Figure 2

Biodistribution of liposomes in zebrafish embryos at different stages of development. (A–H) Representative images of embryos of the Tg(mpeg1:GFP) line injected with AmbiMACs (20% DSPG) at 1 (A,B), 2 (C,D), or 3 dpf (E,F) or with PEGylated liposomes at 3 dpf (G,H). Confocal microscopy images were taken at 2 hpi. Liposomes are shown in red and macrophages in cyan. The tail regions (indicated by dashed boxes in (A,C,E,G)) are shown at higher magnification in (B,D,F,H). Scale bar = 200 μm. (I,J). The number (I) and percentage (J) of macrophages containing liposomes quantified in the whole body of embryos injected with AmbiMACs (20% DSPG) at 1, 2, or 3 dpf or with PEGylated liposomes at 3 dpf. Statistical analysis by one-way ANOVA showed a significantly lower number and percentage of macrophages containing liposomes when injected with PEGylated liposomes at 3 dpf, compared to injection with AmbiMACs (20% DSPG). Upon injection with AmbiMACs (20% DSPG), at 1 and 3 dpf, significantly lower numbers of macrophages containing liposomes were observed than at 2 dpf. Embryos at 3 dpf showed significantly lower percentages of macrophages containing liposomes compared to embryos at 1 and 2 dpf. (K) The ratio between the (fluorescent) signal of liposomes in the CHT/CV area and the dorsal part of the tail (as described in Figure 1I). The ratio at 1 dpf was significantly higher compared to 2 and 3 dpf. No significant difference was observed between the AmbiMACs (20% DSPG) and PEG liposomes at 3 dpf. Statistical analysis was performed by one-way ANOVA with Bonferroni’s post hoc test. Data shown are the mean ± SEM of 3–4 individual embryos of which the individual data are indicated. Statistically significant differences between groups are indicated by: ** p < 0.01; *** p < 0.001; **** p < 0.0001.