Construction Of Magnetic Extracellular Vesicles Synergy With Immunoaffinity/Bioorthogonal Strategy For Efficient Enrichment And Detection Of Circulating Tumor Cells

Circulating tumor cells（CTCs）are tumor cells that shed from tumor sites and enter the bloodstream.As a tumor marker,CTCs plays an important role in non-invasive liquid biopsy.Accurate detection of CTCS will provide a wealth of information for cancer diagnosis and personalized treatment,which is conducive to timely and accurate tumor diagnosis and treatment.It is of great significance for clinical diagnosis and treatment to enrich,detect and analyze CTCs in the blood of cancer patients.Efficient enrichment of CTCs is a prerequisite for effective analysis.Current techniques mainly distinguish CTCs from normal blood cells based on the differences in physical properties（such as density,size,dielectric characteristics,etc.）or biological properties（such as surface antigen,surface characteristic protein,surface glycan,etc.）of CTCs,so as to achieve the purpose of CTCs enrichment.Among these mothods,the immunomagnetic beads could combine with CTCs to realize the separation and enrichment of CTCs under the action of external magnetic field,have the characteristics of convenient operation,efficient separation and cell-friendliness.However,the accurate identification,enrichment and detection of CTCs still have great challenges due to the small number of CTCs in the complex blood circulation system and phenotypic fluctuations of CTCs.On the one hand,the peripheral blood contains abundant proteins and a large number of blood cells,which will be non-specifically adsorbed on the surface of the capture platform（such as immunomagnetic beads）,resulting in a reduction in capture efficiency and purity of CTCs,which affects the reliability of subsequent protein/genomic analysis.On the other hand,the different sources of the organs/tissues and the epithelial-mesenchymal transition（EMT）process occurring with the progression of the disease make the phenotypes of CTCs highly heterogeneous,which greatly restricts the universality and reliability of the immunoaffinity enrichment strategy,thus resulting in missed detection.Therefore,starting from the construction of functional nanomaterials,this study aims to minimize the non-specific adsorption during CTCs enrichment,and implement reliable CTCs recognition and enrichment strategies,so as to carry out highly sensitive cancer-related information detection on the obtained samples.The quantification of CTCs in unit volume of blood can reflect the progression of cancer and establish treatment feedback to a certain extent.Fluorescence spectrum analysis is a convenient and efficient quantitative detection method.The accuracy of quantifying CTCs by fluorescence detection largely depends on the selection of fluorescence reporter molecules.The aggregation-induced emission（AIE）molecule,TPE-IPB（（N-（2-（（4-（4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl）benzyl）oxy）benzylidene）-4-（1,2,2 triphenylvinyl）aniline））can provide a stable and high signal-to-noise ratio reporting model for CTCs detection due to its aggregation induced luminescence behavior and reactive ion response characteristics.Extracellular vesicles（EVs）,as vesicles with a double-layer lipid membrane structure released by cells,not only can be used as carriers of natural signal substances,but also can be used to encapsulate gene,small molecule drugs and nano-particles in a variety of ways.The biogenesis process of extracellular vesicles provides a convenient"top-down"strategy to realize the surface functionalization of magnetic nanoparticles.Through this strategy,magnetic nanoparticles with a biomimetic interface of EVs can not only retain their phospholipid membrane structure to achieve the function of anti-nonspecific adsorption,but also can improve the high-affinity targeting of CTCs through specific membrane proteins on the membrane.In order to deal with the problem of CTCs phenotypic fluctuations caused by EMT process of tumor cells,the establishment of universal“artificial target”driven by the metabolic oligosaccharide engineering of tumor cells may be an important important alternative technique for traditional immunoaffinity relying on known phenotypic antigens on CTCs.Digest the exogenous azide glycosamine through the production pathway of glycans（such as sialic acid）to construct azide groups on the surface of tumor cells as artificial targets,combined with click chemistry is an easible strategy to achieve high specific enrichment of heterogeneous CTCs.Centered on the above content,this paper mainly carried out the following work:（1）Macrophages were used to ingest superparamagnetic Fe₃O₄ nanoparticles,and magnetic extracellular vesicles were prepared through the release pathway of EVs to obtain Fe₃O₄-MVs.Their surface was modified with antibodies to target tumor cells.The prepared Fe₃O₄-MVs had a particle size of about 800 nm with stable cell membrane structure and good magnetic response capability.AIE molecule TPE-IPB with reactive ion response behavior characteristic was synthesized and encapsulated by micellar assembly and polyelectrolyte layer-by-layer self-assembly methods to construct antibodies（anti-Ep CAM antibody）modified AIE nanoparticles（AIE NPs）.AIE NPs（size≈200 nm）had a good fluorescence response behavior under the action of the ONOO^-.Fe₃O₄-MVs and AIE NPs were carried to incubated with tumor cells simultaneously,then CTCs can be enriched with an efficiency of 62%by magnetic separation.（2）Tumor cells（human lung cancer cells-A549 cells,human liver cancer cells-Hep G2 ells）were treated with azidoacetylmannosamine-tetraacylated（Ac₄Man NAz）,and“artificial target”were constructed on the surface of tumor cells through metabolic oligosaccharide engineering.The bionic camouflage of Fe₃O₄nanoparticles were carried out through the top-down procedure of mouse macrophages,magnetic extracellular vesicles with a particle size of～550 nm and a surface potential of～-8.1m V were obtained.The magnetic extracellular vesicle had obvious cell membrane structure and the protein composition on the membrane was basically the same as the protein on parent cell membrane.By introducing the bioorthogonal molecule dibenzocyclooctyne（DBCO）on the magnetic extracellular vesicles,the DBCO-functionalized magnetic extracellular vesicles（Fe₃O₄-MVs-DBCO）were successfully prepared.and Fe₃O₄-MVs-DBCO had a good magnetic response,the recovery rate exceeds 90%within 1 min under the action of an external magnetic field.Fe₃O₄-MVs-DBCO could effectively identify and capture azide labeled tumor cells with a capture efficiency of 84%and a detection limit as low as 5 cells/m L.The xenograft tumor models were constructed by A549 and Hep G2 cells respectively and CTCs were successfully collected from the blood of mice through magnetic extracellular with excellent anti-nonspecific adsorption performance and extremely low amount（≈0.0075（?）-1.1299（?））of non-specific adsorbed white blood cells（WBCs）number.By analyzing the tumor volume,lesion degree and the number of CTCs of tumor-bearing mice,etc.,it is fully proved that the biological orthogonal strategy in this study could avoid the limitation of traditional immune affinity,overcome the different tumor types and the phenotypic fluctuation of CTCs to achieve efficient enrichment of CTCs,has great potential in the clinical application of CTCs.（3）The exosomes from melanoma cell were collected and mixed with liposomes,then magnetic ferroferric oxide nanoparticles were coated in the exosomes by extrusion to form exosomes functionalized magnetic extracellular vesicles（Fe₃O₄@lip/exo）.After modification with DBCO,Fe₃O₄@lip/exo-DBCO were obtained.The magnetic extracellular vesicles with the size of about 380 nm had good magnetic response behavior and stable phospholipid membrane bionic structure on the surface.After incubated with Ac₄Man NAz,a large number of azide groups can be expressed on the cell membrane of mouse breast cancer cells（4T1）,human breast cancer cells（MCF-7）,mouse melanoma cells（B16）and metastatic mouse melanoma cells（B16-F10）,indicating that non-epithelial melanoma cells can still be labeled by metabolism pathways and bioorthogonal chemistry.In the absence of DBCO,Fe₃O₄@lip/exo had a relatively higher capture efficiency（～30%）toward melanoma cells compared with liposome-modified magnetic vesicles（Fe₃O₄@lipsome）.Following the study of CTCs enrichment based on the biological orthogonal strategy in the previous chapter,the anti-Ep CAM antibody-modified magnetic extracellular vesicles（Fe₃O₄@lip/exo-anti-Ep CAM）only had significant capturing ability on two types of epithelial tumor cells（4T1 cells and MCF-7 cells）,but poor performance with melanoma cells（capture efficiency～34%）.However,it was found that Fe₃O₄@lip/exo-DBCO could effectively capture CTCs（capture efficiency>80%）of four models including melanoma（normal melanoma cell B16 and more aggressive melanoma cell B16-F10）without being affected by organ/tissue sources or even the differences of biological species.