Neuroprotective Effects And Mechanisms Of Exos Derived From 3D Culture Loaded With Hydrogel In Acute Ischemic Stroke

Background:Acute ischemic stroke(AIS)is the most common type of stroke and the most common fatal and disabling disease in the world.Currently,the main treatment measures are to quickly restore the blood supply to brain tissue in the ischemic region and reduce neurological damage.However,after receiving high-level vascular recanalization treatment in the acute phase,it may cause severe ischemia-reperfusion injury(CIRI),leading to progressive stroke,symptomatic intracranial hemorrhage,and ischemic edema,further worsening the prognosis of patients.Moreover,the current treatment plan cannot promote the role of endogenous brain repair,and patients still have partial neurological dysfunction in the chronic phase.Studies have found that neuritis caused by AIS is crucial for the prognosis of patients.Among them,microglia are the main participants in the inflammatory response of the central nervous system(CNS).Transforming microglia from M1 phenotype to M2 phenotype in the early stage of AIS can quickly restore immune balance and improve the prognosis of AIS patients.In recent years,mesenchymal stem cells(MSCs)can participate in the treatment process after AIS through immune regulation,angiogenesis,and protection of neural cells.However,MSCs have drawbacks such as abnormal differentiation,thrombotic risk,immune rejection,and poor efficacy consistency,which limit their further clinical application.Mesenchymal stem cells exosomes(MSCs-Exos)derived from MSCs can be used as biological therapeutics for AIS treatment,and can achieve similar therapeutic effects as parental cells.However,the production of MSCs-Exos in vitro is low,the bioavailability in the brain is low,and they cannot stay at the lesion site for a long time to play a role.Therefore,improving the production of therapeutic MSCs Exos and constructing a locally controlled delivery pathway for MSCs-Exos are the current research focus.Objective:The purpose of this study was to explore the neuroprotective effect and mechanism of Exos(3D-Exos)derived from 3D culture MSCs loaded with hydrogel on acute ischemic stroke,and to provide a new idea for the treatment of stroke.In this study,we will use hydrogel materials as the in vitro 3D culture medium of MSCs to maximize the simulation of the in vivo environment,enhance the in vitro culture activity of MSCs,and improve the yield and quality of therapeutic Exos.The anti-inflammatory ability of 3D-Exos was evaluated in combination with in vitro cell experiments.Using experimental rat MCAO model,we observed the neuroprotective and behavioral effects of 3D-Exos loaded hydrogel on ischemic hemisphere,and explored its clinical transformation potential and application challenges.Methods:1.The primary MSCs of rats were isolated and planted in hydrogel materials to realize three-dimensional culture(3D culture),overcome the shortcomings of flat monolayer(2D culture)cell culture,and enhance the in vitro culture activity of MSCs.Using ultra high speed centrifugation technology to separate Exos,combined with Western blot and transmission electron microscopy,the surface markers,morphology,and yield of 3D-Exos were detected.2.Using LPS to induce BV2 to convert to M1 subtype,a stable in vitro inflammatory model was constructed and co cultured with 3D-Exos to evaluate the anti-inflammatory capacity of 3D-Exos in vitro.3.In order to realize the long-term high concentration release of Exos in the ischemic region,polyether F127 thermosensitive hydrogel was used as the local controlled drug delivery platform of Exos in this subject.Select the appropriate concentration of F127 hydrogel and construct the Exos loaded F127 hydrogel complex(F127+Exos),draw the phase change temperature curve,record the degradation performance of F127+Exos in vitro,and draw the slow release capacity curve using the PKH-67 fluorescent labeled Exos.4.Using experimental SD rat MCAO models,F127+3D-Exos was injected stereotactically.The biological safety and therapeutic effects of F127+3D-Exos were verified by behavioral scoring,Western bolt detection of inflammatory related proteins in ischemic hemisphere,and tissue staining.Conclusions:1.3D cultured MSCs can maintain MSCs activity in vitro for a long time and produce highly therapeutic Exos for a long time.2.3D-Exos has stronger anti-inflammatory effect,can significantly reduce the secretion of proinflammatory factors,and is easier to be endocytosed by microglia.3.30%Pluronic-F127 hydrogel has good temperature sensitivity.It is a flowable liquid at low temperature.When it is close to human body temperature,it can quickly transform into a soft gel,and can stably release Exos for more than 6 days while maintaining the gel state.4.30%-F127 loaded with 3D-Exos can be safely injected into the ischemic core area of MCAO rats,effectively reducing the inflammatory response in the ischemic hemisphere,and promoting the recovery of neural function in MCAO rats.