Figure 3.

Factor I conjugation prevents phagocyte association with nanoparticles, including local phagocytes in the target organ of antibody-targeted nanoparticles. a, b) In vitro, conjugation to Factor I reduces serum-treated nanoparticle interactions with neutrophils 2.0-fold. Fluorescent-IgG-liposomes +/− Factor I were incubated with serum for 1 hour, then incubated with neutrophils for 15 minutes. Neutrophils were pelleted and washed to remove unbound nanoparticles. Neutrophils were stained with the specific marker Ly6G (y-axis) and then subjected to flow cytometry. The dot-plots show Factor I changes the relative amount of liposome fluorescence seen in/on the neutrophils, which is quantified as a mean liposome-fluorescence signal (b) in the neutrophils that is half as much when Factor I is conjugated on (blue striped bar vs red striped). c) IV-LPS mice were IV-injected with ¹²⁵I-liposomes conjugated to antibodies that bind to the pulmonary endothelial marker ICAM. As shown for decades, the ICAM-targeted nanoparticles homed to the lungs, but the amount of lung targeting decreased by 31.6% and the blood concentration increased (inset) by 43.7% when the liposomes were conjugated to Factor I. d) Flow cytometry of the lungs of mice similar to (c), but with liposomes traced with fluorescence instead of radioactivity. The left panel (red outline) is from mice treated with ICAM-targeted liposomes, while the right panel (blue outline) received ICAM-targeted liposomes conjugated to Factor I. (e) Quantification of the dot plots in (d) show that Factor I led to a 21.0% lower median liposome-fluorescence in/on the lungs’ local neutrophils (left panel) but a 44.6% increase in endothelial cell-associated liposome fluorescence (right panel). This decrease in neutrophil association with nanoparticles but increase in endothelial-associated nanoparticle fluorescence shows that Factor I improved the targeting specificity of ICAM-targeted liposomes. Statistics: For (b): n=6; *p<1x10⁻¹⁰ via two-way ANOVA with Tukey correction; ‡ = p<1x10⁻¹⁰ via two-way ANOVA with Sidak’s correction. For (c): n=3; * = p=0.0001 by two-way ANOVA with Tukey; ‡ = p=0.04 by one-way ANOVA with Tukey. For (d): n=3; * = p=0.006 by two-way ANOVA with Sidak’s correction; ‡ = p=0.025 by two-way ANOVA with Sidak. For (e): n=3; * = p=0.006 by two-way ANOVA with Sidak’s correction; ‡ = p=0.005 by two-way ANOVA with Sidak’s correction.