摘要
外泌体作为一种细胞外囊泡,其内容物可以反映亲代细胞的重要信息,而自身也具有独特的结构,能够执行特征的生物学功能。基于外泌体的表面化学和生物学特征,制备了不同类型的金属/适配体(Apt)双功能复合磁性纳米材料,并将其应用于外泌体的富集纯化。将适配体和外泌体表面目标膜蛋白的特异性结合性能与以钛、锆为代表的金属氧化物和外泌体磷脂双层膜的特异性亲和作用结合,可极大地提高分离材料对外泌体的分离选择性和富集容量。分别以Fe_(3)O_(4)@Zr-MOFs、Fe_(3)O_(4)@Zr-Ti-MOFs和Fe_(3)O_(4)@TiO_(2)等金属有机框架(MOFs)/金属氧化物磁性纳米材料为基底,制备对应的双功能MOFs/金属氧化物-适配体复合磁性纳米材料Fe_(3)O_(4)@Zr-MOFs-Apt、Fe_(3)O_(4)@Zr-Ti-MOFs-Apt和Fe_(3)O_(4)@TiO_(2)-Apt,并进一步对不同材料的外泌体富集性能加以评价。以超速离心法提取的模型外泌体以及尿液为样品,对修饰相同质量适配体和不同含量金属氧化物的双功能材料的富集性能加以对比。将3种双功能磁性纳米材料应用于尿液外泌体的富集,得到的外泌体裂解后经质谱鉴定,分别得到233、343和832个外泌体蛋白。这一结果也表明双功能磁性纳米材料可以充分结合核酸适配体亲和的高选择性和金属氧化物的高富集容量优势,对于复杂生物样品中外泌体的快速、高效分离纯化具有潜在的应用价值,而针对材料制备和分离纯化方法的设计也为新型外泌体富集材料的设计提供了一条可行的新思路。
Exosomes,which are extracellular vesicles with sizes of 30-150 nm,contain proteins,lipids,RNA,etc.,which can reflect important information about parental cells.They also have unique structures and can perform characteristic biological functions.Although the release of exosomes is a normal process,tumor cells release more exosomes,and the contents can induce cancer progression.Exosomes are widely distributed in body fluids at high concentrations and are easy to obtain;hence,the collection of exosomes released by tumor cells has become one of the main directions in tumor liquid biopsy.In order to ensure the reproducibility and consistency of liquid biopsy results,it is necessary to develop methods for enriching exosomes in sufficient yield and purity from complex samples.Based on the size,hydrophobic proteins,and characteristic proteins of exosomes,various methods for exosome separation and purification have been developed,such as ultracentrifugation,polymer precipitation,and immunoaffinity methods.An aptamer(Apt)is an oligonucleotide chain with a total length of 20-100 nt,which has ligand binding properties and can be used to detect different types of drugs and biomolecules at the nanomolar level.Characteristic proteins on the surface of exosomes such as CD63,CD9,and CD81 are often used as exosomes markers.At present,a variety of aptamer sequences targeting the characteristic proteins of exosomes have been reported.Zirconium and titanium cations as well as the oxides of these metals show high affinity to the phospholipid bilayer on the exosome surface and are used in the separation and purification of exosomes.Metal organic frameworks(MOFs)can provide a wealth of metal oxide affinity sites to interact with the phospholipid bilayer membrane,and their diverse organic ligands can provide numerous modification sites to bind with aptamers.In this study,different metal/aptamer dual-functional composite magnetic nanomaterials were prepared by exploiting the surface chemistry and biological characteristics of exosomes for the enrichment and purification of exosomes.Because of the specific affinity of the aptamers toward the target membrane protein on the exosome surface and the affinity of the titanium or zirconium oxide toward the phospholipid bilayer membrane of exosomes,dual-functional magnetic nanomaterials can greatly improve the enrichment capacity and separation selectivity of exosomes.Fe_(3)O_(4)@Zr-MOFs was used as the substrate to fabricate the dual functional MOFs/metal oxide aptamer composite magnetic nanomaterial Fe_(3)O_(4)@Zr-MOFs-Apt.UiO-66-NH2 was grown in situ on the surface of Fe_(3)O_(4)by a solvothermal method to form a Zr-MOFs layer,and aptamer-CD63 was covalently bonded to the amino group of the organic ligand of the MOFs.The magnetic bimetallic metal organic framework Fe_(3)O_(4)@Zr-Ti-MOFs,which was fabricated via a layer-by-layer assembly approach,was used as the substrate to prepare the dual functional MOFs/metal oxide aptamer composite Fe_(3)O_(4)@Zr-Ti-MOFs-Apt via coordination bond formation between the metal site on the Fe_(3)O_(4)@Zr-Ti-MOFs and the aptamers.The third dual functional MOFs/metal oxide aptamer composite magnetic nanomaterial,Fe_(3)O_(4)@TiO_(2)-Apt,was prepared by using Fe_(3)O_(4)@TiO_(2) as the substrate via coordination bond formation between the metal site on Fe_(3)O_(4)@TiO_(2) and the aptamers.Considering model exosomes extracted by ultracentrifugation and urine as samples,this paper compared the enrichment performance of materials modified with the same quality of aptamers and different levels of metal oxides.The dual-functional composite magnetic nanomaterials modified with different metals/aptamers were used for the enrichment of urine exosomes.The obtained exosomes were lysed and identified by mass spectrometry,and 233,343,and 832 exosomal proteins were identified.This result also shows that dual-functional magnetic nanomaterials can fully combine the high selectivity of the nucleic acid aptamer and the high enrichment capacity of the metal oxides.The rapid,efficient separation and purification of exosomes in biological samples has excellent application potential.The material design and purification methods also provide a new idea for the development of new exosome-enrichment materials.
作者
张维冰
卢睿
张凌怡
ZHANG Weibing;LU Rui;ZHANG Lingyi(Shanghai Key Laboratory of Functional Materials Chemistry, School of Chemistry & Molecular Engineering, East China University of Science and Technology, Shanghai 200237, China)
出处
《色谱》
CAS
CSCD
北大核心
2021年第10期1128-1136,共9页
Chinese Journal of Chromatography
基金
国家自然科学基金(21974045).