Fig. 6

In vivo biodistribution of nanoformulations in zebrafish embryos and acetylcholinesterase activity in the head. (A) Scheme showing the injection site (i.v.) in embryonic zebrafish (48 hpf) and imaging period. NPs-PLA-PEG-Cy5 and EVs-PKH26 were microinjected into the Tg (flk1:EGFP) zebrafish larvae at a dose equivalent to 7.92×105 NPs (4nL). Vascular endothelial cells are in green and labeled nanoformulations are in red. The nanoformulations were screened for in vivo distribution after 24h in the lateral position. Created with BioRender.com. (B and C) Whole embryo (10× magnification) and head-level (20× magnification) views of EVs-PKH26 biodistribution. EVs-PKH26 remains associated with the dorsal region of the embryo. Bright red dots were observed in the brain region of the larvae, corresponding to EVs-PKH26. The gray dots correspond to the presence of nanoformulations in the respective regions. (D and E) Whole embryos and head-level views of biodistribution. NPs-PLA-PEG-Cy5 predominantly circulated freely and was distributed throughout the vasculature of the embryo. High levels of NPs-PLA-PEG-Cy5 were confined to the vasculature but low level in the brain parenchyma (gray). Scale bars: 200µm (whole embryo) and 100µm (head). (F) Schematic representation of the injection and AChE activity analysis. Created with BioRender.com. (G) Effect of nanoformulations on the inhibition of the AChE activity in the zebrafish brain. The percentage of inhibition values was calculated in comparison to the activity of the control group without treatment. Data presented are mean ± SEM (n=3), six fish per sample. ***p <0.001, ****p < 0.0001 vs control, ###p < 0.001, ####p < 0.0001 vs DNZ, and $p < 0.05 vs NPs-PLA-PEG. One-way ANOVA followed by Tukey’s post hoc test.