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Two peptides targeting endothelial receptors are internalized into murine brain endothelial cells

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  • Diána Hudecz
  • Sara Björk Sigurdardóttir, Danmarks Tekniske Universitet
  • ,
  • Sarah Christine Christensen
  • ,
  • Casper Hempel, Danmarks Tekniske Universitet
  • ,
  • Andrew J. Urquhart, Danmarks Tekniske Universitet
  • ,
  • Thomas Lars Andresen, Danmarks Tekniske Universitet
  • ,
  • Morten S. Nielsen

The blood-brain barrier (BBB) is one of the main obstacles for therapies targeting brain diseases. Most macromolecules fail to pass the tight BBB, formed by brain endothelial cells interlinked by tight junctions. A wide range of small, lipid-soluble molecules can enter the brain parenchyma via diffusion, whereas macromolecules have to transcytose via vesicular transport. Vesicular transport can thus be utilized as a strategy to deliver brain therapies. By conjugating BBB targeting antibodies and peptides to therapeutic molecules or nanoparticles, it is possible to increase uptake into the brain. Previously, the synthetic peptide GYR and a peptide derived from melanotransferrin (MTfp) have been suggested as candidates for mediating transcytosis in brain endothelial cells (BECs). Here we study uptake, intracellular trafficking, and translocation of these two peptides in BECs. The peptides were synthesized, and binding studies to purified endocytic receptors were performed using surface plasmon resonance. Furthermore, the peptides were conjugated to a fluorophore allowing for live-cell imaging studies of their uptake into murine brain endothelial cells. Both peptides bound to low-density lipoprotein receptor-related protein 1 (LRP-1) and the human transferrin receptor, while lower affinity was observed against the murine transferrin receptor. The MTfp showed a higher binding affinity to all receptors when compared to the GYR peptide. The peptides were internalized by the bEnd.3 mouse endothelial cells within 30 min of incubation and frequently co-localized with endo-lysosomal vesicles. Moreover, our in vitro Transwell translocation experiments confirmed that GYR was able to cross the murine barrier and indicated the successful translocation of MTfp. Thus, despite binding to endocytic receptors with different affinities, both peptides are able to transcytose across the murine BECs.

OriginalsprogEngelsk
Artikelnummere0249686
TidsskriftPLOS ONE
Vol/bind16
Nummer4
Antal sider20
ISSN1932-6203
DOI
StatusUdgivet - apr. 2021

Bibliografisk note

Funding Information:
The Authors has been supported by a grant from the Lundbeck foundation (grant number R155-2013-14113) The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors acknowledge the AU Health Bioimaging Core Facility and AU Molecular Interactions Core Facility. S?ren K. Moestrup, University of Aarhus, kindly provided purified LRP-1. Claus Pietrzik, University of Mainz, kindly provided anti-LRP-1. Furthermore, Annemette Boe Marnow, University of Aarhus, is acknowledged for her technical assistance.

Publisher Copyright:
© 2021 Hudecz et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Copyright:
Copyright 2021 Elsevier B.V., All rights reserved.

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