Targeted delivery of β-carotene potentially prevents blood-brain barrier breakdown after stroke in mice

Abstract

BackgroundAfter an acute stroke, highly reactive oxygen and nitrogen species (ROS/RNS) lead to microvessel injury and inhibit endogenous mediators to prevent the repair process of endothelial dysfunction and tissue injury. Among them, the peroxynitrite (ONOO−) generation as a result of nitric oxide (NO) and superoxide anion (O2·−) reaction contributes to ischemic brain injury. PurposeTo validate the targeted inhibition of ischemic injury's markers with the delivery of potent antioxidants such as β-carotene (BC) in lipid nanoparticles (LNPs) directly acting with ONOO− compound to prevent the breakdown of blood-brain barrier (BBB). Study designIn the current work, BC in polyethylene glycol (PEG) lipid nanoparticles (PEG-LNPs) with or without attached anti-nitrotyrosine antibody (3-NT) inhibits ONOO− generation in the transient middle cerebral artery occlusion (tMCAO) model. Transmission electron microscopy (TEM) was used to examine structural change in LNPs with or without 3-NT antibody attachment. Fluorescence imaging was employed to study drug encapsulated PEG-LNPs accumulation in mice brain following MCAO and spectrophotometry for the in-vitro drug release profile. Immunoblotting using antibodies specific for spectrin and calcineurin, occludin and ZO-1 and other targets was utilized. The NP3 fluorescent probe was used for the detection of ONOO−generation. Evans Blue was injected 24 h after tMCAO in vehicle and BC PEG-LNPs with 3-NT antibody treated animal groups to observe the tight junction breakdown. ResultsThe fluorescence's data reveals that attachment of 3-NT antibody has directed the delivery of BC to the ischemic ipsilateral region. BC, as well, is found effective to improve infarct volume and neurological deficit by neuronal protection by inhibiting spectrin and calcineurin (CaN) breakdown. Furthermore, inhibition of ONOO−generation is observed in NP3 probe incubation of brain slices and protects BBB from breakdown in Evans blue extravasation. ConclusionThe current work suggests that inhibiting ONOO−at ischemic site protects the brain microvessels from injury. 3-NT/BC/LNPs carrier system is potentially efficient in delivering BC at the site of ONOO−generation, and possibly suitable for ischemic region-targeted therapy.