Supporting Information

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1 Supporting Information Rational Design of Polyphenol-Poloxamer Nanovesicles for Targeting Inflammatory Bowel Disease Therapy Xinyu Wang *, Jun-Jie Yan, Lizhen Wang, Donghui Pan, Runlin Yang, YuPing Xu, Jie Sheng, Qianhuan Huang, Huimin Zhao, Min Yang* Xinyu Wang and Jun-Jie Yan contributed equally on this manuscript.

2 EXPERIMENTAL SECTION Materials. Tannic acid, Pluronic F-127, Pluronic F-68, esterase from porcine liver, IR780 were purchased from Sigma-Aldrich (USA). Dexamethasone and (+)-Catechin were obtained from Macklin Reagent Co., Ltd. (Shanghai, China). (-)-Epigallocatechin gallate was bought from J&K Chemical Ltd. (USA). Dextran sodium sulfate (molecular weight: 36,000 50,000 Da) were purchased from MP Biomedicals Inc. (USA). Tube-O-DIALYZER TM Micro Dialysis System (MWCO 50 kd) were purchased from Sangon Biotech (Shanghai, China). All animal studies were conducted under a protocol approved by the local animal welfare committee and performed according to national regulations. Measurements. Transmission electron microscopy (TEM) analysis was performed on JEM-2100 transmission electron microscopy (JEOL, Japan). Atomic force microscopy (AFM) analysis was performed on Dimension Icon atomic force microscopy (Bruker, German). Dynamic light scattering (DLS) measurements were performed with a Zetasizer Nano ZS (Malvern Instruments, Ltd.). In vivo fluorescence imaging were performed with IVIS spectrum imaging system (Perkin Elmer Inc.) and analyzed by Living Image software (Xenogen, CA). Ultraviolet-visible (UV-Vis) absorption and transmittance of the sample solutions were measured with a Lambda25 UV-visible spectrophotometer (Perkin Elmer Inc.). High-performance liquid chromatography was performed with Waters 1525 equiped with 2998 photodiode array detector. Preparation of PPNP and drug-loaded PPNP. One type of polyphenols (CAT, EGCG or TA) dissolved in DMSO was mixed with one type of polymers (PEG, F-127 or F-68) dissolved in DMSO at a weight ratio of 1:1 (20 mg/ml). (Tips: TA should be dissolved just before it was used.) Afterwards, 400 µl of the solutions were added dropwise to 40 ml DI water under vigorous magnetic stirring. The PPNP solutions were obtained and analyzed by DLS and UV-Vis spectrophotometer immediately. For drug-loaded PPNP preparation, DEX dissolved in DMSO (20 mg/ml) were mixed with TA and F-68 in DMSO (20 mg/ml) at ratios of 1:10 and 1:5 (wt./wt.). Then, 400 µl of the mixed solution was dropped into 40 ml DI water under vigorous magnetic stirring. After that, the solution was stirred for additional 10 min to ensure complete formation of PPNP-DEX. The PPNP were transferred into Tube-O-DIALYZER TM Micro Dialysis System (MWCO 50 kd) and dialyzed against DI water for six times over 24 h. 20 ml of PPNP and PPNP-DEX solutions were lyophilized and weighed to determine the concentration. PPNP-IR780 were obtained with the same procedures of those for PPNP-DEX. The lyophilized PPNP-DEX solutions were dissolved in DMSO and sonicated for 5 min. The DEX concentrations were determined by HPLC analysis. Drug loading efficiency = (weight of DEX in PPNP)/(weight of PPNP-DEX) 100%. Drug encapsulation efficiency = (weight of DEX in PPNP)/(weight of DEX added) 100%. Drug release. To determine the drug release behavior of PPNP-DEX, 5 ml of PPNP-DEX at a concentration of 0.15 mg/ml was loaded into dialysis tubes (MWCO = 4 kd). Simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) without enzyme were prepared according to the previous study. 1 The dialysis tubes were dialyzed against 20 ml SGF buffer (ph = ~1.2) for 4 h and SIF buffer (ph = ~6.8) for another 44 h. For esterase stimulated release, esterase was added into the dialysis tubes to a final concentration of 30 U/mL when changing the dialysis buffer. 0.1 ml of the dialysis solution outside the dialysis tubes was collected every 24 h and then analyzed by HPLC to measure the concentration of released

3 drugs. 0.1 ml fresh PBS buffer was added to the dialysis solution after each measurement to keep the volume in constant. Degradation assay. The absorbance of each sample dialyzed in SIF with or without 30 U/mL esterase was measured at 550 nm. This absorbance corresponds to scattered light from the nanoparticles because TA, F-68, and DEX both do not absorb at 550 nm. Percent degradation was calculated as follows: % degradation = 100 * (A-A )/(A 0 -A ). A is absorbance, A 0 is initial absorbance, and A is absorbance after complete degradation. Antioxidant activities. ABTS and DPPH assays were widely used to access the antioxidant activity of compounds because they can measure both hydrophilic and hydrophobic compounds. 2 ABTS assay was performed with Total Antioxidant Capacity Assay Kit with ABTS method (Beyotime Institute of Biotechnology, Shanghai, China). 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid(abts) can be oxidized into ABTS + with blue color by the oxidant. The experiment was conducted according to the protocol of the kit. The absorbance at 734 nm wavelength of the final solution was detected. DPPH assay was measured according to the method of Brand-Williams et al. 3 Briefly, a 0.06 mm solution of the stable free radical DPPH in ethanol was prepared, and 1.9 ml of this solution was added to 0.1 ml of PPNP-DEX at different concentrations. The absorbance of the solution at 517 nm was determined after 60 min. The antioxidant activity of a series concentrations of trolox was determined at the same time with PPNP-DEX. The antioxidant activity was calculated as Trolox equivalent antioxidant capacity (TEAC). Induction of colitis. Colitis mice model was obtained according to the methods referred in previous study. 4 Animal experiments were carried out in female C57BL/6 mice (6 wk, g). Colitis was induced by replacing the drinking water with 3% (wt./vol) DSS solution. For each of the animal experiments, groups of mice were treated with DSS or normal water for seven days according to the previous study. Mice were observed daily and evaluated for changes in body weight and development of the clinical symptoms of colitis. Cytotoxicity assay. L929 cells were obtained from the Cell Bank in Shanghai Institute of Cell Biology, China. The cells were cultured at 37 C under a humidified 5% CO2 in minimum essential medium (MEM) supplemented with 10% fetal bovine serum and 1% antibiotic solution (Gibco, Invitrogen, USA). The cytotoxicity of PPNP and PPNP-DEX was analyzed on L929 cells by MTT assay. The cells were seeded in 96-well plate with a density of 104 cells per well and incubated overnight at 37 C. PPNPs with different concentrations were added into the wells and incubated with the cells for another 24 hours. After that, the cell viability were tested by MTT assay according to the protocol. In vivo and ex vivo imaging. After receiving DSS solutions for seven days, the mice were orally administered with PPNP-IR780 (200 µl, 0.15 mg/ml). The abdominal hair of mice was removed before fluorescence imaging. The fluorescence images were taken at different set time points. After in vivo images were taken at 24 h, the mice were sacrificed, and the distal colons were separated. The colon contents were removed. The ex vivo images were taken immediately after that. In vivo treatment of DSS induced colitis. After receiving DSS solutions for seven days, the mice were divided into four groups with five mice each group. The drinking water of the mice was water, DEX, PPNP, and PPNP-DEX. The average dosage is 5 ml each day per mouse. The concentrations of DEX and PPNP were 12 µg/ml and 123 µg/ml individually. After drug

4 treatment for 3 days, all the mice were sacrificed and the colons were collected. Half of the fresh colons were isolated for Myeloperoxidase (MPO) activity detection. The MPO Activity was determined by MPO Activity Assay Kit (Abcam, England). The TNF-α activity was determined by ELISA kit (Shanghai Enzyme-linked Biotechnology Co., Ltd.) followed by the protocol. The left colons were fixed in 4% paraformaldehyde and embedded in paraffin. Standard H&E-stained sections were examined. Histology index was determined as the sum of the scores for tissue damage and inflammatory cell infiltration in colons, edema, and erosion; each ranging from 0 to 3. The sum of the two subscores results in a combined score ranging from 0 (no changes) to 6 (widespread cellular infiltrations and extensive tissue damage). 5 Statistical analysis. Statistical analyses were performed using One-way ANOVA with Tukey t-test. P values of * < 0.05, ** < 0.01 and *** < were considered to be statistically significant. All data are presented as mean±sd.

5 Figure S1. Transmittance of the nice PPNP self-assembled by different polyphenols and polymers.

6 Figure S2. FT-IR spectrum of F-68, TA, and PPNP self-assembled by these two compounds.

7 Figure S3. Scattering intensity of PPNP diluted in different ph buffers. The scattering intensity was determined immediately after dilution. Figure S4. UV-Vis spectra (A) and size distribution (B) of PPNP-DEX and PPNP-DEX incubated in SGF for 4 h.

8 Figure S5. The UV-Vis spectrum of F-68, TA, DEX, and PPNP-DEX. Figure S6. Viability of L929 cells incubated with PPNP and PPNP-DEX at various concentrations for 24 h.

9 Figure S7. In vivo fluorescence images of mice after orally delivered PPNP-IR780 for twice. Reference: (1) Valente Nabais, J. M.; Ledesma, B.; Laginhas, C. Removal of amitriptyline from simulated gastric and intestinal fluids using activated carbons. J. Pharm. Sci. 2011, 100, (2) Jiang, H. w.; Yu, C. w.; Yang, T. t.; Hu, J. n.; Liu, R. The evaluation of antioxidant interactions among 4 common vegetables using isobolographic analysis. J. Food Sci. 2015, 80, (3) Brand-Williams, W.; Cuvelier, M.-E.; Berset, C. Use of a free radical method to evaluate antioxidant activity. LWT-Food Sci. Technol. 1995, 28, (4) Wirtz, S.; Neufert, C.; Weigmann, B.; Neurath, M. F. Chemically induced mouse models of intestinal inflammation. Nat. Protoc. 2007, 2, (5) Wirtz, S.; Popp, V.; Kindermann, M.; Gerlach, K.; Weigmann, B.; Fichtner-Feigl, S.; Neurath, M. F. Chemically induced mouse models of acute and chronic intestinal inflammation. Nat. Protoc. 2017, 12,