Tim4-based Strongly Hydrophilic MOFs Immunoaffinity Flake For High-efficiency Capture And Separation Of Exsomes

Exosomes are bio-nanoparticles wrapped in a phospholipid double-layer membrane with a diameter of 30-200 nm.In recent years,because exosomes carry a large amount of genetic material and play an important role in mediating cell-cell communication,they have become the most ideal analysis target for liquid biopsy.However,the low concentration of exosomes makes their effective enrichment still a challenging task.This paper designed and developed a new type of Tim4@ILI-01 immunoaffinity flake material.Based on the Ca²⁺-dependent properties of Tim4 binding to phosphatidylserine on the surface of exosomes,the captured exosomes can be easily eluted and released by adding a chelating agent under neutral conditions.The material has been successfully used to enrich the exosomes in the serum of lung cancer patients.The full text is divided into four chapters:Chapter 1:Firstly,the research background and separation methods of exosomes were summarized.Then,the research progress and related applications of metal-organic framework are summarized.At last,the main content of this thesis is expoundedChapter 2:Designed and developed a new type of metal-organic frameworks（MOFs）material.First,1,3-bis（4-carboxybutyl）-imidazolium bromide（ILI）ionic liquid was synthesized by the reaction of methyl 4-bromobutyrate and 1-（trimethylsilyl）imidazole.The strongly hydrophilic ILI-01 MOFs were prepared with ILI as the organic ligand and zinc nitrate hexahydrate Zn（NO₃）₂·6H₂O.The use of fourier transform infrared spectra,X-ray diffraction signals,scanning electron microscopic and other techniques have proved that the ILI-01 MOFs material has been successfully prepared.Water contact angle shows that the ILI-01 MOFs material has strongly hydrophilic properties.The non-specific adsorption of ILI-01 MOFs material was investigated with bovine serum albumin solution.After washing twice with washing buffer,the non-specific adsorption of standard protein can be reduced to0.7%,which basically eliminates the non-specific adsorption of impurity proteins.The interference of high abundance contaminants during exosomes separation is eliminated.Chapter 3:Based on the abundant carboxyl groups inherent in the ILI-01 MOFs material,the Tim4 antibody was successfully bonded to the ILI-01 MOF material.Fluorescence inverted microscope observed that Tim4@ILI-01 immunoaffinity material has been successfully synthesized,and the results of BCA protein quantitative experiment showed that the binding rate of Tim4 antibody reached 82%.The prepared Tim4@ILI-01 immunoaffinity material was used to separate exosomes in the culture supernatant of lung adenocarcinoma cell line H1299.Exosomes were furture identified by transmission electron microscopy,nanoparticle trace analysis and Western Blotting.The results showed that the size and morphology of exosomes separated by Tim4@ILI-01 immunoaffinity material were consistent with previous studies,and the content of marker proteins in the samples was significantly increased after enrichment.The efficiency of this method to capture exosomes can reach 85.2%,which is 2.2 times and 2.4 times higher than that of CD63@ILI-01 and CD81@ILI-01,respectively.In addition,the prepared Tim4@ILI-01 immunoaffinity material was further compared with the gold standard"ultracentrifugation method".The capture efficiency of this method is 5.2 times that of the ultracentrifugation method.Taking advantage of the Ca²⁺-dependent properties of Tim4 antibody binding to phosphatidylserine on the surface of exosomes,the Tim4@ILI-01 immunoaffinity material captures exosomes by adding EDTA buffer as a chelating agent,and the captured exosomes are easily released.Therefore,the cell wound-healing assay and the cytotoxicity test under the action of cisplatin proved that the exosomes isolated from the Tim4@ILI-01 immunoaffinity material have biological activity and can be used for subsequent biological analysis and clinical experiments.Finally,the Tim4@ILI-01 immunoaffinity material was used for the separation and enrichment of exosomes in human serum samples,and the difference in exosomes secretion between healthy people and patients with lung adenocarcinoma was found.Through experiments to compare the expression level of tumor stem gene CD44 in patients with lung adenocarcinoma at different stages,it was found that the expression of CD44 gene in lung adenocarcinoma patients at different stages changed significantly at the protein level.These results indicate that Tim4@ILI-01 immunoaffinity material is a stable enrichment matrix and has good clinical application potential.Finally,the full text is summarized.In this work,the Tim4@ILI-01 immunoaffinity material with strongly hydrophilicity,low non-specific adsorption performance and non-destructive release of exosomes was developed.It can be used for the effective separation and enrichment of exosomes in cell culture media and serum,and has broad application prospects.