Extracellular Vesicles Loading Research Articles

Enhancing extracellular vesicle cargo loading and functional delivery by engineering protein-lipid interactions

Naturally generated lipid nanoparticles termed extracellular vesicles (EVs) hold significant promise as engineerable therapeutic delivery vehicles. However, active loading of protein cargo into EVs in a manner that is useful for delivery remains a challenge. Here, we demonstrate that by rationally designing proteins to traffic to the plasma membrane and associate with lipid rafts, we can enhance loading of protein cargo into EVs for a set of structurally diverse transmembrane and peripheral membrane proteins. We then demonstrate the capacity of select lipid tags to mediate increased EV loading and functional delivery of an engineered transcription factor to modulate gene expression in target cells. We envision that this technology could be leveraged to develop new EV-based therapeutics that deliver a wide array of macromolecular cargo.

M6A modification inhibits miRNAs’ intracellular function, favoring their extracellular export for intercellular communication

Epitranscriptomics represents a further layer of gene expression regulation. Specifically, N6-methyladenosine (m6A) regulates RNA maturation, stability, degradation, and translation. Regarding microRNAs (miRNAs), while it has been reported that m6A impacts their biogenesis, the functional effects on mature miRNAs remain unclear. Here, we show that m6A modification on specific miRNAs weakens their coupling to AGO2, impairs their function on target mRNAs, determines their delivery into extracellular vesicles (EVs), and provides functional information to receiving cells. Mechanistically, the intracellular functional impairment is caused by m6A-mediated inhibition of AGO2/miRNA interaction, the EV loading is favored by m6A-mediated recognition by the RNA-binding protein (RBP) hnRNPA2B1, and the EV-miRNA function in the receiving cell requires their FTO-mediated demethylation. Consequently, cells express specific miRNAs that do not impact endogenous transcripts but provide regulatory information for cell-to-cell communication. This highlights that a further level of complexity should be considered when relating cellular dynamics to specific miRNAs.

Programmable RNA Loading of Extracellular Vesicles with Toehold-Release Purification.

Synthetic nanoparticles as lipid nanoparticles (LNPs) are widely used as drug delivery vesicles. However, they hold several drawbacks, including low biocompatibility and unfavorable immune responses. Naturally occurring extracellular vesicles (EVs) hold the potential as native, safe, and multifunctional nanovesicle carriers. However, loading of EVs with large biomolecules remains a challenge. Here, we present a controlled loading methodology using DNA-mediated and programmed fusion between EVs and messenger RNA (mRNA)-loaded liposomes. The fusion efficiency is characterized at the single-particle level by real-time microscopy through EV surface immobilization via lipidated biotin-DNA handles. Subsequently, fused EV-liposome particles (EVLs) can be collected by employing a DNA strand-replacement reaction. Transferring the fusion reaction to magnetic beads enables us to scale up the production of EVLs one million times. Finally, we demonstrated encapsulation of mCherry mRNA, transfection, and improved translation using the EVLs compared to liposomes or LNPs in HEK293-H cells. We envision this as an important tool for the EV-mediated delivery of RNA therapeutics.

TRIM25 dictates selective miRNA loading into extracellular vesicles during inflammation

Extracellular vesicles (EVs) such as exosomes are loaded with specific biomolecules in order to perform cell-to-cell communication. Understanding the mechanism of selective cargo loading is important to better understand the physiological and pathological function of EVs. Here we describe a novel target of the E3 ligase TRIM25 and show that inflammation-mediated EV loading of the RNA binding protein FMR1 and its associated microRNA, miR-155, is promoted by TRIM25-mediated K63-ubiquitination of FMR1. This ubiquitination promotes an interaction between FMR1 and the EV loading machinery via the cleavage of the trafficking adaptor protein RILP. These interactions are lost when TRIM25 is knocked down. Loss of TRIM25 also prevents the loading of both FMR1 and miR-155. These findings suggest that inflammation-mediated loading of FMR1 and its associated microRNAs into the EV are dependent on K63-ubiquitination by TRIM25 and provide novel insights and tools to manipulate EV biogenesis for therapeutic benefit.

Imaging of Light-Enhanced Extracellular Vesicle-Mediated Delivery of Oxaliplatin to Colorectal Cancer Cells via Laser Ablation, Inductively Coupled Plasma Mass Spectrometry.

Extracellular vesicles (EVs) are lipid bilayer structures released by all cells that mediate cell-to-cell communication via the transfer of bioactive cargo. Because of the natural origin of EVs, their efficient uptake by recipient cells, capacity to stabilize and transport biomolecules and their potential for cell/tissue targeting and preferential uptake by cancer cells, they have enormous potential for bioengineering into improved and targeted drug delivery systems. In this work, we investigated the use of laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) as a tool to measure the loading of platinum-based chemotherapeutic agents. The EV loading of oxaliplatin via co-incubation was demonstrated, and LA-ICP-MS imaging showed greater efficiency of delivery to colorectal cancer cells compared to free oxaliplatin, leading to enhanced cytotoxic effect. Further, the impact of EV co-loading with a porphyrin (C5SHU, known as 'C5') photosensitizer on oxaliplatin delivery was assessed. Fluorescence analysis using nano-flow cytometry showed dose-dependent EV loading as well as a trend towards the loading of larger particles. Exposure of OXA-C5-EV-treated colorectal cancer cells to light indicated that delivery was enhanced by both light exposure and porphyrins, with a synergistic effect on cell viability observed between oxaliplatin, EVs and light exposure after the delivery of the co-loaded EVs. In summary, this work demonstrates the utility of LA-ICP-MS and mass spectrometry imaging in assessing the loading efficiency and cellular delivery of platinum-based therapeutics, which would also be suitable for agents containing other elements, confirms that EVs are more efficient at delivery compared to free drugs, and describes the use of light exposure in optimizing delivery and therapeutic effects of EV-mediated drug delivery both in combination and independently of porphyrin-based photosensitizers.

PDZ scaffolds regulate extracellular vesicle production, composition, and uptake

Extracellular vesicles (EVs) are membrane-limited organelles mediating cell-to-cell communication in health and disease. EVs are of high medical interest, but their rational use for diagnostics or therapies is restricted by our limited understanding of the molecular mechanisms governing EV biology. Here, we tested whether PDZ proteins, molecular scaffolds that support the formation, transport, and function of signal transduction complexes and that coevolved with multicellularity, may represent important EV regulators. We reveal that the PDZ proteome (ca. 150 proteins in human) establishes a discrete number of direct interactions with the tetraspanins CD9, CD63, and CD81, well-known EV constituents. Strikingly, PDZ proteins interact more extensively with syndecans (SDCs), ubiquitous membrane proteins for which we previously demonstrated an important role in EV biogenesis, loading, and turnover. Nine PDZ proteins were tested in loss-of-function studies. We document that these PDZ proteins regulate both tetraspanins and SDCs, differentially affecting their steady-state levels, subcellular localizations, metabolism, endosomal budding, and accumulations in EVs. Importantly, we also show that PDZ proteins control the levels of heparan sulfate at the cell surface that functions in EV capture. In conclusion, our study establishes that the extensive networking of SDCs, tetraspanins, and PDZ proteins contributes to EV heterogeneity and turnover, highlighting an important piece of the molecular framework governing intracellular trafficking and intercellular communication.

Quantitative and functional characterisation of extracellular vesicles after passive loading with hydrophobic or cholesterol-tagged small molecules

Extracellular vesicles (EVs) are nanosized intercellular messengers that bear enormous application potential as biological drug delivery vehicles. Much progress has been made for loading or decorating EVs with proteins, peptides or RNAs using genetically engineered donor cells, but post-isolation loading with synthetic drugs and using EVs from natural sources remains challenging. In particular, quantitative and unambiguous data assessing whether and how small molecules associate with EVs versus other components in the samples are still lacking. Here we describe the systematic and quantitative characterisation of passive EV loading with small molecules based on hydrophobic interactions - either through direct adsorption of hydrophobic compounds, or by membrane anchoring of hydrophilic ligands via cholesterol tags. As revealed by single vesicle imaging, both ligand types bind to CD63 positive EVs (exosomes), however also non-specifically to other vesicles, particles, and serum proteins. The hydrophobic compounds Curcumin and Terbinafine aggregate on EVs with no apparent saturation up to 106–107 molecules per vesicle as quantified by liquid chromatography – high resolution mass spectrometry (LC-HRMS). For both compounds, high density EV loading resulted in the formation of a population of large, electron-dense vesicles as detected by quantitative cryo-transmission electron microscopy (TEM), a reduced EV cell uptake and a toxic gain of function for Curcumin-EVs. In contrast, cholesterol tagging of a hydrophilic mdm2-targeted cyclic peptide saturated at densities of ca 104–105 molecules per vesicle, with lipidomics showing addition to, rather than replacement of endogenous cholesterol. Cholesterol anchored ligands did not change the EVs' size or morphology, and such EVs retained their cell uptake activity without inducing cell toxicity. However, the cholesterol-anchored ligands were rapidly shed from the vesicles in presence of serum. Based on these data, we conclude that (1) both methods allow loading of EVs with small molecules but are prone to unspecific compound binding or redistribution to other components if present in the sample, (2) cholesterol anchoring needs substantial optimization of formulation stability for in vivo applications, whereas (3) careful titration of loading densities is warranted when relying on hydrophobic interactions of EVs with hydrophobic compounds to mitigate changes in physicochemical properties, loss of EV function and potential cell toxicity.

Extracellular Vesicles Released by Genetically Modified Macrophages Activate Autophagy and Produce Potent Neuroprotection in Mouse Model of Lysosomal Storage Disorder, Batten Disease.

Over the recent decades, the use of extracellular vesicles (EVs) has attracted considerable attention. Herein, we report the development of a novel EV-based drug delivery system for the transport of the lysosomal enzyme tripeptidyl peptidase-1 (TPP1) to treat Batten disease (BD). Endogenous loading of macrophage-derived EVs was achieved through transfection of parent cells with TPP1-encoding pDNA. More than 20% ID/g was detected in the brain following a single intrathecal injection of EVs in a mouse model of BD, ceroid lipofuscinosis neuronal type 2 (CLN2) mice. Furthermore, the cumulative effect of EVs repetitive administrations in the brain was demonstrated. TPP1-loaded EVs (EV-TPP1) produced potent therapeutic effects, resulting in efficient elimination of lipofuscin aggregates in lysosomes, decreased inflammation, and improved neuronal survival in CLN2 mice. In terms of mechanism, EV-TPP1 treatments caused significant activation of the autophagy pathway, including altered expression of the autophagy-related proteins LC3 and P62, in the CLN2 mouse brain. We hypothesized that along with TPP1 delivery to the brain, EV-based formulations can enhance host cellular homeostasis, causing degradation of lipofuscin aggregates through the autophagy-lysosomal pathway. Overall, continued research into new and effective therapies for BD is crucial for improving the lives of those affected by this condition.

Engineering Extracellular Vesicles as Delivery Systems in Therapeutic Applications.

Extracellular vesicles (EVs) are transport vesicles secreted by living cells and released into the extracellular environment. Recent studies have shown that EVs serve as "messengers" in intercellular and inter-organismal communication, in both normal and pathological processes. EVs, as natural nanocarriers, can deliver bioactivators in therapy with their endogenous transport properties. This review article describes the engineering EVs of sources, isolation method, cargo loading, boosting approach, and adjustable targeting of EVs. Furthermore, the review summarizes the recent progress made in EV-based delivery systems applications, including cancer, cardiovascular diseases, liver, kidney, nervous system diseases, and COVID-19 and emphasizes the obstacles and challenges of EV-based therapies and possible strategies.

Placental Extracellular Vesicles Can Be Loaded with Plasmid DNA.

Recently, extracellular vesicles (EVs) have garnered considerable interest as potential vehicles for drug delivery, including gene therapy. Although EVs from diverse sources have been investigated, current techniques used in the field for EV generation limit large-scale EV production. The placenta is essentially a tissue transplant and has unique properties that allow it to avoid the maternal immune system making it likely that placental EVs will not generate inflammatory responses and will avoid clearance by the immune system. We propose that placental EVs produced from explant cultures are an efficient method to produce considerable quantities of EVs that would be safe to administer, and we hypothesize that placental EVs can be loaded with large exogenous plasmids. To this end, we trialed three strategies to load plasmid DNA into placental EVs, including loading via electroporation of placental tissue prior to EV isolation and loading directly into placental EVs via electroporation or direct incubation of the EVs in plasmid solution. We report that the placenta releases vast quantities of EVs compared to placental cells in monolayer cultures. We show successful loading of plasmid DNA into both large- and small-EVs following both exogenous loading strategies with more plasmid encapsulated in large-EVs. Importantly, direct incubation did not alter EV size nor quantity. Further, we showed that the loading efficiency into EVs was dependent on the exogenous plasmid DNA dose and the DNA size. These results provide realistic estimates of plasmid loading capacity into placental EVs using current technologies and showcase the potential of placental EVs as DNA delivery vehicles.

Potential of Mesenchymal Stromal Cell-Derived Extracellular Vesicles as Natural Nanocarriers: Concise Review

Intercellular communication, through direct and indirect cell contact, is mandatory in multicellular organisms. These last years, the microenvironment, and in particular, transfer by extracellular vesicles (EVs), has emerged as a new communication mechanism. Different biological fluids and cell types are common sources of EVs. EVs play different roles, acting as signalosomes, biomarkers, and therapeutic agents. As therapeutic agents, MSC-derived EVs display numerous advantages: they are biocompatible, non-immunogenic, and stable in circulation, and they are able to cross biological barriers. Furthermore, EVs have a great potential for drug delivery. Different EV isolation protocols and loading methods have been tested and compared. Published and ongoing clinical trials, and numerous preclinical studies indicate that EVs are safe and well tolerated. Moreover, the latest studies suggest their applications as nanocarriers. The current review will describe the potential for MSC-derived EVs as drug delivery systems (DDS) in disease treatment, and their advantages. Thereafter, we will outline the different EV isolation methods and loading techniques, and analyze relevant preclinical studies. Finally, we will describe ongoing and published clinical studies. These elements will outline the benefits of MSC-derived EV DDS over several aspects.

Abstract 15005: Tlr4/py14-Caveolin-1-Mediated Endothelial C-iap2 Expression During Schistosomiasis-Associated Pulmonary Arterial Hypertension

Introduction: During schistosomiasis-associated pulmonary arterial hypertension (Sch-PAH), hyperproliferation of abnormal endothelial cells (ECs) contributes to development of severe vascular lesions within the lungs; a life-threatening phenomenon that remains with no target therapy. Hypothesis: Here, we found that antigenic molecules from the major etiological agent of Sch-PAH, i.e., S. mansoni , amplify the secretion of the antiapoptotic protein c-IAP2 via TLR4-pY14Cav-1 signaling pathway, which we hypothesized to contribute to generation of an EC memory of survival and severity of PAH. Methods/Results: Cav-1 deficiency via shedding of extracellular vesicles (EVs) leads to survival of an apoptosis-resistant EC phenotype during idiopathic PAH. Similarly, S. mansoni egg antigens also reduced lung EC-Cav-1 expression, suggesting that Cav-1 also plays a role during pathogen-associated PAH. Cav-1 depletion via EVs precedes its phosphorylation at residue Tyr14 (pY14Cav-1), which in turn, contributes to EV loading. Among apoptotic regulators, IAPs have been found as a cargo of EVs in different conditions, including in TLR4-activated cells. Accordingly, our data indicates that S. mansoni egg antigen p40 also activated TLR4/NFkB-mediated signaling, inducing c-IAP2 expression and a time-dependent phosphorylation of Cav-1 in human lung microvascular EC (HMVEC-L: 228.2 +/- 38.28% of control; p<0.05; n=4). Moreover, although Sm-p40 alone was unable to induce shedding of EVs, combination with NFkB-associated proinflammatory mediators (TNF-α, IL-6, and ATP), significantly increased EV shedding by HMVEC-L, suggesting that S. mansoni may epigenetically prime ECs, but co-factors are required to induce EV shedding. Finally, Sch-PAH model using EC- Cav1 -/- mice ( End-scl.Cre ERT2 ;Rosa mt/mg ;Cav1 fl/fl ) revealed that Cav-1 absence led to accumulation of eggs within the lungs and loss of eGFP+ cells, indicating that EC-Cav-1 phosphorylation may be required to development of Sch-PAH. Conclusions: Our data suggest that S. mansoni -induced pY14Cav-1 is associated to expression of the anti-apoptotic c-IAP2 protein, which may be critical for prolonged survival of a pathogenic EC phenotype during the development of Sch-PAH.

LC3-dependent EV loading and secretion (LDELS) promotes TFRC (transferrin receptor) secretion via extracellular vesicles

LC3-dependent EV loading and secretion (LDELS) is a secretory autophagy pathway in which the macroautophagy/autophagy machinery facilitates the packaging of cytosolic cargos, such as RNA-binding proteins, into extracellular vesicles (EVs) for secretion outside of the cell. Here, we identify TFRC (transferrin receptor), one of the first proteins found to be secreted via EVs, as a transmembrane cargo of the LDELS pathway. Similar to other LDELS targets, TFRC secretion via EVs genetically requires components of the MAP1LC3/LC3-conjugation machinery but is independent of other ATGs involved in classical autophagosome formation. Furthermore, the packaging and secretion of this transmembrane protein into EVs depends on multiple ESCRT pathway components and the small GTPase RAB27A. Based on these results, we propose that the LDELS pathway promotes TFRC incorporation into EVs and its secretion outside the cell.

Synergistic siRNA Loading of Extracellular Vesicles Enables Functional Delivery into Cells.

RNA interference opened new approaches for disease treatment but safe and efficient cell delivery remains a bottleneck. Extracellular vesicles (EVs) are known to naturally shuttle RNA. Due to their potent cell internalization and low-cost scalability, milk-derived EVs in particular are considered promising RNA delivery systems. However, low drug loading currently impedes their use. Here, innovative exogenous loading strategies for small interfering RNA (siRNA) are explored and systematically compared regarding encapsulation efficiency, loading capacity, and loading concentration. Firstly, siRNA is pre-accumulated in liposomes or stabilized calcium phosphate nanoparticles (CaP-NP). The selected systems, which exhibited neutral or negative zeta potentials, are then applied for EV loading. Secondly, EVs are concentrated and applied to protocols known for liposome loading: dehydration-rehydration of vesicles, based on freeze-drying, and mixing by dual asymmetric centrifugation (DAC) after ultracentrifugation. Additionally, DAC after EV ultracentrifugation is combined with CaP-NP leading to a synergistic loading performance. The balance between energy input for siRNA loading and EV integrity is evaluated by monitoring the EV size, marker proteins, and morphology. For the EV-based siRNA formulation via DAC plus CaP-NP, EV properties are sufficiently maintained to protect the siRNA from degradation and deliver cell-death siRNA dose-dependently in Caco-2 cells.

Combinatorial microRNA Loading into Extracellular Vesicles for Increased Anti-Inflammatory Efficacy.

Extracellular vesicles (EVs) have emerged as promising therapeutic entities in part due to their potential to regulate multiple signaling pathways in target cells. This potential is derived from the broad array of constituent and/or cargo molecules associated with EVs. Among these, microRNAs (miRNAs) are commonly implicated as important and have been associated with a wide variety of EV-induced biological phenomena. While controlled loading of single miRNAs is a well-documented approach for enhancing EV bioactivity, loading of multiple miRNAs has not been fully leveraged to maximize the potential of EV-based therapies. Here, an established approach to extrinsic nucleic acid loading of EVs, sonication, was utilized to load multiple miRNAs in HEK293T EVs. Combinations of miRNAs were compared to single miRNAs with respect to anti-inflammatory outcomes in assays of increasing stringency, with the combination of miR-146a, miR-155, and miR-223 found to have the most potential amongst the tested groups.

Biomaterials constructed for MSC-derived extracellular vesicle loading and delivery-a promising method for tissue regeneration.

Mesenchymal stem cells (MSCs) have become the preferred seed cells for tissue regeneration. Nevertheless, due to their immunogenicity and tumorigenicity, MSC transplantation remains questionable. Extracellular vesicles (EVs) derived from MSCs are becoming a promising substitute for MSCs. As a route of the MSC paracrine, EVs have a nano-sized and bilayer lipid-enclosed structure, which can guarantee the integrity of their cargoes, but EVs cannot obtain full function in vivo because of the rapid biodegradation and clearance by phagocytosis. To improve the efficacy and targeting of EVs, methods have been proposed and put into practice, especially engineered vesicles and EV-controlled release systems. In particular, EVs can be cell or tissue targeting because they have cell-specific ligands on their surfaces, but their targeting ability may be eliminated by the biodegradation of the phagocytic system during circulation. Novel application strategies have been proposed beyond direct injecting. EV carriers such as biodegradable hydrogels and other loading systems have been applied in tissue regeneration, and EV engineering is also a brand-new method for higher efficacy. In this review, we distinctively summarize EV engineering and loading system construction methods, emphasizing targeting modification methods and controlled release systems for EVs, which few literature reviews have involved.

Effects of Hypoxia on RNA Cargo in Extracellular Vesicles from Human Adipose-Derived Stromal/Stem Cells.

Mesenchymal stromal/stem cells and their derivates are the most promising cell source for cell therapies in regenerative medicine. The application of extracellular vesicles (EVs) as cell-free therapeuticals requires particles with a maximum regenerative capability to enhance tissue and organ regeneration. The cargo of mRNA and microRNA (miR) in EVs after hypoxic preconditioning has not been extensively investigated. Therefore, the aim of our study was the characterization of mRNA and the miR loading of EVs. We further investigated the effects of the isolated EVs on renal tubular epithelial cells in vitro. We found 3131 transcripts to be significantly regulated upon hypoxia. Only 15 of these were downregulated, but 3116 were up-regulated. In addition, we found 190 small RNAs, 169 of these were miRs and 21 were piwi-interacting RNAs (piR). However, only 18 of the small RNAs were significantly altered, seven were miRs and 11 were piRs. Interestingly, all seven miRs were down-regulated after hypoxic pretreatment, whereas all 11 piRs were up-regulated. Gene ontology term enrichment and miR-target enrichment analysis of the mRNAs and miR were also performed in order to study the biological background. Finally, the therapeutic effect of EVs on human renal tubular epithelial cells was shown by the increased expression of three anti-inflammatory molecules after incubation with EVs from hypoxic pretreatment. In summary, our study demonstrates the altered mRNA and miR load in EVs after hypoxic preconditioning, and their anti-inflammatory effect on epithelial cells.

LINE-1 Cargo and Reverse Transcriptase Activity Profiles in Extracellular Vesicles from Lung Cancer Cells and Human Plasma.

Long Interspersed Element-1 (LINE-1) is an oncogenic human retrotransposon that ‘copies and pastes’ DNA into new locations via reverse transcription. Given that enzymatically active LINE-1 can be exported in extracellular vesicles (EVs), and that LINE-1 mRNA and its two encoded proteins, ORF1p and ORF2p, are required for retrotransposition, the present study examined LINE-1 EV loading patterns relative to reverse transcriptase (RT) activity in vivo and in vitro. Density gradient ultracentrifugation identified conserved patterns of LINE-1 mRNA and protein distribution in EVs, with RT activity readily detected in EV fractions containing both LINE-1 mRNA and protein. Unlike whole cell and tissue lysates, the ORF1p in EVs was detected as a dimer. EVs from ostensibly healthy plasma donors showed variable but consistent ORF1p profiles, with residual levels of LINE-1 mRNA measured in some but not all samples. EVs from cancer cell lines had elevated mean LINE-1 levels and 5–85 times greater RT activity than EVs from normal cells or healthy plasma. EV RT activity was associated with EV LINE-1 mRNA content and was highest in cell lines that also expressed an elevated expression of ORF1p and ORF2p. Given that LINE-1 activation is a hallmark of many cancer types, our findings suggest that an EV LINE-1 ‘liquid biopsy’ may be developed to monitor LINE-1 activity during the course of malignant progression.

Extracellular vesicles: catching the light of intercellular communication in fibrotic liver diseases.

The increasing prevalence of fibrotic liver diseases resulting from different etiologies has become a major global problem for public health. Fibrotic liver diseases represent a redundant accumulation of extracellular matrix, dysregulation of immune homeostasis and angiogenesis, which eventually contribute to the progression of cirrhosis and liver malignancies. The concerted actions among liver cells including hepatocytes, hepatic stellate cells, kupffer cells, liver sinusoidal endothelial cells and other immune cells are essential for the outcome of liver fibrosis. Recently, a growing body of literature has highlighted that extracellular vesicles (EVs) are critical mediators of intercellular communication among different liver cells either in local or distant microenvironments, coordinating a variety of systemic pathological and physiological processes. Despite the increasing interests in this field, there are still relatively few studies to classify the contents and functions of EVs in intercellular transmission during hepatic fibrogenesis. This review aims to summarize the latest findings with regards to the cargo loading, release, and uptake of EVs in different liver cells and clarify the significant roles of EVs played in fibrotic liver diseases.

Potential of on‐chip analysis and engineering techniques for extracellular vesicle bioproduction for therapeutics

AbstractThe clinical interest around extracellular vesicles (EVs) started during the year 2000s, now leading to new clinical diagnostic and therapeutic strategies. Due to their outstanding properties as biogenic drug delivery systems and as alternatives to cell therapy, the need to produce EVs with sufficiently high yield and quality for clinical use expedited the development of analytical techniques and dedicated bioproduction methods. Though preclinical studies revealed the potential of EVs to become next generation subcellular therapies that could lead to major breakthroughs in current therapeutics possibilities, they remain complex objects on both a physicochemical and biological level. Here, we review the capacity of microfluidic technologies to match EV‐based therapeutics need for clinical translation via standardized and intensified bioproduction methods. Indeed, some of the current routine tools used in bioproduction are already achieved on chips such as micromixers or particle sorting and analysis using field flow fraction or nanoparticle tracking analyzer. Also, microfluidics communities have developed a wide set of new techniques to isolate and quantify EVs, but the few that are adopted in a bioproduction workflow are well‐established since the 1990s. We first review the different EV generation and loading methods embedded on chip. We focus on EVs preparation methods, from purification to in‐line separative techniques. We finally describe the on‐chip analytical tools to analyze physicochemistry and phenotypes of EVs.