Abstract
Absorptive- and receptor-mediated transcytosis (AMT/RMT) are widely studied strategies to deliver therapeutics across the blood-brain barrier (BBB). However, an improved understanding of the mechanism surrounding trafficking is required that could promote delivery. Accordingly, we designed a flexible platform that merged AMT and RMT motifs on a single scaffold to probe various parameters (ligand, affinity, valency, position) in a screening campaign. During this process we adapted an in vitro BBB model to reliably rank transcytosis of the vehicle library. Our results demonstrate heightened uptake and trafficking for the shuttles, with a structure-activity relationship for transcytosis emerging. Notably, due to their small size, the majority of shuttles demonstrated increased permeation compared to transferrin, with the highest performing shuttle affording a 4.9-fold increase. Consequently, we have identified novel peptide conjugates that have the capacity to act as promising brain shuttles.
Highlights
A major hurdle hindering the diagnosis and treatment of neurological disorders is the difficulty for biotherapeutics to enter the central nervous system (CNS)
This is due to the blood–brain barrier (BBB), which is comprised of tightly connected polarized endothelial cells that limit the passage of hydrophilic components and prevents the accumulation of material for transport at the BBB.[1]
Whilst there is limited experimental data surrounding the molecular basis of uptake and trafficking for CNS delivery, it has been demonstrated that dissociation from receptors on the brain side is essential for trafficking mediated by the transferrin receptor (TfR), the quintessential receptor for receptor mediated-transcytosis (RMT).[5]
Summary
A major hurdle hindering the diagnosis and treatment of neurological disorders is the difficulty for biotherapeutics to enter the central nervous system (CNS). Both TfRL1 and TfRL2 have been shown to interact with TfR at alternative sites compared to transferrin (Tf), and have either nanomolar (TfRL1; 15.0 nM) or micromolar affinity (TfRL2; 440 mM) towards the receptor.[9] branched BBB-shuttles incorporating dimers of TfRL1 have recently shown a non-linear increase in uptake within cellular models of the BBB.[11] Alternatively, Angiopep[2] (APep2) displays a high transcytosis potential mediated by LRP1.8 While less is known about traf cking of LRP1, APep2-conjugates have shown demonstrable success in clinical trials of neurological disease models.[8] Peptide ligands were synthesised by general SPPS on rink-amide resin (see Table S1† for characterisation data and yields).
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