Microencapsulation technology by nature: Cell derived extracellular vesicles with therapeutic potential

Cell derived extracellular vesicles are submicron structures surrounded by phospholipid bilayer and released by both prokaryotic and eukaryotic cells. The sizes of these vesicles roughly fall into the size ranges of microbes, and they represent efficient delivery platforms targeting complex molecular information to professional antigen presenting cells. Critical roles of these naturally formulated units of information have been described in many physiological and pathological processes. Extracellular vesicles are not only potential biomarkers and possible pathogenic factors in numerous diseases, but they are also considered as emerging therapeutic targets and therapeutic vehicles. Strikingly, current drug delivery systems, designed to convey therapeutic proteins and peptides (such as liposomes), show many similarities to extracellular vesicles. Here we review some aspects of therapeutic implementation of natural, cell-derived extracellular vesicles in human diseases. Exploration of molecular and functional details of extracellular vesicle release and action may provide important lessons for the design of future drug delivery systems

Increased Hematopoietic Extracellular RNAs and Vesicles in the Lung during Allergic Airway Responses.

Extracellular RNAs (exRNAs) can be released by numerous cell types in vitro, are often protected within vesicles, and can modify recipient cell function. To determine how the composition and cellular sources of exRNAs and the extracellular vesicles (EVs) that carry them change in vivo during tissue inflammation, we analyzed bronchoalveolar lavage fluid (BALF) from mice before and after lung allergen challenge. In the lung, extracellular microRNAs (ex-miRNAs) had a composition that was highly correlated with airway-lining epithelium. Using cell type-specific membrane tagging and single vesicle flow, we also found that 80% of detected vesicles were of epithelial origin. After the induction of allergic airway inflammation, miRNAs selectively expressed by immune cells, including miR-223 and miR-142a, increased and hematopoietic-cell-derived EVs also increased >2-fold. These data demonstrate that infiltrating immune cells release ex-miRNAs and EVs in inflamed tissues to alter the local extracellular environment

Extracellular vesicles, ageing, and therapeutic interventions

A more comprehensive understanding of the human ageing process is required to help mitigate the increasing burden of age-related morbidities in a rapidly growing global demographic of elderly individuals. One exciting novel strategy that has emerged to intervene involves the use of extracellular vesicles to engender tissue regeneration. Specifically, this employs their molecular payloads to confer changes in the epigenetic landscape of ageing cells and ameliorate the loss of functional capacity. Understanding the biology of extracellular vesicles and the specific roles they play during normative ageing will allow for the development of novel cell-free therapeutic interventions. Hence, the purpose of this review is to summarise the current understanding of the mechanisms that drive ageing, critically explore how extracellular vesicles affect ageing processes and discuss their therapeutic potential to mitigate the effects of age-associated morbidities and improve the human health span

Distinct RNA profiles in subpopulations of extracellular vesicles: apoptotic bodies, microvesicles and exosomes

Introduction: In recent years, there has been an exponential increase in the number of studies aiming to understand the biology of exosomes, as well as other extracellular vesicles. However, classification of membrane vesicles and the appropriate protocols for their isolation are still under intense discussion and investigation. When isolating vesicles, it is crucial to use systems that are able to separate them, to avoid cross-contamination. Method: EVs released from three different kinds of cell lines: HMC-1, TF-1 and BV-2 were isolated using two centrifugation-based protocols. In protocol 1, apoptotic bodies were collected at 2,000×g, followed by filtering the supernatant through 0.8 µm pores and pelleting of microvesicles at 12,200×g. In protocol 2, apoptotic bodies and microvesicles were collected together at 16,500×g, followed by filtering of the supernatant through 0.2 µm pores and pelleting of exosomes at 120,000×g. Extracellular vesicles were analyzed by transmission electron microscopy, flow cytometry and the RNA profiles were investigated using a Bioanalyzer®. Results: RNA profiles showed that ribosomal RNA was primary detectable in apoptotic bodies and smaller RNAs without prominent ribosomal RNA peaks in exosomes. In contrast, microvesicles contained little or no RNA except for microvesicles collected from TF-1 cell cultures. The different vesicle pellets showed highly different distribution of size, shape and electron density with typical apoptotic body, microvesicle and exosome characteristics when analyzed by transmission electron microscopy. Flow cytometry revealed the presence of CD63 and CD81 in all vesicles investigated, as well as CD9 except in the TF-1-derived vesicles, as these cells do not express CD9. Conclusions: Our results demonstrate that centrifugation-based protocols are simple and fast systems to distinguish subpopulations of extracellular vesicles. Different vesicles show different RNA profiles and morphological characteristics, but they are indistinguishable using CD63-coated beads for flow cytometry analysis

Extracellular Vesicle-Mediated Communication between the Glioblastoma and Its Microenvironment

The glioblastoma is the most malignant form of brain cancer. Glioblastoma cells use multiple ways of communication with the tumor microenvironment in order to tune it for their own benefit. Among these, extracellular vesicles have emerged as a focus of study in the last few years. Extracellular vesicles contain soluble proteins, DNA, mRNA and non-coding RNAs with which they can modulate the phenotypes of recipient cells. In this review we summarize recent findings on the extracellular vesicles-mediated bilateral communication established between glioblastoma cells and their tumor microenvironment, and the impact of this dialogue for tumor progression and recurrence.España MINECO grant number PGC2018-094654-B-10

Three method-combination protocol for improving purity of extracellular vesicles

Extracellular vesicles (EVs) are nanosized structures able to carry proteins, lipids and genetic material from one cell to another with critical implications in intercellular communication mechanisms. Even though the rapidly growing EVs research field has sparked great interest in the last 20 years, many biological and technical aspects still remain challenging. One of the main issues that the field is facing is the absence of consensus regarding methods for EVs concentration from biofluids and tissue culture medium. Yet, not only can classic methods be time consuming, commercialized kits are also often quite expensive, especially when research requires analyzing numerous samples or concentrating EVs from large sample volumes. In addition, EV concentration often results in either low final yield or significant contamination of the vesicle sample with proteins and protein complexes of similar densities and sizes. Eventually, low vesicle yields highly limit any further application and data reproducibility while contamination greatly impacts extensive functional studies. Hence, there is a need for accessible and sustainable methods for improved vesicle concentration as this is a critical step in any EVs-related research study. In this brief report, we describe a novel combination of three well-known methods in order to obtain moderate-to-high yields of EVs with reduced protein contamination. We believe that such methods could be of high benefits for in vitro and in vivo functional studies

Extracellular vesicles: New players in cardiovascular diseases

Extracellular vesicles, particles released by all cell types, represent a new way to convey information between cells such as proteins, second messengers, and genetic information to modify the phenotype and function of the target cells. Recent data suggest that extracellular vesicles play a crucial role in both physiology and pathology, including coagulation, angiogenesis, cell survival, modulation of the immune response, and inflammation. Thus extracellular vesicles participate in the processes of cardiovascular diseases from atherosclerosis, myocardial infarction to heart failure. Consequently, extracellular vesicles can potentially be exploited for therapy, prognosis, and biomarkers for health and disease. This review focuses on the role of extracellular vesicles in the development of cardiovascular diseases, as well as the deleterious and beneficial effects that they may provide in vascular cells and myocardium

Preclinical translation of exosomes derived from mesenchymal stem/stromal cells.

Exosomes are nanovesicles secreted by virtually all cells. Exosomes mediate the horizontal transfer of various macromolecules previously believed to be cell-autonomous in nature, including nonsecretory proteins, various classes of RNA, metabolites, and lipid membrane-associated factors. Exosomes derived from mesenchymal stem/stromal cells (MSCs) appear to be particularly beneficial for enhancing recovery in various models of disease. To date, there have been more than 200 preclinical studies of exosome-based therapies in a number of different animal models. Despite a growing number of studies reporting the therapeutic properties of MSC-derived exosomes, their underlying mechanism of action, pharmacokinetics, and scalable manufacturing remain largely outstanding questions. Here, we review the global trends associated with preclinical development of MSC-derived exosome-based therapies, including immunogenicity, source of exosomes, isolation methods, biodistribution, and disease categories tested to date. Although the in vivo data assessing the therapeutic properties of MSC-exosomes published to date are promising, several outstanding questions remain to be answered that warrant further preclinical investigation