Trans-Endothelial Trafficking in Zebrafish: Nanobio Interactions of Polyethylene Glycol-Based Nanoparticles in Live Vasculature

Trans-endothelial transport of nanoparticles remains poorly characterized in live organisms. The zebrafish is a well-established model for direct <i>in vivo</i> imaging; however, standardized controls have not been consistently applied across studies. Here, we developed a standardized protocol to assess nanoparticle trans-endothelial trafficking in live zebrafish. We identified 2000 kDa dextran as an optimal coinjection control for standardizing microinjections and quantifying nanoparticle transport in a systematic, unbiased manner. Using the standardized protocol, we profiled early physiological trans-endothelial transport pathways in zebrafish embryos using dextran solutes as extravasation markers, showing that steady-state solute extravasation is characterized by paracellular routes selective for solutes <15 nm in diameter, dynamin-mediated endocytosis, and low basal macropinocytosis. We assessed the extravasation of 3, 7, 32, 47, 81, and 109 nm polyethylene glycol (PEG)-based nanoparticles using this protocol. Using a pretreatment protocol with chemical inhibitors, we demonstrate that <7 nm PEG-based hyperbranched polymer (HBP) nanoparticles undergo rapid paracellular extravasation, 32 to 81 nm PEG-based micelles adopt dynamin-dependent and macropinocytic trafficking over paracellular transport, and 109 nm PEG-based micelles extravasate primarily via macropinocytic trafficking. We characterized the emergence of vascular caveolae up to 17 days postfertilization with a <i>cavin1b</i> knock-in zebrafish line. Using <i>cavin1a</i>/<i>cavin1b</i> double knockout zebrafish and tumor-bearing Cavin1 null mice, we showed that caveolae do not contribute to the transvascular transport of these PEG-based nanoparticles. This work demonstrates the rigor of the standardized protocol for assessing trans-endothelial transport in the live zebrafish and provides fundamental cell biology insight into the <i>in vivo</i> behavior of PEG-based nanoparticles.