| The permeability of the blood-brain barrier is a decisive factor affecting the entry of drug molecules into the nervous system.If the permeability of the blood-brain barrier can be improved,the therapeutic effect of drugs for neurological diseases will be greatly improved.The blood-brain barrier is mainly composed of endothelial cells and peripheral structures of brain microvessels(such as basement membrane,astrocyte foot processes,pericytes,etc.).The existence of the blood-brain barrier is a "double-edged sword".On the one hand,it blocks harmful substances from entering the nervous system,actively effluxes metabolic wastes,and regulates the ionic strength of cerebrospinal fluid.On the other hand,it also prevents most therapeutic drugs from actively passing through.Blood-brain barrier,enter the brain tissue to play the corresponding therapeutic function.Therefore,how to safely,effectively and reversibly improve the permeability of the blood-brain barrier,thereby increasing the accumulation of drugs in lesions,has become one of the research hotspots of drug delivery in the nervous system.In order to effectively improve the permeability of the blood-brain barrier,this paper designed a method to improve the permeability of the blood-brain barrier by using superparamagnetic iron oxide nanoparticles(SPIONs)to interact with vascular endothelial cells driven by an external magnetic field.First,SPIONs with good water dispersibility and a diameter of about 200 nm were prepared.The magnetic property test results showed that they have superparamagnetic properties and a maximum magnetic saturation intensity of 166 emu/g.To apply a strong external magnetic field to study the reversible trapping of SPIONs in animals,the results show that they can achieve reversible regulation of magnetic field-driven aggregation in cerebral blood vessels.Subsequently,Evans blue(EB)and SPIONs were co-injected into mice via the tail vein,and different magnetic fields were applied to their brains for different time periods.It is proved that the permeability of the blood-brain barrier can be significantly improved under the action of magnetic field for 4 h.In order to explore the effect of exogenous magnetic force on the leakage and repair of BBB,different modes of action were adopted in this paper: only PBS,only injected SPIONs and EB,only injected EB and applied magnetic field,injected SPIONs and applied magnetic field then injected Evans blue,co-injected EB with SPIONs,then applied magnetic field.The results show that the staining of brain tissue can be observed only when SPIONs and EB are co-injected and then applied with a magnetic field,indicating that the magnetic field has a reversible regulation effect on BBB permeability.Near infrared imaging to further verify this conclusion.In order to clarify whether magnetic-induced BBB leakage displays the properties of size-dependent permeability,Cy5-BSA or PS microspheres(60 nm,120nm)were used as detection reagents in this paper.The accumulated amount of PS microspheres was about2.5 times that of the blank group.Quantitative analysis of the imaging results of PS microspheres showed that 60 nm PS microspheres could effectively accumulate in brain tissue.In summary,exogenous magnetic force acting on BBB endothelial cells can generate reversible gap channels with widths between 60 nm and120 nm,which can be used to improve the brain delivery efficiency of conventional nanomedicines.Next,this paper uses transmission electron microscopy,immunofluorescence staining,transcriptome sequencing,western blotting,RT-q PCR and other techniques to explore the molecular mechanism of magnetic-driven BBB leakage from the tissue level to the molecular level.Through tissue transmission electron microscopy characterization,the experimental results showed that the magnetic force did not permanently destroy the hyperfine structure of the tight junction of the BBB;the immunofluorescence staining results of brain slices showed that the magnetic induction group did not change the continuous expression of the tight junction protein ZO-1;the WB experiment It showed that the exogenous magnetic force did not negatively affect the expression levels of tight junction proteins ZO-1,CD31,Occluddin,etc.during the action.In order to explore whether magnetic nanoparticles are endocytosed by endothelial cells and affect the permeability of BBB,this paper uses magnetic methods to detect the capacity of magnetic nanoparticles in brain tissue.The results show that magnetic nanoparticles do not enter endothelial cells after being dragged by magnetic force.Improved BBB permeability induced by endocytosis of SPIONs.In this paper,we used transcriptome sequencing to explore the effect of magnetism on the overall transcriptome expression of brain tissue at the tissue level.Taking the gene expression level |log2Fold Change|≥1.0 & padj<0.05 as the threshold for significance judgment,the results showed that compared with the control group,there were 565 genes significantly up-regulated and 231 genes were significantly down-regulated in the experimental group.After GSEA analysis,the Hippo-YAP signaling pathway in brain tissue showed a significant up-regulation trend(NES >1.0 & FDR q value<0.05).RT-q PCR technology was used to further detect the expression of key proteins in the Hippo signaling pathway.The results showed that the downstream related genes CCND2 and CYR61 of Hippo-YAP were significantly increased compared with the control group.In addition,the immunofluorescence staining results of YAP1 protein in brain tissue sections showed that the level of YAP1 protein into the nucleus was also significantly increased in the magnetic experiment group.In summary,magnetic force may regulate the permeability of the blood-brain barrier through the Hippo signaling pathway.Finally,this paper uses transcriptome sequencing,immunofluorescence staining,RT-q PCR,blood detection and other techniques to detect the effect of this protocol on the nervous system.Transcriptome sequencing results GSEA analysis showed that inflammation-related pathways were up-regulated,which may be related to endothelial cell inflammation;then RT-q PCR technology was used to detect the expression of major inflammatory factors.The experimental results showed that IL1 A,IL1B,IL6,TNF,NKFB2,SERPINE1 and other major inflammation factors peaked in about 2 to 3 days,and gradually attenuated to normal after 5 days,showing a time-dependent change.The results of immunofluorescence staining of brain slices after magnetic force showed no significant effect on microglia and astrocytes.Compared with the control group,the blood biochemical indexes of the mice were not significantly different after the permeability of the blood-brain barrier was improved by magnetic drive.In summary,this paper provides a powerful,efficient,controllable,and safe method for improving the permeability of the blood-brain barrier,and its main mechanism of action is studied.Efficiency has broad application prospects. |
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