Role of Exosomes in Biological Communication Systems [1st ed.] 9789811565984, 9789811565991

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Faisal A. Alzahrani Islam M. Saadeldin  Editors

Role of Exosomes in Biological Communication Systems

Role of Exosomes in Biological Communication Systems

Faisal A. Alzahrani • Islam M. Saadeldin Editors

Role of Exosomes in Biological Communication Systems

Editors Faisal A. Alzahrani Department of Biochemistry, ESC Research Unit, Faculty of Science King Fahd Medical Research Center King Abdulaziz University Jeddah, Saudi Arabia

Islam M. Saadeldin Department of Animal Production King Saud University Riyadh, Saudi Arabia

ISBN 978-981-15-6598-4 ISBN 978-981-15-6599-1 https://doi.org/10.1007/978-981-15-6599-1

(eBook)

# Springer Nature Singapore Pte Ltd. 2021 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Singapore Pte Ltd. The registered company address is: 152 Beach Road, #21-01/04 Gateway East, Singapore 189721, Singapore

Preface

Exosomes as membrane-bound extracellular vesicles (EVs) are an active area of research and, known as universal minute nanosized vehicles, can be released from all cells in all prokaryotes and eukaryotes to transfer genetic instructions between cells. EVs deliver proteins, mRNAs, miRNAs, lipids, metabolites, and enzymes to alter cell functions on the physiological or pathological levels. There are tremendous increase and thousands of publications related to the isolation, characterization, and functional analysis of EVs. The physiological purpose of generating EVs remains largely unknown and needs investigation. One speculated role is that EVs likely remove excess and/or unnecessary constituents from cells to maintain cellular homeostasis. Recent studies reviewed here also indicate a functional, targeted, mechanism-driven accumulation of specific cellular components in EVs, suggesting that they have a role in regulating intercellular communication. More attention has been given to the regenerative capabilities of stem cells-derived EVs for overcoming the setbacks of cellular therapy and towards a cell-free therapy. EVs are associated with reproductive functions, basic immune responses, parasitic pathogenicity, urinary system diseases, liver diseases, cardiovascular diseases, central nervous system-related diseases, and cancer progression. Proteins, metabolites, and nucleic acids delivered by EVs into recipient cells effectively alter their biological response. These EVs-mediated responses can be disease promoting or restraining. EVs can be artificially engineered to deliver diverse therapeutic cargoes, including siRNAs, antisense oligonucleotides, chemotherapeutic agents, and immune modulators. Additionally, EVs also have the potential to aid in disease diagnosis as they have been reported in all biological fluids and considered as a reliable biomarker for different diseases. In this book we highlight the work from different laboratories and interested researchers regarding the vital aspects of EVs and exosomes including their role in physiological and pathological communications as well as their therapeutic uses in different physiological and pathological levels. Two chapters illustrate the isolation and characterization of EVs. Three chapters discuss the roles of EVs in male and female reproduction as well as the early embryonic life. Two chapters highlight the beneficial roles of EVs derived from stem cells and the regenerative and therapeutic potentials. Moreover, five chapters discuss the EVs in diseases of the urogenital system, nervous system, liver, and stem cells-derived EVs. Four more chapters uncover the critical roles of EVs in different cancers and metastasis. Finally, v

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the trend for generating therapeutic EVs and exosomes is also covered by two chapters. The potentials of EVs to be employed in translational medicine, especially as biomarkers, and therapeutic delivery system are promising for developing novel therapeutic and diagnostic tools for clinical practice. Jeddah, Saudi Arabia Riyadh, Saudi Arabia May 2020

Faisal A. Alzahrani Islam M. Saadeldin

Contents

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Isolation and Characterization of Extracellular Vesicles: Classical and Modern Approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ahmed E. Noreldin, Asmaa F. Khafaga, and Rasha A. Barakat

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Characterization and Fine Structure of Exosomes . . . . . . . . . . . . . Fawzia A. Al-shubaily and Maryam H. Al-Zahrani

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Extracellular Vesicles Mediate the Embryonic-Maternal Paracrine Communication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Islam M. Saadeldin

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The Interplay Between Oviduct-Derived Exosomes and CumulusOocyte Complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Seok Hee Lee and Byeong Chun Lee

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The Interplay Between Exosomes and Spermatozoa . . . . . . . . . . . . Ahmad Yar Qamar, Xun Fang, Seonggyu Bang, Feriel Yasmine Mahiddine, Min Jung Kim, and Jongki Cho

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Mesenchymal Stem Cell–Derived Exosomes and Regenerative Medicine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hoda Elkhenany and Shilpi Gupta

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Therapeutic Potential of Mesenchymal Stem/Stromal Cell–Derived Exosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Fawaz Abomaray

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Exosomes in Neurodegenerative Disorders . . . . . . . . . . . . . . . . . . . Ahmed Osama Elmehrath, Yousef Tarek Sonbol, and Moaz Yahia Farghal

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Differential Expression of Exosomal MicroRNAs in Neurodegenerative Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nesma Elsayed Abdelaal and Mostafa Fathi Abdelhai

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Contents

Urinary Exosomes as a Possible Source of Kidney Disease Biomarkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ahmed Abdeen, Hiroko Sonoda, Ayae Tanaka, and Masahiro Ikeda

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Extracellular Vesicles as Potential Therapeutic Targets and Biomarkers for Liver Disease . . . . . . . . . . . . . . . . . . . . . . . . . . Faisal Abdulrahman Alzahrani

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Implications of Extracellular Vesicles in Blood Protozoan Parasitic Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nagham Gamal Masoud, Nagwa Mostafa El-Sayed, and Manar Ezz Elarab Ramadan

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Cancer Cells–Derived Exosomes and Metastasis . . . . . . . . . . . . . . . Wahaj Alnefaie

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Extracellular Vesicles and Integrins: Partners in Cancer Progression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Wanessa Fernanda Altei, Bianca Cruz Pachane, Patty Karina dos Santos, and Heloisa Sobreiro Selistre de Araújo

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Exosomes: The Crucial Element in Prostate Cancer . . . . . . . . . . . . Mohammed Moulay and Saleh Al-Kareem

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Exosomal microRNAs: Potential Biomarkers for Cancer Diagnosis, Treatment Response and Prognosis . . . . . . . . . . . . . . . . . . . . . . . . Faizah Alotaibi

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Exosomes in Drug Delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Fahad A. Almughem, Abdullah A. Alshehri, and Mohammad N. Alomary

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Exosomes and Supported Lipid Layers as Advanced Naturally Derived Drug Delivery Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . Mahmoud A. Elnaggar and Yoon Ki Joung

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Editors and Contributors

Editors Faisal A. Alzahrani is an Associate Professor of Molecular Biology at King Abdulaziz University. He received his Ph.D. in Medical Sciences from North West Cancer Research Centre at Bangor University, where he studied the mechanisms of genome maintenance in cancer stem cells and stem cells. After that, he joined the university as a faculty member, set up his team and laboratory, and was appointed as deputy head of the stem cell unit. Dr. Alzahrani subsequently focused his research on stem cell-derived exosomes and their therapeutic applications. While working as a visiting lecturer at Aston Medical School in the UK, he became more interested in translating research and joined MirZyme Therapeutics as Vice President for the MENA region. Islam M. Saadeldin is an Associate Professor at King Saud University, Saudi Arabia, and adjunct Associate Professor at Zagazig University, Egypt. He obtained his Ph.D. from Seoul National University, South Korea, in 2012. He was a visiting scholar at Niigata University, Japan (2008), and also served as a postdoctoral fellow at Seoul National University (2013–2014). He received the Asian Universities Alliance (AUA) Scholar Award in 2019 and a visiting professorship to Seoul National University. He holds a patent for embryo transgenesis through PiggyBac transposons. He has published more than 100 research articles on animal cloning, genome editing, assisted reproductive techniques, adult and embryonic stem cells, as well as the roles of exosomes in pathophysiology and embryonic-maternal crosstalk in respected journals. He also serves on the editorial boards of Frontiers in Veterinary Science, Journal of Animal Reproduction and Biotechnology, and Cloning and Transgenesis and as an associate editor for The Open Stem Cell Journal.

Contributors Ahmed Abdeen Department of Forensic Medicine and Toxicology, Faculty of Veterinary Medicine, Benha University, Toukh, Egypt ix

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Nesma Elsayed Abdelaal Biotechnology Program, Chemistry Department, Faculty of Science, Cairo University, Giza, Egypt Fawaz Abomaray Division of Obstetrics and Gynecology, Department of Clinical Science, Intervention and Technology, Karolinska Institutet, Stockholm, Sweden Saleh Al-Kareem Embryonic Stem Cell Research Unit, King Fahd Medical Research, King Abdulaziz University, Jeddah, Kingdom of Saudi Arabia Department of Biology, Faculty of Sciences, King Abdulaziz University, Jeddah, Kingdom of Saudi Arabia Fahad A. Almughem National Centre for Pharmaceutical Technology, King Abdulaziz City for Science and Technology (KACST), Riyadh, Saudi Arabia Wahaj Alnefaie School of Life and Health Sciences, Aston University, Birmingham, UK Mohammad N. Alomary National Centre for Biotechnology, King Abdulaziz City for Science and Technology (KACST), Riyadh, Saudi Arabia Faizah Alotaibi Department of Microbiology and Immunology, Western University, London, ON, Canada London Regional Cancer Program, Lawson Health Research Institute, London, ON, Canada Abdullah A. Alshehri National Centre for Pharmaceutical Technology, King Abdulaziz City for Science and Technology (KACST), Riyadh, Saudi Arabia Fawzia A. Al-shubaily Biochemistry Department, Faculty of Science, King Abdulaziz University, Jeddah, Saudi Arabia Wanessa Fernanda Altei Biochemistry and Molecular Biology Laboratory, Department of Physiological Sciences, Federal University of São Carlos, São Carlos, Brazil Faisal A. Alzahrani Department of Biochemistry, ESC Research Unit, Faculty of Science, King Fahd Medical Research Center, King Abdulaziz University, Jeddah, Saudi Arabia Maryam H. Al-Zahrani Biochemistry Department, Faculty of Science, King Abdulaziz University, Jeddah, Saudi Arabia Seonggyu Bang College of Veterinary Medicine, Chungnam National University, Daejeon, South Korea Rasha A. Barakat Department of Physiology, Faculty of Veterinary Medicine, Damanhour University, Damanhour, Egypt Jongki Cho College of Veterinary Medicine, Chungnam National University, Daejeon, South Korea

Editors and Contributors

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Heloisa Sobreiro Selistre de Araújo Biochemistry and Molecular Biology Laboratory, Department of Physiological Sciences, Federal University of São Carlos, São Carlos, Brazil Patty Karina dos Santos Biochemistry and Molecular Biology Laboratory, Department of Physiological Sciences, Federal University of São Carlos, São Carlos, Brazil Hoda Elkhenany Department of Surgery, Faculty of Veterinary Medicine, Alexandria University, Alexandria, Egypt Ahmed Osama Elmehrath Faculty of Medicine, Cairo University, Cairo, Egypt Mahmoud A. Elnaggar Center for Biomaterials, Biomedical Research Institute, Korea Institute of Science and Technology, Seoul, Republic of Korea Nagwa Mostafa El-Sayed Medical Parasitology Department, Research Institute of Ophthalmology, Giza, Egypt Xun Fang College of Veterinary Medicine, Chungnam National University, Daejeon, South Korea Moaz Yahia Farghal Faculty of Medicine, Cairo University, Cairo, Egypt Mostafa Fathi Abdelhai Biotechnology Program, Faculty of Agriculture, Ain Shams University, Cairo, Egypt Shilpi Gupta Division of Molecular Genetics and Biochemistry Lab, National Institute of Cancer Prevention and Research (NICPR), Noida, India Stem Cell and Cancer Research Lab, Amity Institute of Molecular Medicine and Stem Cell Research (AIMMSCR), Amity University, Noida, Uttar Pradesh, India Masahiro Ikeda Department of Veterinary Pharmacology, Faculty of Agriculture, University of Miyazaki, Miyazaki, Japan Yoon Ki Joung Center for Biomaterials, Biomedical Research Institute, Korea Institute of Science and Technology, Seoul, Republic of Korea Division of Bio-Medical Science and Technology, University of Science and Technology, Daejeon, Republic of Korea Asmaa F. Khafaga Department of Pathology, Faculty of Veterinary Medicine, Alexandria University, Alexandria, Egypt Min Jung Kim Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University, Seoul, South Korea Byeong Chun Lee Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University, Seoul, Republic of Korea Seok Hee Lee Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University, Seoul, Republic of Korea Center for Reproductive Sciences, Department of Obstetrics and Gynecology, University of California San Francisco, San Francisco, CA, USA

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Feriel Yasmine Mahiddine Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University, Seoul, South Korea Nagham Gamal Masoud Faculty of Medicine, Ain Shams University, Cairo, Egypt Mohammed Moulay Embryonic Stem Cell Research Unit, King Fahd Medical Research, King Abdulaziz University, Jeddah, Kingdom of Saudi Arabia Department of Biology, Faculty of Sciences, King Abdulaziz University, Jeddah, Kingdom of Saudi Arabia Ahmed E. Noreldin Histology and Cytology Department, Faculty of Veterinary Medicine, Damanhour University, Damanhour, Egypt Bianca Cruz Pachane Biochemistry and Molecular Biology Laboratory, Department of Physiological Sciences, Federal University of São Carlos, São Carlos, Brazil Ahmad Yar Qamar College of Veterinary Medicine, Chungnam National University, Daejeon, South Korea Department of Clinical Sciences, College of Veterinary and Animal Sciences, Jhang 35200, Sub-campus University of Veterinary and Animal Sciences, Lahore, Pakistan Manar Ezz Elarab Ramadan Parasitology Department, National Hepatology and Tropical Medicine Research Institute, Cairo, Egypt Islam M. Saadeldin Department of Physiology, Faculty of Veterinary Medicine, Zagazig University, Zagazig, Egypt Department of Animal Production, King Saud University, Riyadh, Saudi Arabia Yousef Tarek Sonbol Faculty of Medicine, Cairo University, Cairo, Egypt Hiroko Sonoda Department of Veterinary Pharmacology, Faculty of Agriculture, University of Miyazaki, Miyazaki, Japan Ayae Tanaka Department of Veterinary Pharmacology, Faculty of Agriculture, University of Miyazaki, Miyazaki, Japan

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Isolation and Characterization of Extracellular Vesicles: Classical and Modern Approaches Ahmed E. Noreldin, Asmaa F. Khafaga, and Rasha A. Barakat

Abstract

Extracellular vesicles (EVs) are tiny membrane vesicles containing detailed cellular information. Recently, researchers have been focusing on EVs due to their role in intercellular communication, and prognostic, diagnostic, and therapeutic usage in medical purposes. In this chapter, we summarize the available technologies for EV characterization and describe their limitations and potential. Moreover, we highlight the emerging technologies with their development. Keywords

Extracellular vesicles · Exosomes · Isolation · Electron microscopy · Ultracentrifugation

Abbreviations AF4 AFM AKI

Asymmetric flow field-flow fractionation Atomic force microscopy Kidney injury

A. E. Noreldin (*) Department of Histology and Cytology, Faculty of Veterinary Medicine, Damanhour University, Damanhour, Egypt e-mail: [email protected] A. F. Khafaga Department of Pathology, Faculty of Veterinary Medicine, Alexandria University, Alexandria, Egypt R. A. Barakat Department of Physiology, Faculty of Veterinary Medicine, Damanhour University, Damanhour, Egypt # Springer Nature Singapore Pte Ltd. 2021 F. A. Alzahrani, I. M. Saadeldin (eds.), Role of Exosomes in Biological Communication Systems, https://doi.org/10.1007/978-981-15-6599-1_1

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CCD cryo-EM CSF DLS DUC EM EVs FC FCS FIC Fl-NTA IFC LSPRi LTRS MISEV MSC MVBs NTA PBS PCR PCS PEG PMT Sc-NTA SEA SEC SEM SERS Sp-IRIS SPR SPT TEM TRPS

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Coupling system Cryogenic electron microscopy Cerebrospinal fluid Dynamic light scattering Differential ultracentrifugation Electron microscopy Extracellular vesicles Flow cytometry Fluorescence correlation spectroscopy Fluorescence imaging system Emitted fluorescence Image flow cytometer Localized SPR imaging Laser tweezers Raman spectroscopy Minimal information for studies of extracellular vesicles Mesenchymal stem cell Multivesicular bodies Nanoparticle tracking analysis Phosphate-buffered saline Polymerase chain reaction Photon-correlation spectroscopy Polyethylene glycol Photomultiplier tube Scattered light Fluorescent microscopic analysis Size-exclusion chromatography Scanning electron microscopy Surface enhanced Raman spectroscopy Single-particle IRIS Surface plasmon resonance Single-particle tracking Transmission electron microscopy Tunable pulse resistive sensing

Introduction

Extracellular vesicles (EVs) are phospholipid bilayer vesicles secreted by most cells. EVs have attracted great interest in the field of biomedical research in recent years due to their pivotal biological role in disease and normal physiology (Bank et al. 2015; Colombo et al. 2014; Quek and Hill 2017). Recently, EV-mediated cell-to-cell communication has been highly investigated in cancer, where spreading of EVs to the tumor microenvironment enhances modulating immune, matrix remodeling and angiogenesis (Al-Nedawi et al. 2008; Andreola et al. 2002; Huber et al. 2005; Luga et al. 2012; Skog et al. 2008). On the other hand, tumorigenesis is enhanced by the

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Isolation and Characterization of Extracellular Vesicles: Classical and Modern. . .

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transfer of EVs to tumor cells through elevating tumor cell migration, propagation, resistance to chemotherapy, and epithelial-to-mesenchymal transition (Au Yeung et al. 2016; Leca et al. 2016; Luga et al. 2012; Richards et al. 2017). Moreover, EVs can also move farther from the tumor site where they create a pre-metastatic niche (Alderton 2012; Costa-Silva et al. 2015; Peinado et al. 2012; Somasundaram and Herlyn 2012). Chargaff and West (1946) detected EVs in blood. Then, Wolf (1967) called EVs as “platelet dust.” Later, EVs had been created by rectal adenoma microvillus cells and called “plasma membrane fragments” (De Broe et al. 1977). Moreover, in 1983, detailed investigations showed that vesicles are formed by the union of plasma membrane with multivesicular bodies (MVBs) (Harding et al. 1983). After that, Raposo and colleagues reported that these vesicles, separated from virustransformed B lymphocytes, had the ability to stimulate T cell responses (Raposo et al. 1996). In 2007, EVs attracted more attention as mediators of communication between one cell to another cell due to detection of RNA in EVs (Valadi et al. 2007). Therefore, EVs are a potential origin of biomarkers for various diseases because they indicate the secretion of the cells such as lipids, nucleic acids, and proteins. EV-containing “liquid biopsies” such as urine (Duijvesz et al. 2011), blood (Caby et al. 2005), cerebrospinal fluid (CSF) (Chen et al. 2013), and saliva (Yang et al. 2014) are easily obtained and are considered as a good alternative to common biopsies (Wu et al. 2017). Currently, the biomarkers for EVs are being investigated in many diseases, including cancer (Choi 2015; Merchant et al. 2017; Moon et al. 2016). In addition to prognostic and diagnostic potential, the therapeutic usage of EVs or liposomes as targeted therapy delivery vehicles is being investigated (Crivelli et al. 2017; Usman et al. 2018; van der Meel et al. 2014). In preclinical models, human mesenchymal stem cell (MSC)-derived EVs have proved their therapeutic ability. For instance, the treatment of mice suffering from acute kidney injury (AKI) with MSC-derived EVs supporting the functional recovery of AKI, compared with the administration of MSCs only (Bruno et al. 2009). Furthermore, cardiac function was enhanced by treatment with MSC-derived EVs after myocardial ischemia/reperfusion injury (Arslan et al. 2013). To utilize the biomedical ability of EVs, methods are essential to estimate their concentration in samples and to determine their molecular composition. The complex nature of clinical and biological EV samples and EV heterogeneity hamper EV analysis. The family of EVs can be classified according to their biogenesis into three major categories: apoptotic bodies, microvesicles, and exosomes (Raposo and Stoorvogel 2013). Exosomes have a small diameter (40–100 nm) and are synthesized in endosomal compartments and excreted by the coalition of the plasma membrane with multivesicular bodies. Microvesicles have a large diameter ranging from 100 nm to 1000 nm and are created directly by blebbing of the plasma membrane. The diameter of apoptotic bodies, secreted by membrane budding during programmed cell death, ranges from 50 nm up to 5 μm. The major pathways for EV biogenesis and release are illustrated in Figs. 1.1 and 1.2.

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Fig. 1.1 Major pathways for EV secretion and biogenesis. The exosomes are formed via the inward budding of endosomes and secreted through a fusion of plasma membrane to the MVBs

Recently, the heterogeneity of EVs has been revealed. By utilizing cryogenic electron microscopy (cryo-EM), various shapes of EVs in body fluid samples have been detected (Hoog and Lotvall 2015). About 41% of EVs in human semen are found to be double vesicles, oval vesicles, triple vesicles, double special vesicles, lamellar bodies, and tubules, while the rest are single vesicles (Hoog and Lotvall 2015). The different forms of EVs indicate the presence of various subpopulations, which may have various biochemical characters. Due to the elevated heterogeneity of EVs, it is imperative to arrange them into particular groups to understand their composition and functions in pathological and physiological operations. On the other hand, the means of the analysis of EVs of various intracellular origins are still under development. Recently, a multidimensional EV refining plan has been used to obtain extremely refined EV subgroups for the following analysis of EV cargo. Therefore, more properties in the function and composition of particular EVs and EV-based biomarkers are obtained. This chapter aims to show the novel developments in technologies for EV characterization and quantification and to reveal the recent technologies with a high possibility for more progress. In this chapter, we make our best endeavor to supply the reader with wider aspects of the current status of the field. First, we reveal a general summary of the most utilized methods for EV isolation. Then, we classify the methods of EV characterization.

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Fig. 1.2 Release of EVs. Exosomes and microvesicles are released from live cells constitutively or by activation. The microvesicles are released via direct budding of the plasma membrane, while exosomes are formed from multivesicular bodies. Cells undergoing programmed cell death form apoptotic bodies by membrane budding

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Isolation Techniques

Five main subpopulations of EV isolation techniques have been promoted, namely size-based techniques, differential ultracentrifugation (DUC)-based techniques, polymer-based precipitation, immunoaffinity capture-based techniques, and microfluidic techniques. Different subpopulations of EVs are illustrated in Fig. 1.3.

1.2.1

Ultracentrifugation-Based Techniques

DUC procedures are the most common techniques used for EV isolation. In DUC, particulates are precipitated according to their shape, size, and density. The supernatant is undergone to next centrifugation with high centrifugal force, then the pellet is transferred to a suitable medium. Then, groups of EVs are isolated at various times of centrifugation (Yamashita et al. 2016). Thus, the pelleting time relies on the solvent viscosity and the physical characteristics of the particles. On the other hand, EV pellets procured are mainly contaminated with lipoproteins, protein collections, and

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Fig. 1.3 Different subpopulations of EVs. Various subtypes of EVs showed various sizes and release pathways. Exosomes are released from MVBs to carry protein and mRNA cargo for cell-cell communication; they can be characterized as small and large exosomes. Exomeres are small-size nanoparticles (90 retroelements in the HERV-K band (Booth et al. 2006; Dewannieux and Heidmann 2013; Fang et al. 2007; Subramanian et al. 2011).

2.16.3 Inner Membrane Peripheral Proteins Proteins accumulate inside the exosome cortex connections between exosomal membrane proteins, other related proteins, other partner proteins, and lipids. The inner cortex of exosomes is full of proteins (Pegtel and Gould 2019). Such factor scaffolding includes the defense of ezrin-radixin-moesin (ERM) (Hegmans et al. 2004; Shen et al. 2011; Wubbolts et al. 2003). This is to signal phosphorylation by linking plasma membrane proteins to cytoskeletal proteins and other scaffolds. (Bretscher et al. 2000). Exosomes, including Ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50), CD44, CD43, immunoglobulin superfamily member 8 (IGSF8), and prostaglandin F2 receptor negative regulator (PTGFRN), are not particularly abundant in protein ERM and ERM ligands (Melo et al. 2014; Nakamura et al. 2017; Pisitkun et al. 2004). Syntenin is another exosomal scaffolding element, and it is known to cluster exosomal proteins via its multiple proteinbinding motifs. In the membrane internal leaflet, syntenin also binds phosphatidylinositol-4,5-bisphosphate (PIP2). Interestingly, the relationship between syntenin and exosomal tetraspanin CD63 is complex, as high syntenin expression showed that its plasma membrane retains CD63 by masking its constituent endocytosis signal recognized by AP-2 (Latysheva et al. 2006)). Also, syntenin plays an important role in the exosomal sorting of syndecans (SDCs) (Baietti et al. 2012; Chatellard-Causse et al. 2002) and interaction with Alix proteins (ChatellardCausse et al. 2002).

2.17

Exosomal Enzymes

Exosomes are assigned as secreted, membranous and metabolically active structures. (Pegtel and Gould 2019). In their very earliest characterizations, this view was evident as osteogenic vesicles filled with bone formation enzymes like CD39, CD73, phosphatases, pyrophosphatases, calcium-binding anexins and phosphate transporters. (Anderson 1969; Trams et al. 1981; Bakhshian Nik et al. 2017; Clayton et al. 2011). RNA editing enzymes as lipases, proteases, glycosyltransferases, glycosidases and metabolic enzymes, may alter the contents of exosomes. As a result, exosomes may represent a source to produce macromolecules which are chemically distinctive from their cellular forms. (Li et al. 2016a; Nolte et al. 2016; Rilla et al. 2013; Ronquist et al. 2013; Théry et al. 2001; Thompson et al. 2013; Wubbolts et al. 2003; Pegtel and Gould 2019).

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Characterization and Fine Structure of Exosomes

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Bulk Inclusion and Soluble Proteins

Exosomes contain various cargo proteins and are rich in CD81, CD9, and CD63 (Kleijmeer et al. 1998; Théry et al. 2001). Furthermore, over 3000 proteins in exosomes are present and are released by just one single cell line (Li et al. 2016a); but how many of these proteins are actually enriched in exosomes remains unknown. In exosomes, soluble proteins can only be rarely integrated and will be free to act upon exosome-cell fusion in the receiving cells. (Pegtel and Gould 2019). Because most proteins are among most abundant in the cells (e.g. actin, tubulin, and glycolytic enzymes) and because heterology contributes to exosomal secretion of bacterial or synthetic proteins (Lai et al. 2015; Ridder et al. 2015), exosome biogenesis may include a small subset of exosomal proteins showing signs of active sorting of the cytoplasm and membrane content (Pegtel and Gould 2019).

2.19

Exosomal Glycoconjugates

Many forms of glycoconjugates, including glycoproteins and glycolipids, may be present in eukaryotic cells. Post-translation glycosylation is the most widely studied post-glycosylation modification of proteins (Costa 2017). EVs, especially exosomes, are rich in glycoconjugates (Cao et al. 2020; Xander et al. 2020), specific glycosignatures displayed by EVs. In EV biogenesis and in their association with other cells, surface glycoconjugates play an important role. Changes in glycosylation establish a stamp of diverse cancer cell types; consequently, the glycoconjugates and glycosignatures of EVs are potentially promising candidate molecules that are used to identify novel cancer biomarkers and to enhance the specificity and sensitivity of the existing clinical biomarkers, which are mostly glycosylated (Costa 2017). A glycan canopy bound to surface proteins and certain outer leaflets is the outermost surface of exosomes (Pegtel and Gould 2019). Using lectin panning and other approaches, this canopy has been investigated through lectin panning and other approaches; results have shown an exosomal enrichment for α-2,6-sialic acid and heparan sulfate and an otherwise heterogeneous population of surface carbohydrates. (Batista et al. 2011; Shimoda et al. 2017). Glycosylation has an important role in the EV field. In particular, it is greatly promising for the novel identification and for the improvement of clinical biomarkers in cancer (Costa 2017). Recent research has shown that exosomes derived from biofluids (proteins, nucleic acids, glycoconjugates, and lipids) can be used as biomarkers for the diagnosis, prognosis, and treatment reactions (Jiang et al. 2019). Glycoproteins comprise more than half of the entire markers in existing protein tumor markers approved by the FDA. In addition, the lack of specific oligosaccharides and effective analytical methods impeded the development of newly discovered glycoproteins as disease biomarkers (Jiang et al. 2019).

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Exosomal Lipids

Lipids are important constituents of exosomes, and studies have confirmed that they play a vital role in exosome biology. Compared with donor cells, exosomes are supplemented with sphingolipids, sphingomyelin, and cholesterol, including PS (Skotland et al. 2019a; Subra et al. 2007; Trajkovic et al. 2008; Wubbolts et al. 2003). Interestingly, the lipid content of exosomes shows similarities with that of lipid rafts, and exosomes have a higher order of lipid and more detergent resistance than other EVs (Skotland et al. 2019a). Lipids are not just structural contributors to exosomal membranes but are also important to exosome formation and extracellular release (Skotland et al. 2019b). The best biological structures are phospholipid vesicles whose biogenesis is easily replicated in a tube. Furthermore, new functions have been identified in phospholipid membranes. For example, the phospholipid membranes enveloped by the virus contain a protein for viral recognition and entry into target cells. The phospholipid membrane that surrounds the cells contains proteins that control the inflow and outflow of small molecules, transmit electrical currents, and modulate ligand-receptor interactions (Margolis and Sadovsky 2019). Lipids are essential elements of the exosomal membranes, and exosomes are enriched in specific lipids compared with their cell of origin (Skotland et al. 2017). It is well known that the lipid groups in the plasma membrane are asymmetrically distributed to sphingomyelin, other sphingolipids, and phosphatidyl choline PC (most of it at least) in the outer leaflet, while the others are anticipated to be found within the inner leaflet (Van Meer et al. 2008). The proportions of different lipid groups in several cell types such as oligodendroglial precursor cell line (Oli-neu) have been identified in studies of exosomes (Trajkovic et al. 2008), human B-cells (Wubbolts et al. 2003), mast cell line or RBL 2H3 (Laulagnier et al. 2004), dendritic cells (Laulagnier et al. 2004), and release of vesicles when guinea pig reticulocytes mature in vitro (Vidal et al. 1989). The exosome membrane includes PC, PS, phosphatidylethanolamine (PE), phosphatidylinositols (PIs), phosphatidylic acid (PA), cholesterol, ceramides, sphingomyelin, glycosphingolipids, as well as a variety of small abundant lipids, all of which shape the glycan canopy (Llorente et al. 2013; Skotland et al. 2017). Cholesterol, sphingomyelin, hexosylceramides, PS, and saturated fatty acids form the lipid constituent of exosomes (Colombo et al. 2014), all of these are constituents of plasma membranes (Kalluri 2016).

2.21

Exosomal Nucleic Acids

2.21.1 Exosomal RNAs Exosomes contain RNAs and can functionally transfer these extracellular RNAs to other cells and tissues. Ratajczak et al. first described this phenomenon (Ratajczak et al. 2006) and had documented the movement of octamer-binding transcription

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factor 4 (Oct-4) mRNA into other cells of stem cell exosomes that contain RNA, including Oct-4 mRNA, and its contribution to an increased expression of oct-4 in the cells in receiving the disease. Valadi et al. subsequently verified and expanded the presence of exosomal RNAs and exosome-mediated transfer (Valadi et al. 2007) and had identified the transmission between mast cells of exosome-mediated mRNA. Ratajczak et al. and Skog et al. (Ratajczak et al. 2006; Skog et al. 2008) described the exosome-mediated, intercellular glioblastoma transmission of RNAs. Petgal et al. (2010), provided the first functional background for exosome-mediated miRNA transfer. The amount of exosomal RNA present in individual exosomes is small, but what they contain is a highly skewed cellular RNA subpopulation (Pegtel and Gould 2019). Exosome-transferred miRNAs have a role in intercellular communication by repression of important mRNA targets in nearby or distant cells (Pegtel et al. 2010). MicroRNA cells and viruses control gene expression by repressing mRNA conversion to protein (Bartel 2004; Cullen 2009). The first kind of nucleic acids detected in exosomes were miRNA and mRNA (Gusachenko et al. 2013; Ratajczak et al. 2006; Théry et al. 2002; Valadi et al. 2007). Consequently, exosomes have described all other RNA types, including transmitting RNAs, long noncoding RNAs (lncRNAs), and viral RNA (Bullock et al. 2015; Gusachenko et al. 2013). Different types of the exosomal RNAs like mRNA and noncoding RNAs (e.g., microRNAs and lncRNAs) are efficient and may have an effect on transcriptome of the recipient cells (Crescitelli et al. 2013; Melo et al. 2014; Valadi et al. 2007). The transcriptome of receptor cells in exosomes is functional and can affect (Chen et al. 2014; Deregibus et al. 2007; Valadi et al. 2007) microRNAs and lncRNAs (Crescitelli et al. 2013; Melo et al. 2014; Valadi et al. 2007). It is operational in exosomes and can affect the genetic sequence of receiver cells (Chen et al. 2014; Deregibus et al. 2007; Valadi et al. 2007). The effects of nucleic acids on cell-to-cell contact in exosomes during the development of embryos and organs, standard physiology, and complex diseases are still under investigation (Kalluri 2016). Balaj et al. (2011) show that small pieces of single-stranded DNA occur in exosomes, and Kahlert et al. (2014), have detected large genome fragments, double-stranded DNA (>10 kb) surrounding all the chromosomes within the exosome. Overall, exosomal RNA profiles are enriched in specific RNA species relative to the cellular RNA profile, whereas the RNA profile of microvesicles appears more similar to that of their cell of origin (Wei et al. 2017). A crucial factor in interpreting the current literature is that most studies have examined the RNA composition of exosomes only in bulk rather than on a single-particle level; therefore, they likely underreported the actual complexity of exosomal RNAs (Verweij et al. 2018). Exosomal RNAs show some evidence of specific modification, such as enrichment of miRNAs with nucleotide additions at the 30 end (Koppers-Lalic et al. 2014) and with oligopyrimidine at the 50 end (Baglio et al. 2016), and this phenomenon could be related to as-yet-unknown exosomal sorting and/or RNA quality control mechanisms.

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2.21.2 Exosomal DNA Exosomes include DNA, comprising single-stranded DNA, double-stranded DNA, genomic DNA, mitochondrial DNA, as well as reverse-transcribed complementary DNA (Balaj et al. 2011; Kahlert et al. 2014; Sansone et al. 2017; Thakur et al. 2014). In contrast with exosome RNA (exoRNA), exosome DNA (ExoDNA) is not well known. The cytoplasm of damaged nucleus and mitochondrial DNA also leads to the processing of this damaged exosome DNA using uncertain mechanisms (Sharma and Johnson 2020). It is not clear whether certain DNAs, unlike other exosomal cargoes, can be uniquely inserted into exosomes. Furthermore, several reports show the entire genome sequence of the cell that produced exosomes through DNA sequencing (Kahlert et al. 2014; Thakur et al. 2014). exoDNA is in the organelle and how much the surface is bound is not clear. As for the physiological functions of exoDNA, DNA secretion dependent on exosome can be a useful marker for detecting cancer, viral infection, or chemotherapeutic intensity in the process of regulating the DNA content, probably in inflammation (Pegtel and Gould 2019).

2.21.3 Exosome Functions Exosomes are present in several biological fluids like synovial fluid, breast milk, semen, urine, saliva, amniotic fluid, and multi-cell malignant ascites, such as fibroblasts, intestinal epithelial cells, neurons, adipocytes, tumor cells, blood cells, ECs, immunocytes, platelets, and smooth muscle cells (Bobrie et al. 2011; Fauré et al. 2006; Ge et al. 2012; Hu et al. 2012; Liao et al. 2014; Mignot et al. 2006; Saunderson et al. 2014), but are still known to be extremely stable reservoirs of biomarkers (Boukouris and Mathivanan 2015; Cheng et al. 2014; Kalra et al. 2013). At the time of exosome formation, the morphology, function, and heterogeneity of exosomes depend on the cell of origin and on the status of the originating tissue or cell. At first, exosomes were thought to be involved in a process that eliminates unneeded reticulocyte membrane proteins (Johnstone 2006). Research has shown that exosomes are cellular waste bags that remove nonfunctional cellular components. Endocytic vesicles are also involved in the processing of cell surface proteins and signaling molecules (Pan et al. 1985; Théry et al. 2002). Recent research on the eukaryotic ribosomal RNA processing pathway, which plays a central role in the exact 30 ends of many forms of RNAs, has established a complex consisting of 10 riboexonucleases or exosomes. The exosome eliminates excess ribosomal RNA, unused intermediates, and cytoplasm degradations of poly(A)mRNAs. Such a complex tends to work inside the nucleus with a revised mRNA monitoring system, which disintegrates transcripts in reaction to processing and import pathways with mRNA (Butler 2002). The main role of exosomes is the transmission of their constituent to recipient cells from donor cells, resulting in genetic and phenotypic cell modifications (Ren et al. 2016). Exosome-mediated transfer reported three potential mechanisms: first,

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the exosomes fuse with the cell’s plasma membrane and then release the exosomal cargos into the cytoplasm; then, the exosomes communicate with the target cells via interactions or lipids such as PS through the receptor ligand; and endocytosis or transcytosis is used to internalize exosomes to recipient cells (Nanbo et al. 2013). Exosomes play an important role in various biological processes such as angiogenesis, antigenicity, apoptosis, cellular homeostasis, inflammation, and intercell signaling. Exosomes may transfer and communicate with RNA, proteins, enzymes, and lipids to the receiving cells, thereby influencing the physiological and pathological processes of various diseases such as cancer, neurodegenerative disorder, infections, and autoimmune diseases (Gurunathan et al. 2019; Raposo and Stoorvogel 2013). Exosomes have demonstrated important roles in immune response (Hobbs 2020; Xie et al. 2019), tumor development, and neurodegenerative disorders, according to recent studies (Mäger et al. 2020; Riazifar et al. 2019; Zhang et al. 2015a). Esther et al. reported that activated T cells down-regulate the immune response during interaction between T cells and DCs by recruiting exosomes derived from dendritic cells (DC) that contain MHC class II (Nolte-‘t Hoen et al. 2009). The proliferation and chemical invasions of the adenocarcinoma lung line A549 were induced by exosomes extracted from platelets treated with thrombin and collagen (JanowskaWieczorek et al. 2005). Nevertheless, exosomes derived from SGC7901 induce the proliferation of SGC7901 and another line of gastric cancer cells, BGC823 (Qu et al. 2009). CD147-positive exosomes derived from ovarian epithelial cancer cells were also identified to promote angiogenesis in ECs in vitro (Millimaggi et al. 2007). In vitro, Webber et al. incubated primary fibroblasts with exosomes derived from mesothelioma cell line, a prostate cancer cell line, a bladder cancer cell line, a colorectal cancer cell line, and a breast cancer line. Ironically, they found it possible to turn fibroblasts into myofibroblasts (Webber et al. 2010). A similar incident was reported by Cho et al., who found that tumor-derived exosomes transformed mesenchymal stem cells within tumor stroma into cancer-associated myofibroblasts (Cho et al. 2012). The role of exosomes was recognized in the above-mentioned studies, but whether the recipient cells are affected by a specific class of molecules present in the exosomes remains a matter of debate. The bioactivity of the recipient cells is believed to be regulated by exosomes through the transport of lipids, proteins, and nucleic acids in the extracellular space (Yagci et al. 2019; Zhang et al. 2015a). Intercellular contact is one of the most interesting functions of exosomes; exosomes are thought to act as messengers carrying various effectors or signaling macromolecules between supposedly very similar cells (Vlassov et al. 2012). In addition, they are promising delivery vectors because of their remarkable ability to migrate, target, and selectively internalize into different cells (Betzer et al. 2020). Exosomes transmit specifically integrated compounds, like proteins, lipids, and ribonucleotides, including miRNA, to target cells (Desrochers et al. 2016; Valadi et al. 2007). Exosomes have great potential as therapeutic drug delivery tools due to their cell-specific transport capability to appropriate cells (Pitt et al. 2016). Numerous obstacles including inadequate biodistribution and targeting of diseased tissue/ cells, as well as the underlying immunogenicity, the cytotoxicity of carriers and their

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degradation products, and the rapid blood clearance of traditional delivery systems are facing clinical translation (Bertrand and Leroux 2012; Coviello et al. 2007; Dobrovolskaia and McNeil 2007; Manzoor et al. 2012). Accordingly, for clinical applications, only a limited number of nanoparticles with drug cargo have been approved (Tang et al. 2012). Endogenous drug delivery carriers, compared to synthetic carriers, are more consistent with the dynamic in vivo setting and can target tissues more effectively, thus resulting in increasing drug efficacy and minimizing side effects, as will be expected (Fais et al. 2013; Joseph et al. 2020). The new exosomes from internal cell membrane are being studied (Fais et al. 2013).

2.21.4 Exosomes in Angiogenesis New capillary development from established blood vessels is known as angiogenesis, and it is mediated by a dynamic multi-stage cycle of cellular activities (Bazigou and Makinen 2013). In multicellular eukaryotes, signal-complex integration is an important factor in the intercellular communication during angiogenesis. For example, during recirculation and trans-endothelial migration processes, the vascular ECs and T-lymphocytes closely interact with each other. In support of this theory, Kaur et al. (2014) showed that T-cell-derived EVs modify the vascular endothelial growth factor (VEGF) signaling, tube forming, and gene expression. Remarkably, the EVs derived from JinB8 cells increased the phosphorylation of basal VEGFR2, indicating that VEGF–VEGFR2 signaling in angiogenesis can be indirectly altered by CD47 by targeting ECs through EV trafficking. Additionally, exosomal tumors play a significant role in angiogenesis (Chicón- and Tirado 2020; Hood et al. 2009; Wee et al. 2019). Taking ECs from cancer-derived exosomes promotes angiogenesis under hypoxic conditions by activating ECs’ proangiogenic secretome (Kucharzewska et al. 2013).

2.21.5 Exosomes in Apoptosis Apoptosis is a highly regulated cycle associated with the typical turnover of healthy cells, but it also happens in disease situations as inflammation, infection, autoimmunity and cancer. In malignant conditions such as cell viability, oncogenic mutations induce a homeostatic imbalance (Gurunathan et al. 2019. Apoptotic entities, a large class of EVs, are released as apoptotic cell disassembly products from dying cells (Caruso and Poon 2018). A number of morphological changes occur to apoptotic cells causing a dying cell to dismantle. Recently, apoptotic cell disassembly has been divided into three distinct morphological steps: apoptotic membrane blebbing, thin membrane protrusion, and finally, the development of apoptotic bodies (ranging from 1 μm to 5 μm) (Kerr et al. 1972). Analysis of proteomics of the exosomes and apoptotic vesicles shows an increase in the number of these vesicles in apoptotic cells and a differential enrichment of proteins in each type of vesicle. Tumor apoptotic cells communicate

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with neighboring cells via intercellular interaction and through soluble and EV-encapsulated signal mediators (Gregory and Pound 2010). Critically, an apoptotic body is characterized as a vesicle dependent on apoptosis and encapsulates a wide range of bioactive molecules and cell organelles (AtkinSmith et al. 2015). Apoptotic bodies have a wide-sized heterogeneity between approximately 50 nm and several microns (Atkin-Smith and Poon 2017; Zhu et al. 2019), and they can originate from other organelles, such as endoplasmic reticulum and mitochondria (Kakarla et al. 2020). On the other hand, as a result of cell stress, stromal cell-derived EVs (