PMC

Schematic of the encapsulation strategy and negative-stain TEM. (a) Lipid–DNA conjugates are annealed to outer handles of non-encapsulated DNA nano-octahedron (N-DNO) in a surfactant solution that forms micelles around the conjugates. Liposomes are added, resulting in mixed surfactant–lipid micelles. Dialysis selectively removes the surfactant and results in a fused lipid bilayer around the DNO. (b) TEM images of purified N-DNO, (c) E-DNO, showing a tightly wrapped unilamellar membrane around the nanostructures, and (d) phi12 bacteriophage, showing comparable ultrastructure and dimensions to E-DNO (scale bar = 50 nm).

Bulk encapsulation yield and in vitro immune activation. (a) Encapsulation yield of outer handle DNO variants was estimated by PicoGreen dye membrane exclusion (red), and protection from nuclease was assayed with DNase I (blue). ELISA assay measurements of (b) IL-6 and (c) IL-12 cytokine production by splenocytes after incubation with N-DNO, E-DNO, and 50 nm vesicles for 16 h, as well as nonactivated controls. (d) Flow cytometry measurement of splenocyte mean fluorescence after incubation with Cy5-labeled N-DNO, E-DNO, and negative control. (e) Flow cytometry forward- (cell size) and side-scattering (granularity) properties of splenocytes was used to define two populations. Small, low granularity cells (1) were analyzed separately from large, high granularity cells (2). (f) Histogram of population (2) fluorescence after incubation with Cy5-labeled N-DNO (purple), E-DNO (blue), and negative controls (red). (*a,b: p < 0.05, ANOVA + Dunnet’s test vs control, error bars indicate SEM).

In vivo optical imaging for analysis of pharmacokinetics and biodistribution. Mice were injected with AlexaFluor750-labeled oligonucleotide (orange), N-DNO (blue), or E-DNO dual-labeled with rh-DOPE (green) and imaged for 120 min postinjection. (a) Fluorescence images of mice at 120 min postinjection. E-DNO is seen throughout the body, whereas the other two agents have accumulated in the bladder (calibration bar = 500–20000 afu). (b) Mean fluorescence (measured in a region-of-interest traced around the head and torso) vs time, relative to the signal at t = 8 min (I/I_o). (c) Elimination half-lives estimated from the kinetic analysis. (d) Fluorescence images of organs harvested 120 min postinjection, shown overlaid with photographic images (i) blood, (ii) urine, (iii) lung, (iv) heart, (v) liver, (vi) kidneys, (vii) spleen, calibration bar = 1500–25000 afu). (e) Organ distribution of AlexaFluor750 fluorescence (% of total, afu/g after correcting for calculated blood volumes of organs). (*a,b p < 0.05, ANOVA + Tukey’s test, dashed lines and error bars indicate SEM).