Figure 4. 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 hours of incubation with Nile Red at 37°C. d, Cy5 fluorescence in kidneys, lungs, liver, spleen and heart was measured approximately 1h 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. e, Intravascular Cy5 fluorescence measured through 1h following i.v. delivery of siRNA-lipid conjugate variants (1 mg/kg) was used to calculate siRNA circulation half-life (t_1/2). f, Plasma samples collected from mice approximately 1h 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. For panels C-E and G, each bar represents the average value (± S.D.), and each point represents a value measured for an individual sample. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001