b, Quantification of mean fluorescence intensity of Cy5-CpG 1826 in RAW cells after incubation with cationic materials, calculated by Image J software. Statistical.
SUPPLEMENTARY INFORMATION Cationic Nanoparticles as DNA Induced Inflammatory Inhibitors to Treat Rheumatoid Arthritis Huiyi Liang1, Bo Peng1, Cong Dong1, Lixin Liu1*, Jiaji Mao2, Song Wei3, Xinlu Wang3, Hanshi Xu4, Jun Shen2*, Hai-Quan Mao1,5, Xiaohu Gao1,6, Kam W. Leong1,7*, Yongming Chen1* Table of Contents of Supplementary Information Supplementary Figure 1. Synthesis of PDMA and PLGA-b-PDMA and particle morphology of cNP. ................................................................................................................................................... 2 Supplementary Figure 2. cNP has a lower cytotoxicity than PDMA and the cationic materials alone show no significant stimulus to cells. ...................................................................................... 3 Supplementary Figure 3. Cationic materials could inhibit nucleic acid-mediated activation of TLRs. ................................................................................................................................................ 4 Supplementary Figure 4. MyD88 and TRAF6 enhanced expression with CpG were downregulated by cationic materials in Ramos BlueTM cells. ................................................................... 5 Supplementary Figure 5. Cationic materials can reduce the cellular uptake of immunostimulatory nucleic acid. ................................................................................................................... 6 Supplementary Figure 6. Cationic materials interacted with intracellular nucleic acid. ................ 7 Supplementary Figure 7. Morphology and iconography study of mice treatment by cationic materials. .......................................................................................................................................... 8 Supplementary Figure 8. Morphology and iconography study in both early and established therapeutic treatment of rats. ........................................................................................................... 9 Supplementary Figure 9. Biodistribution of cationic materials in normal rats and CIA rats (score 2) via i.v. injection. .......................................................................................................................... 11 Supplementary Figure 10. Quantification of TNF-a, IL-6 and MMP-3 immunoreactive cells in synovial tissue of normal, model, PDMA, and cNP groups. ........................................................... 12 Supplementary Figure 11. In vivo acute toxicity study of cationic polymers in rats. .................... 13 Supplementary Figure 12. In vivo long-term toxicity of cationic materials in rats. ...................... 14 Supplementary Table 1. Basic information and clinical characteristics of RA patients. ............... 15 Supplementary References ............................................................................................................. 15
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Supplementary Figure 1. Synthesis of PDMA and PLGA-b-PDMA and particle morphology of cNP. a, Chemical structure of PDMA470 and PLGA-b-PDMA463 block copolymer. b, Schematic syntheses of PDMA470 homopolymer and dye-conjugated PDMA470 homopolymer. c, Schematic syntheses of PLGA-b-PDMA463 block copolymer and dye-conjugated PLGA-b-PDMA463 block copolymer. d, 1H NMR spectrum of PDMA470 and PLGA-b-PDMA463 after purification in CDCl3. e, GPC traces of PDMA470 and PLGA-b-PDMA463 in DMF eluent, using PS standard for analysis. f, Number average distribution curve of cNP in PBS measured by DLS. g, TEM micrograph of cNP.
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Supplementary Figure 2. cNP has a lower cytotoxicity than PDMA and the cationic materials alone show no significant stimulus to cells. a, Cell viability of RAW264.7 cells treated with different cationic materials at various concentrations for 24 h incubation. b, TLR activation of Ramos BlueTM cells after adding 50 g/mL cationic materials to the medium for 24 h shows that cationic materials could not activate TLR. c, 25 g/mL cationic materials could not raise TNF-α expression in RAW264.7 cells. d, Cationic materials could not raise TNF-α expression in patient’s SFMC. After incubation with 25 g/mL PDMA or cNP for 24 h, TNF-α expression had no significant increase relative to the control group incubated with Lipofectamine® 2000 (Lipo) only. e, 0.5 g/mL cationic materials could not raise IL-6 expression in patient’s FLS after 24 h incubation. In b, c, d and e, statistical significance was calculated by one-way ANOVA with the LSD post-test, * 0.01
