Extracellular Vesicles Purification Research Articles

Magnetic Nanoparticle-Based Microfluidic Platform for Automated Enrichment of High-Purity Extracellular Vesicles.

Extracellular vesicles (EVs) are available in various biological fluids and have highly heterogeneous sizes, origins, contents, and functions. Rapid enrichment of high-purity EVs remains crucial for enhancing research on EVs in tumors. In this work, we present a magnetic nanoparticle-based microfluidic platform (ExoCPR) for on-chip isolation, purification, and mild recovery of EVs from cell culture supernatant and plasma within 29 min. The ExoCPR chip integrates bubble-driven micromixers and immiscible filtration assisted by surface tension (IFAST) technology. The bubble-driven micromixer enhances the mixing between immunomagnetic beads and EVs, eliminating the need for manual pipetting or off-chip oscillatory incubation. The high-purity EVs were obtained after passing through the immiscible phase interface where hydrophilic or hydrophobic impurities nonspecifically bound to SIMI were removed. The ExoCPR chip had a capture efficiency of 75.8% and a release efficiency of 62.7% for model EVs. We also demonstrated the powerful performance of the ExoCPR in isolating EVs from biological samples (>90% purity). This chip was further employed in clinical plasma samples and showed that the number of GPC3-positive EVs isolated from hepatocellular carcinoma patients was significantly higher than that of healthy individuals. This ExoCPR chip may provide a promising tool for EV-based liquid biopsy and other fundamental research.

Extracellular vesicles selective capture by peptide-functionalized hollow fiber membranes

Recently, membrane devices and processes have been applied for the separation and concentration of subcellular components such as extracellular vesicles (EVs), which play a diagnostic and therapeutic role in many pathological conditions. However, the separation and isolation of specific EV populations from other components found in biological fluids is still challenging. Here, we developed a peptide-functionalized hollow fiber (HF) membrane module to achieve the separation and enrichment of highly pure EVs derived from the culture media of human cardiac progenitor cells. The strategy is based on the functionalization of PSf HF membrane module with BPt, a peptide sequence able to bind nanovesicles characterized by highly curved membranes. HF membranes were modified by a nanometric coating with a copoly azide polymer to limit non-specific interactions and to enable the conjugation with peptide ligand by click chemistry reaction. The BPt-functionalized module was integrated into a TFF process to facilitate the design, rationalization, and optimization of EV isolation. This integration combined size-based transport of species with specific membrane sensing ligands. The TFF integrated BPt-functionalized membrane module demonstrated the ability to selectively capture EVs with diameter < 200 nm into the lumen of fibers while effectively removing contaminants such as albumin. The captured and released EVs contain the common markers including CD63, CD81, CD9 and syntenin-1. Moreover, they maintained a round shape morphology and structural integrity highlighting that this approach enables EVs concentration and purification with low shear stress. Additionally, it achieved the removal of contaminants such as albumin with high reliability and reproducibility, reaching a removal of 93%.

Size-Selective Capturing of Exosomes Using DNA Tripods.

Fractionating and characterizing target samples are fundamental to the analysis of biomolecules. Extracellular vesicles (EVs), containing information regarding the cellular birthplace, are promising targets for biology and medicine. However, the requirement for multiple-step purification in conventional methods hinders analysis of small samples. Here, we apply a DNA origami tripod with a defined aperture of binders (e.g., antibodies against EV biomarkers), which allows us to capture the target molecule. Using exosomes as a model, we show that our tripod nanodevice can capture a specific size range of EVs with cognate biomarkers from a broad distribution of crude EV mixtures. We further demonstrate that the size of captured EVs can be controlled by changing the aperture of the tripods. This simultaneous selection with the size and biomarker approach should simplify the EV purification process and contribute to the precise analysis of target biomolecules from small samples.

Milk Extracellular Vesicles: Natural Nanoparticles for Enhancing Oral Drug Delivery against Bacterial Infections.

Oral drug delivery is typically preferred as a therapeutic intervention due to the complexities and expenses associated with intravenous administration. However, some drugs are poorly absorbed orally, requiring intravenous administration to bypass the gastrointestinal tract and deliver the drug directly into the bloodstream. Thus, there is an urgent need to develop novel drug delivery platforms to overcome the challenges of oral drug delivery with low solubility, low permeability, oral degradation, and low bioavailability. Advances in extracellular vesicles (EVs) as natural carriers have provided emerging approaches to improve potential therapeutic applications. Milk not only contains traditional nutrients but is also rich in EVs. In this Review, we focus mainly on the purification of milk EVs (mEVs), their safety, and the advantages of mEV-based drug carriers in combatting intestinal infections. Additionally, we summarize several advantages of mEVs over conventional synthetic carriers, such as low immunogenicity, high biocompatibility, and the ability to transfer bioactive molecules between cells. Considering the unmet gaps of mEVs in clinical translation, it is essential to review the cargo loading into mEVs and future perspectives for their use as natural drug carriers for oral delivery. This overview of mEV-based drug carriers for oral delivery sheds light on alternative approaches to treat clinical infections associated with intestinal pathogens and the development of novel oral delivery systems.

Effect of Sample Preprocessing and Size-Based Extraction Methods on the Physical and Molecular Profiles of Extracellular Vesicles.

Extracellular vesicles (EVs) are nanometric lipid vesicles that shuttle cargo between cells. Their analysis could shed light on health and disease conditions, but EVs must first be preserved, extracted, and often preconcentrated. Here we first compare plasma preservation agents, and second, using both plasma and cell supernatant, four EV extraction methods, including (i) ultracentrifugation (UC), (ii) size-exclusion chromatography (SEC), (iii) centrifugal filtration (LoDF), and (iv) accousto-sorting (AcS). We benchmarked them by characterizing the integrity, size distribution, concentration, purity, and expression profiles for nine proteins of EVs, as well as the overall throughput, time-to-result, and cost. We found that the difference between ethylenediaminetetraacetic acid (EDTA) and citrate anticoagulants varies with the extraction method. In our hands, ultracentrifugation produced a high yield of EVs with low contamination; SEC is low-cost, fast, and easy to implement, but the purity of EVs is lower; LoDF and AcS are both compatible with process automation, small volume requirement, and rapid processing times. When using plasma, LoDF was susceptible to clogging and sample contamination, while AcS featured high purity but a lower yield of extraction. Analysis of protein profiles suggests that the extraction methods extract different subpopulations of EVs. Our study highlights the strengths and weaknesses of sample preprocessing methods, and the variability in concentration, purity, and EV expression profiles of the extracted EVs. Preanalytical parameters such as collection or preprocessing protocols must be considered as part of the entire process in order to address EV diversity and their use as clinically actionable indicators.

Extracellular-Vesicle Catch-and-Release Isolation System Using a Net-Charge Invertible Curvature-Sensing Peptide.

Extracellular vesicles (EVs) carry various informative components, including signaling proteins, transcriptional regulators, lipids, and nucleic acids. These components are utilized for cell-cell communication between donor and recipient cells. EVs have shown great promise as pharmaceutical-targeting vesicles and have attracted the attention of researchers in the fields of biological and medical science because of their importance as diagnostic and prognostic markers. However, the isolation and purification of EVs from cell-cultured media remain challenging. Ultracentrifugation is the most widely used method, but it requires specialized and expensive equipment. In the present study, we proposed a novel methodology to isolate EVs using a simple and convenient method, i.e., an EV catch-and-release isolation system (EV-CaRiS) using a net-charge invertible curvature-sensing peptide (NIC). Curvature-sensing peptides recognize vesicles by binding to lipid-packing defects on highly curved membranes regardless of the expression levels of biomarkers. NIC was newly designed to reversibly capture and release EVs in a pH-dependent manner. NIC allowed us to achieve reproducible EV isolation from three human cell lines on resin using a batch method and single-particle imaging of EVs containing the ubiquitous exosome markers CD63 and CD81 by total internal reflection fluorescence microscopy (TIRFM). EV-CaRiS was demonstrated as a simple and convenient methodology for EV isolation, and NIC is promising for applications in the single-particle analysis of EVs.

High-Performance Gel-Free and Label-Free Size Fractionation of Extracellular Vesicles with Two-Dimensional Electrophoresis in a Microfluidic Artificial Sieve.

Extracellular vesicles (EVs) are cell-derived particles that exhibit diverse sizes, molecular contents, and clinical implications for various diseases depending on their specific subpopulations. However, fractionation of EV subpopulations with high resolution, efficiency, purity, and yield remains an elusive goal due to their diminutive sizes. In this study, we introduce a novel strategy that effectively separates EV subpopulations in a gel-free and label-free manner, using two-dimensional (2D) electrophoresis in a microfluidic artificial sieve. The microfabricated artificial sieve consists of periodically arranged micro-slit-well structures in a 2D array and generates an anisotropic electric field pattern to size fractionate EVs into discrete streams and steer the subpopulations into designated outlets for collection within a minute. Along with fractionating EV subpopulations, contaminants such as free proteins and short nucleic acids can be simultaneously directed to waste outlets, thus accomplishing both size fractionation and purification of EVs with high performance. Our platform offers a simple, rapid, and versatile solution for EV subpopulation isolation, which can potentially facilitate the discovery of biomarkers for specific EV subtypes and the development of EV-based therapeutics.

Dynamic cultivation of human mesenchymal stem/stromal cells for the production of extracellular vesicles in a 3D bioreactor system

Purpose3D cell culture and hypoxia have been demonstrated to increase the therapeutic effects of mesenchymal stem/stromal cells (MSCs)-derived extracellular vesicles (EVs). In this study, a process for the production of MSC-EVs in a novel 3D bioreactor system under normoxic and hypoxic conditions was established and the resulting EVs were characterized.MethodsHuman adipose-derived MSCs were seeded and cultured on a 3D membrane in the VITVO® bioreactor system for 7 days. Afterwards, MSC-EVs were isolated and characterized via fluorescence nanoparticle tracking analysis, flow cytometry with staining against annexin V (Anx5) as a marker for EVs exposing phosphatidylserine, as well as CD73 and CD90 as MSC surface markers.ResultsCultivation of MSC in the VITVO® bioreactor system demonstrated a higher concentration of MSC-EVs from the 3D bioreactor (9.1 × 109 ± 1.5 × 109 and 9.7 × 109 ± 3.1 × 109 particles/mL) compared to static 2D culture (4.2 × 109 ± 7.5 × 108 and 3.9 × 109 ± 3.0 × 108 particles/mL) under normoxic and hypoxic conditions, respectively. Also, the particle-to-protein ratio as a measure for the purity of EVs increased from 3.3 × 107 ± 1.1 × 107 particles/µg protein in 2D to 1.6 × 108 ± 8.3 × 106 particles/µg protein in 3D. Total MSC-EVs as well as CD73−CD90+ MSC-EVs were elevated in 2D normoxic conditions. The EV concentration and size did not differ significantly between normoxic and hypoxic conditions.ConclusionThe production of MSC-EVs in a 3D bioreactor system under hypoxic conditions resulted in increased EV concentration and purity. This system could be especially useful in screening culture conditions for the production of 3D-derived MSC-EVs.

Density-based lipoprotein depletion improves extracellular vesicle isolation and functional analysis

BackgroundBlood plasma is the main source of extracellular vesicles (EVs) in clinical studies aiming to identify biomarkers and to investigate pathophysiological processes, especially regarding EV roles in inflammation and thrombosis. However, EV isolation from plasma has faced the fundamental issue of lipoprotein contamination, representing an important bias since lipoproteins are highly abundant and modulate cell signaling, metabolism, and thromboinflammation. ObjectivesHere, we aimed to isolate plasma EVs after depleting lipoproteins, thereby improving sample purity and EV thromboinflammatory analysis. MethodsDensity-based gradient ultracentrifugation (G-UC) was used for lipoprotein depletion before EV isolation from plasma through size-exclusion chromatography (SEC) or serial centrifugation (SC). Recovered EVs were analyzed by size, concentration, cellular source, ultrastructure, and bottom-up proteomics. ResultsG-UC efficiently separated lipoproteins from the plasma, allowing subsequent EV isolation through SEC or SC. Combined analysis from EV proteomics, cholesterol quantification, and apoB-100 detection confirmed the significant reduction in lipoproteins from isolated EVs. Proteomic analysis identified similar gene ontology and cellular components in EVs, regardless of lipoprotein depletion, which was consistent with similar EV cellular sources, size, and ultrastructure by flow cytometry and transmission electron microscopy. Importantly, lipoprotein depletion increased the detection of less abundant proteins in EV proteome and enhanced thromboinflammatory responses of platelets and monocytes stimulated in vitro with EV isolates. ConclusionCombination of G-UC+SEC significantly reduced EV lipoprotein contamination without interfering in EV cellular source, gene ontology, and ultrastructure, allowing the recovery of highly pure EVs with potential implications for functional assays and proteomic and lipidomic analyses.

Novel insights into the isolation of extracellular vesicles by anion exchange chromatography.

Extracellular vesicles (EVs) are membrane structures enclosed by a lipid bilayer that are released into the extracellular space by all types of cells. EVs are involved in many physiological processes by transporting biologically active substances. Interest in EVs for diagnostic biomarker research and therapeutic drug delivery applications has increased in recent years. The realization of the full therapeutic potential of EVs is currently hampered by the lack of a suitable technology for the isolation and purification of EVs for downstream pharmaceutical applications. Anion Exchange Chromatography (AEX) is an established method in which specific charges on the AEX matrix can exploit charges on the surface of EVs and their interactions to provide a productive and scalable separation and purification method. The established AEX method using Eshmuno® Q, a strong tentacle anion exchange resin, was used to demonstrate the principal feasibility of AEX-based isolation and gain insight into isolated EV properties. Using several EV analysis techniques to provide a more detailed insight into EV populations during AEX isolation, we demonstrated that although the composition of CD9/63/81 remained constant for tetraspanin positive EVs, the size distribution and purity changed during elution. Higher salt concentrations eluted larger tetraspanin negative vesicles.

Tandem SERS and MS/MS Profiling of Plasma Extracellular Vesicles for Early Ovarian Cancer Biomarker Discovery.

Extracellular vesicles (EVs) are vectors of biomolecular cargo that play essential roles in intercellular communication across a range of cells. Protein, lipid, and nucleic acid cargo harbored within EVs may serve as biomarkers at all stages of disease; however, the choice of methodology may challenge the specificity and reproducibility of discovery. To address these challenges, the integration of rigorous EV purification methods, cutting-edge spectroscopic technologies, and data analysis are critical to uncover diagnostic signatures of disease. Herein, we demonstrate an EV isolation and analysis pipeline using surface-enhanced Raman spectroscopy (SERS) and mass spectrometry (MS) techniques on plasma samples obtained from umbilical cord blood, healthy donor (HD) plasma, and plasma from women with early stage high-grade serous carcinoma (HGSC). Plasma EVs were purified by size exclusion chromatography and analyzed by surface-enhanced Raman spectroscopy (SERS), mass spectrometry (MS), and atomic force microscopy. After determining the fraction of highest EV purity, SERS and MS were used to characterize EVs from HDs, pooled donors with noncancerous gynecological ailments (n = 6), and donors with early stage [FIGO (I/II)] with HGSC. SERS spectra were subjected to different machine learning algorithms such as PCA, logistic regression, support vector machine, naïve Bayes, random forest, neural network, and k nearest neighbors to differentiate healthy, benign, and HGSC EVs. Collectively, we demonstrate a reproducible workflow with the potential to serve as a diagnostic platform for HGSC.

Nanoscale sorting of extracellular vesicles via optically-induced dielectrophoresis on an integrated microfluidic system.

We reported a microfluidic system for sorting of extracellular vesicles (EVs), which can house DNAs, RNAs, lipids, proteins, and metabolites that are important in intercellular communication. Their presence within bodily fluids has demonstrated potential in both clinical diagnostic and therapeutic applications. Furthermore, EVs exhibit distinct subtypes categorized by their sizes, each endowed with unique biophysical properties. Despite several existing techniques for EV isolation and purification, diminished purity and prolonged processing times still hamper clinical utility; comprehensive capture of EVs remains an ongoing pursuit. To address these challenges, we devised an innovative method for automated sorting of nano-scale EVs employing optically-induced dielectrophoresis on an integrated microfluidic chip. With this approach, EVs of three distinct size categories (small: 100-150 nm, medium-sized: 150-225 nm, and large: 225-350 nm) could be isolated at a purity of 86%. This new method has substantial potential in expediting EV research and diagnostics.

Engineered extracellular vesicles: A new approach for targeted therapy of tumors and overcoming drug resistance.

Targeted delivery of anti-tumor drugs and overcoming drug resistance in malignant tumor cells remain significant clinical challenges. However, there are only few effective methods to address these issues. Extracellular vesicles (EVs), actively secreted by cells, play a crucial role in intercellular information transmission and cargo transportation. Recent studies have demonstrated that engineered EVs can serve as drug delivery carriers and showed promising application prospects. Nevertheless, there is an urgent need for further improvements in the isolation and purification of EVs, surface modification techniques, drug assembly processes, and precise recognition of tumor cells for targeted drug delivery purposes. In this review, we summarize the applications of engineered EVs in cancer treatment and overcoming drug resistance, and current challenges associated with engineered EVs are also discussed. This review aims to provide new insights and potential directions for utilizing engineered EVs as targeted delivery systems for anti-tumor drugs and overcoming drug resistance in the near future.

Economical large-scale purification of extracellular vesicles from urine

Research on extracellular vesicles (EVs), both in general laboratory methods and in novel therapeutic approaches, is limited by the difficulty of obtaining large quantities of EVs. This is due to the fact that EVs are only present at low concentrations in most starting samples and common methods for concentrating EVs from large volumes have decisive disadvantages. In particular, ultracentrifugation (UC) and tangential flow filtration (TFF) are used in EV bulk production. However, while UC leads to low EV-yield with high levels of impurities and damaged EVs, TFF is suitable for enriching EVs from large volumes, but requires expensive equipment. In addition, both methods require pre-purification of the EV material and downstream EV isolation steps. To circumvent all these obstacles, we evaluated various methods for their applicability to EV mass purification, optimized both their implementation and the workflow, and thus developed the following sequential protocol for EV bulk production: First, a preclearing step is performed by 1 µm filtration, followed by PEG precipitation to reduce total volume. Larger debris and apoptotic bodies are removed by centrifugation and filtration prior to enrichment and pre-purification of EVs by ultrafiltration or using the ExoEasyMaxi kit. Finally, EVs are purified by size exclusion chromatography. Since the availability of suitable starting material to obtain pure EVs is also a challenge, we compared different biological fluids, with urine proving to be the most suitable source. We determined the purity and quantity of the EV samples using nanotracking analysis, dynamic light scattering, phospholipid-, RNA- and protein-concentration measurement, electron microscopy and western blot.In summary, we have developed procedures to obtain about 1012 pure EVs in less than a day, with a handling time under 2 h, and at a cost of approximately €24. This method should facilitate further scientific and technical developments in the field of EV biology.

First insight into extracellular vesicle-miRNA characterization in a sheep in vitro model of inflammation

Extracellular vesicles (EVs) and their microRNA (miRNA) cargoes have garnered attention in the veterinary field for their regulatory role in various biological processes. This study aimed to (i) evaluate two techniques of EV isolation from sheep peripheral blood mononuclear cell (PBMC) supernatants using the ultracentrifugation (UC) and reagent (REA) methods and (ii) characterize the EV-miRNA profiles after an in vitro inflammatory environment mediated by lipopolysaccharides (LPS). Sheep peripheral blood was collected, and PBMCs were separated using a density gradient reagent. Subsequently, PBMCs were cultured at 37°C for 24 h (5% CO2), and the supernatants were collected to perform the EV isolation. The presence of CD81+ extracellular vesicle marker was determined, and the purity of isolated EVs was calculated as a ratio between the number of isolated EVs and the protein concentration. Moreover, the morphological characterization revealed mainly round-shaped structures with average sizes of 211 nm for EVs isolated by the UC method and 99 nm for EVs isolated by the REA method. Illumina NextSeq sequencing in a single-end mode was used to characterize the miRNA profile, and the differentially expressed (DE) miRNAs were analyzed using a combination of bioinformatics tools. The results revealed that the REA method is reliable for EV isolation from sheep supernatants. It was considered an improvement of the recovery rate and purity of EVs with the enhancement of the number and the expression levels of characterized miRNAs. The EVs isolated by the UC method after an LPS challenge showed 11 DE miRNAs, among which eight miRNAs were upregulated and three were downregulated. On the other hand, the REA method revealed an EV cargo in which eight DE miRNAs were upregulated and 21 DE miRNAs were downregulated. The master miRNA regulators of the biological process were identified by performing the MIRNA-mRNA network analysis, showing that, among the higher representative miRNAs based on the centrality and betweenness, the miR-26a-5p could have a crucial role in the resolution of inflammation. Moreover, the identification of the let-7 miRNA family in all the EVs showed potential targeted genes that regulate the inflammation and immune responses.

Enhancing Extracellular Vesicle Analysis by Integration of Large-Volume Sample Stacking in Capillary Electrophoresis with Asymmetrical Flow Field-Flow Fractionation.

Extracellular vesicles (EVs) play important roles in cell-cell communication and pathological development. Cargo profiling for the EVs present in clinical specimens can provide valuable insights into their functions and help discover effective EV-based markers for diagnostic and therapeutic purposes. However, the highly abundant and complex matrix components pose significant challenges for specific identification of low-abundance EV cargos. Herein, we combine asymmetrical flow field-flow fractionation (AF4) with large-volume sample stacking and capillary electrophoresis (LVSS/CE), to attain EVs with high purity for downstream protein profiling. This hyphenated system first separates the EVs from the contamination of smaller serum proteins by AF4, and second resolves the EVs from the coeluted, nonvesicular matrix components by CE following LVSS. The optimal LVSS condition permits the injection of 10-fold more EVs into CE compared to the nonstacking condition without compromising separation resolution. Collection and downstream analysis of the highly pure EVs after CE separation were demonstrated in the present work. The high EV purity yields a much-improved labeling efficiency when detected by fluorescent antibodies compared to those collected from the one-dimension separation of AF4, and permits the identification of more EV-specific cargos by LC-MS/MS compared to those isolated by ultracentrifugation (UC), the exoEasy Maxi Kit, and AF4. Our results strongly support that AF4-LVSS/CE can improve EV isolation and cargo analysis, opening up new opportunities for understanding EV functions and their applications in the biomedical fields.

One-Pot Analytical Pipeline for Efficient and Sensitive Proteomic Analysis of Extracellular Vesicles.

Extracellular vesicle (EV) proteomics emerges as an effective tool for discovering potential biomarkers for disease diagnosis, monitoring, and therapeutics. However, the current workflow of mass spectrometry-based EV proteome analysis is not fully compatible in a clinical setting due to inefficient EV isolation methods and a tedious sample preparation process. To streamline and improve the efficiency of EV proteome analysis, here we introduce a one-pot analytical pipeline integrating a robust EV isolation approach, EV total recovery and purification (EVtrap), with in situ protein sample preparation, to detect urinary EV proteome. By incorporating solvent-driven protein capture and fast on-bead digestion, the one-pot pipeline enabled the whole EV proteome analysis to be completed within one day. In comparison with the existing workflow, the one-pot pipeline was able to obtain better peptide yield and identify the equivalent number of unique EV proteins from 1 mL of urine. Finally, we applied the one-pot pipeline to profile proteomes in urinary EVs of bladder cancer patients. A total of 2774 unique proteins were identified in 53 urine samples using a 15 min gradient library-free data-independent acquisition method. Taken altogether, our novel one-pot analytical pipeline demonstrated its potential for routine and robust EV proteomics in biomedical applications.

Rapid and Highly Efficient Purification of Extracellular Vesicles Enabled by a TiO2 Hybridized Spongy-like Polymer.

We developed a novel purification medium of extracellular vesicles (EVs) by constructing a spongy-like monolithic polymer kneaded with TiO2 microparticles (TiO2-hybridized spongy monolith, TiO2-SPM). TiO2-SPM was applied in a solid-phase extraction format and enabled simple, rapid, and highly efficient purification of EVs. This is due to the high permeability caused by the continuous large flow-through pores of the monolithic skeleton (median pore size; 5.21 μm) and the specific interaction of embedded TiO2 with phospholipids of the lipid bilayers. Our method also excels in efficiency and comprehensiveness, collecting small EVs (SEVs) from the same volume of a cell culture medium 130.7 times more than typical ultracentrifugation and 4.3 times more than affinity purification targeting surface phosphatidylserine by magnetic beads. The purification method was completed within 1 h with simple operations and was directly applied to serum SEVs. Finally, we demonstrated flexibility toward the shape and size of our method by depleting EVs from fetal bovine serum (FBS), which is a necessary process to prevent contamination of culture cell-derived EVs with exogenous FBS-derived EVs. Our method will eliminate the tedious and difficult purification processes of EVs, providing a universal purification platform for EV-based drug discovery and pathological diagnosis.

Rigorous Comparison of Extracellular Vesicle Processing to Enhance Downstream Analysis for Glioblastoma Characterization.

Extracellular vesicles (EVs) are highly sought after as a source of biomarkers for disease detection and monitoring. Tumor EV isolation, processing, and evaluation from biofluids is convoluted by EV heterogeneity and biological contaminants and is limited by technical processing efficacy. This study rigorously compares common bulk EV isolation workflows (size exclusion chromatography, SEC; membrane affinity, MA) alongside downstream RNA extraction protocols to investigate molecular analyte recovery. EV integrity and recovery is evaluated using a variety of technologies to quantify total intact EVs, total and surface proteins, and RNA purity and recovery. A comprehensive evaluation of each analyte is performed, with a specific emphasis on maintaining user (n = 2), biological (n = 3), and technical replicates (n≥3) under in vitro conditions. Subsequent study of tumor EV spike-in into healthy donor plasma samples is performed to further validate biofluid-derived EV purity and isolation for clinical application. Results show that EV surface integrity is considerably preserved in eluates from SEC-derived EVs, but RNA recovery and purity, as well as bulk protein isolation, is significantly improved in MA-isolated EVs. This study concludes that EV isolation and RNA extraction pipelines govern recovered analyte integrity, necessitating careful selection of processing modality to enhance recovery of the analyte of interest.

CRISPR delivery with extracellular vesicles: Promises and challenges

AbstractThe CRISPR gene editing tool holds great potential for curing genetic disorders. However, the safe, efficient, and specific delivery of the CRISPR/Cas9 components into cells and tissues remains a challenge. While many currently available delivery methods achieve high levels of gene editing effects in vivo, they often result in genotoxicity and immunogenicity. Extracellular vesicles (EVs), which are cell‐derived lipid nanoparticles, are capable of transferring protein and nucleic acid cargoes between cells, making them a promising endogenous alternative to synthetic delivery methods. This review provides a comprehensive analysis of the currently available strategies for EV‐mediated delivery of CRISPR/Cas9. These strategies include cell‐based, passive loading obtained by overexpression of CRISPR/Cas9, active loading involving protein or RNA dimerization, and loading into already purified EVs. All these approaches suggest that EV‐based CRISPR/Cas9 delivery is useful for achieving both in vitro and in vivo gene editing. Despite that, substantial variations in cellular uptake and gene editing efficiencies indicate that further improvement and standardization are required for the therapeutic use of EVs as a CRISPR/Cas9 delivery vehicle. These improvements include, but is not limited to, the high‐yield purification of EVs, increased loading and release efficiencies, as well as improved tissue‐ or cell‐specific targeting specificities.