Study On CXCR4 Overexpressed Cell Membrane Coated With ROS-responsive Nanomedicine For Targeted Treatment Of Ischemic Stroke

As a disease characterized by cerebral ischemia and hemorrhagic injury,stroke has a very high mortality and disability rate.Ischemic stroke subjects represented up to 80%of the stroke population,which mostly caused by interruption of blood flow in the brain due to thrombosis or embolus.If occlusion persists for more than 5 to 10 minutes,leading to the formation of characteristic infarcts surrouding surrouding the ischemic necrosis.The longer the disrupted lasts,the larger the ischemic core area will continue to transform into a penumbra,with the amount of salvageable nerve tissue will gradually decrease.The current approved treatment is mainly to remove the occlusions by thrombolytic drug or endovascular thrombectomy,so as to prevent the further deterioration of the cerebral ischemic injury.However,the therapeutic window is short and only the low percentage stroke patients could actually receive these treatment.Unfortunately,there still remains almost rarely perfect therapy strategy to reverse the recovery of damaged nerous cells and restoration of the connections between injured nerves.Although the mature brain has been proved to maintain the ability of moderate internal regeneration,it cannot fully achieve functional recovery because of its limited endogenous repair ability.Therfore,the developing therapies of enhanced nerve regeneration and improved functional recovery should be an evaluable alternative.With the advancement of nanotechnology and bioengineering,recently,major innovations in stroke treatment has been occured:the prolonging circulation time of drugs in vivo;the blood-brain barrier crossing,which can be achieved by improved size,shape and surface charge of the nanocarrier.Modifying the surface of nanomaterials results in the targeted specificity of stroke therapy,that,can be further elevated according to the specificity proteins at ischemic sites.Of note,the excessive oxidative stress caused by cerebral ischemia can be acted as the stimulus for controlled release of nanomedicine.Importantly,the strengthening neuroprotection may make contribution to the prevation of parenchymal injury.This study focused on a bioengineered CXCR4 overexpression cell membrane coated ROS-responsive nanoparticles,the scientific interest is in designing and preparation for the treatment of cerebral ischemic stroke.Firstly,the particle size,zeta potential and related physical and chemical properties were verified by nanomaterial characterization.next,its targeting,biosafety,ROS scavenging effect and anti-inflammatory effect were evaluated by in vitro cell experiment;meanwhile,the therapeutic effect of nano drug in vivo was comprehensively estimated by using MCAO mice models;finally,the biocompatibility,hepatorenal toxicity and drug pharmacokinetics of nanomedicine were validated.The main content and results are as follows:?After Lentivirus transcription,the CXCR4 overexpression mouse primary thoracic aorta cells was obtained.The 8^th passage primary cells were identified by q-PCR and WB,which was confirmed that CXCR4 could still be stably and highly expressed.Cell experiments showed that the CXCR4 overexpression endothelial cells could migrate specifically through the CXCR4/SDF-1 pathway.The CXCR4overexpression primary endothelial cell membrane was collected by physical extrusion method,the results of WB and SDS-PAGE demonstrated that there was still significant CXCR4 overexpression protein on the surface of the coated nanoparticles.?In light of the up-regulation of reactive oxygen species(ROS),neuroinflammation occur within neurovascular units in the ischemic neuron involved in the events of ischemia-reperfusion.Herein,a multi-target drug delivery system including specific targeting to the ischemic site,good controllability of drug release and long circulation lives is developed to achieve therapeutic efficacy.The ROS-responsive nanocarrier is composed of p-Hydroxybenzyl alcohol(HBA)and PEG₂₀₀₀ through condensation reaction of oxalyl chloride(OC),against oxidative damage consume and eliminate of excessive ROS.The ROS-sensitive polymer loaded with rapamycin(RAPA)then encapsulated into a bioengineered overexpression of CXCR4 cell membrane,target to the stroke ischemic brain damage location of SDF-1 promoted expression.These targeted?core-shell?nanoparticles(designated as RAPA@BMHOP)could thus have controlled release of nanomedicine triggered by high intracellular ROS in ischemic neurons after homing to ischemic brain tissues.The potential of the RAPA@BMHOP for ischemic stroke therapy was systematically evaluated in vitro.The results indicated that the chemical binding of HBA with PEG₂₀₀₀ could be observed by ¹H NMR.The morphology features of nanoparticles was about 200 nm are determined by TEM scanning and DLS measurement respectively.The stability of nanoparticles could be last for 7 days,and ROS responsive chemical bond breaking could occur rapidly in PBS buffer containing H₂O₂,which has high entrapment efficiency.?HUVEC and RAW264.7 cells were used to explore the effect of RAPA@BMHOP in vitro.The results of HUVEC co-culture with BMHOP implied that no adverse effects on cell proliferation at a concentration?100 g/mL.RAPA@BMHOP could effectively reduce the intracellular ROS level and repair the growth activity of cells damaged by peroxide.Using of induced-HUVEC with high expression of SDF-1under the stimulation of ox-LDL,the results of cell flow cytometry and confocal microscopy also confirmed that DiD@BMHOP had good targeting ability;and RAPA@BMHOP could effectively inhibit the growth of inflammatory cells when they co-cultured with RAW264.7;and the encapsulated DiD@BMHOP could well cross the transwell simulated blood–brain barrier(BBB)in vitro.?The potential of the RAPA@BMHOP for ischemic stroke therapy was systematically evaluated in middle cerebral artery occlusion(MCAO)mouse model by tail vein injection.The drug targeting ability,BBB transmembrane properties,drug biocompatibility and stroke therapy effect were evaluated.Ex vivo IVIS imaging showed that RAPA@BMHOP with red fluorescent could accumulated into ischemic location within 2 hours,and it would be well maintained until 24 hours.The permeability of the BBB were investigated by using the traditional Evans Blue(EB)staining assay,which demonstrated that RAPA@BMHOP can maintain the integrity of the BBB.DHE staining brain tissue slides indicated that the ROS level significantly decreased,and the expression of pro-inflammatory factors decreased,and the content of anti-inflammatory factors increased.According to the TTC staining showed that RAPA@BMHOP exerted an obvious protective effect on the salvageable ischemic penumbra.Finally,the good pharmacokinetic properties of DiD@BMHOP were confirmed by the determination of fluorescence content in blood,which could be attributed to the immune evasion ability of the?don‘t eat me?protein CD47 on the bioengineered cell membranes;the H&E staining of heart,liver,spleen,lung and kidney showed no evidence of abnormal and inflammatory cell infiltration,demonstrating the good biocompatibility of BMHOP in vivo.There is no significant abnormality in the estimated serum parameters by biochemical detection.All ultimately further demonstrated enhanced ischemic injury repairation,good biocompatibility and prolong systemic circulation of RAPA@BMHOP.In conclusion,this strategy gives a proof of concept that RAPA@BMHOP can serve as an integrated ischemic stroke treatment,enhance the active targeting of the ischemic area in the MCAO models,and reduce ischemic brain damage.This nanoparticle was aimed at enhancing the active targeting of therapeutic agents,achieved by the interaction of the chemokine receptor,CXCR4 with its ligands SDF-1,with prolonged in vivo circulation time.In vitro evaluation showed that direct reparation of ischemic stroke were achieved by combined ROS scavenging and anti-inflammatory effects,indicating RAPA@BMHOP‘s potential to provide protective effects against ischemia-perfusion injury.Furthermore,ex vivo fluorescent imaging study showed that RAPA@BMHOP exhibited remarkable active targeting functionalities.Importantly,RAPA@BMHOP greatly ameliorated neuroscores and infarction volumes in response to surgical MCAO injury.Therefore,compared with current neuroprotectants,our findings suggest that RAPA@BMHOP could provide more options for a systemic control of multiple events during disease cascades,and may be utilized as a potential formulation strategy to enhance the clinical treatment of ischemic stroke.