Supporting Information. Osteoblast-Targeting Peptide-Modified Nanoparticle for sirna/microrna Delivery

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1 Supporting Information Osteoblast-Targeting Peptide-Modified Nanoparticle for sirna/microrna Delivery Yao Sun 2,4,5*, Xiongzhen Ye 1*, Mingxiang Cai 2*, Xiangning Liu 3*, Jia Xiao 1, Chenyang Zhang 2,Yayu Wang 1, Li Yang 1, Jiafan Liu 1,Shannai Li 1, Chen Kang 5, Bin Zhang 5, Qi Zhang 2, Zuolin Wang 2,4#, An Hong 1#,Xiaogang Wang 1# 1 Department of Cell Biology & Institute of Biomedicine, College of Life Science and Technology, Jinan University, Guangzhou, China. 2 Department of Oral Implantology, School of Stomatology, Tongji University, Shanghai, China. 3 The First Affiliated Hospital of Jinan University, Guangzhou,China. 4 Shanghai Engineering Research Center of Tooth Restoration and Regeneration, Shanghai, China 5 Sino-Russian Institute of Hard Tissue Development and Regeneration, the SecondAffiliated Hospital of Harbin Medical University, Harbin, China *All these authors contribute equally to this work. #Correspondence to Xiaogang Wang Department of Cell Biology & Institute of Biomedicine, Jinan University 601 Huangpu Avenue. West, Guangzhou, , China Phone: Fax: txg_wang@jnu.edu.cn Or #Correspondence to An Hong Department of Cell Biology & Institute of Biomedicine, Jinan University 601 Huangpu Avenue. West, Guangzhou, , China Phone: Fax: tha@jnu.edu.cn Or #Correspondence to Zuolin Wang Department of Oral Implantology School of Stomatology, Tongji University 399 Middle Yanchang Road, Shanghai, China Phone: Fax: zuolintongji@126.com

2 Results of protein binding of SDSSD peptide in human osteoblasts are performed in supplementary table 1. Results of protein binding of SDSSD peptide in mouse osteoblasts are performed in supplementary table 2. Figure S1 Cytotoxicity assay of SDSSD peptide in vitro. (A-B) Results of mouse and human osteoblasts viability assays after incubation with 1mg/mL SDSSD peptide, DSS6 or control peptide for 24 hours by using the CellTiter-Blue Reagent. Data was shown as mean ± s.d., N=3 per group. (C-D) Results of mouse and human bone microenvironment cells viability assays after incubation with 1mg/mL SDSSD peptide for 24 hoursdetected by thecelltiter-blue Reagent. Data was shown as mean ± s.d., N=3 per group.

3 Figure S2 Design, synthesis and characterization of SDSSD-PU. (A) Schematic procedure of PU synthesis. (B) Fourier transforminfrared spectroscopy (FI-TR) spectra of PU, SDSSD and SDSSD-PU. Arrow points SDSSD covalent linkage to PU. (C) UV-Vis absorption spectra of PU, SDSSD peptide and SDSSD-PU in water. (D) The standard curve for monitoring of SDSSD peptide at the wavelength of 214 nm. (E) H NMR spectrum of PU, SDSSD peptide and SDSSD-PU in D2O.

4 Figure S3 Cytotoxicity assay of SDSSD-PU in vivo. (A-B) Results of mouse and human bone microenvironment cell viability assays after incubation with SDSSD-PU for 24 hours by using the CellTiter-Blue Reagent. Data was shown as mean ± s.d., N=6 per group. (C) Hemagglutination assay: mouse blood cells were incubated with PU or SDSSD-PU for 1hour. Scale bar, 50 µm. (D) H&E staining of heart, liver, spleen and kidney collected from mice with tail vein injected of PU or SDSSD-PU. Scale bar, 20 µm. (E) Quantification of serum CK-MB, ALT, AST and BUN level by a clinical chemistry analyzer. CK-MB: creatine kinase-mb; ALT: alanine aminotransferase; AST: aspartate aminotransferase; BUN: blood urea nitrogen. Data was shown as mean ± s.d., N=6 per group. (F) Quantification analysis of serum IL-6, TNF-α and IFN-α by ELISA. IL-6: interleukin-6; TNF-α: tumor necrosis factor-α; IFN-α: interferon-α. Data shown as mean ± s.d., N=6 per group.

5 Figure S4 SDSSD-PU uptake by human endothelial cells line (HUVEC). (A) Fluorescence micrographs of endothelial cell line (HUVEC) and osteoblasts incubated with PU-Cy3-miRNA or SDSSD-PU-Cy3-miRNA for 6 hours. (B) Q-PCR analysis of cel-mirna-67 level in endothelial cells line (HUVEC) and osteoblasts incubated with PU-cel-miRNA-67 or SDSSD-PU-cel-miRNA-67 for 6 hours.

6 Figure S5 The stability and degradation of SDSSD-PU. (A)The particle size distribution of SDSSD-PU in aging time determined by DLS analysis. (B) Zeta-potential of SDSSD-PU in aging time determined by dynamic light scattering. (C) Localization of mirna in mouse bone tissue after injection of SDSSD-PU-Cy3-miRNA or naked Cy3-miRNA for 48 hours. (D)Colocalization analysis of SDSSD-PU-Cy3-miRNA and endosome marker Rab5 by confocal microscopy in osteoblasts.