Supporting Information. Self-supplied tumor oxygenation through separated liposomal delivery of H 2 O 2

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1 Supporting Information Self-supplied tumor oxygenation through separated liposomal delivery of H 2 O 2 and catalase for enhanced radio-immunotherapy of cancer Xuejiao Song 1,2, Jun Xu 2, Chao Liang 2, Yu Chao 2, Qiutong Jin 2, Chao Wang 2, Meiwan Chen 1 *, Zhuang Liu 2 * 1, State Key Laboratory of Quality Research in Chinese Medicine Institute of Chinese Medical Sciences University of Macau, Macau , China 2, Institute of Functional Nano & Soft Materials (FUNSOM) & Collaborative Innovation Center of Suzhou Nano Science and Technology Soochow University, Suzhou , China mwchen@umac.mo zliu@suda.edu.cn Experimental Section Materials Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) was purchased from NOF America Corporation. 1,2-distearoyl-sn-glycero-3-phosphoethanolamineN-[methoxy(polyethylene glycol)-5000] (DSPE-PEG) was purchased from Laysan Bio Inc. Cholesterol (CH), hydrogen peroxide solution (30%) were purchased from J&K Co. Ltd. Potassium iodide (KI, 99.5%) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, C18H16BrN5S, 97.5%) were purchased from Sigma-Aldrich. Catalase solution ( 35,000 units/mg protein) was purchased from Aladdin. Preparation of CAT@Liposome and H 2 O To prepare CAT@Liposome or H 2 O DPPC (17.6 mg), cholesterol (6.17 mg) and DSPE-PEG (5k) (10 mg) at a 6: 4: 0.5 molar ratio were dissolved in 2 ml chloroform. The solution was blow-dried by N 2 to form a lipid film. For fabrication of CAT@Liposome, a PBS solution containing 5 mg catalase was used for hydration. After being extruded through a 200 nm polycarbonate filter for 20 times, the suspension was purified by Sephacryl S-300 high resolution

2 column (GE Healthcare). For fabrication of H 2 O 1 M hydrogen peroxide containing PBS solution was added to the lipid film for hydration. H 2 O was obtained also by purification using Sephacryl S-300 column. DIR labeled liposomes (DiR-CAT@Liposome / DiR-H 2 O was prepared according to our previously developed method 1 by adding DIR to prepare the lipid film (molar ratio of DPPC: cholesterol: DSPE-PEG (5k): DiR = 6: 4: 0.5: 0.5). Characterization The dynamic light scattering (DLS) measurement was carried out with a Malvern Zetasizer (Nano Z90). The morphology was observed under a TEM (Tecnai F20, FEI) after staining liposomes by phosphotungstic acid (1 wt.%) according to the standard procedure. The encapsulation efficiency of CAT was measured by using the standard bicinchoninic acid (BCA) protein assay (Thermo Scientific). Quantitation of H 2 O 2 loaded in Liposome was determined by mixing H 2 O 2 solutions with KI to generate I 3, which could be quantified by UV vis measurements. Cell experiments Murine breast cancer 4T1 cells were cultured under standard conditions. Relative cell viabilities were evaluated using the MTT assay following the standard protocol. To evaluate the cytotoxicity of H 2 O in particular, 4T1 cells ( cells/well) were incubated with H 2 O for 24 h, or incubated with CAT@Liposome for 4 h first and then added with different concentrations of H 2 O for another 20 h of incubation, before the standard MTT assay. The crystal violet staining (CVS) assay is also carried out to determine the cytotoxicity of various liposomes. 4T1 cells ( cells/well) were incubated with CAT@Liposome, free H 2 O 2, H 2 O or CAT@Liposome plus H 2 O After 24 h, each well was carefully washed with PBS solution for 3 times before methanol was added to fix the cells for 15 min. 0.1% crystal violet solution was added for cell staining for 20 min. After removing the crystal violet solution, the plate was washed with water and dried. 0.2 ml 2% SDS (sodium dodecyl sulfate) solution was added to each well to lyse the cells. The absorbance at 570 nm was measured by the microplate reader. Intracellular ROS generation was examined using the DCFH-DA (Sigma-Aldrich) probe following the recommended protocol. Briefly, 10 mmol/l DCFH-DA stock solution (in methanol)

3 was diluted 500-fold in DMEM medium to yield a 20 µmol/l working solution. After incubation with the DCFH-DA working solution for 30 min, CAT@Liposome, H 2 O or CAT@Liposome plus H 2 O were added and incubated for another 2 h. After washing with PBS solution for 3 times, fluorescence was then determined by a confocal and flow cytometry. Animal tumor models Female Balb/c mice and female nude mice were bought from Nanjing Sikerui Biological Technology Co. Ltd and used under protocols approved by Soochow University Laboratory Animal Center. In order to avoid the X-ray caused damages to important organs such as heart and lung, the tumor was inoculated subcutaneously on the back of each mouse in our experiments. For 4T1 tumor model, the 4T1 tumors were generated by subcutaneous injection of 50 µl tumor cell suspension ( cells) onto the back of each female Balb/c mouse. For the PDX tumor model, the primary prostate tumor was directly collected from a patient in SuZhou Municipal Hospital after obtaining informed consent and used under the Good Clinical Practice (GCP) approved by China Food and Drug Administration (CFDA). After mechanically treatment, tumor fragments were homogeneously separated into fractions and subcutaneously implanted on the back of 5 immunodeficient nude mice. When the tumors grow up to about 500 mm 3, each tumor was collected, fragmented, and further implanted subcutaneously on the back of ~8 immunodeficient nude mice, to obtain a sufficient number of mice for the following experiment (two passages for the PDX tumor model). In vivo behaviors of liposome nanoparticles The blood circulation behavior of DiR-CAT@Liposome/ DiR- H 2 O was examined by drawing blood samples from mice at different time points after i.v. injection with DiR-CAT@Liposome or DiR- H 2 O (200 µl of 0.6 mg/ml DiR). Each collected blood sample was solubilized by 0.1 ml of 1% Triton X-100 solution. The fluorescence intensity of DiR was then measured to determine the blood concentrations of DiR, whose unit was presented as the percentage of injected dose per gram tissue (%ID/g). For in vivo biodistribution, tumor-bearing mice were sacrificed at 24 h post i.v. injection of DiR-CAT@Liposome or DiR- H 2 O (200 µl of 0.6 mg/ml DiR). Their main organs and tumors were collected and imaged under a Maestro in vivo optical imaging system (Cambridge Research & Instrumentation, Inc).

4 To determine the catalase activity in blood at different time after injection, blood samples were draw from mice at different time points after i.v. injection with The blood sample was solubilized by 0.1 ml of 1% Triton X-100 solution before being added to 0.5 ml H 2 O 2 solution (50 mm). After reacted at 37 o C for 1 min, the reaction was terminated by adding 0.5 ml ammonium molybdate (32.4 mm) by reacting with the residual H 2 O 2 to form stable primrose complexes. After been cooling down to 25 o C, the blood solution was centrifuged at 5000 rmp for 3 min. The relative catalytic activity was calculated by using the following equation: [Absorbance (Total H 2 O 2 ) - Absorbance (blood sample)]/ (blood weight) - [Absorbance (Total H 2 O 2 ) - Absorbance (blank blood sample)]/ (blood weight). To determine the catalase activity in tumors, tumors with the same weight were obtained and homogenated after i.v injection with CAT@Liposome. The tumor homogenates were added with H 2 O 2, and the dissolved oxygen concentrations in those solutions were measured by the oxygen probe. Evaluation of tumor hypoxia relief Six mice bearing 4T1 tumors were i.v injected with 200 µl CAT@Liposome (CAT=1.5 mg/ml) and randomly divided into two groups. For the second group, the mice were i.v. injected with 200 µl H 2 O (H 2 O 2 = 50 mm) at 4 h post CAT@Liposome injection. In vivo photoacoustic imaging was carried out for these two groups of mice utilizing the Oxy-hem mode (excitation wavelength = 750 nm and 850 nm), with tumor boundaries outlined according to the ultrasound images. For ex vivo immunofluorescence staining analysis, twelve mice bearing 4T1 tumors were randomly divided into four groups: 1, control group without any treatment; 2, mice with i.v. injection of H 2 O (200 µl, H 2 O 2 = 50 mm); 3, mice with i.v injection with CAT@Liposome (200 µl, CAT= 1.5 mg/ml); 4, mice with the i.v. injection of CAT@Liposome (200 µl, CAT= 1.5 mg/ml) followed by i.v. injection of H 2 O (200 µl, H 2 O 2 = 50 mm) 4 h later. Mice were intraperitoneal injected with pimonidazole hydrochloride (0.6 mg per mouse) and sacrificed 90 min later to collect tumors at 24 h post injection of the first agent. The obtained tumors were prepared with optimum cutting temperature (OCT) compound (Sakura Finetek) and then cut into 4 µm slices for immunofluorescence staining according to our previously experimental

5 procedure 2. Finally, confocal microscopy was utilized to analyze the stained slices. In vivo radiotherapy For in vivo radiotherapy, mice bearing 4T1 tumors or prostatic PDX tumors were randomly divided into 5 group (six mice each group): 1, control group with PBS injection (Control); 2, i.v injection of CAT@Liposome (200 µl, CAT = 1.5 mg/ml) plus the subsequent injection of H 2 O (200 µl, H 2 O 2 = 50 mm) 4 h later (Both, X-ray -); 3, PBS injection plus X-ray radiation (X-ray +); 4, i.v injection of CAT@Liposome (200 µl, CAT=1.5 mg/ml) and then irradiated by X-ray (CAT@Liposome, X-ray +); 5, subsequent i.v injection of CAT@Liposome plus H 2 O (4 h later), and with X-ray radiation (Both, X-ray +). The X-ray radiation dose at 8 Gy was given 24 h post injection of the first agent. During X-ray radiation, the mouse was covered by a lead plate to avoid X-ray damages to its important organs, and only a part of its body with the tumor was directly exposed to the X-ray source. After various treatments, the tumor sizes were monitored every 2 days for 14 days and tumor volume was calculated according to the following equation: width 2 length/2. In vivo radiotherapy and immunotherapy To evaluate the immunological effects after i.v injection of Liposome@CAT and Liposome@H 2 O 2, CT26 tumor-bearing mice were randomly divided into 4 groups (four mice each group): 1, control group with PBS injection (Control); 2, i.v injection of CAT@Liposome (200 µl, CAT=1.5 mg/ml) (CAT@Liposome); 3, i.v injection of H 2 O (200 µl, H 2 O 2 = 50 mm) (H 2 O 4, i.v injection of CAT@Liposome (200 µl, CAT = 1.5 mg/ml) plus the subsequent injection of H 2 O (200 µl, H 2 O 2 = 50 mm) 4 h later (Both). At day 3 after injection, mice were sacrificed and tumors were collected for the immunological evaluations. The obtained tumor tissues were stored in PBS solution and cut into small pieces. Then, the single cell suspension was prepared by gentle pressure with the homogenizer without addition of digestive enzyme 55. Finally, IL-10 and IL-12p40 levels were determined by ELISA assay (ebioscience) utilizing the supernatant of tumors. On the other hand, after removal of red blood cells (RBCs) by the RBC lysis buffer, cancer cells were collected and stained with anti-cd206-fitc, anti-cd11b-pe and anti-f4/80-apc (ebioscience) antibodies according to the manufacturer s protocols to evaluate the

6 macrophage polarization. CD11b + F4/80 + and CD11b + F4/80 + CD206 + cells were defined as macrophages and M2 phenotype macrophages, respectively. To carry out in vivo combined radiotherapy and immunotherapy, CT26 tumor-bearing mice were established. The hair on the right flank of the mice was first removed. Balb/c mice subcutaneous tumor models were established by subcutaneous injection of 1x10 6 CT26 cells into the mice. CT26 tumor (~60 mm 3 ) bearing mice were randomly divided into six group (5 mice each groups): 1, control group with PBS injection (Control); 2, i.v injection of anti-ctla-4 (α-ctla-4); 3, X-ray radiation (X-ray); 4, X-ray radiation plus i.v injection of anti-ctla-4 (X-ray, α-ctla-4); 5, subsequent i.v injection of CAT@Liposome plus H 2 O (4 h later), and with X-ray radiation (Both, X-ray); 6, subsequent i.v injection of CAT@Liposome plus H 2 O (4 h later), and with X-ray radiation and i.v injection of anti-ctla-4 (Both, X-ray, α-ctla-4). Anti-CTLA-4 antibody at a dose of 500 µg kg 1 was i.v. injected at day 1, 3, and 5 post X-ray irradiation. Tumor sizes and body weights were monitored every 2 days. At day 7 post radiotherapy, the tumors from different groups were collected, homogenized into single-cell suspensions, and stained with respective antibodies for flow cytometry analysis following the manufacturer s protocols. CD3 + CD4 + FoxP3 + and CD3 + CD8 + cells were defined as Treg and CTL, respectively. In vivo toxicology examinations Healthy Balb/c mice were i.v injected of CAT@Liposome (200 µl, CAT = 1.5 mg/ml) plus the subsequent injection of H 2 O (200 µl, H 2 O 2 = 50 mm) 4 h later. Major organs from these mice were harvested at 1, 3, 7 days post injection for the standard H&E staining. Meanwhile, blood samples were collected (~0.8 ml for each mouse) for blood biochemistry assay and complete blood panel test, which were conducted in Shanghai Research Center for Biomodel Organism. Reference 1. Feng, L. Z.; Gao, M.; Tao, D. L.; Chen, Q.; Wang, H. R.; Dong, Z. L.; Chen, M. W.; Liu, Z. Adv. Funct. Mater. 2016, 26, (13), Gong, H.; Chao, Y.; Xiang, J.; Han, X.; Song, G. S.; Feng, L. Z.; Liu, J. J.; Yang, G. B.; Chen, Q.; Liu, Z. Nano Lett 2016, 16, (4),

7 Supporting Figures Figure S1. (a) TEM images of H 2 O and CAT@Liposome stained with phosphotungstic acid. (b) Hydrodynamic diameters of H 2 O and CAT@Liposome nanoparticles. (c) The relative enzymatic activity changes of free catalase and CAT@liposome under protease K digestion for different periods of times. (d) Time-dependent changes of dissolved oxygen concentrations in different groups including free CAT, H2O2@Liposome, H2O2@Liposome plus CAT.

8 Figure S2. (a-d) Relative viabilities of 4T1 cells after incubation with free H 2 O 2 and H 2 O at various concentrations for 24 h determined by crystal violet staining assays. Figure S3. (a) The catalase activity in blood at different time after injection. (b) Time-dependent changes of dissolved oxygen concentrations in tumor homogenate with or without CAT@Liposome injected.

9 Figure S4. Distribution of H 2 O and CAT@Liposome nanoparticles in the tumor after separately injection. H 2 O were labeled with DiD, while CAT@Liposome were labeled with FITC. Blood vessels were stained with anti-cd31 antibody.

10 Figure S5. The magnified views of the full-scale immunofluorescence images shown in Figure 3f after different treatments. Figure S6. The relative viable areas in the H&E staining tumor slices in Figure 4d calculated by the Image-Pro Plus software.

11 Figure S7. Ex vivo immunofluorescence images of PDX tumor slices. (a) Representative immunofluorescence images of tumor slices after different treatments. The cell nuclei, blood vessels and hypoxic areas were stained with DAPI (blue), anti-cd31 antibody (red), and anti-pimonidazole antibody (green), respectively. (b) Quantification of tumor hypoxia and blood vessel densities from the images shown in (a) (3 mice per group). Figure S8. The weight of mice in different groups after treatments.

12 Figure S9. (a&b) Gating strategies and representative flow cytometry data of cytotoxic T lymphocytes (CTL) infiltration in tumors. CD3 + CD8 + cells were defined as CTLs. (c&d) Gating strategies and flow cytometry plots of CD4 + FoxP3 + Treg cells in tumors after various treatments indicated.

13 Figure S10. H&E staining slices of major organs (Liver, spleen, kidney, heart and lung) after being subsequently treated by plus H 2 O (4 h later). No noticeable damages could be observed in all major organs.