Journal: Molecular Therapy. Nucleic Acids

Article Title: Immunogenicity Testing of Lipidoids In Vitro and In Silico : Modulating Lipidoid-Mediated TLR4 Activation by Nanoparticle Design

doi: 10.1016/j.omtn.2018.02.003

Figure Lengend Snippet: Lipidoid-Induced TLR Activation Assessed Using HEKblue Human TLR Reporter Cell Lines (A) Incubation of the TLR2-expressing cell line for 16 hr at 37°C with L 5 and the positive control PAM 3 CSK 4 . (B) Incubation of the TLR2-expressing cell line with L 4 , L 5 , L 6 , and L mix in the presence of PAM 3 CSK 4 , 100 ng/mL, is shown. (C) Incubation of the TLR3-expressing cell line with L 5 and positive control poly(I:C) is shown. (D and E) TLR4-expressing cells were stimulated with L 4 , L 5 , L 6 , and L mix concentrations ranging from 0.001 to 10 μM (D), as well as with L 5 or DOTAP in the concentration range from 0.01 to 300 μM (E). (F) Activation of the TLR4-expressing cell line by L 5 and DOTAP, respectively, in the presence of LPS, 10 ng/mL, is shown. Data have been corrected for background measured for solvent-stimulated cells and are shown relative to the maximal response (100%) with the respective selective agonists: PAM 3 CSK 4 (100 ng/mL for TLR2; A and B), poly(I:C) (5 μg/mL for TLR3; C), and LPS (10 ng/mL for TLR4; D–F). Data represent mean values ± SD (n = 3; technical replicates) and represent results of one of two independent experiments. Statistically significant differences from L 5 are marked with x and from solvent are marked with y: **p < 0.01 and ***p < 0.001.

Article Snippet: The human TLR reporter cell lines (HEK-BluehTLR2, -hTLR3, -hTLR4, -hTLR7, and -hTLR9 reporter cells) were cultured according to the manufacturer's instructions (Invivogen, Toulouse, FR).

Techniques: Activation Assay, Incubation, Expressing, Positive Control, Concentration Assay, Solvent

Journal: Molecular Therapy. Nucleic Acids

Article Title: Immunogenicity Testing of Lipidoids In Vitro and In Silico : Modulating Lipidoid-Mediated TLR4 Activation by Nanoparticle Design

doi: 10.1016/j.omtn.2018.02.003

Figure Lengend Snippet: Immune Activation by Lipidoid-Based SNALPs and Lipoplex and Not by Lipidoid-Modified LPNs (A and B) Activation of the hTLR4 receptor cell line (A) and pAPC maturation (B) by lipoplexes composed of L 4 , L 5 , L 6, or L mix with siRNA-EGFP. (C and D) Activation of the hTLR4 receptor cell line (C) and pAPC maturation (D) by SNALPs prepared with L 5 and L mix , respectively, is shown. (E and F) Activation of the hTLR4 receptor cell line (E) and pAPC maturation (F) induced by LPNs prepared with L 4 , L 5 , L 6 , L mix , or DOTAP is shown. Sample OD was corrected for OD value measured for the negative control (solvent 1:10) and divided by the maximum OD (LPS 10 ng/mL). Professional APC maturation was tested by stimulating murine bone-marrow-derived, granulocyte macrophage colony-stimulating factor (GM-CSF)-differentiated pAPCs. Maturation was measured as the upregulation of the activation marker CD86 in CD11c + MHCII + cells and compared to the levels on BM-APCs stimulated with LPS (10 ng/mL) and PBS, as quantified by flow cytometry (see also Figure S2 ). Data represent mean values ± SD (n = 3 for A, C, and E and n = 2 for B, D, and F technical replicates).

Article Snippet: The human TLR reporter cell lines (HEK-BluehTLR2, -hTLR3, -hTLR4, -hTLR7, and -hTLR9 reporter cells) were cultured according to the manufacturer's instructions (Invivogen, Toulouse, FR).

Techniques: Activation Assay, Modification, Negative Control, Solvent, Derivative Assay, Marker, Flow Cytometry