Functional DNA Nanoprobes-Enabled Exosome Detection And Its Application In Monitoring Regulation Of Active Ingredients Of Traditional Chinese Medicine On Tumoral Exosomes

Background and Objectives:As the treasure of Chinese traditional culture,traditional Chinese medicine(TCM)has made great contributions to Chinese people's health for thousands of years with its unique theoretical framework as well as definite clinical efficacy.In recent years,with the change of people's health concept and the overturning of medical model,TCM has shown unique advantages in the prevention and treatment of various diseases.Tumor,especially malignant tumor,is one of the most serious diseases endangering human life.According to the theory of TCM,tumors are caused by the imbalance of Yin and Yang,the disorder of qi and blood,and the disorder of qi in the five internal organs(including heart,liver,spleen,lungs and kidneys).On the basis of syndrome differentiation,TCM treatment gives the centralizer thrift,soft firm fights,promoting blood circulation to remove blood stasis,qi stagnant,heat-clearing and detoxifying methods for the tumor treatment of the overall view.In recent years,with the in-depth development of anti-tumor experiments and clinical studies on TCM,TCM and its active ingredients have been increasingly recognized and accepted by the international community for their increasing effectiveness and reducing toxicity in assisting tumor radiotherapy and chemotherapy,improving the overall immune function of patients,clinical symptoms and the quality of life.Modern clinical researchers have made extensive efforts on the TCM anti-tumor effect from the perspective of molecular biology and molecular pharmacology,and found that the TCM anti-tumor has the characteristics of multi-target,multi-effect and multi-link.However,the complex mechanism of TCM anti-tumor is not completely clear,especially the regulation of TCM on tumor microenvironment,tumor immune escape and tumor drug resistance needs to be further studied.Exosomes are widely found in body fluids and are involved in tumor cell growth,invasion and migration,drug resistance,immune escape and apoptosis as a messenger of intercellular material exchange and information transmission.At present,Exosomes as ideal liquid biopsy markers and drug carriers have been used in the diagnosis and treatment of various tumors.Many anti-tumor TCM monomers,compounds and active ingredients have been found to play anti-tumor roles by regulating exosome release,which makes exosomes a promising new target for tumor treatment.Therefore,it will be of great significance to study how the TCM regulate the tumoral exosomes to affect the development of tumors.When using TCM to treat tumor,how to monitor the changes of tumor exosomes quickly and accurately is the first issue to which researchers need to pay attention.Currently,common methods such as ELISA and Western blot have been used to detect exosomes.Although these methods are classic and reliable,the sensitivity and specificity of the methods are limited,the instruments and reagents required for the experiment is costly,and the operation steps are tedious and time-consuming.Therefore,it is particularly important to develop simple,low-cost,reliable,sensitive and specific methods for exosome detection.Biosensor detection has been widely used in the environmental health and clinical medicine fields due to its high sensitivity,specificity,rapidity,accuracy and convenience.At present,a variety of biosensors have been used for exosome detection,such as electrochemistry,surface plasmon resonance,surface acoustic wave,field effect transistor,etc.Although these exosome biosensors based on interface modification have high sensitivity,the preparation of biosensors usually requires a complex nanometer preparation process or tedious interface engineering,and most biosensors use expensive antibodies as exosome recognition components.However,reliable,effective and cost-effective exosome testing remains a difficult task in routine clinical laboratories,with the main problem being the lack of a simple and rapid enough testing platform.Deoxyribonucleic acid(DNA)is an important carrier of genetic information in living organisms.With the development of solid phase DNA synthesis technology,the cost of synthetic DNA has been greatly reduced.DNA synthesized in vitro can perform many important functions as functional molecules or nanomaterials.DNA can be used as a nucleic acid probe for specific recognition of its complementary sequence,and also as a nucleic acid aptamer to target and identify various targets including small organic compounds,inorganic metal ions,proteins,peptides,microvesicles,cells and even tissues.By combining the biometric properties of DNA with the excellent sensor properties and signal transduction mechanisms of some nanomaterials(such as graphene oxide,carbon nanotubes,etc.),the highly sensitive detection of various biological samples including exosomes can be realized through a variety of conventional laboratory testing platforms(such as fluorescence analysis,colorimetric analysis,etc.).It is worth noting that the DNA itself can be self-assembled into an ordered and functionally controllable DNA nanomaterial through strict complementary base pairs between complementary chains,and DNA nanomaterial can be further constructed into a DNA nanomolecular machine with signal response and signal amplification functions for exosomes.Since that the nanoscale of exosomes,functional DNA components and exosomes are limited in the same nanoscale space,and the effective concentration of reactants will be greatly increased,which can realize the rapid amplification of detection signals.This study intends to use the biometric characteristics of functional DNA,combined with the signal transduction and signal amplification functions of nanomaterials or DNA nanomolecular machines,to develop the functional DNA nanobiosensors for the exosomal quantification and phenotyping in a homogeneous system based on fluorescence analysis.The application of this nanobiosensor in the analysis of exosomes in the blood of patients can not only reflect the abundance and phenotype of exosomes at the time of tumor occurrence,but also dynamically monitor the changes of exosomes before and after drug treatment.It is of great significance to study the anti-tumor effect and anti-tumor mechanism of TCM.Methods and Results:In this work,Graphene Oxide(GO)-aptamer nanoprobes catalyzed by Deoxyribonuclease I(DNase I)have been developed for high sensitivity detection and phenotypic analysis of tumor exosomes.Then,an exosome-driven multivalent DNA nanomolecular machine with enzyme-free amplification was constructed to realize the bicolor detection of exosome surface molecules,and can be used for tumor immunotherapy monitoring.On this basis,the constructed sensor was used to monitor the regulation of TCM on tumor exosomes and exosomes PD-L1.The main content of this project includes the following three parts:Part I:ExoAPP:exosome-oriented,aptamer nanoprobe-enabled surface proteins profiling and DetectionTumor exosomes that inherit molecular markers from their parent cells are emerging as cellular“surrogates”in cancer diagnostics.Molecular profiling and detection of exosomes offer a noninvasive access to the state of cancer progression,yet are still technically challenging.Here we report an exosome-oriented,aptamer nanoprobe-based profiling(ExoAPP)assay to phenotype surface proteins and quantify cancerous exosomes in a facile mix-and-detect format.The GO nanoribbons bind to the flurescent aptamer by?-?stacking to form the"Off"nanoprobe complex.When the complex recognizes and binds to exosomal prtoteins,the spatial structure of aptamer changes,and the adsorption between with GO turns to weak,resulting in the release of aptamer and change of fluorescence signal"On".The aptamer bingding on the exosome surface will be hydrolyzed by DNaseI due to the lack of GO protection,which will lead to the nanoprobe complex repeatedly identifying the target exosomes and continuously amplifing the fluorescence signal.Our ExoAPP interfaces GO with target-responsive aptamers to profile exosomal markers across 5 cell types by complementing with enzymeassisted exosome recycling,revealing a heterogeneous pattern.This assay achieves a detection limit down to 1.6×10~5 particles/mL,lowered by several orders of magnitude over other homogeneous protocols.Such a sensitive ExoAPP assay allows for monitoring epithelial-mesenchymal transition through heterogeneous exosomes without involving cellular internalization that often occurs in GO-based cargo delivery.Using ExoAPP to analyze blood samples from prostate cancer patients,we find that target exosome can be identified by surface PSMA,suggesting their potential in clinical diagnosis.Part?:Exosome-actuated multivalent DNA nanomachines(ExoADM)for bicolor exosomal phenotyping and anti-PD-1immunotherapy GuidingInspired by the efficient molecular machines running in cells,we combine the multivalent molecular recognition between exosomes and aptamers with the Toehold-mediated strand displacement recycling reaction to construct a bioinspired exosome-activated DNA molecular machine(ExoADM)with multivalent cyclic amplification that enables highly sensitive detection and phenotyping of circulating exosomes.ExoADM consists of 3 core components:a recognition probe that responds to exosome surface markers,a signal probe with 2 Toehold domains that is activated by exosome molecular recognition events to initiate a Toehold-mediated strand displacement reaction,and a fuel probe that can catalyze a recycling amplification reaction.In the presence of target exosomes,initiation chain can be released from the target-responsive recognition probe,due to the specific and thermodynamically more favorable binding between the exosomal marker and the corresponding aptamer.The free initiation chain then hybridizes to Toehold?in the signal probe and displaces the quench chain through branch migration,which allows the exposure of Toehold?and meanwhile separates the donor-acceptor pairs for signaling.Next,the fuel probe binds to the exposed Toehold?domain and liberates the initiation chain again.The released initiation chain can further hybridize to another Toehold?domain of the signal probes,activating a next round of strand displacement reaction via DNA nanomachines.Thus,a single molecular recognition event can initiate consecutive release of abundant fluorescent reporters for signal amplification.By recording the rapid signal change of the increased fluorescent signal,the target exosomes can be quantified.This self-powered ExoADM shows high sensitivity(3.3×10~4 particles/mL)and selectivity against specific exosomes.Importantly,our ExoADM can synergize with another DNA nanomachine targeting different exosomal surface markers for dual-color phenotype detection.Using bicolor strategy,we can simultaneously track the dynamic changes of PD-L1 and CD63 expression on the exosomal surface of PC-3 cells induced by IFN-?and Resveratrol.Further,we find that their expression levels on circulating exosomes can well differentiate cancer patients from the healthy ones.More importantly,we find that ExoPD-L1 level can reflect the efficacy of different treatments(e.g.,radiotherapy,chemotherapy and anti-PD-1 immunotherapy)and guide the anti-PD-1 immunotherapy,suggesting the potential of ExoPD-L1 in clinical diagnosis and treatment monitoring.Part?:Monitoring the regulation of the active ingredients of TCM on tumoral exosomes and exosomal PD-L1 by exosome-driven multivalent DNA nanomachine.Based on the above-mentioned work,4 active ingredients of TCM are selected to treat with the non-small cell lung cancer(NSCLC)A549 cell line.They are resveratrol,shikonin,platycodonin D and paclitaxel,respectively.First,CCK-8 assay,flow cytometry and Western blotting assay are used to investigate the effects of the 4 active ingredients on the proliferation and apoptosis of A549 cells.The results show that the 4 drugs have time-and dose-dependent inhibitory effect on A549 cell proliferation and induce apoptosis.Subsequently,exosomes are isolated from the supernatant of A549cells treated with 4 drugs for 48 h respectively,and the level of CD63 and PD-L1 on exosomes surface before and after drug treatment are detected by ExoADM.It is found that the 4 active ingredients of TCM can affect the signal pathways related to exosome synthesis,content assembly,and transport release.The results show that the dugs can induce or inhibit extracellular release of exosomes and the expression of biological information molecules carried by exosomes.Among the 4 active ingredients we select,except for the inhibiting effect of shikonin on exosome release in A549 cells,the other 3show inducing effect of the secretion of exosomes.At the same time,the 4active ingredients upregulate the expression of exosomal PD-L1 to varying degrees.We further verify our experimental results by using NTA,BCA and Western blotting assays.These results suggest that the PD-L1/PD-1 pathway may be activated by upregulation of exosomal PD-L1 level to promote the immune escape and metastasis of tumor although these 4 active ingredients of TCM can be used in the treatment of non-small cell lung cancer by inhibiting the proliferation of primary tumor cells.It is also possible to increase the sensitivity of immunosuppressive therapy by upregasing tumor PD-L1 level,which suggests that the combination of TCM active ingredients will become an important development direction of tumor immunotherapy.Our experimental results further confirm the inhibitory effect of TCM on the proliferation of tumor cells,and also reveal the complexity of the regulation of TCM on tumor exosomes and the signal molecules carried by exosomes,which reflect the multi-target,multi-effect and multi-link regulation characteristics of TCM on tumor.Conclusion:1.We have developed an ExoAPP assay based on nucleic acid aptamer nanoprobe to enable highly sensitive detection and phenotypic analysis of tumor exosomes,providing a simple,fast and reliable biosensor platform for tumor liquid biopsy based on exosomes.2.An ExoADM assay based on DNA nanomolecular machine has been constructed to achieve enzyme-free ultra-sensitive exosome detection,dual-color phenotype analysis of exosomal proteins,and tumor immunotherapy(anti-PD-1)monitoring.3.Resveratrol,shikonin,platycodonin D and paclitaxel show the ability to inhibit proliferation and induce apoptosis of A549 cells,respectively.4.The regulation of the active ingredients of TCM on tumor exosomes and exosomal PD-L1has been monitored by ExoADM assay.The release of A549 exosomes can be inhibited by shikonin,while resveratrol,platycodon D and paclitaxel all induce the release of A549 exosomes.The 4 active ingredients of TCM promote the expression of PD-L1 on A549 exosomes to varying degrees.