Protein Nanoparticle Osmotic Pressure Modifies Nonselective Permeability of the Blood Brain Barrier by Increasing Membrane Fluidity

Abstract

BackgroundIntracellular tension plays a crucial role in the destruction of the blood brain barrier (BBB) in response to lesion stimuli. Tight junction structure could be primarily affected by tension activity. In this study, we aimed to determine the effects of extracellular BBB damage on intracellular tension activity, and elucidate the mechanism underlying the effects of intracellular protein nanoparticle-dependent osmotic pressure on BBB permeability.MethodsThe intracellular tension for tight junction proteins occludin and ZO1 were evaluated using the fluorescence resonance energy transfer (FRET)-based tension probes and cpstFRET analysis. The efficiency of the probes was examined by acceptor photobleaching FRET (FRET-AB) analysis. The changes in mobility ratios of the transmembrane protein occludin were evaluated via the fluorescence recovery after photobleaching (FRAP) test. The cytoplasmic osmotic pressure (OP) was measured using the Osmomat 3000 Freezing Point Osmometer and 050 Membrane Osmometer. The count rate of cytoplasmic nanoparticles was detected by Nanosight NS300. The activation of cofilin and stathmin was examined by Western blot analysis. The BBB permeability in vivo was determined via investigating the changes of Evans Blue (EB) injected into the SD rats. The tight junction formation was assessed by the measurement of transendothelial electrical resistance (TEER). Intracellular calcium or chloride ions were measured using Fluo-4 AM or MQAE dyes. ResultsBBB lesions were accompanied by changes in occludin/ZO1 tension. Increases in intracellular osmotic pressure were involved in alteration of BBB permeability, possibly through the depolymerization of microfilaments or microtubules and mass production of protein nanoparticles according to the Donnan effect. Recovery of protein nanoparticle osmotic pressure could effectively reverse the effects of changes in occludin/ZO1 tension and BBB lesions. Outward tension of intracellular osmotic potential also caused upregulation of membrane fluidity, which promoted nonselective drug influx.ConclusionsOur results suggest a crucial mechanical mechanism underlying BBB lesions, and protein nanoparticle osmotic pressure could be a novel therapeutic target for BBB lesion-related brain diseases. Our results also provide a basis for further studies on the regulation of intracellular tension activity and its effect on the permeability of the BBB, and development of novel drugs that cross the blood-brain barrier.