Extracellular vesicles as personalized medicine.

The past two decades have seen major advancements in technologies that enable improved understanding and characterization of EVs. Before EVs are released from cells, they acquire intracellular cargo that is often comprised of a mixture of proteins, metabolites, and nucleic acids (usually RNA). A variety of ‘omic’ analyses of EV populations isolated from patient blood in a number of disease conditions have revealed that EVs carry unique molecular signatures during disease ( de Miguel Perez et al., 2020 ; Hendrix, 2021 ; Hood, 2019 ; Kinoshita et al., 2017 ; Marleau et al., 2012 ; Szabo and Momen-Heravi, 2017 ; Thompson et al., 2016 ; Wu et al., 2020 ; Xu et al., 2020 ). This has accelerated the scientific community’s understanding of the significance of EVs in the context of disease and how these biogenic nanoparticles can be used to phenotype various disease conditions, treat degenerative conditions, or deliver drugs. The contents of EVs change during the pathogenesis of disease, and technological advancements in the analysis of RNA, proteins, glycans, and lipid profiles allows more comprehensive signature detection than previously possible ( Cheng et al., 2015 ; Saugstad et al., 2017 ; Walker et al., 2020 ). A biomarker is optimal if it is specific and sensitive to a particular disease, and EVs offer this opportunity in stable packaging ( Bei et al., 2017 ; Rodrigues et al., 2018 ). Another advantage is that EVs are highly abundant in many bioavailable fluids, offering several sources from which to diagnose or track disease ( El-Shennawy et al., 2022 ; Zhong et al., 2021 ). The heterogeneity of these populations present opportunities for truly tailored personalized medicine approaches, but are also extremely challenging to fully characterize ( Zhong et al., 2021 ). Emerging methods for more detailed characterization of EVs include multiplexed super resolution microscopy via directed stochastic reconstruction microscopy ( dSTORM ) ( Fig. 4 ) and flow-cytometry that allow single EV analysis. These methods are not yet available for use clinically, but demonstrate progress in approaches to understand and characterize specific EV populations in the context of disease ( Thery et al., 2018 ). Currently, some EV biomarker panels have been developed and approved by the Food and Drug Administration ( FDA ) for use in patients-the first for cancer reached the market in 2016 ( Sheridan, 2016 ). While these panels show great potential, their use in the clinic is hindered by poor reproducibility, which is affected by the lack of widely accepted guidelines or standardized protocols for isolation, handling, and storage of EVs ( Khalaj et al., 2019 ). Choosing the appropriate biofluid to study is important in determining the mechanistic contribution of EVs to regeneration or disease; for example, urine may not be a good biofluid to identify EVs related to lung injury. Understanding the nuances of EV derivation is essential for appropriate biomarker characterization and development of clinical tests. Blood is the most common source of biofluid used for biomarker studies due in part to accessibility. Studies characterizing pathogenic RNA and protein content of EVs from blood have been reviewed for several diseases such as hepatitis ( Szabo and Momen-Heravi, 2017 ), cancer ( de Miguel Perez et al., 2020 ; Hood, 2019 ; Kinoshita et al., 2017 ; Marleau et al., 2012 ; Nawaz et al., 2014 ), neurodegeneration ( Thompson et al., 2016 ; You and Ikezu, 2019 ), autoimmune disease ( Wu et al., 2020 ; Xu et al., 2020 ), and cardiovascular disease ( Bei et al., 2017 ; Chong et al., 2019 ; Dickhout and Koenen, 2018 ; Fu et al., 2020 ). These studies represent the next generation of biomarker development. Developing early and rapid detection of diseases using EVs has led to the concept of EV depletion as a potential therapeutic option ( Marleau et al., 2012 ). Aethlon Medical Incorporated is in recruitment stages for an early feasibility study to use their Hemopurifier ™ for EV depletion for patients with head and neck squamous cell cancers. The study is expected to be completed by 2023 ( Clinical Trials.gov website). Side effects from a reduction in total EV numbers may impact underlying physiological processes that rely on them to maintain health or allow other disease conditions to emerge ( Hill, 2019 ). Simple depletion may not be the answer; instead, targeting EVs from specific cell types or pathways has been suggested ( Hill, 2019 ; Khalaj et al., 2019 ). This specific targeting strategy is limited by current technical ability/knowledge, as it is challenging to differentiate pathological and physiological EVs ( Rodrigues et al., 2018 ). This issue is further complicated in early timepoints or stages of disease, when EV disease signatures can only be found in relatively rare EV populations compared to other physiological populations, which range in the trillions ( Hill, 2019 ; Rodrigues et al., 2018 ). A potential solution to this rarity is being assessed in neurological disease settings where antibody-meditated pull down for non-pathogenic EVs is being performed on blood in the periphery, allowing more efficient isolation of rarer populations for biomarker discovery, a technique that could also be applied to pathogenic EVs for specific depletion of pathogenic messengers ( Hill, 2019 ). While stem cell therapy has traditionally dominated the regenerative space, there is increasing evidence that the restorative functions once assumed to be due to stem cells are mediated by released products from stem cells termed the ‘secretome’, which includes EVs ( Ding et al., 2021 ; Tao et al., 2018 ; Whittaker et al., 2020 ). Some advantages to using the secretome or EVs instead of cells include easier handling and storage, for example, freeze-drying can be performed to produce an “off the shelf” product that is readily available when needed. Additionally, EVs are unable to form cancerous growths, which is a risk with cell therapy ( Feng et al., 2020 ; Qi et al., 2020 ; Szwedowicz et al., 2022 ; Tao et al., 2018 ; Wan et al., 2022 ; Wellings et al., 2021 ; Willis et al., 2020 ). The small size of EVs compared to cells also makes vascular obstructions less likely following intravenous administration and results in different biodistribution profiles ( Ali et al., 2020 ). Finally, cells are more responsive to environmental conditions than EVs, and can change characteristics, which may be advantageous or disadvantageous depending on the context. EVs can also be modified by altering surface and/or internal components, allowing modular component design ( Tao et al., 2018 ), and several EV-based drug delivery applications have been developed ( S. Walker et al., 2019 ; Witwer, 2021 ). The reparative functions of EVs are numerous, for example, EVs are able to reduce the effects of aging on cells ( Feng et al., 2020 ; Mensa et al., 2020 ; Prattichizzo et al., 2019 ; Y. Yin et al., 2021 ). EVs obtained from the plasma of young mice have been found to reduce aging when administered to old mice ( Iannotta et al., 2021 ; Prattichizzo et al., 2019 ; Sahu et al., 2021 ; Yoshida et al., 2019 ). Cellular senescence that occurs with age has been linked to EV signaling, providing new targets to mitigate the deteriorative effects of aging ( Yin et al., 2021 ). Altering inflammatory responses ( Grieco et al., 2021 ), tissue/wound healing ( Bray et al., 2021 ; Costa et al., 2022 ), and brain remodeling ( Gualerzi et al., 2021 ) are all active areas of research with EV products for both tracking and inducing regenerative processes ( Gualerzi et al., 2021 ). Inhibiting inflammation is an avid area of EV research. EVs are known to participate in the cross-talk between the immune system and other cells of the body, and are altered in a variety of disease states ( Grieco et al., 2021 ). Fibrosis often leaves scar tissue which is an endpoint of tissue damage that has been irreparable with medication or surgery. Thus, great interest has been placed on determining whether EVs are able to prevent or return scar tissue to a healthy state ( Qi et al., 2020 ; Wan et al., 2022 ; Wellings et al., 2021 ). Complete tissue regeneration via EV therapy is being actively tested in bone, skin, and cardiac muscle ( Kost et al., 2022 ; Pishavar et al., 2021 ; Thankam and Agrawal, 2020 ; Yin et al., 2021 ). Aside from their capacity as biomarkers and endogenous therapeutics, EVs can also be used for delivery of exogenous therapeutic agents, including small molecules, peptides/proteins, and RNA. Nanodelivery improves the site-specific accumulation of free drugs, resulting in increased therapeutic efficacy and less side effects ( Khalid et al., 2017 ; Shen et al., 2017 ; Wolfram et al., 2015 ). Additionally, nanoparticles enable protection of RNA and protein therapeutics that are sensitive to degradation by extracellular and intracellular enzymes ( Shen et al., 2015 ). Nanoformulations also have advantages over micro-formulations, including larger surface area to volume ratio, which can improve interactions with targets, and reduced risk of vascular obstructions ( Martin et al., 2005 ). There has been considerable interest in the use of EVs as medication carriers, especially for chemotherapeutics ( Busatto et al., 2019 ; S. Walker et al., 2019 ). Although laboratory-created simple nanocarriers, such as liposomes, can be easily made through well-established methods and have been in clinical use for decades ( Gentile et al., 2013 ), biologically-derived nanoparticles like EVs have the potential to outperform conventional delivery systems. Currently, the recognition and clearance of intravenously injected EVs by the innate immune system is much faster than that of synthetic nanoparticles ( Couch et al., 2021 ). However, studies in reporter mice indicate that endogenous EVs can avoid immunological clearance and reach target tissues over long distances ( Luo et al., 2020 ). Therefore, selecting the optimal EV subtype, preserving endogenous characteristics (i.e., minimizing damage from isolation, drug loading, and labeling), and lowering the infusion rate could overcome rapid immunological recognition. Clinically approved nanoparticles have simple surfaces that lack protein and glycan decorations, and attempts to develop targeted delivery systems have repeatedly failed in clinical trials ( Wolfram and Ferrari, 2019 ). It is likely that the aforementioned failures are partially due to overly simplistic strategies (one surface ligand) to target highly complex biological surfaces with thousands of biomolecules. EVs demonstrate specificity for recipient cells through complex surface interactions that involve multiple molecules in optimal orientations, spatial arrangements, and ratios, and may therefore, be more equipped than synthetic nanoparticles to mediate site-specific delivery. Additionally, EVs have been shown to cross the blood brain barrier, a major roadblock for many neuro-therapies ( Hill, 2019 ). EVs can also be harnessed in infectious diseases for their specificity and ability to target pathogens ( Schorey and Harding, 2016 ). Many EV-based drug delivery systems are currently in the translational pipeline ( Lener et al., 2015 ; Rodrigues et al., 2018 ), and the upcoming decade is likely to reveal the utility of these intercellular messengers as drug carriers. Many challenges need to be overcome before EVs can successfully be used as biomarkers or therapies for diseases in the clinical setting. A critical challenge is the multitude of isolation methods that vary widely in EV enrichment capabilities. Specifically, over 190 different isolation methods and over 1000 unique protocols have been reported for EV isolation ( EV-TRACK Consortium et al., 2017 ). Isolation and storage methods substantially impact structure and function of EVs, which remains a fundamental issue in the field. Additionally, there is a lack of controls that can be used to standardize conditions between laboratories. There are a number of technical challenges in isolating EVs from biological samples, in particular, separation from similar sized contaminants. Improved tools, such as those based on flow cytometry and super resolution microscopy, are needed for an improved understanding of EV heterogeneity, which is also a limiting factor for clinical translation. Data derived from super-resolution microscopy, for example, can be altered based on the chemistry of the imaging mediums used ( Arsic et al., 2020 ). Although EVs have been studied for almost 60 years, many aspects of EV biology remain largely unknown ( Couch et al., 2021 ; De Tkaczevski, 1968 ; Feller and Chopra, 1968 ; Sun, 1966 ; Wolf, 1967 ). The study of EVs is further complicated by host factors that alter the phenotype of EVs, including donor age, biological sex, current or previous pregnancy, menopause, pre/postprandial status (fasting/non-fasting), time of day of collection (circadian variations), exercise level and time of last exercise, diet, body mass index, specific infectious and noninfectious diseases, medications, and other factors ( Thery et al., 2018 ). EV characteristics are also affected by sample collection conditions, such as collection volume, first tube discard, type of container(s), time to processing, choice of anticoagulant (for blood plasma), mixing or agitation, temperature (of both storage and processing), type of transport (if any), whether the tube remained upright before processing, exact centrifugation or filtration procedures, degree of hemolysis, possible confirmation of platelet and lipoprotein depletion prior to storage, and so on ( Thery et al., 2018 ). Overall, there are a vast number of factors contributing to variation in EV characteristics and functions that need to be resolved in order to create a consistent product that can be used for personalized medicine.

EVs are promising biological nanoparticles for use as biomarkers to diagnose disease, to monitor progression of disease (or pre-disease) and to use as therapies to prevent or reverse disease. The ability of EVs to communicate directly with recipient cells in a cell/tissue, sex, age, and disease-specific manner places them at the forefront of candidates for personalized medicine. EV research over the past 60 years has laid the foundation and resulted in critical insights, yet a great deal remains to be understood. EV research holds the promise of changing how we understand, monitor, and treat disease, and researchers are racing forward to make those discoveries.

Extracellular vesicles ( EVs ) are released from all cells and form a critical intercellular communication mechanism ( Couch et al., 2021 ). The rich diversity of EVs supports a growing list of functions in maintaining health and promoting disease. EVs are defined and characterized by their size (nanoparticles), the presence of a phospholipid bilayer that contains certain distinguishing markers (e.g., tetraspanins CD9, CD63, CD81), and functional ability (e.g., anti-inflammatory), as described in the most recent guidelines from the International Society for Extracellular Vesicles ( ISEV ) ( Thery et al., 2018 ). The EV membrane contains bioactive lipids, carbohydrates, and proteins, while nucleic acids, such as DNA and RNA, and proteins (for example, cytokines) can be present in the EV interior. These EV-associated biomolecules reflect the cell of origin, enabling diagnostic and therapeutic applications ( Thery et al., 2018 ). Cells continuously release EVs using both intracellular endocytic pathways and direct budding from the plasma membrane. EVs circulate in the blood and extracellular space, where they act in a paracrine or long-distance manner on recipient cells ( Rodrigues et al., 2018 ). Depending on the context, EVs can have favorable (therapeutic) or unfavorable (pathological) effects, and much remains unknown regarding the role of EVs in homeostasis and various disease states ( Yates et al., 2022b ). Understanding the contribution of EVs to disease is complicated by the heterogeneity of EVs in biological samples. A variety of terms and definitions have been used over time for EVs, leading to some confusion in the field ( Bazzan et al., 2021 ; Couch et al., 2021 ; Thery et al., 2018 ). Here, we use the term EVs to refer to all extracellular, lipid bilayer, sub-cellular particles and their functional contents with sizes ranging from 30 nm to 1 μm. This definition includes the widely recognized major subgroups termed exosomes, microvesicles, and apoptotic bodies ( Fig. 1 ). Distinctions between these groups are based primarily on their origin ( Rodrigues et al., 2018 ). Exosomes are released by endocytic pathways within cells and range from approximately 30 to 150 nm ( DeLeo and Ikezu, 2018 ). Microvesicles or ectosomes are shed from the plasma membrane into the extracellular space and range from approximately 100 to 1000 nm ( Colombo et al., 2014 ; Janas et al., 2016 ). Apoptotic bodies arise from degrading cells and range from approximately 100 nm to several micrometers, sometimes large enough to contain entire cellular organelles ( Buzas et al., 2014 ; Gyorgy et al., 2011 ). The collective term EV is used in this article, as subtypes have overlapping size ranges and biomolecular content, and current technology is unable to accurately separate or distinguish exosomes from microvesicles ( Bazzan et al., 2021 ; Crescitelli et al., 2013 ; Gyorgy et al., 2011 ; Khalaj et al., 2019 ; Rodrigues et al., 2018 ; Thery et al., 2018 ; Yates et al., 2022a ). Historically, the main role of EVs was thought to be as nano-sized “trash bags”, eliminating unwanted waste from the cell ( Couch et al., 2021 ; Szwedowicz et al., 2022 ). Today, they are known to play vital roles in cellular communication ( Yanez-Mo et al., 2015 ). Regardless of the origin of EVs (i.e., plasma membrane, intraluminal vesicle), the membranous and inner contents of EVs are influenced by the parent cell. Factors that may affect EV characteristics include donor sex, age, and cellular stress. All components of the lipid membrane and internal compartment of EVs can impact recipient cells ( Thery et al., 2018 ). Larger EVs may contain sub-cellular contents such as mitochondria or other functional macromolecules that can affect recipient cells ( Dean et al., 2009 ; Gasecka et al., 2019 ). EV-mediated transfer of functional products enables a signaling axis between donor and recipient cells that can be useful in a variety of situations to promote physiological homeostasis or pathology (e.g., stress responses, host cell responses to pathogens, and tumor microenvironment modulation) ( Couch et al., 2021 ; Yanez-Mo et al., 2015 ; Yates et al., 2022a , 2022b ). Once EVs are internalized by recipient cells, intracellular signaling cascades can be initiated by EV-associated biomolecules, such as microRNAs ( miRNAs ) and proteins, that are released into the cytoplasm ( Pant et al., 2012 ; Rodrigues et al., 2018 ). In addition to intracellular uptake, EVs may also activate signaling cascades in recipient cells through surface interactions without subsequent uptake, that is, a “kiss-and-run” approach ( Morris and Witwer, 2022 ). All cells release EVs, which are present in biological secretions, excretions, and tissues ( Fig. 1 ), ( Robbins and Morelli, 2014 ) including ejaculate ( Hoog and Lotvall, 2015 ), lipoaspirate ( Tian et al., 2020 ; Wang et al., 2021 ), synovial fluid ( B. Yin et al., 2022 ), breast milk ( Zhong et al., 2021 ), amniotic fluid ( Costa et al., 2022 ), saliva ( Li et al., 2022 ; Yuana et al., 2015 ), urine ( Barreiro and Holthofer, 2017 ; Minkler et al., 2021 ; Yuana et al., 2015 ), cerebrospinal fluid ( Welton et al., 2017 ), blood/plasma ( Yuana et al., 2015 ), lymph ( Milasan et al., 2016 ), and mucus ( Pastor et al., 2021 ). Other types of nanoparticles are also found in biofluids, such as lipoproteins ( Feingold, 2000 ) and exomeres ( H. Zhang et al., 2018 ). New sources of EVs continue to be characterized as technology limitations surrounding isolation and authentication improve; however, a comprehensive understanding of the functional properties of EVs in health and disease remains to be determined. Difficulties in assigning a particular function to EVs stems from their heterogeneity. Without the technical ability to independently isolate subpopulations, it is challenging to accurately assess individual contributions of EVs to homeostasis and disease ( Ramirez et al., 2018 ; Thery et al., 2018 ; Veerman et al., 2021 ; Yates et al., 2022a ). Limitations in EV separation and subsequent characterization stem from overlapping biomolecular content and size ranges, which can also be affected by isolation methods, storage conditions, and measurement parameters ( Ramirez et al., 2018 ; Thery et al., 2018 ; Veerman et al., 2021 ; Yates et al., 2022a ). Although there are reports of EVs containing distinct cargo, leading to a specific response in a disease model, such findings are often difficult to replicate due to differences in characterization methods and/or isolation techniques (see Challenges below) ( Raposo and Stoorvogel, 2013 ; Tans et al., 1991 ; Yates et al., 2022a ). Many of the qualities that make EVs difficult to characterize also imbue them with incredible versatility and applicability as promising candidates for personalized medicine. Because of their specificity for certain tissues and disease states, EVs can be used as personalized diagnostic biomarkers and allogenic or autologous biotherapeutics and/or biogenic drug delivery vehicles. EVs offer promise for a personalized precision medicine approach to healthcare, where therapy can be tailored to the sex, age, and condition/disease of the individual.