| About 15 million people worldwide have a stroke each year,and about 5 million die from strokes.In China,one person has a stroke every 12 seconds and one person dies from a stroke every 21 seconds.Ischemic stroke is the most common type of stroke,caused by massive infarction of brain tissue due to blockage of the middle cerebral artery,and to date intravenous tissue-type fibrinogen activator has been the only treatment for acute ischaemic stroke.However,the use of this drug in clinical practice is limited by the very short treatment window(3-4.5 hours)and the risk of haemorrhagic transformation.Neutrophils as immune cells in vivo and their interaction with cerebrovascular endothelial cells is an important pathological feature of ischaemic stroke.Neutrophil migration towards the blood-brain barrier and infiltration into the brain promote the ischaemia-reperfusion cascade response,exacerbating brain tissue damage and neuroinflammation.Therefore,preventing neutrophil migration to the site of ischaemic lesions is a potential approach to treating ischaemic stroke.In this study,we designed and prepared a rod-shaped poly(lactic acid-hydroxyacetic acid)(PLGA)nanoparticle loaded with paclitaxel(Pic)to target and interfere with neutrophil interactions with brain endothelial cells in stroke infarct foci.Firstly,spherical PLGA nanoparticles(Pic@Spheres)were prepared and rodshaped nanoparticles with aspect ratios of 2,3,5 and 6(Pic@AR2,Pic@AR3,Pic@AR5,Pic@AR6)were obtained by thin film thermal stretching.The size morphology,drug loading,drug loading efficiency and drug release of the nanoparticles were characterized by Malvern particle size meter,scanning electron microscope and UV spectrophotometer.Secondly,the phagocytic ability of neutrophils and macrophages on nanoparticles of different morphologies was investigated.Characterisation by laser confocal microscopy and flow cytometry showed that neutrophils phagocytosed Pic@AR5 at 87.50%,while spherical nanoparticles were phagocytosed at 35.9%.Moreover,Pic@AR5 avoided phagocytosis by macrophages.In the competitive phagocytosis assay in blood,50.23% of neutrophils phagocytosed Pic@AR5 and only14.31% of macrophages showed a fluorescence positive signal.Subsequently,Pic@AR5 was found to inhibit transendothelial cell migration of neutrophils and suppress the p-Syk inflammatory signalling pathway in an in vitro blood-brain barrier(BBB)model,and an in vitro inflammation model.In addition,Pic@AR5 exerted anti-inflammatory effects in a microglia glyoxylate deprivation(OGD/R)model,inhibiting microglia p-Syk pathway levels and reducing reactive oxygen species(ROS)release by 28.4%,suggesting that Pic@AR5 can act on microglia through a small fraction of neutrophils mediating entry into the brain.Finally,Pic@AR5 was used to study the treatment of a mouse model of transient middle cerebral artery occlusion(t MCAO).The experimental results of brain tissue immunofluorescence sections,brain cell flow,and intravital fluorescence microscopy showed that compared with the normal saline group,Pic@AR5 could prevent about2.5-fold the degree of neutrophil infiltration.In the exploratory cerebral infarction experiment,Pic@AR5 reduced the infarct volume by 60.3±3.2% at 28 days.In addition,long-term motor neurological function in stroke mice was examined by rotating barometer and Barnes maze,and Pic@AR5 improved motor sensory,spatial memory and cognitive abilities by inhibiting neutrophil infiltration.In experiments on mice with in vivo labeled vessels,Evans blue staining assay and transmission electron microscopy,Pic@AR5 was found to attenuate blood-brain barrier damage,protect tight junction proteins on the blood-brain barrier and improve survival and prognosis of stroke mice. |
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