Research On Exosome-based Cancer Diagnosis And Drug Delivery

Exosomes are nano-scale vesicles secreted by normal and pathological cells,which are widely present in various body fluids.Exosomes regulate local and systemic intercellular communication through the proteins,m RNAs and mi RNAs they carry,and induce pathological changes in recipient cells.Tumor cell-derived exosomes can provide autocrine,paracrine,endocrine and other signals that promote tumor invasion and metastasis.The types and levels of surface proteins and internal nucleic acids of exosomes are closely related to tumor types and their occurrence and development.Sensitive and accurate detection of disease-related markers presented by exosomes is the key to studying the occurrence and development of tumor diseases.At the same time,as a natural excellent carrier,exosomes can be transformed into drug carriers with targeted functions to assist anti-tumor drugs or biological macromolecules to play an anti-tumor effect and achieve high-efficiency and low-toxicity cancer therapy effects.Based on this,this article has developed analysis techniques for cancer diagnosis and drug-loading strategies based on exosomes.The details are as follows:(1)An integrated microfluidic device named PS-ED chip for rapid plasma separation and quantitative analysis of exosomes in blood samples was developed.The device contained two modules,PS module was for the separation of blood cells and plasma,Diluted blood samples run through the spiral channel under an optimal flow rate of 1.5 m L min^-1.It was found that the separation efficiency of blood cells was 100%with hematocrit(HCT)of 1%under the inertia force.ED module was for quantitative detection of exosomes and high-throughput exosome protein analysis based on chemiluminescence ELISA assay.This device could be used to detect 10²?10⁸particles?L^-1 of exosomes,compared to commercial immunoassays,our PS-ED chip offers high detection sensitivity and a wide dynamic range.Furthermore,ED module contains four channels,each channel can detect different exosomal protein markers.Using this device to analyze blood samples from ovarian cancer patients,we found that exosomes derived from ovarian cancer cells can be identified by the expression of CD24 and Ep CAM from healthy controls,and the protein level of CD24,Ep CAM per exosome could be used for therapy monitoring,which was decreased among responding patients but increased in nonresponding patients.These results demonstrate the potential of the PS-ED chip in exosomes quantitative detection in blood for clinical disease diagnosis and therapy monitoring.(2)A double-channel microfluidic platform was developed with 3D structure,which combined with quantum dots(QDs)labeling and vesicle fusion technology,to achieve rapid capture of exosomes and simultaneous determination of three surface proteins and three mi RNAs.The channel contained Y-shaped micropost arrays and was coated with multilayer biotinylated gelatin film,streptavidin and polyethylene glycol(PEG)modified polystyrene spheres(PS)were immobilized on the film to construct nanostructure.All these strategies have significantly improved the efficiency of exosome capture.On one hand,three QDs modified with different specific antibodies realized the simultaneous detection of multiple protein markers in one channel.On the other hand,virus-mimicking fusogenic vesicles(FVs)encapsulating three molecular beacons(MBs)were constructed for the rapid detection of exosomal mi RNAs via membrane fusion with exosome.We applied the chip for pancreatic cancer diagnosis with exosomes using only 2?L plasma.The combined assessment of four markers(Eph A2,mi RNA-451a,mi RNA-21 and mi RNA-10b)could improve accuracy in discriminating early and late-stage cancer.Thus,by replacing antibodies labeled with QDs and MBs in FVs,this chip could be a useful tool to facilitate the clinical use of exosomes for non-invasive disease diagnosis.(3)An exosome targeted drug delivery system with lactic acid oxidase(LOD)on the surface and the photosensitizer PpIX and siRNA inside was constructed,combined with metabolic therapy and photodynamic therapy,to achieve effective treatment of breast cancer.Among them,PpIX,which was biosynthesized from donor cells,had the characteristics of high encapsulation efficiency and low biological toxicity.Also,compared with commercial transfection reagents,for the delivery of siRNA,the performance of exosomes was better in the delivery efficiency and the protein silencing effect.At the same time,the modification of the aptamer(Apt _{Ep CAM})not only improved the targeting of exosomes but also increased the efficacy of the drug.In addition,the partial complementary chain attached to the LOD(c DNA-LOD)fell off into the tumor microenvironment,consumed lactic acid,and improved the acidic conditions of the tumor microenvironment.The PpIX that entered the cell produces a large amount of ROS under laser irradiation;siRNA targeted and silenced the monocarboxylic acid transporter MCT4,the lactic acid transport was inhibited,and the accumulation of lactic acid in the cell caused cell acidosis and promoted cell apoptosis.As an important signal molecule,the breakdown of lactic acid homeostasis caused the down-regulation of factors related to cell promotion of tumor metastasis and immune resistance,such as the angiogenic factor AREG secreted by tumor cells and the programmed death ligand PD-L1.Therefore,our targeted drug delivery system based on exosomes could effectively achieve the purpose of targeted killing of cancer cells,regulating tumor microenvironment,inhibiting tumor metastasis,and regulating immune resistance.According to the internal and external drug loading strategy of exosomes we use,the construction of targeted drug loading systems for other cancers or diseases can be achieved by changing the targeted molecules and internal drug types.