Research Of Extracellular Vesicle-based Cancer Diagnosis And Drug Delivery

Extracellular vesicles(EVs)are various phospholipid bilayer membrane vesicles formed and actively released by cells,which are widely found in cell culture supernatants and various body fluids(blood,saliva,urine,lymph,ascites,milk,etc.),carrying a variety of active substances from mother cells,such as proteins,lipids,DNA,m RNA,mi RNA,etc.They can directly or indirectly involve in biological processes such as intercellular communication,induction of angiogenesis,promotion of cell migration and proliferation,and mediating immune regulation.Among them,the relevant extracellular vesicles secreted by cancer cells can deliver their contents to local or distant sites,interact with recipient cells,directly or indirectly involve in the regulation of numerous physiological and pathological processes in the bod,and promote tumor initiation and progression,invasion and metastasis.The types and expression levels of extracellular vesicle surface marker proteins and nucleic acids contained within are closely related to the type and extent of diseases,and have been recognized as promising novel biomarkers for sensitive diagnosis and prognosis of diseases.In addition,extracellular vesicles can also be used as delivery tools for active drugs,anticancer materials,vaccines and gene carriers,so as to achieve targeted killing of cancer cells and the effect of treating cancer.Based on this,this paper developed an extracellular vesicle-based cancer diagnosis and drug delivery technology.The details are as follows:(1)Secretory autophagosome is an extracellular vesicle produced during autophagy.The expression level of marker proteins on their surface are closely related to autophagy process,which can indirectly reflect disease status.Based on this,we designed a three-dimensional(3D)microfluidic chip for efficient enrichment and sensitive detection of tumor cell-derived secretory autophagosomes surface biomarkers LC3 B and HSP60.LC3 B is a marker protein of autophagosomes,which can reflect the autophagy level of tumor cells.HSP60 is one of the heat shock family proteins and is closely related to disease,inflammation,immunity,etc.The designed 3D microfluidic chip channel adopted a vertically staggered and uniformly distributed fusiform micropillar array structure to increase the collision probability of secreted autophagosomes.;the biochemical gelatin and avidin were assembled and modified layer by layer to increase the binding sites of captured antibodies,thus improving the capture efficiency of autophagosomes.The secretory autophagosomes of ovarian cancer cell line SKOV-3 were used as the detection model.When secretory autophagosomes are captured on the chip by the fusiform micropillar array modified by the capture antibody,two quantum dots with the same excitation wavelength and different emission wavelengths specifically labeled LC3 B and HSP60 on the surface of secretory autophagosomes,respectively,which could realize the simultaneous detection of multiple secretory autophagosomes surface proteins.The results showed that the two specific proteins could be used as quantitative markers for the secretion of autophagosomes by tumor cells.Secondly,the difference in expression of the two proteins can effectively distinguish cancer patients from healthy people,providing a new idea for clinical diagnosis of cancer.(2)Exosomes are extracellular vesicles with a size of 30-150 nm,which have high biocompatibility and low immunogenicity,and are able to cross various biological barriers and evade clearance by the immune system.It is widely used in the construction of novel drug delivery systems.Based on this,a novel anti-tumor exosome drug delivery system was constructed with the synergistic effect of mi RNA autophagy inhibition and coumarin derivatives.In this drug-loading system,the surface of exosomes were modified with magnetic ferric oxide nanoparticles,which achieved efficient enrichment of exosomes and maintained the integrity of exosomes;canceractive coumarin derivatives and autophagy-inhibiting mi RNAs could synergistically achieve effective treatment of ovarian cancer.Among them,coumarin derivatives had good pharmaceutical activities,such as antibacterial,anti-inflammatory,anticancer,etc.In addition,coumarin derivatives had their unique optical properties,which can realize intracellular visualization and tracking.At the same time,the encapsulated mi RNAs could participate in the regulation of autophagy process and reduce the autophagic flux.By studying the interaction between the drug-loading system and tumor cells,the inhibition of autophagy by mi RNA and the apoptosis of tumor cells caused by drugs were successfully achieved.Under the joint action of the two materials,the apoptosis rate of tumor cells was much greater than that of the two alone,which verified the synergistic effect of the drug-loading system and provided a new idea for the treatment of tumors.