Recent advances and challenges in the recovery and purification of cellular exosomes

Exosomes are nanovesicles secreted by most cellular types that carry important biochemical compounds throughout the body with different purposes, playing a preponderant role in cellular communication. Because of their structure, physicochemical properties and stability, recent studies are focusing in their use as nanocarriers for different therapeutic compounds for the treatment of different diseases ranging from cancer to Parkinson's disease. However, current bioseparation protocols and methodologies are selected based on the final exosome application or intended use and present both advantages and disadvantages when compared among them. In this context, this review aims to present the most important technologies available for exosome isolation while discussing their advantages and disadvantages and the possibilities of being combined with other strategies. This is critical since the development of novel exosome‐based therapeutic strategies will be constrained to the effectiveness and yield of the selected downstream purification methodologies for which a thorough understanding of the available technological resources is needed.     

Epitope Imprinting of Phospholipids by Oriented Assembly at the Oil/Water Interface for the Selective Recognition of Plasma Membranes

Phospholipids, as fundamental building blocks of the cell membrane, play important roles for molecule transportation, cell recognition, etc. However, due to the structural diversity and amphipathic nature, there are few methods for the specific recognition of lipids as compared to other biomolecules such as proteins and glycans. Herein, we developed a molecular imprinting strategy for controllable imprinting toward the polar head of phospholipid exposed on the surface of cellular membranes for recognition. Phosphatidylserine, as unique lipid on the outer membrane leaflet of exosome and also hallmark for cell apoptosis, was imprinted with the developed method. The phosphatidylserine imprinted materials showed high efficiency and specific targeting capability not only for apoptotic cell imaging but also for the isolation of exosomes. Collectively, the synthesized molecularly imprinted materials have great potential for selective plasma membrane recognition for targeted drug delivery and biomarker discovery.     

Exosome aggregation mediated stop-flow paper-based portable device for rapid exosome quantification

Exosome quantification is important for estimation of informative messengers (e.g., proteins, lipids, RNA, etc.) involving physiological and pathological effects. This work aimed to develop a simple and rapid distance-based paper portable device using exosome-capture vesicles (polydiacetylene conjugated with antiCD81) for exosome quantification in cell cultures. This novel concept relied on distinct aggregation of exosomes and exosome-capture vesicles leading to different solvent migration. Distances of the migration were used as signal readouts, which could be detected by naked eye. PDA-antiCD81 as exosome-capture vesicles were optimized (e.g., size, reaction ratio, and concentration) and the paper designs were investigated (e.g., diameter of sample reservoir and lamination layer) to enhance the solvent stop-flow effects. Finally, exosome screening on three cell culture samples (COLO1, MDA-MB-231, and HuR-KO1 subclone) was demonstrated. The method could linearly measure exosome concentrations in correlation with solvent migration distances in the range of 10⁶–10¹⁰ particles/mL (R² > 0.98) from the cell culture samples. The exosome concentration measurements by the developed device were independently assessed by nanoparticle tracking analysis. Results demonstrated no statistically significant difference (p > 0.05) by t-test. This low-cost and rapid device allows a portable platform for exosome quantification without the requirement of expensive equipment and expertise of operation. The developed device could potentially be useful for quantification of other biomarker-related extracellular vesicles.     

Molecularly Engineered Macrophage-Derived Exosomes with Inflammation Tropism and Intrinsic Heme Biosynthesis for Atherosclerosis Treatment

Atherosclerosis (AS) is a major contributor to cardiovascular diseases worldwide, and alleviating inflammation is a promising strategy for AS treatment. Here, we report molecularly engineered M2 macrophage-derived exosomes (M2 Exo) with inflammation-tropism and anti-inflammatory capabilities for AS imaging and therapy. M2 Exo are derived from M2 macrophages and further electroporated with FDA-approved hexyl 5-aminolevulinate hydrochloride (HAL). After systematic administration, the engineered M2 Exo exhibit excellent inflammation-tropism and anti-inflammation effects via the surface-bonded chemokine receptors and the anti-inflammatory cytokines released from the anti-inflammatory M2 macrophages. Moreover, the encapsulated HAL can undergo intrinsic biosynthesis and metabolism of heme to generate anti-inflammatory carbon monoxide and bilirubin, which further enhance the anti-inflammation effects and finally alleviate AS. Meanwhile, the intermediate protoporphyrin IX (PpIX) of the heme biosynthesis pathway permits the fluorescence imaging and tracking of AS.     

Rapid Detection of Exosomal MicroRNAs Using Virus‐Mimicking Fusogenic Vesicles

Exosomal microRNAs (miRNAs) are important biomarkers for clinical diagnosis and disease treatment monitoring. However, most approaches for exosomal miRNA detection are time‐consuming, laborious, and expensive. Herein, we report a virus‐mimicking fusogenic vesicle (Vir‐FV) that enables rapid, efficient, and high‐throughput detection of exosomal miRNAs within 2 h. Fusogenic proteins on Vir‐FVs can specifically target the sialic‐acid‐containing receptors on exosomes, inducing efficient fusion of Vir‐FVs and exosomes. Upon vesicle content mixing, the molecular beacons encapsulated in Vir‐FVs specifically hybridize with the target miRNAs in the exosomes, generating fluorescence. Combined with flow cytometry, the Vir‐FVs can not only detect exosomal miRNAs but also distinguish tumor exosomes from normal exosomes by sensing the tumor‐related miRNAs, paving the way towards the rapid and efficient detection of exosomal miRNAs for diagnosis and prognosis prediction of diseases.     

基于核酸适体的外泌体分子识别研究进展

自研究者证实外泌体承担了细胞外RNA等物质的运输功能以来, 关于外泌体来源与功能的研究一直备受关注. 近年来外泌体被发现具有作为疾病生物标志物的潜力, 使得拥有特定表面蛋白以及特定装载物的外泌体成为分析化学领域有价值的检测对象. 从化学本质角度来说, 外泌体的获取与分析需要依赖特异性的分子识别过程. 核酸适体作为分子识别单元, 因其特异性强、 亲和力高、 生物活性稳定、 易于合成和保存、 而且其序列和结构上具有可编程性, 易于设计和修饰, 已成功地用在外泌体相关的生物传感体系中. 本文从外泌体的化学组成及其具有生理、 病理意义的组分出发, 从外泌体通用生物标志物识别、癌细胞来源外泌体的检测及外泌体蛋白谱的分析这3个方面综述了以核酸适体作为分子识别单元在外泌体分析领域的代表性工作, 总结了现有的靶向外泌体的核酸适体序列信息以及应用场景, 阐述了利用化学合成与修饰以及DNA自组装等化学调控手段增强核酸适体分子识别性能的最新进展, 并从适用于外泌体分子识别的核酸适体的筛选以及化学修饰的角度, 对未来的研究方向进行了展望.     

Restoration of full-length APC protein in SW480 colon cancer cells induces exosome-mediated secretion of DKK-4

Mutations in the adenomatous polyposis coli (APC) tumor suppressor gene are common in both inherited and sporadic forms of colorectal cancer (CRC), and are associated with dysregulated Wnt signaling. Colon carcinoma SW480 cells restored with stable expression of wild-type APC (SW480APC cells) exhibit attenuated Wnt signaling, and reduced tumorigenicity, including increased cell adhesion. We performed a comparative proteomic analysis of exosomes isolated from SW480 and SW480APC cells to examine the effects of restored APC on exosome protein expression. A salient finding of our study was the unique expression of the Wnt antagonist Dickkopf-related protein 4 (DKK4) in SW480APC, but not parental SW480 cell-derived exosomes. Upregulation of DKK4 in SW480APC cells was confirmed by semiquantitative RT-PCR, immunoblotting, and immunogold electron microscopy. Analysis of the DKK4 gene promoter by methylation-specific PCR revealed reduced methylation in SW480APC cells, while RT-PCR demonstrated the downregulation of DNMT-3a, compared to the parental cell line. Our discovery of exosome-mediated secretion of DKK4 opens up the possibility that exosomal DKK4 may be a mechanism used by epithelial colon cells to regulate Wnt signaling which is lost during CRC progression.     

非小细胞肺癌患者尿中Exosomes蛋白质组的差异表达分析

除血浆之外,尿液是另一种寻找潜在生物标志物的重要生物材料。本研究以200000×g超速离心法分离正常人和非小细胞肺癌(NSCLC)患者尿液中的Exosomes,运用1DSDS-PAGE对Exosomes蛋白质组进行分组,从电泳胶上切取正常组和疾病组的20～31kDa条带,胰蛋白酶酶解后,进行HPLC-CHIP-MS/MS分析,并通过UniProtKB/SWISS-PORT数据库搜索鉴定了24种蛋白质,其中在NSCLC患者尿液Exosomes蛋白质组中发现了8种差异表达蛋白,包括免疫球蛋白κ的3个片段、2种Ras相关蛋白、谷胱甘肽S转移酶A2、血清淀粉样P成分前体和磷脂酰乙醇胺结合蛋白1。     

A Pretreatment‐Free,Polymer‐Based Platform Prepared by Molecular Imprinting and Post‐Imprinting Modifications for Sensing Intact Exosomes

Exosomes are small (30–100 nm) membrane vesicles that serve as regulatory agents for intercellular communication in cancers. Currently, exosomes are detected by immuno‐based assays with appropriate pretreatments like ultracentrifugation and are time consuming (>12 h). We present a novel pretreatment‐free fluorescence‐based sensing platform for intact exosomes, wherein exchangeable antibodies and fluorescent reporter molecules were aligned inside exosome‐binding cavities. Such antibody‐containing fluorescent reporter‐grafted nanocavities were prepared on a substrate by well‐designed molecular imprinting and post‐imprinting modifications to introduce antibodies and fluorescent reporter molecules only inside the binding nanocavities, enabling sufficiently high sensitivity to detect intact exosomes without pretreatment. The effectiveness of the system was demonstrated by using it to discriminate between normal exosomes and those originating from prostate cancer and analyze exosomes in tear drops.