Surface-engineered Cascade-targeting Exosomes Contribute To Protection Of Ischemic Stroke Via Modulating Polarization Of Microglia

ObjectiveStroke is a neurological disease with a poor prognosis.After stroke,there is strong inflammation and immune response,leading to serious sequelae and extremely high mortality.Currently,drug treatment for stroke includes antithrombotic drugs,thrombolytic drugs and neuroprotective drugs,but their efficacy is generally limited.Neuroprotective drugs such as neurotrophins contribute to functional reconstruction of infarcts,but do not enrich in ischemic areas.Therefore,there IS an urgent need for effective treatment of ischemic stroke.When IS occurs,microglia cells can be converted into pro-inflammatory M1 and produce cytokines and chemokines,exacerbating neuronal damage In addition,activated M1 microglia induce neurotoxic reactive astrocytes,and activated microglia lead to blood-brain barrier damage in ischemic stroke by down-regulating tight junction protein(TJPs)expression such as occludin and claudin-5.IS often causes NVU disturbance,manifested by blood-brain barrier injury glial cell changes and neuronal death Luteolin(lut),which has various biological activities such as antioxidant,anti-inflammatory,anti-atherosclerosis and anti-tumor In particular,lut has a beneficial neuroprotective effect,inhibiting neuroinflammation by inhibiting oxidative stress and regulating the activation of microglial cells in cerebral ischemia models.However,its inability to target the brain,poor solubility,and low bioavailability have severely limited its use in food and medicine.Platelet-derived exosomes(pEXO)have emerged as promising vectors for brain drug delivery due to their nanosize and ability to breach the blood-brain barrier(BBB).They can accept a wide variety of cargo,have good biocompatibility,and are easy to incorporate drugs In particular,thanks to platelets’ innate role in pathological thrombus,pEXO binds to platelets and effectively targets damaged blood vessels Based on the potential regulation of microglia polarization by lut and the inherent targeting and loading capability of pEXOs,we designed surface-engineered lut/ Man-Pexo-supported plateletderived exosomes(lut/man-pEXO)to regulate microglia polarization by enhancing sequence targeting and contribute to neuroprotection of NVU.MethodsFirst,pEXO was isolated and extracted from platelet-rich plasma by overspeed centrifugation,and lut was loaded into platelet-derived exosomes by ultrasound co-incubation to obtain lut/pEXO.Then,DSPE-PEG-mannose was modified on the surface of lut/pEXO to obtain lut/man-pEXO by physical addition The parameters such as the release of morphometric particle size marker proteins were investigated by Western Blot and UV.The ability of lut/man-pEXO to enter the ischemic side of the brain in middle cerebral artery ischemia reperfusion(MCAO/R)rats was evaluated by immunofluorescence.MTT TUNEL kit and Western Blot were used to verify the mechanism of action and anti-apoptosis of lut/man-pEXO in OGD/R model.In vitro experiments,immunofluorescence and Western Blot tests were used to prove the inhibitory effect of lut/man-pEXO on OGD/R model.In vivo,the neuroprotective effects of lut/man-pEXO on MCAO/R rats were evaluated by classical 2,3,5-triphenyltetrazole chloride(TTC)staining and neurofunctional scores;The effect of lut/man-pEXO on the inflammation of MCAO/R model was further investigated at the tissue level by immunofluorescence and Western Blot lut/man-pEXO alleviated blood-brain barrier deterioration and inhibited astrocyte activation to improve the function and integrity of NVU using Evans Blue,immunofluorescence,and Western Blot assay.ResultsThe results of particle size analysis by transmission electron microscopy showed that the prepared lut/pEXO had a complete structure,regular spherical shape,uniform particle size of about 100 nm.With the help of pEXO,lut showed a stable and lasting release mode Furthermore,in the MCAO/R model,platelets adhere to and accumulate in the injured cerebral vessels,and lut/manpEXO specifically migrates to the injured cerebral vessels by binding to platelets and effectively targeting the ischemic site Subsequently,lut/manpEXOs targets microglia and promotes drug accumulation through binding of lut/man-pEXOs surface-modified mannose to mannose receptors in microglia as a potential targeting guide.The results of MTT,TUNEL and Western Blot showed that lut/man-pEXO effectively alleviated OGD/ R-induced cell damage and greatly reduced the degree of apoptosis TTC staining and neurofunctional scores showed that lut/man-pEXO could reduce cerebral infarction volume and improve neural function in MCAO/R rats.The results of Nissl staining and immunofluorescence staining showed that lut/man-pEXO could significantly maintain neuronal structure and increase MCAO/R.The number of neuronpositive cells in the ischemic area of rats,These results indicate that lut/manpEXO therapy can effectively reduce the damage of neurons in ischemic area by immunofluorescence and Western Blot,Evans Blue experiments showed that lut/man pEXO inhibited the inflammatory response by shifting microglia polarization from the harmful pro-inflammatory M1 phenotype to the beneficial anti-inflammatory M2 phenotype,thereby alleviating blood-brain barrier deterioration by saving neuronal survival and inhibiting astrocyte activation to improve the function and integrity of NVU.ConclusionsThe lut/man-pEXOs potentiated efficient sequential targeted delivery via platelet-mediated accumulation at ischemic blood vessels and microglial targeting of lut based on the interaction between man-pEXO-delivered mannose and the mannose receptors expressed in microglia.Therefore,they exerted enhanced neuroprotective effects on the neurovascular unit,which included the alleviation of BBB damage,reducing neuronal cell death and inhibiting glial reaction in ischemic stroke.This protection against dysfunction of the neurovascular unit was attributed to the beneficial anti-inflammatory effects of lut/man-pEXOs,which promoted the polarization of microglia from the M1 phenotype to the M2 phenotype.Taken together,these encouraging results indicated that lut/man-pEXOs provide a potentially extensive and clinically applicable effective treatment strategy for reducing the disorder of NVU and protecting against ischemic stroke.