Stab2-Mediated Clearance of Supramolecular Polymer Nanoparticles in Zebrafish Embryos

Abstract

Supramolecular polymers are attractive scaffolds for use as nanocarriers in drug delivery thanks to their modularity and easy fabrication; however, a molecular view into their in vivo behavior is lacking. Herein, we prepare fluorescent squaramide-based supramolecular polymer nanoparticles that range from fibers to spheres while maintaining their surface chemistry and near-neutral surface charge by a co-assembly approach involving a sulfo-cyanine-labeled monomer to track their in vivo biodistribution behavior and clearance in optically transparent zebrafish embryos. Evasion of macrophages, localization of the fibrillar aggregates in the caudal vein, and association with scavenger endothelial cells are observed. The interaction of the fibrillar supramolecular nanoparticles with the caudal vein is abrogated in gene-edited zebrafish lacking Stabilin-2, a receptor analogously found in the mammalian liver, providing a molecular view into their interaction with scavenger endothelial cells. We further show that this interaction can be tuned based on the choice of monomer and its resultant self-assembly.

Figure 1

Squaramide-based bolaamphiphiles used to prepare supramolecular polymer nanoparticles for in vivo biodistribution studies. (a) Chemical structure of bolaamphiphile monomers 1, 2, and 3 employed in the study to form fibrillar (1), mixed rod and spherical (2), or spherical (3) nanoparticles, respectively, upon self-assembly. The fluorescently labeled squaramide-based monomer with a sulfo-Cy3 dye (4) is used for tracking of the various supramolecular polymer nanostructures in vivo, while the sulfo-Cy5 dye (5) is used in combination with 4 for fluorescence resonance energy transfer (FRET) experiments. (b) Schematic representation of the morphology of the co-assembled native (1, 2, or 3) with dye-labeled monomer 4 (2 mol %) into fluorescently labeled squaramide-based supramolecular polymer nanoparticles before injection into a zebrafish embryo model. Representative cryo-TEM images of co-assembled squaramide-based supramolecular polymer nanoparticles from native monomers (2 mM) 1, 2, and 3 with 4 in water. Samples were prepared using the native squaramide-based bolaamphiphile monomer co-assembled with 2 mol % 4, displaying fiberlike structures for 1, a mixture of spherical and rodlike structures for 2 and spherical aggregates for 3. The scale bars represent 100 nm.

Figure 2

Spectroscopic studies of the self-assembled squaramide-based supramolecular polymer nanoparticles (30 μM). (a) UV–vis spectra of 1 (blue), 2 (red), and 3 (green) co-assembled with 4 (2 mol %). (b) Static measurements of 1 and 3 with an equimolar amount of 4 and 5 (2 mol % total of the dye-labeled monomers); FRET signal observed at 670 nm. (c) Dynamic measurements of 1, 2, and 3 co-assembled with 4 or 5 (2 mol % of each dye-labeled monomer) independently and then mixed together in an equimolar ratio. FRET ratios are compared over time (6 h) for 1 (blue squares), 2 (red circles), and 3 (green triangles).

Figure 3

Squaramide-based supramolecular polymer nanoparticles avoid macrophage uptake after 1 h of intravenous injection. (a) Whole-embryo view (10× magnified) after 1 h of injection in the Duct of Cuvier of 1 (2 mM) co-assembled with 4 (2 mol %) in an transgenic zebrafish (mpeg1:EGFP^gl22) expressing EGFP in macrophages (green) at 56 hpf. The arrow represents the site of injection (Duct of Cuvier). Scale bar represents 200 nm. (b) Higher magnification (40×) of the boxed region in (a) showing the dorsal aorta (DA), caudal hematopoietic tissue (CHT), and the caudal vein (CV). Scale bar represents 50 nm. (c) Biodistribution of fluorescently labeled squaramide-based supramolecular polymer nanoparticles 1 h post-injection. Left panel represents an overlay of macrophage and fluorescently labeled squaramides (magenta) of 1 (top), 2 (middle), and 3 (bottom) (2 mM) with 4 (2 mol %).

Figure 4

Biodistribution of the fluorescently labeled squaramide-based supramolecular polymer nanoparticles in the Tg(kdrl:GFP) zebrafish expressing GFP in endothelial cells. (a) Whole-embryo view 1 h post-injection in the Duct of Cuvier of 1 (2 mM) with 4 (2 mol %) in embryonic zebrafish at 56 hpf. (b) Higher magnification (40×) of the boxed region in (a) (caudal vein region) showing the dorsal aorta (DA), caudal hematopoietic tissue (CHT), and the caudal vein (CV). Scale bar represents 200 nm. (c) Biodistribution of 1, 2, and 3 fluorescently labeled 1 h post-injection. In the left panel, schematic representation of 1 (top), 2 (middle), and 3 (bottom) (2 mM) with 4 (2 mol %). (d) Biodistribution of 1 (top), 2 (middle), and 3 (bottom) (2 mM) with 4 (2 mol %) in the zebrafish mutant stab2^ibl2 (stab2^–/–).