Fig. 4: Phosphorothioate linkages on the 5’ end of the sense strand and within the linker region contribute to albumin association in plasma and tumor gene silencing activity.

a Schematic structural representations of the siRNA-lipid conjugates with varied inclusion of PS bonds. b Cy5-labeled lipid-siRNA conjugates were delivered i.v. to mice at 1 mg/kg. Intravascular Cy5 fluorescence measured through ~1 h following delivery was used to calculate siRNA circulation half-life (left panel) (n = 3–4). Size exclusion chromatographic calculation of fraction of Cy5 fluorescence associated with albumin-containing plasma fractions (right panel) (n = 3). c Cy5 fluorescence in kidneys, lungs, liver, spleen and heart was measured 1 h after treatment. Fluorescence values in each organ relative to the total fluorescent values of all organs combined is shown. d–f Mice bearing orthotopic MDA-MB-231.Luc tumors were treated i.v. with siRNA-lipid conjugates, using a siRNA sequence against luciferase (si_Luc). d Schematic showing treatment schedule for i.v. delivery of siRNA-lipid conjugates. e Cy5 fluorescence in kidneys, liver, and tumor for each mouse was measured 18 h after treatment (n = 5–6). f Tumors were collected on day 7 and either dissociated to assess cell-associated Cy5 fluorescence by flow cytometry (left panel) or lysed to assess for luciferase activity (right panel). Bars show the average (± S.D.) value per group, and points are the values for each tumor analyzed. All relative values shown are calculated relative to the average value of saline-treated controls, which was set to a value of 1. Each bar represents the average value (± S.D.), and each point represents values measured for each individual sample (n = 5–6). For (b, f), significance was assigned using one-way ANOVA with Tukey’s multiple comparisons test. For (c, e), significance was assigned by two-way ANOVA with Tukey’s multiple comparisons test. For (e) and (f), n = 5–6.