Visualization of CaP-lipid NPs after tail vein injection in transgenic zebrafish. Sterile filtered non ASO-loaded CaP-lipid NPs containing LissRdB-DSPE (stock, 1:5 or 1:10 v/v) was injected into the vein of zebrafish expressing a green fluorescent reporter in their vasculature (Tg(fli1a:EGFP)). Schematic illustration of the route of injection (A). Expression of EGFP in transgenic fish highlighting the blood vessels (B). Visualization of CaP-lipid NP in 6-day-old transgenic zebrafish 2 h after injection (C). Zoomed image of the treated zebrafish from area indicated by white box, showing distribution and accumulation of CaP-lipid NP in and around blood vessels (D). Overlay of transgenic EGFP expression and CaP-lipid NP distribution (E).

Visualization of CaP-lipid NPs and neurons in the spinal cord and brain of a 6-dpf zebrafish. Sterile filtered non ASO-loaded CaP-lipid NPs containing LissRdB-DSPE (red) were micro injected into the zebrafish spinal cord (A) and brain (B). (A) Visualization of CaP- lipid NPs containing LissRdB-DSPE (red; injections) within the spinal cord neurons (green; transgenic expression) of a 6-day-old transgenic zebrafish. Zoomed image of the treated zebrafish from area indicated by white box, showing distribution of the CaP-lipid NPs within GFP-expressing spinal cord neurons. (B) Expression of brain-injected CaP-lipid NPs (red) in a transgenic zebrafish expressing astrocyte-specific GFP [green; Tg(GFAP:GFP)] to highlight the brain-specific delivery of these particles. The bottom image overlays the bright-field channel for better visualization of the CNS. The schematic inserts in panels depict the orientation of the fish and outline the presented area.

Visualization of control LissRdB injections in the zebrafish. LissRdB was injected into the vasculature (A-C), the spinal cord (D-F) and the brain (G-I) to compare distribution and brightness to CaP-lipid NPs containing LissRdB-DSPE. (A) Schematic representation of zebrafish and vein injection. (B) Transgenic zebrafish expressing EGFP (green) in the vasculature highlighting blood vessels. (C) Minimal expression of control-LissRdB throughout the vessels two hours after injection. (D) Schematic representation of zebrafish and spinal cord injection. (E) Minimal expression of control-LissRdB throughout the neurons of the spinal cord (green) two hours after injection. (F) Control-LissRdB (red) channel only. (G) Schematic representation of zebrafish and brain injection. (H) Minimal expression of control-LissRdB throughout the brain of a transgenic zebrafish (green) two hours after injection. (F) Control-LissRdB (red) channel only.