Fig. 5: Proximal placement of the divalent lipid branch point immediately adjacent to the siRNA drives albumin binding.
a Schematic structural representations of the siRNA-lipid conjugates with varied branchpoint placement and length of the linker. b Representative in vitro binding responses of siRNA conjugates to human serum albumin (400 nM) measured by biolayer interferometry. c Critical micelle concentration of siRNA conjugates observed after 2 h of incubation with Nile Red at 37 °C (n = 3). d Cy5 fluorescence in kidneys, lungs, liver, spleen and heart was measured ~1 h after i.v. delivery of siRNA-lipid conjugates (1 mg/kg). Fluorescence values in each organ relative to the total fluorescent values of all organs combined is shown (n = 3–4). e Intravascular Cy5 fluorescence measured through 1 h following i.v. delivery of siRNA-lipid conjugate variants (1 mg/kg) was used to calculate siRNA circulation half-life (t1/2) (n = 4–5). f Plasma samples collected from mice ~1 h after treatment (1 mg/kg) were fractionated and quantitated for Cy5 fluorescence by size exclusion chromatography. A representative trace is shown of fluorescence signal within each plasma fraction for each siRNA structure; the albumin-containing plasma fractions are highlighted. g Cy5 fluorescence within the albumin-containing fractions was quantitated relative to total plasma fluorescence (n = 3). For (c–e, g), each bar represents the average value (± S.D.), and each point represents a value measured for an individual sample. For (c, e, g), significance was assigned using one-way ANOVA with Tukey’s multiple comparisons test. For (d), significance was assigned using two-way ANOVA with Tukey’s multiple comparisons test.
