基于亲和作用力的外泌体高效分离方法的经典策略和研究进展

doi:10.3724/SP.J.1123.2024.11004

摘要/Abstract

摘要：

外泌体是大多数细胞分泌的细胞外囊泡的一个特殊亚类,存在于血液、尿液、唾液、羊水、乳汁等各种生物体液中,也存在于各种组织和细胞间隙中,是细胞间通讯的重要介质。越来越多的研究表明,基于外泌体的液体活检有望成为疾病早期诊断、治疗监测以及预后评估的重要手段,高质量外泌体的分离则是保障后续相关诊断及治疗结果准确性和可重复性的前提和保障。然而,由于外泌体在样本中丰度较低,且与体液中的类似成分(如细胞碎片、蛋白质)共存,如何在复杂的生物样本中实现其高效的分离仍具有挑战性。目前,基于外泌体的物理、化学和生物特性,已发展出多种外泌体分离技术。近年来,基于亲和作用力的外泌体高效分离方法克服了传统分离方法的局限性和弊端,在科研和临床中得到了广泛应用。本文聚焦外泌体分离富集领域,系统综述了近年来基于亲和作用力的外泌体分离富集方法,并对外泌体分离富集方法的发展前景进行了分析和展望,期望为外泌体分离方法新策略的构建及应用提供参考。

关键词: 外泌体, 生物标志物, 亲和作用, 分离富集, 研究进展

Abstract:

Exosomes form a subclass of extracellular vesicle that are secreted by most cells and found in nearly all body fluids, including blood, urine, saliva, amniotic fluid, and milk, as well as in various tissues and intercellular spaces. Exosomes have recently been recognized as crucial intercellular communication mediators, and an increasing number of studies have shown that exosomes are important liquid-biopsy tools that play irreplaceable roles in the diagnosis, prognosis, and treatment of diseases. The ability to isolate high-quality exosomes is a prerequisite for diagnosing and subsequently treating diseases in an accurate and repeatable manner. However, efficiently isolating exosomes from complex biological samples is challenging owing to their relatively low abundances and interference from non-vesicular macromolecules (such as cell debris and proteins). To date, various isolation techniques based on the physical, chemical, and biological characteristics of exosomes have been developed. Indeed, efficient affinity-interaction-based methods have recently overcome the limitations and drawbacks of traditional exosome isolation methods and are widely used in scientific research and clinical applications. This review focuses on exosome isolation and enrichment, and systematically reviews recent research progress on efficient isolation methods based on affinity interactions. Developmental prospects of exosome isolation and enrichment directions are analyzed with the aim of providing a reference for the construction and use of new exosome-isolation strategies.

Key words: exosomes, biomarkers, affinity interaction, isolation and enrichment, research progress

中图分类号:

O658

王海燕, 谢沛涓, 乔晓强, 张丽媛. 基于亲和作用力的外泌体高效分离方法的经典策略和研究进展[J]. 色谱, 2025, 43(5): 413-423.

WANG Haiyan, XIE Peijuan, QIAO Xiaoqiang, ZHANG Liyuan. Typical strategy and research progress of efficient isolation methods of exosomes based on affinity interaction[J]. Chinese Journal of Chromatography, 2025, 43(5): 413-423.

图/表 7

图1 外泌体的生物功能

Fig. 1 Biological functions of exosomes ESE: early-sorting endosome; MVBs: multivesicular bodies.

图2 (a)Tim4@ILI-01和(b)Fe3O4@SiO2-ILI-0l@Tim4材料用于外泌体富集及下游分析的示意图[21,22]

Fig. 2 Schematic of (a) Tim4@ILI-01 and (b) Fe3O4@SiO2-ILI-0l@Tim4 for exosome enrichment and downstream analysis[21,22]

图3 外泌体分离纳米系统富集外泌体[24]

Fig. 3 Exosome isolation by pH-responsive host-guest-based nanosystem (pH-HGN)[24]

图4 (a)基于TiO2-BSA-SPM的外泌体分离机制[25]; (b)外泌体通过高亲和力的CP-PC相互作用黏附在MB@CP上[27]; (c)Fe3O4@SiO2@Eu2O3纳米材料富集血浆中的外泌体[28]

Fig. 4 (a) Exosome isolation mechanism of TiO2-BSA-SPM[25]; (b) exosome adhesion on MB@CP via high-affinity choline phosphate-phosphatidylcholine (CP-PC) interactions[27]; (c) exosome enrichment in plasma using Fe3O4@SiO2@Eu2O3 nanomaterials[28]

图5 (a)SiO2@BSA@Fe-TA6在外泌体分离及下游分析中的应用[29]; (b)基于PS-MIPs-NELISA-SERS的外泌体检测平台[31]; (c)基于g-C3N4纳米片(BCNNS)的外泌体分离检测平台[32]

Fig. 5 (a)Applications of SiO2@BSA@Fe-TA6 for exosome isolation and downstream analysis [29]; (b) an exosome detection platform based on the phospholipids-molecularly imprinted polymers-nanozyme-linked immunosorbent assay-surface-enhanced Raman scattering (PS-MIPs-NELISA-SERS)[31]; (c) an exosome isolation and detection platform based on g-C3N4 nanosheets (BCNNS)[32]

表1 本课题组发展的基于亲和作用的外泌体富集方法比较

Table 1 Comparison of exosome-enrichment methods developed by our group, based on affinity-interaction

Approach	Capture efficiency/%	Advantages	Insufficiencies	Applications	Ref.
Tim4@ILI-01	85.2	high capture efficiency; easy nondestructive release; good biological activities of the captured exosomes	potentially exosome loss due to multiple mechanical centrifugation steps	sera of healthy persons and lung adenocarcinoma patients	[22]
Fe₃O₄@SiO₂- ILI-0l@Tim4	90.3	high-efficiency and high-throughput exosome capture and release; simple and quick sample handling	increased economic cost due to the employment of Tim4	high-throughput exosomal PD-L1 screening for accurately predicting anti-PD-1 response	[21]
pH-HGN	90.2	highly efficient homogeneous capture and rapid recovery of intact exosomes	biological interference of enriched exosome in clinical treatment due to the conjugation of anti-CD63 antibody	differentiating lung-cancer patients from healthy persons	[24]
SiO₂@BSA@ Fe-TA₆	85.4	label-free, universal, low-cost, and easy to scale-up exosome capture	difficulty in exosome elution	lung-cancer diagnosis and typing	[29]

图6 (a)基于微流控芯片的外泌体分过程和苝四羧酸二亚胺衍生物(PTCDI)-适配体荧光信号转导策略用于外泌体检测[36];(b)唾液外泌体蛋白质组学分析和哮喘生物标志物的高通量阵列检测示意图[37]; (c)支持脂质膜(SLM)捕获外泌体方案[38]; (d)蛇形通道法分离纯化外泌体[39]

Fig. 6 (a) Exosome-isolation process by a microfluidic chip and a perylene tetracarboxylic acid diimide derivative (PTCDI)-aptamer fluorescence signal transduction strategy for detecting exosomes[36]; (b) schematic depicting salivary exosome proteomic analysis and the high-throughput array detection of asthma biomarkers[37]; (c) scheme depicting exosome capture by a supporting lipid membrane (SLM)[38]; (d) exosome isolation and purification procedure using serpentine channels[39]

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