IntroductionThe amyloid-β42 (Aβ42) peptide plays a crucial role in the pathogenesis of Alzheimer’s disease (AD), the most common neurodegenerative disorder affecting the elderly. Over the past years, several approaches and compounds developed for the treatment of AD have failed in clinical studies, likely in part due to their low penetration of the blood–brain barrier (BBB). Since nanotechnology-based strategies offer new possibilities for the delivery of drugs to the brain, this technique is studied intensively for the treatment of AD and other neurological disorders.MethodsThe Aβ42 lowering drug flurbiprofen was embedded in polylactide (PLA) nanoparticles by emulsification-diffusion technique and their potential as drug carriers in an in vitro BBB model was examined. First, the cytotoxic potential of the PLA-flurbiprofen nanoparticles on endothelial cells and the cellular binding and uptake by endothelial cells was studied. Furthermore, the biological activity of the nanoparticulate flurbiprofen on γ-secretase modulation as well as its in vitro release was examined. Furthermore, the protein corona of the nanoparticles was studied as well as their ability to transport flurbiprofen across an in vitro BBB model.ResultsPLA-flurbiprofen nanoparticles were endocytosed by endothelial cells and neither affected the vitality nor barrier function of the endothelial cell monolayer. The exposure of the PLA-flurbiprofen nanoparticles to human plasma occurred in a rapid protein corona formation, resulting in their decoration with bioactive proteins, including apolipoprotein E. Furthermore, luminally administered PLA-flurbiprofen nanoparticles in contrast to free flurbiprofen were able to modulate γ-secretase activity by selectively decreasing Aβ42 levels in the abluminal compartment of the BBB model.ConclusionsIn this study, we were able to show that flurbiprofen can be transported by PLA nanoparticles across an in vitro BBB model and most importantly, the transported flurbiprofen modulated γ-secretase activity by selectively decreasing Aβ42 levels. These results demonstrate that the modification of drugs via embedding in nanoparticles is a promising tool to facilitate drug delivery to the brain, which enables future development for the treatment of neurodegenerative disorders like AD.
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.
This study was performed to explore the feasibility of tracing nanoparticles for drug transport in the healthy rat brain with a clinical MRI scanner. Phantom studies were performed to assess the R1 ( = 1/T1) relaxivity of different magnetically labeled nanoparticle (MLNP) formulations that were based on biodegradable human serum albumin and that were labeled with magnetite of different size. In vivo MRI measurements in 26 rats were done at 3T to study the effect and dynamics of MLNP uptake in the rat brain and body. In the brain, MLNPs induced T1 changes were quantitatively assessed by T1 relaxation time mapping in vivo and compared to post-mortem results from fluorescence imaging. Following intravenous injection of MLNPs, a visible MLNP uptake was seen in the liver and spleen while no visual effect was seen in the brain. However a histogram analysis of T1 changes in the brain demonstrated global and diffuse presence of MLNPs. The magnitude of these T1 changes scaled with post-mortem fluorescence intensity. This study demonstrates the feasibility of tracking even small amounts of magnetite labeled NPs with a sensitive histogram technique in the brain of a living rodent.
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Since nanoparticles (NPs) have shown great potential in various biomedical applications, live cell response to NPs should be thoroughly explored prior to their in vivo use. In the current study, live cell array (LCA) methodology and unique cell-based assays were used to study the interaction of magnetite (HSA-Mag NP) loaded human serum albumin NPs with phagocytic cells. The LCA enabled cell culturing during HSA-Mag NP accumulation and monolayer or spheroid formation, concomitantly with on-line monitoring of NP internalization. These platforms were also utilized for imaging intercellular links between living cells preloaded with HSA-Mag NP in 2D and 3D resolution. HSA-Mag NP uptake by cells was quantified by imaging, and analyzed using time-resolved measurements. Image analysis of the individual cells in cell populations showed accumulation of HSA-Mag NP by promonocytes and glial cells in a dose- and time-dependent manner. High variability of NP accumulation in individual cells within cell populations, as well as cell subgroups, was evident in both cell types. Following 24 h interaction, uptake of HSA-Mag NP was about 10 times more efficient in glial cells than in activated promonocytes. The presented assays may facilitate detection and analysis of the amount of NPs within individual cells, as well as the rate of NP accumulation and processing in different subsets of living cells. Such data are crucial for estimating predicted drug dosage delivered by NPs, as well as to study possible mechanisms for NP interference with live cells
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