| Ischemic stroke is one of the most common inflammatory encephalopathies,characterized by high morbidity,high disability rate and high fatality rate,which seriously threatens human health and causes a heavy social burden.The treatment of ischemic stroke currently faces two major bottlenecks.One is the existence of the blood-brain barrier,whose high selectivity and low permeability maintain the homeostasis in the brain and hinder the delivery of drugs to the brain.Without an effective targeted delivery system,it is difficult to precisely deliver drugs to ischemic lesions.Second,after stroke occurs,it is often accompanied by a severe inflammatory response.If the development of inflammation cannot be controlled in time,it is easy to cause irreversible brain damage.Therefore,using the inflammatory properties of ischemic lesions to construct a highly efficient drug delivery system carrier with brain targeting ability to achieve efficient treatment of ischemic stroke has gradually become a research hotspot.Common nanoscale delivery carriers targeting ischemic stroke lesions include polymer micelles,liposomes and inorganic nanoparticles,among which liposomes are relatively common nanoscale delivery carriers with good biocompatibility and strong modification potential.system.In recent years,biologically derived delivery systems have shown unique advantages in stroke targeting and treatment.Among them,stem cells have the ability to target and migrate to the focal part of stroke,so they can be used as an efficient drug/gene carrier for inflammatory brain targeting.The stem cell membrane is one of the material bases for the targeting ability of stem cells,retaining a large number of receptors and ligands,making it inherit the inflammatory targeting ability of stem cells.Compared with stem cells,stem cell membrane modification is more flexible and the size range is easier to control,so it can be used for nanoparticle biomimetic modification and the construction of inflammatory brain targeted delivery systems.Firstly,the preparation method of biomimetic targeting carrier based on stem cell membrane was explored,and the membrane of mesenchymal stem cells(MSCs)was fused with phospholipid material.Using the fusion principle of similar fusion of phospholipids,stem cell membranes were fused with phospholipid materials to prepare biomimetic targeting carriers,and suitable fusion methods and prescription ratios were screened,and the fusion ability with different electrical phospholipid materials was investigated.Then,the loading capacity and release mode of the stem cell membrane derived biomimetic targeted delivery system for the lipid-soluble anti-inflammatory drug curcumin and the water-soluble anti-inflammatory drug metformin were explored,and the drug-loading performance of the drug delivery system was investigated.The in vivo and in vitro biocompatibility of the constructed stem cell membrane biomimetic targeted delivery system was also investigated.On this basis,the in vivo distribution and targeting ability of brain inflammatory regions were tracked,and the stem cell membrane derived biomimetic targeted delivery system loaded with anti-inflammatory drugs was investigated on the phenotypic changes and inflammation regulation of microglia in the brain of ischemic stroke model mice to investigate its therapeutic effect in the treatment of ischemic stroke.Further,the specific recognition between the stem cell membrane derived biomimetic targeted delivery system and the injured brain microvascular endothelial cells was preliminarily explored,and the targeting mechanism was analyzed.Finally,it is verified that the preparation method obtained by screening has certain generality.Using the principle of similar compatibility,the mesenchymal stem cell membrane and the phospholipid material are fused.According to the preparation method of biomimetic modification strategy,the application of mechanical co-extrusion method and ultrasonic fusion method in stem cell membrane derived biomimetic targeted delivery system was explored.By comparing the particle size and yield of the biomimetic targeted delivery system prepared by the two methods,the fluorescence co-localization of stem cell membranes and phospholipid materials,etc.,the ultrasonic fusion method was selected as the appropriate preparation method.The ratio of stem cell membrane and phospholipid material was refined and screened.From the particle size,zeta potential and fluorescence co-localization results of stem cell membrane and phospholipid material,phospholipid to stem cell membrane protein weight ratio of 1:0.5 was selected as the appropriate preparation ratio.The prepared biomimetic targeted delivery system was characterized,and the stem cell membrane was successfully grafted into the liposome,and its surface total protein and characteristic protein markers were successfully grafted into the biomimetic targeted delivery system.Through the combination of different electrical phospholipid materials and mesenchymal stem cell membranes,biomimetic targeting carriers with different electrical properties were constructed,indicating that this method broke the mechanical strength and electrical limitations of cell membrane-coating nanotechnology.This study firstly investigated the drug loading capacity and release mode of the mesenchymal stem cell membrane biomimetic targeted delivery system.Mesenchymal stem cell membrane biomimetic targeted delivery system has better loading capacity for lipid-soluble anti-inflammatory drug curcumin and water-soluble anti-inflammatory drug metformin.Compared with simple liposomes,its drug loading capacity has no effect.It has controlled release capability for both drugs,and can achieve rapid drug release in an acidic environment.Then,the biocompatibility in vitro and in vivo of the prepared mesenchymal stem cell membrane biomimetic targeted delivery system was investigated.Compared with simple liposomes,the in vitro cytotoxicity of the mesenchymal stem cell membrane biomimetic targeted delivery system to brain microvascular endothelial cells and microglia was significantly reduced.And the hemolytic ability is reduced,and the blood safety is further improved.After a single injection through the tail vein,during the two-week observation period,the mice had no obvious toxic reactions,maintained an increase in body weight,and all physiological indicators were normal.Two weeks later,the blood routine and blood biochemical indexes of the mice were detected,and the brain,heart,lung,liver,spleen and kidney were analyzed by sectioning and H&E staining.The results showed that there was no change in the blood routine and blood biochemistry.The quality is stable and no abnormality is seen.It shows that the mesenchymal stem cell membrane biomimetic targeted delivery system has good biocompatibility in vitro and in vivo.After injection through the tail vein,the mesenchymal stem cell membrane derived biomimetic targeted delivery system has the ability of long circulation in vivo,and can achieve efficient targeting and long retention in the brain lesions of middle cerebral artery occlusion(MCAO)mice.According to the drug loading and in vitro release,the curcumin-loaded mesenchymal stem cell membrane derived biomimetic targeted delivery system was selected to treat MCAO mice.Compared with the drug-loaded liposome treatment,a single injection could improve the survival of MCAO mice significantly.The rate has increased from 30% to over 90%.Microglia have strong affinity for the mesenchymal stem cell membrane derived biomimetic targeted delivery system,which can promote the repolarization of M1 type to M2 type in vivo,and significantly reduce the release level of inflammatory factors.In addition,behavioral exploration of mice showed that MCAO mice treated with curcumin-loaded mesenchymal stem cell membrane biomimetic targeted delivery system had better neurological and motor recovery.This study further preliminarily explores the inflammatory brain targeting mechanism of the stem cell membrane biomimetic targeted delivery system.After mesenchymal stem cell membrane transfer,very late antigen-4(VLA-4),a specific ligand of vascular cell adhesion molecule-1(VCAM-1),was It was successfully grafted into the stem cell membrane biomimetic targeted delivery system,and the VCAM-1 binding and targeting ability on the surface of damaged cerebral vascular endothelial cells was achieved through the VCAM-1/VLA-4 pathway.After the stem cell membrane derived biomimetic targeted delivery system was endocytosed into endothelial cells,it hardly entered lysosomes,and could cross the blood-brain barrier and penetrate deep into the tissue in an intact form.It was suggested that the VCAM-1/VLA-4 pathway was a key molecular pathway for the inflammatory brain targeting of the stem cell membrane derived biomimetic targeted delivery system.In order to verify the versatility of the preparation method of the stem cell membrane biomimetic targeted delivery system,a biomimetic targeted delivery system based on neural stem cell membrane was constructed.The drug-loading ability of the neural stem cell membrane biomimetic targeted delivery system was investigated by using the lipidsoluble anti-inflammatory drug curcumin and the water-soluble anti-inflammatory drug metformin.After the stem cell membrane was added,the loading capacity of the two drugs was not affected,and it had controlled release capacity.After neural stem cell membrane fusion,the neural stem cell membrane derived biomimetic targeted delivery system has good in vitro biocompatibility with cerebral vascular endothelial cells and microglia,and inherits the in vivo long-circulation and brain inflammation targeting capabilities of neural stem cells,realizing the simple integration of complex biological functions.In conclusion,this study utilized stem cell membrane and phospholipid materials to construct a biomimetic targeted delivery system based on stem cell membrane.The versatility of the preparation method and the platform of the delivery system were verified by stem cell membranes from different sources and drugs of different properties.The stem cell membrane derived biomimetic targeted delivery system had high affinity with brain microvascular endothelial cells and microglia,and could target injured brain microvascular endothelial cells in the lesion area through the VCAM-1/VLA-4 pathway.Loading and targeting delivery of the lipid-soluble anti-inflammatory drug curcumin achieved effective treatment of MCAO mice,regulated the phenotype of microglia,inhibited the release of pro-inflammatory inflammatory factors,and improved the survival rate of MCAO mice.This paper proposes a new strategy for biomimetic functional modification of cell membranes,which provides new ideas and experimental basis for the construction of stem cell membrane-based biomimetic targeted delivery systems and the treatment of ischemic stroke. |
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