Liquid biopsy refers to the sampling and molecular analysis of the biofluids of circulating tumor cells, extracellular vesicles, nucleic acids, and so forth. Exosomes are small extracellular vesicles with sizes between 30–150 nm. They are secreted by multivesicular bodies through exocytosis in live cells and can participate in intercellular communication due to their contents, including nucleic acids, proteins, and lipids. Herein, we investigate publication frequencies on exosomes over the past 10 years, and review recent clinical studies on liquid biopsy of exosomes in the fields of oncology, pregnancy disorders, cardiovascular diseases, and organ transplantation. We also describe the advantages of exosomes as an effective liquid biopsy tool and the progression of exosome extraction methods. Finally, we depict the commercial development of exosome research and discuss the future role of exosomes in liquid biopsy.

/pdf/application-of-exosomes-as-liquid-biopsy-in-clinical-4dw9yvenlc.pdf

Application of exosomes as liquid biopsy in clinical diagnosis.

Modern isolation and separation techniques for extracellular vesicles.

Exosomes are a subgroup of nanosized extracellular vesicles enclosed by a lipid bilayer membrane and secreted by most eukaryotic cells. They represent a route of intercellular communication and participate in a wide variety of physiological and pathological processes. The biological roles of exosomes rely on their bioactive cargos, including proteins, nucleic acids, and lipids, which are delivered to target cells. Their distinctive properties—innate stability, low immunogenicity, biocompatibility, and good biomembrane penetration capacity—allow them to function as superior natural nanocarriers for efficient drug delivery. Another notably favorable clinical application of exosomes is in diagnostics. They hold various biomolecules from host cells, which are indicative of pathophysiological conditions; therefore, they are considered vital for biomarker discovery in clinical diagnostics. Here, we use data from the CAS Content Collection and provide a landscape overview of the current state and delineate trends in research advancement on exosome applications in therapeutics and diagnostics across time, geography, composition, cargo loading, and development pipelines. We discuss exosome composition and pathway, from their biogenesis and secretion from host cells to recipient cell uptake. We assess methods for exosome isolation and purification, their clinical applications in therapy and diagnostics, their development pipelines, the exploration goals of the companies, the assortment of diseases they aim to treat, development stages of their research, and publication trends. We hope this review will be useful for understanding the current knowledge in the field of medical applications of exosomes, in an effort to further solve the remaining challenges in fulfilling their potential.

https://pubs.acs.org/doi/pdf/10.1021/acsnano.2c08774

Exosomes—Nature’s Lipid Nanoparticles, a Rising Star in Drug Delivery and Diagnostics

Extracellular vesicles (EVs) are increasingly tested as therapeutic vehicles and biomarkers, but still EV subtypes are not fully characterised. To isolate EVs with few co-isolated entities, a combination of methods is needed. However, this is time-consuming and requires large sample volumes, often not feasible in most clinical studies or in studies where small sample volumes are available. Therefore, we compared EVs rendered by five commonly used methods based on different principles from conditioned cell medium and 250 μl or 3 ml plasma, that is, precipitation (ExoQuick ULTRA), membrane affinity (exoEasy Maxi Kit), size-exclusion chromatography (qEVoriginal), iodixanol gradient (OptiPrep), and phosphatidylserine affinity (MagCapture). EVs were characterised by electron microscopy, Nanoparticle Tracking Analysis, Bioanalyzer, flow cytometry, and LC-MS/MS. The different methods yielded samples of different morphology, particle size, and proteomic profile. For the conditioned medium, Izon 35 isolated the highest number of EV proteins followed by exoEasy, which also isolated fewer non-EV proteins. For the plasma samples, exoEasy isolated a high number of EV proteins and few non-EV proteins, while Izon 70 isolated the most EV proteins. We conclude that no method is perfect for all studies, rather, different methods are suited depending on sample type and interest in EV subtype, in addition to sample volume and budget.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/jev2.12128

Molecular evaluation of five different isolation methods for extracellular vesicles reveals different clinical applicability and subcellular origin.

Cells release nanometer-scale, lipid bilayer-enclosed biomolecular packages (extracellular vesicles; EVs) into their surrounding environment. EVs are hypothesized to be intercellular communication agents that regulate physiological states by transporting biomolecules between near and distant cells. The research community has consistently advocated for the importance of RNA contents in EVs by demonstrating that: (1) EV-related RNA contents can be detected in a liquid biopsy, (2) disease states significantly alter EV-related RNA contents, and (3) sensitive and specific liquid biopsies can be implemented in precision medicine settings by measuring EV-derived RNA contents. Furthermore, EVs have medical potential beyond diagnostics. Both natural and engineered EVs are being investigated for therapeutic applications such as regenerative medicine and as drug delivery agents. This review focuses specifically on EV characterization, analysis of their RNA content, and their functional implications. The NIH extracellular RNA communication (ERC) program has catapulted human EV research from an RNA profiling standpoint by standardizing the pipeline for working with EV transcriptomics data, and creating a centralized database for the scientific community. There are currently thousands of RNA-sequencing profiles hosted on the Extracellular RNA Atlas alone (Murillo et al., 2019), encompassing a variety of human biofluid types and health conditions. While a number of significant discoveries have been made through these studies individually, integrative analyses of these data have thus far been limited. A primary focus of the ERC program over the next five years is to bring higher resolution tools to the EV research community so that investigators can isolate and analyze EV sub-populations, and ultimately single EVs sourced from discrete cell types, tissues, and complex biofluids. Higher resolution techniques will be essential for evaluating the roles of circulating EVs at a level which impacts clinical decision making. We expect that advances in microfluidic technologies will drive near-term innovation and discoveries about the diverse RNA contents of EVs. Long-term translation of EV-based RNA profiling into a mainstay medical diagnostic tool will depend upon identifying robust patterns of circulating genetic material that correlate with a change in health status.

/pdf/characterizing-extracellular-vesicles-and-their-diverse-rna-24264zaps0.pdf

Characterizing Extracellular Vesicles and Their Diverse RNA Contents.

Extracellular vesicles (EVs) are nano-sized vesicles containing nucleic acid and protein cargo that are released from a multitude of cell types and have gained significant interest as potential diagnostic biomarkers. Human serum is a rich source of readily accessible EVs; however, the separation of EVs from serum proteins and non-EV lipid particles represents a considerable challenge. In this study, we compared the most commonly used isolation techniques, either alone or in combination, for the isolation of EVs from 200 µl of human serum and their separation from non-EV protein and lipid particles present in serum. The size and yield of particles isolated by each method was determined by nanoparticle tracking analysis, with the variation in particle size distribution being used to determine the relative impact of lipoproteins and protein aggregates on the isolated EV population. Purification of EVs from soluble protein was determined by calculating the ratio of EV particle count to protein concentration. Finally, lipoprotein particles co-isolated with EVs was determined by Western blot analysis of lipoprotein markers APOB and APOE. Overall, this study reveals that the choice of EV isolation procedure significantly impacts EV yield from human serum, together with the presence of lipoprotein and protein contaminants.

/pdf/a-comparison-of-methods-for-the-isolation-and-separation-of-4ncrg5sw37.pdf

A comparison of methods for the isolation and separation of extracellular vesicles from protein and lipid particles in human serum.

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A comparison of methods for the isolation and separation of extracellular vesicles from protein and lipid particles in human serum.