Engineered Extracellular Vesicles as a Targeted Delivery Platform for Precision Therapy.

Extracellular vesicles (EVs) are a kind of heterogeneous vesicles released by essentially all cell types, which plays an important role in intercellular communication and in the regulation of physiological process (or pathology). EVs mainly includes microvesicles (50–1000 nm), apoptotic bodies (50–5000 nm) and exosomes (30–150 nm), based on distinct biogenesis pathways. Microvesicles are formed by direct budding of the plasma membrane, in which ARF6 and RhoA are important regulators. In addition, oncosomes are also a kind of microvesicles, which are specifically released by tumor cells and contain many specific molecules of tumor cells. When cells undergo apoptosis, they could also release a population of EVs called apoptotic bodies. This kind of EVs is formed through outward blebbing and fragmentation of the cell membrane. Exosomes are derived from multivesicular bodies (MVBs), and released by cells upon fusion of MVBs with the plasma membrane. Exosomes have complex formation and regulatory mechanisms, including endosomal sorting complex dependent and non-dependent mechanisms. Many researchers have shown that EVs with different types and different cell sources may have different components and functions. EVs mainly carry the bioactive molecules of donor cells, such as proteins, DNA or RNA, and target nearby or remote cells to perform different biological functions, thus affecting the physiological and pathological processes of recipient cells. Cells can also discharge their waste by EVs. By transporting cargoes to extracellular, EVs can regulate the amount of intracellular cargoes, thus affecting the survival status of cells. This approach can be beneficial to cells (removing harmful substances) or harmful (transporting drugs outside the cell). Secondly, some key molecules in EVs can be regulated by donor cells according to their state. So, EVs can also reflect the state changes of donor cells, and have the potential as a biomarker for the diagnosis and treatment of diseases. Due to their stable existence in body fluids and derived from various tissues of the body, EVs can not only be a new biomarker of liquid biopsy, but also reflect the heterogeneity and the genesis or development of diseases. As a natural endogenous material, EVs can also serve as a potential drug delivery carrier, and have the advantages of low immunogenicity and crossing the blood brain barrier. In addition, EVs have many other applications. For example, they can be an alternative to cells as a therapeutic agent, as well as targets of disease treatment. This review summarizes the following parts: Firstly, biological origin, biological composition, biological characteristics, and biological functions of EVs. Secondly, the isolation and characterization of EVs. Thirdly, the application of EVs in the diagnosis and treatment of diseases were introduced. At present, the heterogeneity, biological composition and the formation mechanism of EVs need to be further studied, so that it can be better applied in the diagnosis and treatment of diseases. The effective isolation method is also necessary for its clinical application. It is believed that with the development of various biological technologies, these problems will be gradually solved, the biological function of EVs will be also clearly understood, and EVs based application in diseases will make new breakthroughs.

PURPOSE: To elucidate novel mechanisms of peritoneal dissemination by focusing on extracellular vesicles BACKGROUNDS AND SUMMARY: Metastasis to the abdominal cavity is frequently observed in ovarian cancer patients and is one of the reasons for the unfavorable outcomes. Extracellular vesicles (EVs), including exosomes and microvesicles, are small membrane vesicles that contain various bioactive molecules, such as microRNAs, mRNAs and proteins, and they are released from all cell types and play key physiological roles in intercellular communication. Recent evidence has demonstrated that cancer cells secrete EVs to both proximal surrounding cells and distal sites, thereby enabling the development of a cancer microenvironment that in turn promotes cancer metastasis. We established orthotopic mouse models by using 4 ovarian carcinoma cell lines with different metastatic traits and investigated the functions of cancer-derived EVs. The EVs from highly metastatic cells strongly induced metastatic behavior in moderately metastatic tumors. Notably, the metastatic cancer EVs efficiently induced apoptotic cell death in mesothelial cells in vitro and in vivo, thus resulting in the destruction of the peritoneal mesothelium barrier. Whole transcriptome analysis showed that MMP1 was significantly elevated in mesothelial cells treated with highly metastatic cancer EVs, and intact MMP1 mRNAs were selectively packaged in the EVs. Importantly, MMP1 expression in ovarian cancer is tightly correlated with a poor prognosis, particularly in stage I patients. Moreover, we found MMP1 mRNA-carrying EVs in the ascites of cancer patients, and these EVs also induced apoptosis in mesothelial cells. CONCLUSIONS: Our findings provide a promise for the understanding of a previously unknown mechanism for peritoneal dissemination that is associated with cancer-derived EVs, and which may be novel biomarkers for prognosis and can provide a new therapeutic strategy for inhibiting metastasis by disrupting the EVs. Citation Format: Akira Yokoi and Takahiro Ochiya. MALIGNANT EXTRACELLULAR VESICLES CAUSE MESOTHELIAL CELL DAMAGE TOWARD PERITONEAL DISSEMINATION IN OVARIAN CANCER [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr TMEM-042.

Extracellular vesicles are lipid bilayer-enclosed extracellular membrane vesicles secreted from cells that have gradually shown promise in cancer treatment. As an essential intercellular communication mediator, extracellular vesicles carry bioactive molecules including proteins, lipids, and nucleic acids, which aid in the control and reprogramming of cell signals and ultimately accelerate the spread of cancer. Simultaneously, extracellular vesicles have become a highly desirable option for drug delivery carriers because of the special capacity to cross boundaries and low immunogenicity. Bypassing the endosomal pathway and lysosomal degradation, they can provide medications directly, increasing their effectiveness and decreasing their negative effects. We review the extracellular vesicles’ development, drug loading techniques, and discuss their potential in clinical applications to provide new avenues for precise and targeted treatment strategies. Additionally, engineering modifications of extracellular vesicles can enhance their ability to target specific cells, improving the stability and effectiveness of cargoes, further driving the development of cancer therapy. In this review, we particularly highlight new therapeutic applications of extracellular vesicles, including the latest progress in extracellular vesicle vaccines in tumors. Nonetheless, there are still a lot of obstacles to be overcome before extracellular vesicles may be used to treat cancer. Future research needs to focus on the characteristics of extracellular vesicles from various sources and explore their specific roles in disease treatment to fully leverage the potential of extracellular vesicles in clinical applications such as drug delivery.

BackgroundGlioblastoma (GBM) is the most lethal primary brain tumor in adult, characterized by highly aggressive and infiltrative growth. The current therapeutic management of GBM includes surgical resection followed by ionizing radiations and chemotherapy. Complex and dynamic interplay between tumor cells and tumor microenvironment drives the progression and contributes to therapeutic resistance. Extracellular vesicles (EVs) play a crucial role in the intercellular communication by delivering bioactive molecules in the surrounding milieu modulating tumor microenvironment.MethodsIn this study, we isolated by ultracentrifugation EVs from GBM stem-like cell (GSC) lines and human microvascular endothelial cells (HMVECs) exposed or not to ionizing irradiation. After counting and characterization, we evaluated the effects of exposure of GSCs to EVs isolated from endothelial cells and vice versa. The RNA content of EVs isolated from GSC lines and HMVECs exposed or not to ionizing irradiation, was analyzed by RNA-Seq. Periostin (POSTN) and Filamin-B (FLNB) emerged in gene set enrichment analysis as the most interesting transcripts enriched after irradiation in endothelial cell-derived EVs and GSC-derived EVs, respectively. POSTN and FLNB expression was modulated and the effects were analyzed by in vitro assays.ResultsWe confirmed that ionizing radiations increased EV secretion by GSCs and normal endothelial cells, affected the contents of and response to cellular secreted EVs. Particularly, GSC-derived EVs decreased radiation-induced senescence and promoted migration in HMVECs whereas, endothelial cell-derived EVs promoted tumorigenic properties and endothelial differentiation of GSCs. RNA-Seq analysis of EV content, identified FLNB and POSTN as transcripts enriched in EVs isolated after irradiation from GSCs and HMVECs, respectively. Assays performed on POSTN overexpressing GSCs confirmed the ability of POSTN to mimic the effects of endothelial cell-derived EVs on GSC migration and clonogenic abilities and transdifferentiation potential. Functional assays performed on HMVECs after silencing of FLNB supported its role as mediator of the effects of GSC-derived EVs on senescence and migration.ConclusionIn this study, we identified POSTN and FLNB as potential mediators of the effects of EVs on GSC and HMVEC behavior confirming that EVs play a crucial role in the intercellular communication by delivering bioactive molecules in the surrounding milieu modulating tumor microenvironment.

Extracellular vesicles (EVs) are membrane-enclosed particles containing a vast array of biomolecules from the parental cells, such as proteins, lipids, various nucleic acids, and soluble small molecules. There is increasing evidence that EVs play active roles in intercellular communication in various biological and pathological processes and offer promise as novel biomarkers and therapeutic agents. EVs were identified as important carriers of bioactive paracrine factors in the regenerative stem and progenitor cell secretome. Especially for patients suffering from end-stage heart failure caused by myocardial infarction and ischemia-reperfusion injury, for which treatment options are limited to heart transplantation, these newly discovered therapeutic entities have opened novel treatment possibilities. EVs isolated from cardiac progenitor cells (CPC-EVs) were demonstrated to reduce infarct size and improve cardiac function in several preclinical models. Their functionality was attributed to stimulating different cardiac repair processes in the ischemic myocardium, such as inducing angiogenesis. In this thesis, various aspects to improve the application of CPC-EVs as biological therapeutics and as natural endogenous drug delivery vehicles are addressed. We identified the functional protein components of CPC-EVs involved in endothelial cell activation and migration by employing in-depth EV characterization techniques. This provides insights into the localization of functional content in EV preparations and mechanistic insights into CPC-EV-mediated recipient cell activation. Moreover, we demonstrated that interfering with endolysosomal trafficking in donor cells leads to an increase in EV yield with maintained bioactivity. This provides openings for increasing CPC-EV production for their therapeutic application. Furthermore, we compared the therapeutic potential of EVs released from different donor cell types and purified with different isolation techniques in myocardial infarction mouse models and demonstrated the influence of these factors on EV functionality. Another important objective before using EVs in clinical trials is determining the biodistribution and retention of EVs after local and systemic administration. We showed that EVs injected in the myocardium displayed no direct flush-out from the myocardium and were retained in the heart up to days after administration. CPC-EVs delivered intravenously were rapidly cleared and mainly distributed to the liver. This is a challenge for the delivery of EVs to the heart. We showed that EV engineering techniques can be used to direct EVs to the endothelium and to achieve simultaneous cargo protein loading inside EVs. These strategies could further improve the targeted delivery of therapeutic EV cargo to the injured heart. 
\nTaken together, the results in this thesis highlight the broad applicability and complexity of EVs as therapeutic agents. Further improvements, such as developing standardized EV separation and characterization techniques to safeguard batch-to-batch variations and prevent problems with reproducibility, will lead to the development of EV therapeutics for cardiac repair.

[This retracts the article DOI: 10.21037/sci.2017.08.07.].

Exosomes are extracellular vesicles with nano size range. The use of exosomes as drug delivery vehicle has useful advantages compared to other vesicular systems. Exosomes are nonimmunogenic, nontoxic, and stable with good composition and potency for crossing blood brain barrier. They can be used as theranostic agents and vaccine therapy. The exosomes are a new choice for nonimmunogenic targeted drug delivery vehicle. Introduction : Exosomes are nano size vesicles secreted by different cell types. The main role of exosomes in the body is a cell to cell communication. They are not immunogenic due to similar structure as body cells. They can cross blood brain barrier, stable in the blood circulation, biocompatible, with low toxicity, suitable size, and structure. These endogenous vesicles can be used for drug delivery, immunevaccines and as diagnostic agents. Methods: The following databases were reviewed for bibliography of using exosomes as delivery agents: Web of science, Scopus, Medline, and Embase. Results : Exosomes can deliver different kinds of cargos (RNA, proteins and small molecules) to the target cells. Exosome as a biomarker of diseases (cancer, cardiovascular disease, MS, etc) personalized therapy with the exosomes that generated from induced pluripotent stem cell (iPSC) derived that used for patient specific and disease specific cell therapy. They have specific role in tissue repair and regeneration, vaccines for immunotherapy (phase II of clinical trial). The strategies to introduce drugs into exosomes are active and passive loading. Artificial exosome mimetics can be isolated from exosome secreting cell lines but these exosomes in comparison with autologous are immunogenic. Isolation techniques of exosomes are ultracentrifugation, electron microscopy, and SDS page. Exosomes were mainly taken up by macrophages in the liver after IV administration. Exosomes characterization methods are biophysical, molecular and microfluidics. Conclusion: Exosomes are good carriers for delivery of drugs and genes to the target without immunogenic reaction. They can be used as immunovacines for the cancer treatment by activating immune system against tumor cells. Exosomes can substitution the liposomes because of their size, structure, non-immunogenicity and their natural composition. Key words : Exosomes, Extracellular vesicles, Drug delivery, Nano carrier, Immunovaccine

Proteomic and Biological Analysis of Myeloma Cell Derived Extracellular Vesicles

Exposure of Cord Blood Hematopoietic Stem Cells to Bone Marrow Mesenchimal Cells-Derived Microvesicles Induces Cell Survival and Inhibition of Differentiation

In recent years, exosomes have emerged as crucial mediators of intercellular communication, fundamentally altering our understanding of biological signaling networks. These nano-sized extracellular vesicles (EVs), derived from multivesicular bodies and secreted by nearly all cell types, carry a diverse cargo, including proteins, lipids, RNA, and DNA. The special issue "Exosome-Mediated Intercellular Communications: Immune Stimulation and Suppression" aims to explore the multifaceted roles of exosomes in modulating immune responses, highlighting their potential in immunotherapy, diagnostics, and pathophysiology. This editorial provides an overview of the advances presented in this collection, underscores the significance of these findings, and sets the stage for future research directions.The issue opens with a study exploring the diagnostic potential of exosomes in trauma. Schindler et al. identify novel EV biomarkers specific to traumatic brain injury (TBI), demonstrating how EVs with distinct protein profiles (e.g., CD13 and MOG) correlate with neurological outcomes. This work emphasizes the clinical value of EVs as minimally invasive tools for early diagnostics in polytrauma settings, illustrating the interplay between injuryspecific exosome-mediated signaling and immune modulation.Exosomes have recently been recognized as significant contributors to disease pathogenesis. In this context, the study conducted by Kobiela et al. highlights the role of keratinocyte-derived small extracellular vesicles (sEVs) in allergic skin inflammation, specifically regarding filaggrin insufficiency. Their study reveals that sEVs act as antigenic sources for CD1arestricted T cells, promoting a type 2 immune bias that exacerbates atopic dermatitis. By linking lipidomic alterations in sEVs to immune dysregulation, the authors provide a mechanistic understanding of how exosome-mediated communication perpetuates skin inflammation, presenting opportunities for targeted interventions in allergic disorders.Guo et al. delve into the immune-regulatory roles of exosomal microRNAs (miRNAs) in heart failure (HF). These miRNAs mediate the crosstalk between cardiac and immune cells, modulating processes like inflammation, fibrosis, and cardiac dysfunction. Highlighted as potential biomarkers and therapeutic agents, exosomal miRNAs could redefine diagnostic and therapeutic paradigms in HF, paving the way for more personalized approaches in cardiovascular medicine.Exosomes have emerged as a promising candidate for various therapeutic applications across a range of diseases. Moreover, they possess the essential characteristics of an effective drug delivery system, enhancing their potential in medical treatments. In this context, a study by Wang et al. provided compelling evidence on the role of miR-146a-enriched fibroblast-like synoviocyte-derived exosomes (FLS-Exos) in alleviating osteoarthritis (OA). By targeting the Toll-like receptor 4/TRAF6/NF-κB signaling pathway, these exosomes reduced cartilage degradation and shifted synovial macrophages from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype. This innovative approach highlights the potential of miR-146a as a therapeutic agent in OA, emphasizing the role of exosome-associated microRNAs in immune regulation and tissue repair.Menay et al. explore the immunogenic potential of EVs derived from antigen-presenting cells (APCs) pulsed with foot-and-mouth disease virus (FMDV) antigens. These EVs carry viral proteins capable of stimulating both B and T cell responses, providing a new avenue for vaccine development. The ability of these vesicles to induce lymphocyte expansion, particularly in antigen-primed splenocytes, underscores their role as a platform for next-generation antiviral vaccines.In another review, Mei et al. explore the therapeutic potential of mesenchymal stem cells (MSCs) as vehicles for drug delivery and immune modulation. The authors emphasize MSCs' tumor tropism and homing ability, qualities that render them effective carriers for targeted therapies. They further discuss the promise of MSC derivatives, such as MSC-derived EVs and MSC cell membrane-coated nanoparticles, highlighting how these innovative strategies can enhance precision and efficacy in the treatment of inflammatory, fibrotic, and oncologic conditions. Despite the promise, challenges such as scalability and reproducibility must be addressed to ensure clinical translation.The review by Xiu et al. sheds light on the crucial roles of bacterial membrane vesicles (MVs) in regulating bacterial physiology, environmental adaptation, and pathogenic processes. MVs are key players in interkingdom communication, influencing microbial community dynamics and host-pathogen interactions. The authors highlight their potential applications in developing novel antimicrobial strategies and vaccines, underscoring the importance of understanding bacterial MVs in both environmental and clinical contexts.The collection collectively demonstrates the multifaceted nature of exosomes, from their ability to stimulate immune activity to their role in suppressing inflammatory pathways. The therapeutic and diagnostic innovations explored here highlight the transformative potential of EV research, paving the way for novel applications in immunology and beyond.We hope this special issue inspires future investigations into the intricate mechanisms of exosome-mediated communication and their implications for immune health and disease management. As the field continues to evolve, the convergence of clinical insights and translational innovations promises to redefine therapeutic paradigms.For more details on these contributions, explore the articles in this issue on the Frontiers in Immunology website.

Recently, stem cells and their secretomes have attracted great attention in biomedical applications, particularly extracellular vesicles (EVs). EVs are secretomes of cells for cell-to-cell communication. They play a role as intercellular messengers as they carry proteins, nucleic acids, lipids, and therapeutic agents. They have also been utilized as drug-delivery vehicles due to their biocompatibility, low immunogenicity, stability, targetability, and engineerable properties. The therapeutic potential of EVs can be further enhanced by surface engineering and modification using functional molecules such as aptamers, peptides, and antibodies. As a consequence, EVs hold great promise as effective delivery vehicles for enhancing treatment efficacy while avoiding side effects. Among various cell types that secrete EVs, stem cells are ideal sources of EVs because stem cells have unique properties such as self-renewal and regenerative potential for transplantation into damaged tissues that can facilitate their regeneration. However, challenges such as immune rejection and ethical considerations remain significant hurdles. Stem cell-derived EVs have been extensively explored as a cell-free approach that bypasses many challenges associated with cell-based therapy in cancer therapy and tissue regeneration. In this review, we summarize and discuss the current knowledge of various types of stem cells as a source of EVs, their engineering, and applications of EVs, focusing on cancer therapy and tissue engineering.

Extracellular Vesicles: Biology and Their Use as Potential Biomarkers in Hematopoietic Post-Transplant Complications

Although pioneering preclinical research on the use of cell therapy for cardiac regeneration was conducted in the last quarter of the 20th century,1,2 a preponderance of advances have occurred in the 21st century, making this a relatively young field. In the first important clinical trial of cardiac cell therapy, begun in 2001, Menasche et al3 implanted autologous skeletal myoblasts into postinfarct scar at the time of coronary artery bypass surgery. Although the transplanted cells remained viable and exhibited contraction, they formed the nidus for serious ventricular tachyarrhythmias, which led to premature discontinuation of the trial. Despite this outcome, the trial energized the field, accelerating both preclinical and clinical research, albeit not with skeletal myoblasts. The extensive progress in cardiac regeneration is reviewed in this Compendium, and as occurs frequently in science, important observations have led to more questions and challenges (Table). View this table: Table. Important Challenges to Cell Therapy for Cardiac Regeneration Many cell types have been evaluated as candidates for cardiac regeneration. Among the earliest clinical trials, Zeiher’s group infused autologous bone marrow-derived progenitor cells into the coronary arteries of patients with acute,4 as well as healed myocardial infarction (MI)5 and reported improvements in left ventricular (LV) function. However, these results have not been fully confirmed by later studies, as pointed out in the review in the Compendium by Banarjee, Bolli, and Hare.6 Pittenger et al7 were among the first to direct attention to bone marrow-derived (stromal) mesenchymal stem cells (MSCs), emphasizing that these cells proliferated extensively in culture and suggesting that they could be attractive candidates for transplantation. In 2004, Chen et al8 reported that intracoronary infusion of autologous bone marrow-derived MSCs improved cardiac function. Zimmet and Hare pointed out that MSCs lack histocompatibility type II markers and elude rejection by …

HSP70 Is Increased in Circulating Extracellular Vesicles from Transfusion-Dependent β-Thalassemia Patients: Potential Biomarker for Ineffective Erythropoiesis

Simple SummaryExtracellular vesicles represent a heterogeneous family of lipid bilayer-enclosed particles naturally released by all cytotypes into the extracellular space. They can act as mediators of cell–cell communication. Recently, the applications of extracellular vesicles (EVs) have been of great interest in translational cancer research. The advances in next-generation omics technologies allow us to discover EVs’ selective cargo packaging and as EV roles in horizontal transport, non-invasive biomarkers, and as new therapeutic options. Herein, discussing recent studies, information on extracellular vesicle (EV) biogenesis, morphological characteristics, isolation, and current detection methods is summarized.In recent years, knowledge of cell-released extracellular vesicle (EV) functions has undergone rapid growth. EVs are membrane vesicles loaded with proteins, nucleic acids, lipids, and bioactive molecules. Once released into the extracellular space, EVs are delivered to target cells that may go through modifications in physiological or pathological conditions. EVs are nano shuttles with a crucial role in promoting short- and long-distance cell–cell communication. Comprehension of the mechanism that regulates this process is a benefit for both medicine and basic science. Currently, EVs attract immense interest in precision and nanomedicine for their potential use in diagnosis, prognosis, and therapies. This review reports the latest advances in EV studies, focusing on the nature and features of EVs and on conventional and emerging methodologies used for their separation, characterization, and visualization. By searching an extended portion of the relevant literature, this work aims to give a summary of advances in nanomedical applications of EVs. Moreover, concerns that require further studies before translation to clinical applications are discussed.