Development and optimisation of a human in vitro blood brain barrier model for high throughput screening of drug delivery vehicles

Abstract

The blood brain barrier (BBB) is a highly restrictive barrier which regulates the transportation of compounds into the brain. The high integrity of the BBB is an obstacle to the development of novel therapeutics for the treatment of neurological disorders, such as Parkinson’s Disease (PD). The experimental work described within this thesis successfully developed and optimised a human 3D in vitro BBB model, which has the potential to be used for high throughput screening of novel therapeutics. The in vitro BBB model was developed to mimic the in vivo BBB by demonstrating high integrity (230 Ω/cm2), and low permeability. This was achieved by optimisation of cell positioning, the addition of substrata and the application of shear stress.

Poly (lactic-co-glycolic acid) (PLGA) nanoparticles (NP) which encapsulated Fitc-dextran (70 kDa) (65 % encapsulation efficiency (EE)) were chemically modified with DAS ligand. DAS binds to the alpha 7 nicotinic receptors found on brain endothelial cells which facilitate the traversing of the in vitro BBB model via receptor-mediated transcytosis (RMT). It was demonstrated that DAS-Fitc-dextran-PLGA NP could transverse our in vitro model more efficiently than the non-conjugated Fitc-dextranPLGA NP. This demonstrates that our optimal model (Dynamic, endothelial pericyte+ astrocyte, fibronectin+ gelatin, MgSO4 (EP+A F+G Mg2+)), is an effective model which could be used for screening of delivery of novel therapeutics to the brain.

The accumulation of α-synuclein and tau protein aggregates, which form Lewy bodies (LB) and neurofibrillary tangles (NFT) respectively, are known to be a causative factor in PD development. It is hypothesised that by targeting these causative proteins with their respective antibodies, we could reduce or prevent the accumulation of the aggregates, slowing down PD progression. PLGA NP were effective in encapsulating α-synuclein and tau antibodies (99% EE). Antibodies were successfully released from the PLGA NP (66.75±10.43% at 96 h), and it was shown that they retained their activity by binding to their respective proteins, as demonstrated by dot blot analysis. The released antibodies significantly reduced protein aggregation, as supported by a significant difference in comparison to the untreated protein group, with p-values ranging from P<0.05 to P<0.001 over a 24 to 72-hour period.

PLGA NP with anti-tau and anti-a-synuclein antibody payloads, conjugated to the targeting ligand DAS may have potential for the treatment of PD. The utility of this potential therapeutic in vivo must be investigated but the initial stages of testing using our in vitro BBB model are promising.

Thesis embargoed until 31 October 2026

Date of Award	Oct 2024
Original language	English
Sponsors	Dowager Countess Eleanor Peel Trust
Supervisor	Susan Hawthorne (Supervisor) & Bridgeen Callan (Supervisor)