Research Study On The Detection Of Tumor Exosomes And Drug Delivery

Exosomes are a type of vesicle structure secreted by all cells with a diameter of approximately 30-150 nm.Because it is rich in potential biomarkers,such as proteins,mRNA and miRNA,it is considered to have broad clinical application prospects.Detection of exosomes can help early diagnosis,efficacy evaluation and prognosis analysis of tumors.In addition,since the exosomes are endogenous vesicles of cells,they have the advantages of low immunogenicity,good biodegradability,low toxicity,and ability to cross the blood-brain barrier.Exosomes are good carriers for drug delivery.Based on this,the main research contents of this thesis are:1.Magnetic-based microfluidic device for on-chip isolation and detection of tumor-derived exosomes.We combine an easy-to-automate and integrated microfluidic chip with a fast,sensitive,and low-cost electrochemistry to design an exosome analysis platform that combines mixing,separation,enrichment,detection,and release(ExoPCD-chip).The ExoPCD-chip consists of an exosome magnetic capture region consisting of a Y-shaped microcolumn array and an etched ITO glass electrode.Exosomes are captured by a magnetic bead-modified phosphatidylserine(PS)binding protein that specifically binds to exosome-exposed phosphatidylserine.Electrochemical detection uses a hairpin structure having a CD63 aptamer sequence and a G-quadruplex sequence for signal output.The chip requires only 30 p,L of sample and is tested within 3.5 hours.The detection limit is as low as 4.39×103 exosomes/mL,and the linear range spans 5 orders of magnitude.Moreover,we analyze human serum exosomes through the ExoPCD chip and successfully distinguish liver cancer patients from healthy controls.2.Correlation study of CPPs surface modified exosomes for drug delivery.R9 is modified to form R9-EXO on the surface of exosomes by amide reaction.G3139 binds to R9-EXO under the dual action of electrostatic adsorption and cholesterol anchoring to form R9-EXO-G3139 complex.After co-incubation with the liver cancer cells,the complex enters the cells by endocytosis and releases the G3139 chain under the action of the cell lysosome.G3139 down-regulates Bcl-2 protein expression by complementing Bcl-2 mRNA.R9 is modified to the surface of the exosomes,which changes the exosome entry into the cell pathway,from membrane fusion to clathrin-mediated endocytosis,and better delivery of G3139 into the cell.Its delivery efficiency is increased by 6.03 times compared to unmodified.The data of qRCR and Western blot show that R9-EXO-G3139 could down-regulate Bcl-2 mRNA by 72.80%and Bcl-2 protein by 69.69%,which could achieve the purpose of silencing tumor cell Bcl-2 gene expression.