Fletcher, N. L. | bioRxiv Science

bioRxiv2025

A Standardized Protocol to Investigate Trans-Endothelial Trafficking in Zebrafish: Nano-bio Interactions of PEG-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 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.

cell biology↗

bioRxiv2022

Self-cyclisation as a general and efficient platform for peptide and protein macrocyclisation

Macrocyclisation of proteins and peptides results in a remarkable increase in structural stability, making cyclic peptides and proteins of great interest in drug discovery--either directly as drug leads or as in the case of cyclised nanodiscs (cNDs), as tools for studies of trans-membrane receptors and membrane-active peptides. Various biological methods have been developed that are capable of yielding head-to-tail macrocyclised products. Such enzymatic methods require careful optimisation of cyclisation over polymerisation. Here, we describe the engineering of self-cyclising "autocyclase" proteins, where an intramolecular rearrangement can be triggered to yield a monomeric cyclic product in high yields. We characterise the self-cyclisation reaction mechanism and demonstrate how the unimolecular reaction path can circumvent existing challenges of enzymatic cyclisation. We use the method to produce several notable cyclic peptides and proteins, demonstrating how autocyclases offer a simple and scalable way to access a vast diversity of macrocyclic biomolecules.

biochemistry↗