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 211166, China 2. Sir Run Run Hospital, Nanjing Medical University, Nanjing 211166, China 3. Jiangsu Jiangyin People's Hospital, Jiangyin 214400, China # These authors contributed equally to this manuscript. Corresponding author: Hongliang Xin (Tel.: +86-25-86868476, Fax: +86-25-86868467, E-mail: xhl@njmu.edu.cn). 1
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 1 10 4 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
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, 118-25. (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, 799-806. (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, 12651. (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, 385-394. (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, 49-62. 3
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
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
Figure. S4. The hydrodynamic diameters of SHp-RBC-NP in PBS and PBS containing 10% FBS over two weeks at 25 C. 6
Figure. S5. The hydrodynamic diameters of SHp-RBC-NP in PBS and PBS containing 10% FBS over two weeks at 37 C. 7
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
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
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 167.33±4.16 0.07±0.02 21.10±1.35 60.00±3.58 2.91±0.55 RBC-NP/NR2B9C 198.65±2.52 0.10±0.04 12.33±1.02 62.13±2.61 3.16±0.73 SHp-RBC-NP/NR2B9C 197.67±3.51 0.17±0.06 12.70±0.57 62.33±3.07 3.23±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) 265.43±43.96 515.11±52.84 1272.79±219.18 1312.21±188.21 T 1/2 0.47±0.12 0.77±0.12 1.32±0.33 1.38±0.29 CL (L/h) 0.33±0.06 0.16±0.02 0.06±0.02 0.06±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±1.65 3.28±0.87 5.60±0.12 44.30±3.60 NP 10.65±2.55 3.40±2.12 5.15±0.94 44.53±3.75 RBC-NP 10.74±2.56 3.96±1.32 5.66±0.65 45.93±0.77 SHp-RBC-NP 10.70±2.54 3.94±0.64 5.88±0.44 45.93±4.96 10
Scheme 1. Synthesis and Degradation of PHB-Dextran. 11