A Standardized Protocol to Investigate Trans-Endothelial Trafficking in Zebrafish: Nano-bio Interactions of PEG-based Nanoparticles in Live Vasculature

Lim, Y.-W.; Bell, C. A.; Fletcher, N. L.; Condon, N. D.; Akhter, D. T.; Rae, J.; Ferguson, C.; Martel, N.; Humphries, J.; Lo, H.; Wu, Y.; Houston, Z. H.; Lagendijk, A. K. A.; Hall, T. E.; Thurecht, K. J.; Parton, R. G.

doi:10.1101/2025.07.23.666282

bioRxiv · 10.1101/2025.07.23.666282

A Standardized Protocol to Investigate Trans-Endothelial Trafficking in Zebrafish: Nano-bio Interactions of PEG-based Nanoparticles in Live Vasculature

Lim, Y.-W.·Bell, C. A.·Fletcher, N. L.·Condon, N. D.·Akhter, D. T.·Rae, J.·Ferguson, C.·Martel, N.·Humphries, J.·Lo, H.·Wu, Y.·Houston, Z. H.·Lagendijk, A. K. A.·Hall, T. E.·Thurecht, K. J.·Parton, R. G.

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Abstract

Trans-endothelial transport of nanoparticles remains poorly characterized in live organisms. The zebrafish is a well-established model for direct in vivo imaging; however, standardized controls are not consistently applied across studies. Here, we developed a standardized protocol to assess nanoparticle trans-endothelial trafficking in live zebrafish. We tested a range of tracers and identified 2000 kDa dextran as an optimal control for standardizing microinjections and quantifying nanoparticle transport in a systematic unbiased manner. As validation, we identified the early physiological trans-endothelial transport pathways in zebrafish embryos using 40 kDa dextran as a nanoparticle surrogate. Using our workflow, we assessed the extravasation of 3, 7, 32 and 47 nm polyethylene glycol (PEG)-based nanoparticles. Using cAMP stimulation to restrict paracellular routes, and inhibitors to impede dynamin-dependent endocytosis, we demonstrated that PEG-based hyperbranched polymer (HBP) nanoparticles of 3 to 7 nm traverse the endothelial barrier via paracellular routes and 32 and 47 nm PEG micelles adopt dynamin- dependent endocytic trafficking over paracellular transport. We characterized the emergence of caveolae in the vasculature up to 17 days post-fertilization (dpf) using a cavin1b knock-in zebrafish line. Using cavin1a/cavin1b double knockout (DKO) zebrafish, and tumor-bearing Cavin-1 null mice, we showed that caveolae do not contribute to the transvascular transport of these PEG-based nanoparticles. This work highlights the rigour of the standardized protocol for assessing nanoparticle trans-endothelial transport in the live zebrafish, providing a systematic approach for quantification using a standardized control.

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Lim, Y.-W., Bell, C. A., Fletcher, N. L., Condon, N. D., Akhter, D. T., Rae, J., Ferguson, C., Martel, N., Humphries, J., Lo, H., Wu, Y., Houston, Z. H., Lagendijk, A. K. A., Hall, T. E., Thurecht, K. J., Parton, R. G.. 2025-07-23. A Standardized Protocol to Investigate Trans-Endothelial Trafficking in Zebrafish: Nano-bio Interactions of PEG-based Nanoparticles in Live Vasculature. https://doi.org/10.1101/2025.07.23.666282

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