oxy-hem mode (750 and 850 nm). 10. Ex vivo immunofluorescence staining For hypoxia staining, 4T1 tumor bearing mice were treated with PBS, free CAT, BSA@S/Ce6-CTPP/DPEG, or CAT@S/Ce6-CTPP/DPEG (CAT = 3.8 mg kg -1, Ce6 = 5 mg kg -1 ). 5

6 The hypoxia staining of tumor slices was conducted following the reported method In vivo treatment Mice bearing subcutaneous 4T1 tumors were randomly divided into 6 groups (5 mice per group). 200 μl of PBS, free Ce6, CAT@S/Ce6/DPEG, BSA@S/Ce6-CTPP/DPEG, CAT@S/Ce6-CTPP/SPEG, or CAT@S/Ce6-CTPP/DPEG (CAT = 3.8 mg kg -1, Ce6 = 5 mg kg -1 ) was i.v. injected into mice. After 24 h, the tumors of mice were exposed to 660 nm light at the power density of 5 mw cm -2 for 1 h. The weights and tumors of mice were measured every two days for following two weeks. The tumor volume was calculated by the following equation: width 2 length/2. The tumors collected from different groups of mice were stained with H&E and TUNEL. To design the bilateral tumor model, mice were subcutaneously injected with 4T1 cells into the left and right flanks. After the tumor volume reached ~100 mm 3, 200 L of PBS (Group 1) or CAT@S/Ce6-CTPP/DPEG (CAT = 3.8 mg kg -1, Ce6 = 5 mg kg -1 ) (Group 2, 3, 4) were i.v. injected into mice. At 24 h after injection, the left tumors of group 3 and group 4 were irradiated with 660-nm light at the power density of 5 mw cm -2 for 1 h. The left tumor of the mouse was only exposed to the outside. All other parts of the mouse were wrapped with tinfoil in the process of light irradiation. The mice were anesthetized by pentobarbital sodium in the process of light irradiation. Although it would be difficult to rule out the possibility of a small amount of light scattering from the first tumor to the second through the mouse tissues, the optical dose to the second tumor should be several orders of magnitude lower than that for the first tumor under direct light exposure. Then, anti-pd-l1 antibody (BioXcell, product number: BE0101, clone number: 10F.9G2) was i.v. injected into mice for group 2 and 4 at a dose of 750 g/kg at day 1, 3, 5. The length and width of each tumor were monitored 6

7 every two days. At day 7 post irradiation, the sera of mice were collected to evaluate interferon gamma (IFN-γ) by using ELISA assay (ebioscience). At last, cytotoxic T lymphocytes (CTL) infiltration in primary and distant tumors at day 18 post various treatments was evaluated by flow cytometry after staining with anti-cd3-apc (BD Biosciences) and anti-cd8-pe (BD Biosciences). References 1. Wang, C.; Cheng, L.; Liu, Y.; Wang, X.; Ma, X.; Deng, Z.; Li, Y.; Liu, Z. Adv. Funct. Mater. 2013, 23, Feng, T.; Ai, X.; An, G.; Yang, P.; Zhao, Y. ACS Nano 2016, 10, Liu, J.; Yang, G.; Zhu, W.; Dong, Z.; Yang, Y.; Chao, Y.; Liu, Z. Biomaterials 2017, 146, Luo, S.; Tan, X.; Fang, S.; Wang, Y.; Liu, T.; Wang, X.; Yuan, Y.; Sun, H.; Qi, Q.; Shi, C. Adv. Funct. Mater. 2016, 26, Kong, X.; Su, F.; Zhang, L.; Yaron, J.; Lee, F.; Shi, Z.; Tian, Y.; Meldrum, D. R. Angew. Chem. Int. Ed. 2015, 54, Hu, Q.; Gao, M.; Feng, G.; Liu, B. Angew. Chem. Int. Ed. 2014, 53, Song, X.; Feng, L.; Liang, C.; Yang, K.; Liu, Z. Nano Lett. 2016, 16,

8 Figure S1. UV-vis-NIR absorption spectra of free CTPP and 8

9 Figure S2. Average size changes of incubated in H 2 O, PBS and cell medium for 24 h. 9

10 Figure S3. The production of singlet oxygen determined by the increased SOSG fluorescence for free Ce6, and under exposure to the 660-nm LED light (5 mw/cm 2 ) with or without addition of H 2 O 2. 10

11 Figure S4. (a) Blood circulation of after i.v. injection into mice. (b) Biodistribution of in mice. The data were determined by ICP-AES measured Si concentrations. 11

12 Figure S5. H&E stained images of major organs collected from healthy mice and mice after PDT treatment with 12

13 Figure S6. The photos to show the setup for a mouse under light irradiation. Only one tumor from each mouse was exposed to light irradiation, while the rest parts of the mouse body are fully covered during PDT. 13