The blood-brain barrier (BBB) is the most extensive and restrictive barrier to brain delivery for therapeutic agents. A low proportion of low molecular-weight agents can cross into the CNS. This decreases further as the molecular weight increases, meaning therapeutic antibodies, oligonucleotides and other supramolecular entities effectively cannot reach therapeutic levels within the CNS. Targeting ligands against receptors thought to undergo transcytosis across the brain microvascular endothelial cells (BMECs), can boost CNS delivery of therapeutics. Understanding these mechanisms, in an in-vitro setting, has proved challenging, due to the constraints of cell culture systems and the difficulty to replicate the in-vivo environment. With even the most extensively studied targeting receptor, transferrin receptor, not producing clear evidence to suggest the occurrence of transcytosis. To understand in-vitro trafficking of brain targeting ligands a pulse-chase assay, in combination with sub-cellular localisation microscopy was developed and compared to the current permeability-based assay method. The characterisation was done by comparison of transferrin receptor ligands; native holo-transferrin, the 8D3 antibody and a low-affinity variant; with the non-specific uptake probe, dextran. The method could distinguish between the two endocytosis methods, with concentration-dependent efflux efficiency observed with the targeted probes. The combination of techniques was then applied to the novel targeting ligand, Rabies-Virus Glycoprotein (RVG) peptide, to assess its suitability as a brain delivery. Studies were performed to confirm the target receptor of the RVG peptide, including competitive uptake, siRNA knockdown methods. The RVG peptide demonstrated desirable delivery characteristics, and the target receptor was confirmed as the α7 nicotinic acetylcholine receptor. Finally, attempts were made to develop a total internal reflection fluorescence (TIRF) microscopy assay for the assessment of ligand arrival at the basolateral membrane of BMECs. Initial work for this was performed with the transferrin receptor and transferrin, using both labelled ligand and photoswitchable receptor constructs. In summary, the pulse-chase assay provides a complementary technique to permeability assays for the assessment of brain targeting ligand trafficking in BMEC cell-lines in-vitro.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:753549 |
| Date | January 2018 |
| Creators | Sim, Jack |
| Publisher | Cardiff University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://orca.cf.ac.uk/112529/ |
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