A zebrafish
model for liposome biodistribution. (a) Schematic of
liposome injection and quantification in zebrafish. Fluorescently
labeled liposomes (1 mM total lipids containing 1 mol % Rhod-PE) were
injected into the duct of Cuvier at 54 hpf. Confocal microscopy is
performed in a defined region (boxed) caudal to the yolk extension
at 1, 8, 24, and 48 h after injection. (b) Whole-embryo view of liposome
distribution in kdrl:GFP transgenic embryos, 1 hpi
with three different liposome formulations (AmBisome, EndoTAG-1, and
Myocet). (c) High-resolution imaging allows quantification of liposomes
in circulation (measured in the lumen of the dorsal aorta (white box))
and liposome association with different blood vessel types (see Supporting Information). CHT-EC: caudal hematopoietic
tissue endothelial cells, DLAV: dorsal longitudinal anastomotic vessel.
ISV: intersegmental vessel. (d) Tissue level view of liposome distribution
in kdrl:gfp transgenic embryos, 1 h and 8 h after
injection with three different liposome formulations and a single
confocal section through the dorsal aorta (DA) at 1 h after injection.
(e) Quantification of liposome levels in circulation based on mean
rhodamine fluorescence intensity in the lumen of the dorsal aorta
at 1, 8, 24, and 48 h after injection (error bars: standard deviation.) n = 6 individually injected embryos per formulation per
time point (in two experiments). (f) Quantification of liposome levels
associated with venous vs arterial endothelial cells
based on rhodamine fluorescence intensity associated with caudal vein
(CV) vs DA at 8 h after injection. (g) Quantification
of extravascular liposome levels based on rhodamine fluorescence intensity
outside of the vasculature between the DLAV and DA at 8 h after injection.
(h) Quantification of liposome levels associated with the vessel wall
based on rhodamine fluorescence intensity associated with all endothelial
cells relative to rhodamine fluorescence intensity in circulation
at 1h after injection. (f–h) Bar height represents median values,
dots represent individual data points, brackets indicate significantly
different values (*: p < 0.05, **: p < 0.01, ***: p < 0.001) based on Kruskal–Wallis
and Dunn’s tests with Bonferroni correction for multiple testing. n = 12 individually injected embryos per group (in 2 experiments).
(i) Whole-embryo view of liposome distribution in kdrl:GFP transgenic embryos, 1 h after injection with DOPG and DSPC liposomes.
Liposome accumulation for both formulations is observed in the primitive
head sinus (PHS), common cardinal vein (CCV), posterior cardinal vein
(PCV), and caudal vein (CV). (j) Tissue level view of liposome distribution
in kdrl:GFP transgenic embryos, 1 h after injection
with DOPG and DSPC liposomes at 102 hpf. Liposome accumulation is
observed in the entire caudal vein (CV), but only on the dorsal side
of the PCV (dPCV, arrows).