Protein Enables Conformation Transition of a Hydrogel Based on Pentapeptide and Boosts. Immune Response in vivo. Supporting Information

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1 Protein Enables Conformation Transition of a Hydrogel Based on Pentapeptide and Boosts Immune Response in vivo Yune Zhao ab*, Zhen Wang c*, Chenyang Mei b*, Zhengxuan Jiang d, Yifan Feng e, Rongrong Gao ab, Qinmei Wang ab#, Jinhai Huang ab# a School of Ophthalmology and Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China. b Key Laboratory of Vision Science, Ministry of Health Peoples Republic of China, Wenzhou, Zhejiang, China. c Education Ministry Key Laboratory of Laboratory Medicine, Zhejiang Provincial People's Hospital, People's Hospital of Hangzhou medical College, Hangzhou, China. d Department of Ophthalmology, The Second Affiliated Hospital of Anhui Medical University, Hefei, Anhui, China. e Department of Ophthalmology, Zhongshan Hospital, Fudan University, Shanghai, China. Supporting Information Materials and general methods Chemicals and animals: 2-Cl-trityl chloride resin ( mmol/g), Fmoc-OSu and other Fmoc-amino acids were obtained from GL Biochem (Shanghai, China). Chemical reagents and solvents were purchased from Alfa (China) and used as received; Paraformaldehyde was obtained from Beijing Solarbio Science & Technology Co., Ltd. Dulbecco s modified Eagle s medium (DMEM), fetal bovine serum (FBS) and penicillin/streptomycin were purchased from Gibco Corporation. Horseradish peroxidaseconjugated goat anti-mouse IgG, IgG1, IgG2a, or IgG2b were obtained from Southern Biotechnologies (AL, USA). Recombinant mouse GM-CSF and IL-4 were purchased from Peprotech (Rocky Hill, USA). Mouse IL-5 and IFN-γ ELISA kits were obtained from Biolegend (CA, USA). Fluorochrome-labeled anti-mouse monoclonal antibodies (FITC-CD40, PE-CD80) were purchased from Biolegend (CA, USA). EndoFit Ovalbumin (endotoxins < 1EU/mg) was purchased from InvivoGen (CA, USA). Alum was purchased from Pierce Biotechnology (IL, USA). Six- to eight-week-old C57BL/6J mice were purchased from Academy of Military Medical Science (Beijing, China), and maintained in specific pathogen-free conditions in the animal facility at Wenzhou Medical University, China. General methods: Compounds were characterized by 1 H NMR (Bruker ARX-400) using DMSO-d6 as the solvent. HPLC was conducted at LUMTECH HPLC (Germany) system using a C18 RP column with MeOH (0.05% of TFA) and water (0.05% of TFA) as the eluents. LC-MS was performed at the

2 LCMS-20AD (Shimadzu) system. HR-MS was got at the Agilent 6520 Q-TOF LC/MS using ESI-L low concentration tuning mix (Lot No. LB60116 was from the Agilent Tech.). Preparation of peptide Synthetic of peptide follows standard solid phase peptide synthesis (SPPS) method by using 2-chlorotrityl chloride resin and the corresponding N-Fmoc protected amino acids with side chains properly protected. Firstly, the C-terminal of the first amino acid was conjugated on the resin. Anhydrous N, N -dimethyl formamide (DMF) containing 20% piperidine was used to de-protect Fmoc group. Next, we use O-Benzotriazol-1-yl-N, N, N, N -tetramethyluronium hexafluorophosphate (HBTU) to couple the next amino acid to the free amino group. Peptides chain was extended according the standard SPPS protocol. Nap was used at the final step. Finally, we use the regent of [TFA (95%): H 2 O (2.5%): TIS (2.5%)] in volume ratio to cleave peptides from resin and the mixture was filtered. Ice cold diethylether was poured into filtrate concentrated by rotary evaporation. The precipitate was centrifuged at 5000 rpm for 10 min. The solid was dried by vaccum pump and then purified by HPLC. Hydrogel formation and hydrogel-based vaccine preparation 5 mg peptide of Nap-HHFF and 2 equiv. Na 2 CO 3 were dissolved in 1 ml PBS (ph=7.4), and then 20 µl NaOH (2 M) was add to the solution to adjust the ph value up to 8.5 at room temperature. After ultrasonication for 5 min, the solution was clear and was placed at room temperature for 3 h resulting in hydrogel. For preparation of gel-based vaccine, 100 µg of OVA solution was added into 100 µl hydrogel and then mixed gently by shaking or vortex. The hydrogel was formed after placing for h at room temperature. Rheology experiment Rheology experiment were done on AR1500ex (TA instrument) system, 40 mm parallel plates were used during the experiment at the gap of 300 µm. The dynamic time sweep was conducted at the frequency of 1 rad s-1 and the strain of 0.5%. Dynamic strain sweep was performed and the strain values within the linear range were chosen for the following dynamic frequency sweep. The gels were also characterized by the mode of dynamic frequency sweep in the region of rad s-1 at the strain of

3 0.5%. The different mechanical properties of the two hydrogels were compared at the parameters of strain of 0.5% and frequency of 1 rad/s. Circular dichroism measurement CD spectra were recorded ( nm) using a BioLogic(MOS-450) spectrometer under a nitrogen atmosphere. The sample was placed evenly on the 1 mm thick quartz cuvette and scanned with 0.1 nm interval for three times. Transmission Electron Microscopy experiment Transmission Electron Microscopy (TEM) samples were prepared at room temperature. A micropipette was to load 5 µl of hydrogel to a carbon coated copper grid. Using 2% of UA to stain the sample for 30 seconds, the excess solution was removed by a piece of filter paper. The samples were conducted by a Tecnai G2 F20 system, operating at 200 kv. Cytotoxicity assay of hydrogels The cytotoxicity was determined by the viability of cells using CCK-8 assay. Raw cells lines were seeded in 96-well plates at cells/well for 12 h (splenocytes, cells/well), and subsequently added different concentration of the gels. After 24 h of incubation, 10 μl of CCK-8 solution was added to each well and incubated at 37 for another 4 h. The absorbance of each well at 450 nm (with reference wavelength of 620 nm) was measured by a multimode microplate reader (BioTek, Synergy 4). The results were calculated as cell viability percentage relative to untreated cells. The cytotoxicity assay was performed by three times and the average value of the three measurements was taken. The effect of hydrogel on antigen uptake by dendritic cells and of DCs maturation BMDCs were obtained and cultured as previously described. Briefly, Bone marrow cells were isolated from C57BL/6J mouse femur and tibia, and then cultured in X-vivo 15 medium containing GM-CSF (20 ng/ml) and IL-4 (10 ng/ml) at 37 for 5-6 day to obtain immature DCs. The immature DCs were treated with free OVA-FITC (1.25 µg/ml) or hydrogel encapsulated OVA-FITC (1.25 µg/ml) at 37 for 0.5 h and then the cells were collected and washed by PBS by three times. Finally, the cellular uptake of

4 OVA-FITC was quantified using flow cytometry. For confocal experiment, 2.5 µg OVA-FITC was added into 12.5 µg Nap-HHFF hydrogel. Then the mixture was mixed and incubated at 37 C for 0.5 h. BMDCs were incubated with free OVA-FITC (1.25 µg/ml) or hydrogel-encapsulated OVA-FITC (1.25 µg/ml) at 37 for 2 h or 4 h, and then stained with lysotracker and hochest. The fluorescence image was recorded by confocal microscope. For BMDCs maturation study, BMDCs were treated with medium (Med), Nap-HHFF hydrogel (80 µg/ml) or LPS (500 ng/ml) at 37 for 24 h. Then, the cells were collected and stained with FITC-CD40 and PE-CD80 antibody at 4 for 0.5 h. The cells were collected and washed with PBS. The expression of CD40 and CD80 on BMDCs were detected by flow cytometry. The cytokines (IFN-γ and IL-6) in the supernatants were measured using ELISA according to the manufacture. The effect of Nap-HHFF hydrogel-based vaccine on antibody production and splenocytes proliferation C57 BL/6J female mice (6-8 week) were s. c immunized with free OVA (20 µg/mouse/dose), NapHHFF hydrogel/ova (5:1, W/W), or Alum/OVA (25:1, W/W) on day 0 and day 14 (schematic graph). Seven days after the last immunization, the blood was collected and anti-ova antibody titers (IgG, IgG1, IgG2b, IgG2c) in the serum were detected using ELISA. Anti-OVA titer was defined as the reciprocal sample dilution corresponding to a two-fold higher OD than that of the negative sera. The animal facility operates under the Government license no. SYXK All animals were treated according to the regulations of Chinese law and the local Ethical Committee. At seven days after the second immunization, splenocytes were seeded in a 24-well plate at density of cells/ml, and then incubated with OVA (50 µg/ml) at 37 for 96 h. The production of IFN-γ and IL-5 in the supernatants was measured using ELISA kit. For splenocytes proliferation, splenocytes were seeded in 96-well plate at density of cells/ well in total 100 µl, and then were treated with soluble OVA (50 µg/ml), Medium, or Bovine serum albumin (BSA, 50 µg/ml) at 37 for 72 h. After that, 10 µl CCK-8 solution (Genebio, Shanghai, China) was added into each well and incubated at 37 for another 4 h. The absorbance at 450 nm was detected using Spectra Max MR-5 reader.

5 Schematic graph of animal experiment. Supplementary Figures Fig. S1: 1 H-NMR of molecule 1. 1 H NMR (400MHz, DMSO-d6) δ (s, 2H), (dd, 2H), (dd, 2H), (m, 3H), (s, 1H), (m, 2H), (m, 14H), (m, 4H), (m, 3H), (m, 9H).

6 Fig. S2: MS spectrum of molecule 1. Fig. S3. Strain sweep of hydrogel formed by molecule 1.

7 Fig. S4. Thixotropic properties of hydrogel formed by molecule 1. Fig. S5. Optical image of hydrogel G2 that formed by 1 and OVA. Fig. S6. CD spectrum of OVA (0.5 mg/ml) in PBS buffer

8 Fig. S7. The cytotoxicity of molecule 1 on L929 cells and Raw cells. Bars shown are mean ± SEM. Fig. S8. CLSM images of BMDC cells that were treated with free OVA-FITC at 37 C for 2 h or 4 h, and then were stained with Lysotracker and hochest. Magnification: 63.

9 Fig. S9. The effect of G1 on BMDCs maturation. Bars shown are mean ± SEM. 80 IFN-γ 60 pg/ml OVA G2 Alum Fig. S10. The effect of G2-based vaccine on IFN-γ production by splenocytes. The production of IFN-γ in splenocytes culture supernatants was measured by ELISA. Bars shown are mean ± SEM.

10 OD (450 nm) 0.4 OVA G2 Alum Medium BSA Fig. S11. The effect of different vaccines on splenocytes proliferation. The splenocytes proliferation was measured using CCK-8 analysis ex vivo. Bars shown are mean ± SEM.