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1 Supporting information Bioengineered Boronic Esters Modified Dextran Polymer Nanoparticles as Reactive Oxygen Species Responsive Nanocarrier for Ischemic Stroke Treatment Wei Lv 1, 3#, Jianpei Xu 1#, Xiaoqi Wang 1, Xinrui Li 2, Qunwei Xu 1, Hongliang Xin 1 * 1. Department of Pharmaceutics, School of Pharmacy, Nanjing Medical University, Nanjing , China 2. Sir Run Run Hospital, Nanjing Medical University, Nanjing , China 3. Jiangsu Jiangyin People's Hospital, Jiangyin , China # These authors contributed equally to this manuscript. Corresponding author: Hongliang Xin (Tel.: , Fax: , xhl@njmu.edu.cn). 1

2 Experimental Section Synthesis and characterization of SHp-PEG-DSPE: SHp-PEG-DSPE was synthesized with the similar procedure as previously reported. 1 Briefly, maleimide-peg-dspe (Mal-PEG-DSPE, MW of 2000 Da, 4 mg) and SHp (10 mg) were dissolved in N,N-Dimethylformamide (DMF) and PB (0.2 M, ph 7.4), respectively. Then, under the protection of nitrogen, the SHp peptide was covalently conjugated to the maleimide group of Mal-PEG-DSPE by the thiol of the cysteine. 2 The SHp-PEG-DSPE copolymer was dialyzed with a dialysis bag (MWCO of 3.5 kda) against deionized wate. Finally, the resulting SHp-PEG-DSPE was lyophilized and characterized by 1 H-NMR. 3 Hydrolysis of the nanoparticles in 10% FBS:Hydrolysis of the nanoparticles triggered by ROS was performed in 0.01 M PBS (containing 10% FBS and various concentrations of H 2 O 2 ) at 37 ºC. Quantitative experiments were conducted by the measurement of the UV absorbance of nanoparticles at 294 nm and particle size was monitored by DLS at pre-determined time intervals. In vitro cytotoxicity. Cell-based cytotoxicity testing for different nanoparticle formulations was determined using MTT assay. 4,5 Briefly, the BCECs were cultured in 96-well plates at a density of cells/well. After 24 h incubation, the culture medium was replaced by the medium containing a series of dilutions of NP, RBC-NP and SHp-RBC-NP in DMEM with at a polymer concentration of 0.1, 1, 10, 100, 1000 µg/ml, respectively. The UV absorption intensity was measured by a microplate reader (ELX800, Biotek, USA) at 570 nm. 2

3 Statistical analysis: All the results were expressed as mean ± standard deviation (SD). Statistical analysis was performed with SPSS 20.0 software. Statistical analysis was used one-way ANOVA test. Differences were considered significant when *P<0.05, **P<0.01, ***P<0.001, respectively. References (1) Yan, Z.; Wang, F.; Wen, Z.; Zhan, C.; Feng, L.; Liu, Y.; Wei, X.; Xie, C.; Lu, W. LyP-1-Conjugated PEGylated Liposomes: a Carrier System for Targeted Therapy of Lymphatic Metastatic Tumor. J Control. Release 2011, 157, (2) Mulder, W.J.; Strijkers, G.J.; Griffioen, A.W.; van Bloois, L.; Molema, G.; Storm, G.; Koning, G.A.; Nicolay, K. A Liposomal System for Contrast-Enhanced Magnetic Resonance Imaging of Molecular Targets. Bioconjugate Chem. 2004, 15, (3) Wang, Z.; Zhao, Y.; Jiang, Y.; Lv, W.; Wu, L.; Wang, B.; Lv, L.; Xu, Q.; Xin, H. Enhanced Anti-Ischemic Stroke of ZL006 by T7-Conjugated PEGylated Liposomes Drug Delivery System. Sci. Rep. 2015, 5, (4) Jiang, X.; Xin, H.; Sha, X.; Gu, J.; Jiang, Y.; Law, K.; Chen, Y.; Chen, L.; Wang, X.; Fang, X. PEGylated Poly(Trimethylene Carbonate) Nanoparticles Loaded with Paclitaxel for the Treatment of Advanced Glioma: in vitro and in vivo Evaluation. Int. J. Pharm. 2011, 420, (5) Martins, S.; Tho, I.; Reimold, I.; Fricker, G.; Souto, E.; Ferreira, D.; Brandl, M. Brain Delivery of Camptothecin by Means of Solid Lipid Nanoparticles: Formulation Design, in vitro and in vivo Studies. Int. J. Pharm. 2012, 439,

4 Supplementary Figures Figure. S1. 1 H NMR spectra of PHB-Dextran. Figure. S2. 1 H NMR spectra of Mal-PEG-DSPE (A) and SHp-PEG-DSPE (B). 4

5 Figure. S3. The ROS-triggered hydrolysis curves of SHp-RBC-NP in PBS containing 10% FBS (A) and free FBS (B) at various concentration of H 2 O 2 (n=3). The particle size changes of SHp-RBC-NP in PBS containing 10% FBS and at the H 2 O 2 concentration of 1 mm (C), 0.5 mm (D), 0.25 mm (E), 0.1Mm (F), 0.025Mm(G), and 0 mm (H). 5

6 Figure. S4. The hydrodynamic diameters of SHp-RBC-NP in PBS and PBS containing 10% FBS over two weeks at 25 C. 6

7 Figure. S5. The hydrodynamic diameters of SHp-RBC-NP in PBS and PBS containing 10% FBS over two weeks at 37 C. 7

8 Figure. S6. In vitro cytotoxicity of different nanoparticles against BCECs. Statistical analysis was used one-way ANOVA test. Differences were considered significant when *P<0.05, **P<0.01, ***P<0.001, respectively (n = 5). 8

9 Figure. S7. The biodistribution of various nanoparticles in major organs after intravenous injection at A) 1h, B) 2h and C) 6 h. (n = 3) 9

10 Table S1. (The characterizations of NP or RBC-NPs. Data are represented with mean ±SD (n =3)) Zeta Particle Polydispersity Formulation Potential EE % LC % size (nm) index (PDI) (-mv) NP/NR2B9C ± ± ± ± ±0.55 RBC-NP/NR2B9C ± ± ± ± ±0.73 SHp-RBC-NP/NR2B9C ± ± ± ± ±0.58 Table S2. (Pharmacokinetic parameters of free NR2B9C, NP, RBC-NP and SHp-RBC-NP. Data are represented with mean ±SD (n =3)) Parameters NR2B9C NP/NR2B9C RBC-NP/NR2B9C SHp-RBC-NP/NR2B9C AUC(ng/ml h) ± ± ± ± T 1/2 0.47± ± ± ±0.29 CL (L/h) 0.33± ± ± ±0.02 Table S3. (Mice serum level of biochemical variables after intravenous treatment with saline, NP, RBC-NP and SHp-RBC-NP at concentration of 100 mg/kg for 7 days (n=5)) Groups AST ALT BUN Creatinine (U/L) (U/L) (mmol/l) (mmol/l) Saline 10.30± ± ± ±3.60 NP 10.65± ± ± ±3.75 RBC-NP 10.74± ± ± ±0.77 SHp-RBC-NP 10.70± ± ± ±

11 Scheme 1. Synthesis and Degradation of PHB-Dextran. 11