# Fileset

[biomolecules-2613451-supplementary.pdf](https://mdr.nims.go.jp/filesets/7ed9bbd1-2dd8-4b47-98de-98a531f50a09/download)

## Creator

[Nguyen Bui Thao Le](https://orcid.org/0000-0002-4797-2339), [Anh Thi Tram Tu](https://orcid.org/0000-0002-7243-2086), Dandan Zhao, [Chiaki Yoshikawa](https://orcid.org/0000-0002-6589-387X), [Kohsaku Kawakami](https://orcid.org/0000-0002-3466-9365), [Yoshihisa Kaizuka](https://orcid.org/0000-0002-8019-0873), [Tomohiko Yamazaki](https://orcid.org/0000-0003-2136-8042)

## Rights

[Creative Commons BY Attribution 4.0 International](https://creativecommons.org/licenses/by/4.0/)

## Other metadata

[Influence of the Charge Ratio of Guanine-Quadruplex Structure-Based CpG Oligodeoxynucleotides and Cationic DOTAP Liposomes on Cytokine Induction Profiles](https://mdr.nims.go.jp/datasets/2b3a1789-6f50-41e8-b5ff-8fca72765bf4)

## Fulltext

Supporting Information  Influence of the Charge Ratio of Guanine-Quadruplex Structure-Based CpG Oligodeoxynucleotides and Cationic DOTAP Liposomes on Cytokine Induction Profiles  Nguyen Bui Thao Le 1, 2, Anh Thi Tram Tu 1, 3, 4, Dandan Zhao 1, Chiaki Yoshikawa 1, 2, Kohsaku Kawakami1, Yoshihisa Kaizuka1, Tomohiko Yamazaki 1, 2* 1 Research Center for Macromolecules and Biomaterials, National Institute for Materials Science (NIMS), 1-2-1 Sengen, Tsukuba 305-0047, Japan; le.buithaonguyen@nims.go.jp (N.B.T.L.); tuthitramanh@gmail.com (A.T.T.T.); amberdiary@gmail.com (D.Z.); yoshikawa.chiaki@nims.go.jp (C.Y.); kawakami.kohsaku@nims.go.jp (K.K.); kaizuka.yoshihisa@nims.go.jp (Y.K.) 2 Division of Life Science, Hokkaido University, Kita 10, Nishi 8, Kita-ku, Sapporo 060-0808, Japan 3 Department of Magnetic and Biomedical Materials, Faculty of Materials Science and Technology, VNUHCM-University of Science, 227 Nguyen Van Cu street, Ward 4, District 5, Ho Chi Minh City 70000, Vietnam 4 Ho Chi Minh City Campus, Vietnam National University, Linh Trung, Thu Duc, Ho Chi Minh City 70000, Vietnam * Correspondence: yamazaki.tomohiko@nims.go.jp; Tel.: +81-29-859-2345; Fax: +81-29-859-2449   Figure S1. Polyacrylamide gel electrophoresis analysis of G4-CpG ODNs in complex with DOTAP. Electrophoresis was performed using a 10–20% linear gradient polyacrylamide gel in tris-glycine buffer supplemented with 4 mM KCl. Linear ODN is the ssODN with the same length (30mer) as GD2.   Figure S2. CD spectra of naked GD2 and GD2-DOTAP complexes were drawn from the original raw data. Figure S3. Intensity-, volume-, and number-weighted particle size distributions of GD2-DOTAP at various charge ratios. Distribution was calculated using the CONTIN algorithm.  Figure S4.  Hydrodynamic size (nm) reported as a function of time for all complexes at different charge ratios. Data are presented as mean ± SD (n=3).  Figure S5. (a) Hydrodynamic size, polydispersity index, and (b) zeta potential of GD3-DOTAP complexes. Data are presented as mean ± SD (n=3).   Figure S6. Characterization of linear CpG ODN-DOTAP liposome complexes of different charge ratios. (a, c) Hydrodynamic size, polydispersity index, and (b, d) Zeta potential. Data are presented as mean ± SD (n=3).   Figure S7. Cell viability of human PMBCs after stimulation with 0.5 μM Nacked GD2 and GD2-DOTAP for 48 h. Data are presented as mean ± SD (n=5). Statistical significance was calculated compared to non-treated cells (D-PBS). ****p<0.0001, *p<0.05, ns (not significantly different) p>0.05 (one-way analysis of variance, followed by Tukey’s multiple comparisons test).  Figure S8. Cytokine induction by the GD2-DOTAP complex at a charge ratio of 1.5 in human PBMCs at different stimulation times. The final ODN concentration in the cell medium was 0.5 μM. Data are represented as mean ± SD (n=5). #, lower than the detection limit (3.9 pg/mL).    Figure S9. Cytokine induction by GD3-DOTAP complexes in (a) Namalwa and (b) PMDC05 cells. Relative mRNA levels of IL-6 and IFN-α in cells were examined after 6 h (in PMDC05 cells) and 4 h (in Namalwa cells) of stimulation with naked GD3 and GD3-DOTAP. The final ODNs concentration in the cell medium was 1 μM. Data are represented as mean ± SD (n=5). Statistical significance was calculated in comparison to bare GD3-treated cells (D-PBS). ****p<0.0001, ***p<0.001, *p < 0.05, ns (not significantly different) p>0.05 (one-way analysis of variance, followed by Tukey's multiple comparisons test).  Figure S10. Internalization of naked GD3 and GD3-DOTAP complexes in Namalwa cells, after 2 h of stimulation. Non-treated cells served as the control. Cy5 (red), MemBrite™ (green), and DAPI (blue) represent GD3-DOTAP, cell membrane, and nuclei, respectively. Scale bar: 10 μm.     Figure S11. Internalization of naked GD3 and GD3-DOTAP complexes in PMDC05 cells, after 2 h of stimulation. The low-charge ratio complexes were localized inside the cells, while high-charge ratio complexes were still bound to the cell membrane. Non-treated cells served as the controls. Cy5 (red), MemBrite™ (green), and DAPI (blue) represent GD3-DOTAP, cell membrane, and nuclei, respectively. Scale bar: 10 μm.    Figure S12. IL-1β production by GD2-DOTAP complexes in human PMBCs. The complexes at high-charge ratios (2 and 2.5) significantly induced IL-1β, which triggers the induction of pro-inflammatory cytokines, including IL-6. Data are represented as mean ± SD (n=5). *p<0.05, ns (not significantly different) p>0.05 (one-way analysis of variance, followed by Tukey's multiple comparisons test).