Flurbiprofen-loaded Nanoparticles Can Cross a Primary Porcine In vitro Blood-brain Barrier Model to Reduce Amyloid-β42 Burden

Abstract

Elevated amyloid-β42 (Aβ42) in the brain is expected to cause Alzheimer's Disease (AD). Reducing Aβ42 is therefore a cornerstone in causal drug development. Nevertheless, many promising substances failed in clinical trials, because reaching the target organ in vivo is difficult. The brain is protected by the Blood-Brain Barrier (BBB) that shields off most molecules to maintain the brain homeostasis. Brain-targeted nanoparticles are one successful tool to bypass this problem: by acting as Trojan horses they carry embedded drugs across the BBB for brain disorder treatment.Here, flurbiprofen, a γ-secretase modulator, was embedded in Poly(Lactic Acid) (PLA) nanoparticles. We tested if the drug-loaded nanoparticles affected the integrity of our advanced in vitro BBB model in transendothelial electrical resistance measurements and permeability assays, and investigated the nanoparticle-cell interaction in flow cytometry and confocal laser scanning microscopy. Furthermore, we assessed the drug transport capacity by highperformance liquid chromatography and the biological efficacy of the embedded drug in an Aβ42-detecing ELISA. We also verified the viability of the AD model cells by a cellular viability assay.After adding flurbiprofen-loaded nanoparticles to the blood compartment of a Transwell ® model, the drug was detectable in the brain compartment, where it induced an Aβ42 lowering effect. Flurbiprofen from nanoparticles crossed the BBB without impairing barrier integrity, whereas the free drug was highly cytotoxic and destroyed the barrier. Ligand coupling of apolipoprotein E3 to the nanoparticles increased cellular uptake. Hence, we expect an even more pronounced Aβ42 reducing effect for apolipoprotein-modified, flurbiprofen-loaded nanoparticles.In conclusion, we enabled transport of a hardly permeable drug across an advanced in vitro BBB model, opening opportunities in the treatment and prevention of AD and other brain disorders. Using a primary porcine BBB model that displays excellent barrier characteristics, we show that flurbiprofen-loaded nanoparticles reduce Aβ42 burden without impairing barrier function.

Mechanism of drug extrusion by brain endothelial cells via lysosomal drug trapping and disposal by neutrophils

“…For isolation of human blood-derived neutrophils, all subjects gave informed consent for blood sampling and analyses, and respective experiments were approved by the Ethics Committee (agreement 3295-2016) of the Hannover Medical School. Primary cultures of pBCECs and porcine neutrophils were prepared as described ( 76 , 77 ). All experiments with human or porcine cell cultures were performed after cells reached confluency.…”

A face-to-face comparison of claudin-5 transduced human brain endothelial (hCMEC/D3) cells with porcine brain endothelial cells as blood–brain barrier models for drug transport studies

“…Primary porcine brain capillary endothelial cells (pBCECs) were isolated as described earlier [23], with slight modifications. In brief, fresh porcine brain hemispheres from Sus scrofa domestica (domestic pig) were kindly provided by the local slaughterhouse in Hannover (Germany) and stored on ice for transport.…”

Evaluation of the Transport and Binding of Dopamine-Loaded PLGA Nanoparticles for the Treatment of Parkinson’s Disease Using In Vitro Model Systems

“…To achieve this, specific substances, peptides or proteins that are recognised by these processes have to be present on the nanoparticle surface. While the direct linking of these substances to the particle surface is widely used [22,43,44], simpler surface coatings are also able to attract plasma proteins, which will then facilitate the uptake at the blood-brain barrier [34,45]. This shortens the nanoparticle production process and thus allows fewer possibilities to lose some amount of the encapsulated dopamine at each of the steps.…”