124,99 €
Mehr erfahren.
- Herausgeber: Wiley-VCH GmbH
- Kategorie: Wissenschaft und neue Technologien
- Sprache: Englisch
Biomedical Applications of Extracellular Vesicles
Unique resource focusing on biomedical applications and clinical translation of extracellular vesicles in science and medicine
Focusing on key points to better understand extracellular vesicles (EVs) and their development, Biomedical Applications of Extracellular Vesicles describes in detail the biogenesis of EVs, the mechanism of intercellular communication, and the treatment of various diseases of EVs and the EV-based drug delivery platforms. An application-oriented resource, the work presents rapidly newer biomedical and clinical applications of natural and engineered EVs such as drug delivery, diagnosis, prognosis monitoring, immunotherapy, and more.
The first part of this book provides a basic background on EVs. Next, the book introduces the excellent therapeutic effects of EVs themselves and the underlying mechanisms, followed by how EVs from different sources were used to construct drug delivery platforms. The latest research on EVs from leading groups around the world is presented.
Sample topics covered in Biomedical Applications of Extracellular Vesicles include:
- Biogenesis of various EVs
- Pros and cons of the different instrumental and methodological developments for analytical strategies applied to EVs
- EVs in treatment of major diseases, such as cancer, cardiovascular and respiratory diseases
- Current methods of engineering EVs, and a comparison of the advantages and disadvantages of each method
- Biomaterials, such as hydrogels, scaffolds, and microneedles, that have been developed to further enhance the therapeutic efficacy of EVs
- Key challenges, such as quality control, scalability, and biosafety, that limit the clinical and industrial translation of EVs
Explaining in detail how extracellular vesicles are produced and engineered, along with potential applications and commercial developments of EVs in science and industry, Biomedical Applications of Extracellular Vesicles is an essential resource on the subject for chemists, cell biologists, and molecular physicists.
Sie lesen das E-Book in den Legimi-Apps auf:
Seitenzahl: 389
Veröffentlichungsjahr: 2023
Ähnliche
Table of Contents
Cover
Table of Contents
Title Page
Copyright
Preface
1 Extracellular Vesicles and Their Biomedical Applications: An Overview
1.1 Introduction
1.2 Biogenesis and Composition of Extracellular Vesicles
1.3 Biological Functions of Extracellular Vesicles
1.4 Extracellular Vesicles Isolation and Limitations
2 Biogenesis and Identification of Extracellular Vesicles
2.1 Biogenesis of Extracellular Vesicles
2.2 Identification of Extracellular Vesicles
References
3 Therapeutic Potential of Extracellular Vesicles from Different Cell Sources
3.1 Extracellular Vesicles Derived from Stem Cells (SCs)
3.2 Extracellular Vesicles Derived from Immune Cells
3.3 Extracellular Vesicles Derived from Cancer Cells
3.4 Extracellular Vesicles Derived from Plants
References
4 Biomedical Applications of Extracellular Vesicles in Treatment of Disease
4.1 Tissue Engineering and Regenerative Medicine
4.2 Metabolic Diseases
4.3 Cardiovascular Diseases
4.4 Respiratory Diseases
4.5 Cancers
4.6 Conclusion and Perspectives
References
5 Applications of Engineered Extracellular Vesicles
5.1 Engineering EVs for Cargo Loading
5.2 Engineering EVs for Surface Modification
References
6 Current Technology for Production, Isolation, and Quality Control of Extracellular Vesicles
6.1 Production of EVs
6.2 Extraction of EVs
6.3 Quality Control of EVs
References
7 Prospects and Limitations of Clinical Application of Extracellular Vesicles
7.1 Application of Exosomes as Liquid Biopsy in Clinical Diagnosis
7.2 Exosomes—It has Become a Star Molecule in Disease Diagnosis
7.3 The Commercial Application of Exosomes
7.4 Commercial Development of Exosomes
7.5 Issues and Challenges
References
8 Conclusion and Future Perspectives
8.1 Summary and Conclusions
8.2 General Trends and Developments
8.3 Challenges for Future Research
Index
End User License Agreement
List of Tables
Chapter 7
Table 7.1 Exosome as potential predictive biomarkers in different cancers.
List of Illustrations
Chapter 2
Figure 2.1 (a) Exosomes in the secondary phloem of woody plants.(b) SEM ...
Figure 2.2 TEM picture of EVs.
Figure 2.3 EV surface structure observed by AFM.
Figure 2.4 Picture of inflammatory EVs.
Figure 2.5 The size of EVs was identified by NTA. (a) Nanotracking analysis ...
Figure 2.6 The size of EVs was identified by DLS. (a) the size of MDA‐MB‐231...
Figure 2.7 WB result of representative exosomal proteins.
Figure 2.8 nFCM result of EVs. (a) Dose‐dependent uptake profiles of human s...
Figure 2.9 Droplet digital ExoELISA 3 calibration results showing that the d...
Figure 2.10 ERP current−time recordings obtained with a CNP positioned near ...
Figure 2.11 2‐dimensional tSNE mapping of individual EVs analyzed for protei...
Figure 2.12 The microfluidic system for on‐chip detection of circulating EVs...
Chapter 3
Figure 3.1 A proposed model for mechanisms underlying stem cell EVs therapeu...
Figure 3.2 Various therapeutic effects of MSCs.(a) MSCs have the ability...
Figure 3.3 The therapeutic effect of MSCs‐derived EVs on MI/RI.(a) The p...
Figure 3.4 Efficacy of neural stem cell‐derived EVs in animal models of trau...
Figure 3.5 EVs derived from immune cells play distinct roles in regulating i...
Figure 3.6 Exosomes derived from M2 macrophages target areas of inflammation...
Figure 3.7 The schematic figure of DCs‐derived exosomes therapeutic effects ...
Figure 3.8 Cancer‐derived EVs directly regulate tumor progression. (a) Cance...
Figure 3.9 Exosome derived from edible tea flowers induced mitochondrial dam...
Chapter 4
Figure 4.1 Schematic demonstration of osteoporosis treatment mediated by Mu‐...
Figure 4.2 Development of small EVs with nanomorphology memory for the promo...
Figure 4.3 Schematic illumination of milk EVs‐mediated anti‐inflammatory wou...
Figure 4.4 Scheme of Schwann cells‐derived EVs released from hydrogel for de...
Figure 4.5 Microparticles containing gingival mesenchymal stem cells‐derived...
Figure 4.6 Schematic demonstration of small intestinal submucosa membrane fu...
Figure 4.7 Oral administration of insulin‐loaded milk‐derived exosomes indic...
Figure 4.8 Exosomes derived from human umbilical cord mesenchymal stem cells...
Figure 4.9 Schematic illumination of reverse of diabetic β‐cell dedifferenti...
Figure 4.10 Intrarenal delivery of MSC‐derived EVs for myocardial injury att...
Figure 4.11 Schematic demonstration of hypoxia‐pretreated small EVs (sEVs) f...
Figure 4.12 Treatment of
Akkermansia muciniphila
and derived EVs promotes in...
Figure 4.13 EVs with surface modification with CD47 for treatment of myocard...
Figure 4.14 Schematic demonstration of therapeutic effect of human pluripote...
Figure 4.15 Schematic illumination of mitochondria‐rich EVs derived from iPS...
Figure 4.16 Schematic demonstration of exosome‐eluting stents for the treatm...
Figure 4.17 Alteration of miRNA expression in cardiac‐derived exosomes by he...
Figure 4.18 Schematic representation of Treg induction by MSC‐exosomes via F...
Figure 4.19 Schematic illumination of exosomes derived from Atorvastatin‐pre...
Figure 4.20 Schematic demonstration of the angiogenesis promotion effects of...
Figure 4.21 Schematic illumination of antibody‐functionalized magnetic nanop...
Figure 4.22 Schematic demonstration of exosomes derived from M2‐like macroph...
Figure 4.23 Schematic illumination of heart homing peptide‐modified exosomes...
Figure 4.24 Schematic representation of the functions of miR‐155‐rich exosom...
Figure 4.25 Schematic illumination of airway epithelial cells‐derived EVs (A...
Figure 4.26 Schematic illumination of the preparation of synthetic bacterial...
Figure 4.27 Schematic representation of bioinspired artificial exosomes for ...
Figure 4.28 Schematic demonstration of exosomes derived from M1 macrophage f...
Figure 4.29 Schematic illumination of tumor cell‐derived EVs for the deliver...
Chapter 5
Figure 5.1 Engineering strategies for EVs.
Figure 5.2 Cargo loading strategies for EVs.
Chapter 6
Figure 6.1 Schematic and photograph of the 2D and 3D culture system.(a) ...
Figure 6.2 Physically triggered EV release in blood vessels due to physiolog...
Figure 6.3 Schematic diagram of exosome separation based on differential ult...
Figure 6.4 Tangential Flow Analyte capture (TFAC) technique for particle sep...
Figure 6.5 EVs separation principle based on size exclusion chromatography....
Figure 6.6 Schematic diagram of EVs microfluidic separation method.
Figure 6.7 Schematic diagram of (a) EV‐FISHER synthesis and (b) plasma EVs s...
Figure 6.8 TEM image of cup‐shaped EVs with a diameter smaller than 100 nm....
Figure 6.9 Schematic diagram of the Exotest—an ELISA used for EVs detection ...
Figure 6.10 ATR spectrum obtained from diluting samples of REVs.
Figure 6.11 NTA measured the mean value, concentration, and average particle...
Figure 6.12 Schematic diagram of the nFCM constructed in the laboratory.
Chapter 7
Figure 7.1 Biogenesis and identification of exosomes.
Figure 7.2 Exosomes as a new target for liquid biopsy.
Figure 7.3 ExoFlo sourced from the Directbiologics' website (https://directb...
Figure 7.4 engEx™ PLATFORM sourced from the Codiak BioSciences.
Figure 7.5 ASCEplus sourced from the ExoCoBio's website (http://www.exocobio...
Guide
Cover
Table of Contents
Title Page
Copyright
Preface
Begin Reading
Index
End User License Agreement
Pages
iii
iv
xi
xii
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
140
141
142
143
144
145
146
147
148
149
150
151
152
153
154
155
156
157
158
159
160
161
162
163
164
165
166
167
168
169
170
171
172
173
174
175
176
177
178
179
181
182
183
184
185
186
187
188
189
190
191
192
193
194
195
196
197
Biomedical Applications of Extracellular Vesicles
Edited by Zhenhua Li, Xing-Jie Liang, and Ke Cheng
Editors
Prof. Zhenhua LiThe Tenth Affiliated Hospital of Southern Medical UniversityDongguanChina
Prof. Xing‐Jie LiangNational Center for Nanoscience and TechnologyBeijingChina
Prof. Ke ChengColumbia UniversityNew YorkNY, US
Cover: © Jason Drees/Shutterstock; Marcus Millo/Getty Images/iStockphoto
All books published by WILEY‐VCH are carefully produced. Nevertheless, authors, editors, and publisher do not warrant the information contained in these books, including this book, to be free of errors. Readers are advised to keep in mind that statements, data, illustrations, procedural details or other items may inadvertently be inaccurate.
Library of Congress Card No.: applied for
British Library Cataloguing‐in‐Publication Data A catalogue record for this book is available from the British Library.
Bibliographic information published by the Deutsche Nationalbibliothek The Deutsche Nationalbibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data are available on the Internet at <http://dnb.d-nb.de>.
© 2024 WILEY‐VCH GmbH, Boschstraße 12, 69469 Weinheim, Germany
All rights reserved (including those of translation into other languages). No part of this book may be reproduced in any form – by photoprinting, microfilm, or any other means – nor transmitted or translated into a machine language without written permission from the publishers. Registered names, trademarks, etc. used in this book, even when not specifically marked as such, are not to be considered unprotected by law.
Print ISBN: 978‐3‐527‐35212‐8ePDF ISBN: 978‐3‐527‐84213‐1ePub ISBN: 978‐3‐527‐84214‐8oBook ISBN: 978‐3‐527‐84215‐5
Preface
Extracellular vesicles (EVs) are a type of nano‐sized particles that are secreted by cells and widely found in tissues, serum, and body fluids. In the past decades, EVs have drawn increasing interest because of their involvement in various physiological and pathological activities, such as cell–cell communication, cell death, and immune regulation. The understanding of EV features and biofunctions also facilitates the progress of biomedical applications of EVs. For instance, EVs have been used as drug delivery systems due to their inherent ability of substance transportation. In addition, scientists have conducted various attempts aiming to achieve the clinical and industrial translation of EV‐based therapeutics. The purpose of this book is to provide a detailed image of the present understanding of EVs and derived artificial nanovesicles and their biomedical applications.
This book includes eight chapters that focus on different aspects of EVs. Each chapter introduces the current knowledge of EVs in a specific area of interest and discusses the possible future direction. In Chapter 1, the authors provide a brief introduction to EVs, including their nature and biological functions. In Chapter 2, the authors focus on EV biogenesis and identification. Specifically, the biogenesis of various EVs, including exosomes, microvesicles, apoptotic bodies, and large oncosomes, are systematically summarized. In addition, current techniques in the identification of EVs, such as electron microscopy, nanoparticle tracking analysis, and dynamic light scattering are introduced. Chapter 3 mainly discusses the biological functions of EVs from different sources, such as stem cells, immune cells, tumor cells, plants, and microorganisms. Their applications in anti‐inflammation, tissue regeneration, and neuroprotection are also discussed. In Chapter 4, the authors summarize the recent achievements in the applications of EVs in disease therapy. Several major diseases, including cardiovascular diseases, tissue engineering and regenerative medicine, cancer, respiratory diseases, and metabolic diseases, are selected to reveal their therapeutic efficacy. To further enhance the therapeutic efficiency and reduce the side effects of EVs, researchers have also been devoted to developing novel engineered EVs via various techniques. The engineered EVs for biomedical applications are discussed in Chapter 5. In Chapter 6, the authors introduce the current approaches for the production, isolation, and quality control of EVs. Chapter 7 summarizes the current progress of EVs in clinical applications. In addition, the potential challenges and future trends of clinical translations of EV are discussed. In Chapter 8, a summary of the main contents of this book is provided as a concluding chapter.
Taken together, this book summarizes the current knowledge and potential applications of EVs across various basic and technical disciplines. We further discussed the challenges in the current EV research, and their translation in clinical and industrial research. We hope this book could act as a handbook to help scientists in the field of EV study and promote the development of EV research. We would like to give our sincere gratitude to all the authors, the reviewers, and the editors who have contributed and assisted in this book. Without their valuable time and efforts, this book would never be possible.
Dongguan, Guangdong, China, Zhenhua Li
Beijing, China, Xing‐Jie Liang
New York, NY, USA Ke Cheng
1Extracellular Vesicles and Their Biomedical Applications: An Overview
Xing‐Jie Liang1, Ke Cheng2, and Zhenhua Li3,4
1CAS Key Laboratory for Biological Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology, Beijing, 100190, China
2Department of Biomedical Engineering, Columbia University, New York, 10032, USA
3The Tenth Affiliated Hospital of Southern Medical University, 78 Wanjiang Avenue, Dongguan, Guangdong, 523059, China
4Guangdong Provincial Key Laboratory of Shock and Microcirculation, Southern Medical University, Shatai South Avenue Guangzhou, Guangdong, 510080, China
1.1 Introduction
Extracellular vesicles (EVs) are small membrane‐bound vesicles that are secreted by a variety of cells, including stem cells, immune cells, and cancer cells. These vesicles contain a range of molecules, including lipids, proteins, and nucleic acids, that reflect the cellular and physiological state of the parent cell, and play a variety of physiological and pathological roles in the body. They can act as signaling molecules, carrying bioactive molecules such as proteins, lipids, and nucleic acids, between cells and tissues. They can also act as vehicles for the transfer of genetic material, such as microRNAs, between cells. The biomedical applications of EVs have been widely explored in recent years, and they are emerging as a promising tool for diagnosis, therapy, and drug delivery.
1.2 Biogenesis and Composition of Extracellular Vesicles
EVs are released by cells into the extracellular space and can be classified into three main types based on their biogenesis and size: exosomes, microvesicles, and apoptotic bodies. Exosomes are small vesicles (30–150 nm) that are released by cells through the endosomal pathway, while microvesicles (100–1000 nm) are formed by budding of the plasma membrane. Exosomes and microvesicles are collectively referred to as small EVs. Exosomes are enriched in endosomal markers such as CD63 and Alix, while microvesicles are enriched in plasma membrane markers such as phosphatidylserine and CD31. Apoptotic bodies are referred to as large EVs (1–5 μm) that are released by cells undergoing programmed cell death. The biogenesis of each type of EV is distinct and involves the selective packaging of different molecules.
The composition of EVs varies depending on the type of vesicle, the cell of origin, and the physiological or pathological condition. EVs can contain a range of biological molecules, including proteins, nucleic acids (RNA and DNA), lipids, and carbohydrates. Proteins present in EVs include membrane proteins, cytosolic proteins, and extracellular matrix proteins. Nucleic acids in EVs include mRNAs, miRNAs, and long noncoding RNAs. Lipids in EVs include phospholipids, sphingolipids, and cholesterol.
1.3 Biological Functions of Extracellular Vesicles
EVs have been shown to regulate a range of cellular processes, including proliferation, differentiation, and apoptosis. EVs have been shown to play a role in many biological processes, including immune regulation, inflammation, angiogenesis, tissue repair, and cancer progression. They have also been implicated in the pathogenesis of many diseases, including cardiovascular disease, neurological disorders, and infectious diseases. Given their diverse functions, EVs have the potential to be used for a range of biomedical applications, especially in the fields of diagnostics, therapeutics, and regenerative medicine.
In the area of diagnostics, EVs have great potential as diagnostic biomarkers for a variety of diseases. They are stable in biological fluids, and their cargo can reflect the physiological state of the cell of origin, making them attractive targets for disease diagnosis and monitoring. Cancer is one area where EVs have shown particular promise as diagnostic biomarkers. Cancer cells release large numbers of EVs into the circulation, which can be detected in blood samples. These EVs contain specific biomolecules that can be used to detect and monitor the progression of cancer. For example, circulating tumor cells (CTCs) shed EVs that contain proteins and nucleic acids that are specific to the cancer cells. These biomolecules can be used to develop noninvasive diagnostic tests for cancer. In addition, EVs released by cancer cells can provide information about the molecular profile of the tumor, which can be used to develop personalized cancer treatments. EVs have also been explored as diagnostic biomarkers for other diseases, such as cardiovascular disease, neurological disorders, and infectious diseases. For example, EVs released by damaged or diseased heart tissue contain specific proteins and nucleic acids that can be used to diagnose and monitor cardiovascular disease.
Recent research has shown that EVs have enormous potential as therapeutic agents for a variety of diseases, including cancers, neurodegenerative diseases, and cardiovascular disease. Here are some of the key ways that EVs are being investigated for therapeutic use. In cancer therapy, EVs derived from stem cells have been shown to inhibit tumor growth and metastasis in several animal models of cancer. EVs have also been explored as a means of drug delivery, as they can be engineered to carry therapeutic molecules directly to tumor cells. In neurodegenerative diseases, EVs derived from stem cells have shown promise in treating neurodegenerative diseases such as Parkinson's and Alzheimer's. These EVs contain factors that can protect neurons from damage and promote their survival. In cardiovascular disease, EVs derived from endothelial cells have been shown to improve heart function after a heart attack in animal models. EVs can also carry proteins and genetic materials that promote angiogenesis, which can help to repair damaged blood vessels. In autoimmune diseases, EVs have been shown to play a role in regulating immune responses, and EVs derived from stem cells have been explored as a potential treatment for autoimmune diseases such as multiple sclerosis and rheumatoid arthritis. Overall, EVs offer a promising avenue for the development of novel therapeutics for a wide range of diseases. While there are still many challenges to overcome, including standardization of isolation and characterization techniques, early results suggest that EVs may be a powerful tool in the fight against disease.
EVs have been shown to have tremendous potential in regenerative medicine. EVs are small, membrane‐bound structures secreted by cells that contain a variety of biomolecules, including lipids, proteins, and nucleic acids. They play a critical role in intercellular communication and have been shown to have therapeutic effects in a variety of preclinical models of disease. Here are some of the key ways that EVs are being investigated for regenerative medicine. In tissue repair, EVs derived from stem cells have been shown to promote tissue repair in a variety of preclinical models, including wound healing, bone regeneration, and cartilage repair. These EVs contain a variety of growth factors and other molecules that can promote cell proliferation and differentiation, as well as regulate the immune response. In cardiovascular regeneration, EVs have been shown to have beneficial effects on cardiac function following a heart attack in animal models. EVs can carry proteins and genetic material that promote angiogenesis, or the growth of new blood vessels, which can help repair damaged tissue and improve blood flow. In neuroregeneration, EVs have shown promise in promoting neuronal survival and regeneration in preclinical models of neurological disease and injury, including stroke and traumatic brain injury. These EVs can carry neuroprotective factors, and promote the growth and differentiation of new neurons. Overall, EVs offer a promising approach to regenerative medicine, while there are still challenges to overcome, including standardization of isolation and characterization techniques.
1.4 Extracellular Vesicles Isolation and Limitations
EVs have attracted considerable attention as potential biomarkers for various diseases, including cancer, cardiovascular disease, and neurodegenerative disorders. However, the isolation of EVs is a complex and challenging process, and there are several limitations in the manufacturing and clinical translation of EV‐based therapeutics.
Isolation of EVs is a crucial step in their study and application. There are several methods for isolating EVs, including ultracentrifugation, size‐exclusion chromatography, immunoaffinity capture, and microfluidics‐based techniques. Each method has its advantages and limitations in terms of purity, yield, and scalability. Ultracentrifugation is the most commonly used method for EV isolation, but it can result in coisolation of non‐EV contaminants, such as protein aggregates and lipoproteins. Size‐exclusion chromatography can provide higher purity, but it is less efficient and requires specialized equipment. Immunoaffinity capture can specifically isolate EVs expressing certain surface markers, but it can result in low yields and requires specific antibodies. Microfluidics‐based techniques have the potential for high throughput and precise isolation, but they are still in the development stage and not yet widely used.
There are also some limitations in both manufacturing and clinical translation. One of the major limitations in manufacturing EV‐based therapeutics is the variability in EV isolation methods that can lead to variations in the size, content, and purity of EVs. This can impact the efficacy and safety of EV‐based therapeutics. Moreover, the scalability of EV isolation methods is also a challenge, as large‐scale production of EVs is currently not feasible. Another challenge in manufacturing EV‐based therapeutics is the lack of standardized protocols for EV characterization, which can lead to inconsistencies in the reporting of EV‐related data. This can make it difficult to compare data between different studies and hinder the development of EV‐based therapeutics. As for the clinical translation of EV‐based therapeutics is the lack of a regulatory framework for EVs, there is currently no FDA‐approved EV‐based therapy, and the regulatory pathway for EVs is not well defined. This can make it challenging for researchers and companies to develop and commercialize EV‐based therapeutics. Another challenge in clinical translation is the heterogeneity of EVs, which can make it difficult to define a specific population of EVs for therapeutic use. Moreover, the biodistribution and pharmacokinetics of EVs are not well understood, and there is limited data on the safety and efficacy of EV‐based therapeutics in humans.
In conclusion, while EVs hold great potential as biomarkers and therapeutics for various diseases, there are several challenges in the isolation, manufacturing, and clinical translation of EV‐based products. These challenges highlight the need for standardized protocols, regulatory guidance, and further research to fully harness the potential of EVs for clinical use.
2Biogenesis and Identification of Extracellular Vesicles
Dandan Ding1,2, Xing Zhang3, Yu Zhao3, Xiaoya Li3, Qingqing Leng3, and Zhenhua Li1,2
1The Tenth Affiliated Hospital of Southern Medical University, 78 Wanjiang Avenue, Dongguan, Guangdong, 523059, China
2Guangdong Provincial Key Laboratory of Shock and Microcirculation, Shatai South Avenue, Guangzhou, Guangdong, 510080, China
3Hebei University, College of Pharmaceutical Science, Key Laboratory of Pharmaceutical Quality Control of Hebei Province and Key Laboratory of Medicinal Chemistry and Molecular Diagnosis of Ministry of Education, Chemical Biology Key Laboratory of Hebei Province, Institute of Life Science and Green Development, Baoding, 071002, China
Extracellular vesicles (EVs) play an important role in physiological and pathological processes as widely available bioactive substances involved in intercellular information exchange [1–3]. Compared with conventional nanomaterials, EVs are one of the most promising candidates in nanomedicine because of their biocompatibility, biodegradability, low toxicity, and non‐immunogenicity. As an emerging research field in recent years, it has been found that EVs can be involved in the occurrence and development of a variety of diseases and have great potential as markers of diseases [4, 5]. With in‐depth research, the exploration of their biogenesis is important for the early diagnosis and treatment of diseases. In addition, since the existing isolation techniques are based on the size and structure of EVs, and the capture of some membrane proteins, it is difficult to completely distinguish them from other vesicles and macromolecular protein complexes, which requires their identification. Chapter 2 focuses on EVs through biogenesis mechanism and identification.
2.1 Biogenesis of Extracellular Vesicles
On the basis of their biogenesis, cargo, and size, EVs are generally divided into four broad categories: exosomes, microvesicles (MVs), apoptotic bodies (ApoBDs), and large oncosomes (LOs). Exosomes (30–200 nm in size) and MVs (200–2000 nm in size) are produced by almost all kinds of healthy living cells, derived from the fusion of multivesicular bodies (MVBs) with the plasma membrane, and formed by a direct outward budding of the plasma membrane, respectively [6]. ApoBDs (500–2000 nm in size) are released by apoptotic cells during programmed cell death [7]. LOs (1–10 μm in size) are cancer cell‐derived microvesicles [8].
2.1.1 Biogenesis of Exosome
Exosomes are usually generated through the endosome pathway [9]. First, the invagination of the plasma membrane leads to the formation of endosomes. Then, intraluminal vesicles (ILVs) were further formed by the inward budding of the limiting membrane inside endosomes. ILVs further accumulate in the lumen to form multivesicular bodies (MVBs) [1, 10]. Eventually, MVBs may be degraded by fusion with lysosomes [11]. Alternatively, MVB fuses with the plasma membrane and releases ILVs into the extracellular environment in the form of exosomes [12].
The transformation of endosomes into MVBs containing ILVs is an important step of exosome biogenesis. The main mechanism of ILVs' formation is the endosomal sorting complex required for transports (ESCRTs) [13]. ESCRTs include four different protein complexes (ESCRT‐0, ESCRT‐I, ESCRT‐II, and ESCRT‐III) [14–17]. In the process of ILVs formation, ESCRT complexes are gradually recruited to the endosomal membrane. First, ESCRT‐0 is recruited to the early endosomal membrane via direct interaction of the hepatocyte growth factor‐regulated tyrosine kinase substrate (HRS) with phosphatidylinositol 3‐phosphate (PIP3) on the endosomal membrane [18–21]. HRS can recognize and internalize the ubiquitination proteins [22, 23]. Then, ESCRT‐0 recruits ESCRT‐I to endosomal membranes by HRS binding to tumor susceptibility gene 101 (TSG101) of ESCRT‐I [24, 25]. ESCRT‐0 and ESCRT‐I are jointly responsible for the sorting of ubiquitinated proteins [26]. ESCRT‐II binds to ubiquitin, and ESCRT‐I binds to Vps36 subunit [27–29]. ESCRT‐I/ESCRT‐II complex has been thought to play a role in the inward budding of the endosomal membrane [27, 30, 31]. ESCRT‐II activates ESCRT‐III to bind to the budding neck [32, 33]. Finally, AAA‐ATPase Vps4 drives the plasma membrane separation, generating ILVs, and leading to the formation of MVBs [34–36].
Several other ESCRT mechanisms have been demonstrated in the formation of ILVs. (i) The syndecan–syntenin–ALIX exosome biogenesis pathway has been proposed for ILVs formation without the involvement of ubiquitination and ESCRT‐0, but dependent on ESCRT‐III [37, 38]. In this pathway, biogenesis of the exosome and loading of proteins are regulated by heparanase, syndecan heparan sulfate proteoglycans, the small GTPase ADP ribosylation factor 6 (ARF6), and phospholipase D2 (PLD2) [38–40]. (ii) His domain protein tyrosine phosphatase (HD‐PTP) acts as a scaffold to continuously recruit ESCRT‐0, ESCRT‐I, and ESCRT‐III without the involvement of ESCRT‐II [41–43]. (iii) ALIX can directly bind protease‐activated receptor 1 (PAR1) or lysobisphosphatidic acid (LBPA) to recruit ESCRT‐III independent of ESCRT‐0, ESCRT‐I, and ESCRT‐II [44–47].
In addition, it has been shown that the existence of mechanisms for exosome biogenesis is not dependent on ESCRT. For example, the formation of MVB cannot be inhibited in the absence of the ESCRT complex [48]. Trajkovic et al. find that the formation of ILVs is independent of ESCRT but requires the sphingolipid ceramide [49]. The cone‐shaped structure of ceramide promotes membrane invagination of the endosomal membrane [50]. The release of exosomes is decreased after inhibition of neutral sphingomyelinase (nSMase), which can produce ceramides [51–53]. Another ESCRT‐independent exosome biogenesis is mediated by tetraspanins. In this mechanism, exosome secretion is not inhibited even in the absence of ESCRTs and ceramides [54]. In a tetraspanin‐dependent mechanism, tetraspanins can aggregate related molecules to endosomal membranes, leading to the invagination of the endosomal membrane to form ILVs [55, 56]. Tetraspanin CD63 plays an important role in exosome formation and release [57–59]. In addition, CD9, CD81, and CD82 have also been shown to be involved in the formation of exosomes [60, 61].
After the formation of ILVs, MVBs can be degraded by fusing with lysosomes. In this mechanism, ISGylation of ESCRT‐I component TSG101 can reduce exosome secretion by promoting the fusion of MVBs with lysosomes [62, 63]. Another fate of MVBs is promoting exosome release into the extracellular environment by fusing with the plasma membrane. The RAB GTPases, including RAB7, RAB11, RAB27, RAB31, and RAB35, are thought to play a crucial role in the translocation of MVBs to the plasma membrane [64, 65]. It was shown that deletion of RAB7 reduced the secretion of exosomes containing syntenin and ALIX in MCF‐7 cells, but did not affect the release of exosomes from Hela cells [37, 66]. Recently, Fei et al. demonstrated that RAB7 could be recruited to MVBs by binding directly to neddylated Coro1a, promoting degradation of MVBs and leading to a decrease in exosome secretion [67]. RAB11 promotes the release of exosomes containing transferrin receptor (TfR) and heat shock cognate 71 kDa protein (Hsc70) in K562 cells [68]. Both subtypes of RAB27— RAB27A and RAB27B—affect exosome release from cancer cells [69–71]. Silencing of RAB27A leads to decreased secretion of exosomes containing the traditional markers CD63, TSG101, ALIX, and HSC70, without affecting the secretion of exosomes containing CD9 and Mfge8 [72]. RAB31—an ESCRT‐independent exosome biogenesis pathway—drives the formation of ILVs and prevents the fusion of MVBs with lysosomes [73]. RAB35 regulates the release of exosomes from oligodendrocytes [74]. Inhibition of RAB35 reduced the release of proteolipid protein (PLP)‐containing exosomes from oligodendrocytes [74].
The final step in exosome secretion is controlled by the soluble NSF attachment protein receptor (SNARE) protein. SNARE protein can mediate membrane fusion of different intracellular compartments [75–79]. Vesicle‐associated membrane protein 7 (VAMP7) is involved in Ca2+‐regulated exocytosis of traditional lysosomes and regulates exosome release from K562 cells [80–82]. Syntaxin 1A (Syx1A) is a necessary SNARE protein for evenness interrupted (Evi)‐containing exosome secretion [83]. In non‐small cell lung cancer, the SNARE protein YKT6 is essential for regulating Wnt‐containing exosome secretion [84, 85]. The target membrane SNARE protein SYX‐5 also participates in regulating exosome secretion; MVBs aggregate under the plasma membrane when SYX‐5 is deficient [86]. In addition, SNAP‐23 promotes exosome secretion in tumor cells by regulating pyruvate kinase type M2 (PKM2)‐mediated phosphorylation [87, 88].
2.1.2 Biogenesis of Microvesicle
Microvesicles (MVs) are generated mainly by direct outward budding and division of the plasma membrane, and have multiple biogenesis and release mechanisms. The redistribution of phospholipids and contraction of the cytoskeleton through actin–myosin interactions together lead to the formation of MVs [6,89–92]. Recent studies have shown that ESCRT is involved in the biogenesis of MVs [93, 94]. The arrestin domain‐containing protein 1 (ARRDC1) facilitates the production of MVs containing TSG101, ARRDC1, and intracellular proteins by binding to ESCRT‐I subunit TSG101 and recruiting it to the plasma membrane [95]. Vps4 ATPase is also required for the generation of such MVs [95]. Activation of acid sphingomyelinase induces ceramide‐dependent production of MVs from astrocytes [96]. Similarly, acidic sphingomyelinase also enhances the production of MVs in erythrocytes [97]. The formation of MVs is also correlated with cholesterol levels, and the production of MVs significantly decreases when cholesterol is depleted [98]. Caveolin‐1 (cav‐1) has been reported to be involved in the formation of miRNA‐containing MVs [99]. Phosphorylation of cav‐1 tyrosine 14 (Y14) leads to the interaction of cav‐1 with hnRNPA2B1, which induces hnRNPA2B1 to bind miRNAs, resulting in selection of miRNAs into MVs [99]. Other than the above mechanism, small GTPase proteins, such as PhoA, ARF1, and ARF6, can also regulate production of MVs [92, 100, 101]. In addition, external stimuli can also induce the release of MVs. In erythrocytes and platelet cells, calcium influx induces phospholipid redistribution and promotes the release of MVs [102, 103]. Hypoxia also facilitates the release of MVs from breast cancer by the HIF‐dependent expression of PAB22A [104].
2.1.3 Biogenesis of Apoptotic Bodies
Apoptotic bodies (ApoBDs) are generated only during programmed cell death [6]. ApoBDs originate from the condensation of nuclear chromatin, undergo nuclear division and membrane blebbing, and eventually the cell contents split into distinct membrane‐encapsulated vesicles referred to as ApoBDs [105, 106]. At present, applied studies on ApoBDs are scarce, mainly attributed to the limited understanding of the biogenesis of ApoBDs. Membrane blebbing is a key morphological step in the formation of ApoBDs. Studies suggest that membrane blebbing may be mediated by actin–myosin interactions and is thought to be regulated by a number of kinases, including Rho‐associated kinase 1 (ROCK1) [107–109], p21 activated kinase 2 (PAK2) [110, 111], and Lim domain kinase 1(LIMK1) [112]. In addition, in T cells and thymocytes, the formation of ApoBDs is regulated by the cystatin‐activated pannexin 1 (PANX1) channel. Blocking the activity of PANX1 channel promotes the formation of ApoBDs [113]. ApoBDs can promote phagocytosis and clearance of apoptotic cells by macrophages [114–116]. During apoptosis, membrane lipids undergo rearrangement, transferring phosphatidylserine located in the inner leaflet of the plasma membrane to the outer leaflet [117], facilitating the binding of phosphatidylserine to Annexin V of macrophages [118], and achieving the clearance of apoptotic cells.
2.1.4 Biogenesis of Large Oncosomes
Large oncosomes (LOs) are secreted by cancer cells in an amoeboid motile manner. Di Vizio et al. revealed that amoeboid migration of DU145 and LNCaP human prostate cancer cells triggered the generation of giant EVs referred to as Los [119]. It was shown that extracellular matrix degradation products, such as elastin degradation products (EDPs), affect intracellular calcium flow and cytoskeleton reorganization, inducing a tumor amoeboid phenotype [120]. LOs are byproducts of nonapoptotic cell plasma membrane blebbing, and their shedding process is regulated by various proteins [121]. The shedding of LOs can be promoted by silencing of the cytoskeletal regulator diaphanous‐related formin‐3 (DIAPH3) protein, overexpression of oncoproteins including caveolin‐1 (Cav‐1), myristylated Akt1 (MyrAkt1) and heparin‐binding epidermal growth factor (HB‐EGF), or activation of EGFR [8, 119,122–124]. In addition, cytokeratin 18 (CK18) can be used as a marker of tumor‐derived LOs [124].
2.2 Identification of Extracellular Vesicles
2.2.1 Electron Microscopic Identification
Electron microscopy has a high resolution and can directly observe the morphology and structure of nanoparticles in a sample, which can therefore be used to identify the presence and integrity of EVs; but the electron micrographs of nanoparticles such as lysosomes, mycoplasma, polyethylene glycol protein aggregates, and ferritin aggregates are very similar to those of exosomes, and the electron microscope can only show a partial view. Therefore, it cannot distinguish between EVs and nanoparticles with similar morphology, nor does it reflect the number of EVs in the sample. Moreover, electron microscopy instruments are expensive, the method requires more stringent sample preparation, and the results are subject to subjectivity [125].
2.2.1.1 Scanning Electron Microscopy
Scanning electron microscope (SEM) is the use of secondary electron signal imaging to observe the surface morphology of a sample. SEM is a microscopic morphology observation tool between transmission electron microscopy and optical microscopy, which can directly image through the surface properties of the material. The images obtained by SEM are stereoscopic and can be used to observe various morphological features of biological samples, mainly for observing the surface morphology of nanomaterials, analysis of material fractures, direct observation of the original surface, observation of thick specimens, and observation of the details of each area. The resolution of advanced SEM is less than 1 nm, which can meet the requirements of EV size observation. The EVs observed using SEM show a distorted cup‐like morphology, which may be due to the deformation of the vesicles as a result of chemical fixation and dehydration treatment required for sample preparation.
As we can see from Figure 2.1A, Chukhchin et al. studied EVs from the phloem and xylem of woody plants by SEM, which revealed the cup‐like structural features of EVs in the dry state [126]. Vijayarathna et al. observed the generation and morphological features of apoptotic vesicles by scanning electron microscopy (Figure 2.1B) [127]. Beekman et al. analyzed EVs of tumor origin using scanning electron microscopy and studied the size and size distribution of individual EVs levels (Figure 2.1C) [128].
Figure 2.1 (a) Exosomes in the secondary phloem of woody plants.
Source: Chukhchin et al. [126]/with permission of Springer Nature.
(b) SEM micrographs of surface ultrastructure characteristics of HeLa cells.
Source: Vijayarathna et al. [127]/with permission of Elsevier.
(c) SEM image of a selected ROI.
Source: Beekman et al. [128]/with permission of Royal Society of Chemistry.
2.2.1.2 Transmission Electron Microscopy
Transmission electron microscope (TEM) is the most widely used type of electron microscope. TEM is used to observe and study the internal submicroscopic structure of cells and the morphological structure of viruses, proteins, nucleic acids, and other biological macromolecules. [129]
TEM is an electro‐optical instrument with high resolution and high magnification that can be used to determine whether the isolated and purified sample has a more typical structure of EVs, distinguishing EVs from nanoparticles. Currently, the resolution of the most advanced TEM has reached 0.1 nm. However, the number of particles observed at one time is limited and is not suitable for large sample sizes. EVs observed by TEM appear as cups with a double‐layer membrane structure. Similar to SEM, the process of fixation and dehydration of the sample prior to observation leads to unpredictable changes in the structure of EVs.
Figure 2.2 TEM picture of EVs.
Source: (a) Wang et al. [130]/Journal of Immunology Research/CC BY 4.0.; (b) Yuan et al. [131]/with permission of Taylor and Francis group.; (c) Vijayarathna et al. [127]/with permission of Elsevier.
As we can see from Figure 2.2a, Wang et al. verified the morphological and structural characteristics of EVs from PC cells, and their internalization by human umbilical vein endothelial cells (HUVEC) by TEM [130]. Yuan et al. prepared a self‐assembled vesicle in aqueous solution, and confirmed that the vesicles were spherical in shape using TEM and had an average particle size of ∼33.7 nm as measured by dynamic light scattering (Figure 2.2b) [131]. Vijayarathna et al. used TEM to confirm the extrusion of cytoplasm and the formation of apoptotic vesicles (Figure 2.2c) [127].
2.2.1.3 Atomic Force Microscopy
Atomic force microscopy (AFM) can be used to study the surface structure of solid materials, including insulators. It studies the surface structure and properties of materials by detecting extremely weak interatomic interaction forces between the surface of the sample to be measured and a micro‐force‐sensitive element [132].
Compared to other electron microscopes, AFM has been found to have some significant advantages in the identification of EVs. Firstly, the operating condition of AFM is not harsh; it can work at atmospheric pressure and even in liquid environments. This allows it to be used to study macroscopic characteristics of organisms and even living biological tissues. Secondly, AFM can form a true three‐dimensional structure, which is more convenient for observation. Finally, AFM can be observed without destructive processing of the sample, which ensures the accuracy of the observation results. Compared with SEM, the disadvantages of AFM are that the imaging range is too small, the speed is slower, and the influence of the probe is too great.
As Figure 2.3A shows, Sharma et al. used AFM to demonstrate the structure of EVs that could not be clearly identified under electron microscopy and correlated the data with field emission scanning electron microscopy (FESEM) and AFM images to explain the nanoscale structure of EVs under different forces [133]. Single EVs showed reversible mechanical deformation, displaying distinctive 70–100 nm elastic trilobal membranes with substructures bearing specific transmembrane receptors (Figure 2.3B). Ayat et al. used AFM to detect changes in cell surface morphology, granulation, and mean surface roughness, and found cell shrinkage and increased cytoplasmic organelles, confirming the generation of apoptotic vesicles [134]. Sharma et al. compared the size, structure, and surface properties of small EVs (sEVs) derived from breast cancer cells using AFM (Figure 2.3C) [135].
Figure 2.3 EV surface structure observed by AFM.
Source: (a) Sharma et al. [133]/with permission of American Chemical Society.; (b) Ali et al. [134]/with permission of Elsevier.; (c) Sharma et al. [135]/Springer Nature/CC BY 4.0.
2.2.1.4 Cryo‐Electron Microscopy
Cryo‐electron microscopy (Cryo‐M) is an ultra‐low temperature frozen sample preparation and delivery technique for scanning electron microscopy, which could provide direct observation of liquid, semiliquid, and beam‐sensitive samples, such as biological and polymeric materials. Cryo‐M can analyze the morphology of EVs without extensive processing. When using Cryo‐M for observation of samples, the vacuum environment and the impact of electron energy cause rapid deterioration of the sample, and the sample becomes glassy without forming ice crystals by rapid freezing. In this state, the sample deteriorates much more slowly in order to facilitate the study of EV structure [136].
Some undeformed EVs were identified using Cryo‐M by Bairamukov's team at the Petersburg Institute of Nuclear Physics (Figure 2.4a) [137]. Talmon et al. studied the appearance of EV through Cryo‐electron microscopy. The darker area was irradiated longer than the lighter area, exposing the CNTs after etching away some of the CSA, showing clearly bundles of CNTs, forming a nematic liquid–crystalline phase. The bright spots are nanoparticles of the iron catalyst exposed by etching (Figure 2.4b) [138]. The morphological structure, size distribution, and membrane thickness of vesicles were distinctly determined via Cryo‐M imaging by Jiang et al. (Figure 2.4c) [139].
Figure 2.4 Picture of inflammatory EVs.
Source: (a) Bairamukov et al. [137]/with permission of Elsevier.; (b) Matatyaho Ya'akobi and Talmon [138]/American Chemical Society/CC BY 4.0.; (c) Jiang et al. [139]/with permission of Oxford University Press.
2.2.2 Particle Size Detection
The morphology of EVs can be observed by electron microscopy, but the particle size distribution and overall concentration of all EVs in a sample cannot be represented. Particle size detection allows rapid and accurate analysis of the particle size distribution and concentration of EVs, providing strong evidence for the identification of EVs. Measuring the particle size distribution of EVs has long been an important part of EV characterization. However, since the size of EVs is only 30–200 nm, some special detection methods must be used to observe these invisible particles under the light microscope. The typical methods include nanoparticle tracking analysis (NTA) and dynamic light scattering (DLS).
2.2.2.1 Nanoparticle Tracking Analysis
NTA nanoparticle tracking analysis technology has been recognized as one of the means of exocrine characterization in the field of exocrine research. The principle is to track and analyze the Brownian motion of each particle and calculate the Fluid mechanics diameter and concentration of nanoparticles. Sample processing is simpler, more able to ensure the original state of exosomes, and detection speed is faster. NTA techniques can be used to determine the size and concentration of particles, and to measure the zeta potential; the higher the potential, the less likely it is that particles in solution will aggregate and precipitate [140]. Smaller and less‐numerous particles are easily overlooked by traditional measurement methods. The NTA is capable of measuring particle sizes between 10 and 1000 nm in diameter.
The NTA tracks each particle in the image directly during the measurement process, so the NTA has a very high resolution for complex samples and can clearly distinguish between particles of different sizes, which is important for the detection of EVs. The NTA uses a measurement technique that eliminates the need for prior knowledge of the mass, refractive index, and hydrodynamic diameter of the particle material, allowing the instrument to skip the tedious preparatory work and move directly into the analysis, which not only saves a lot of time and speeds up the entire research process but also saves valuable manpower costs. Its unique concentration measurement technique provides reliable concentration data directly to researchers of EVs. Prior to NTA analysis of EVs in biological fluids, such as blood, it is essential to separate and purify the vesicles by centrifugation or other methods in order to remove lipoprotein particles, protein complexes, and other particles that may be similar in size to EVs and may exceed the number of EVs in the blood.
For example, Kenneth Gouin et al. used NTA to count and characterize the exocysts of different types of cardiomyocyte‐derived cells in terms of particle size. The experimental results showed that the exocysts secreted by different cell donors were similar in size, and the particle size was mainly distributed between 70 and 90 nm (Figure 2.5a) [141]. Zhang Jinchao et al. developed a tumor antigen‐carrying exosome (tDC‐Exo) and obtained antibody‐engineered exosomes (Exo‐OVA‐aCD3/aEGFR) by modifying anti‐CD3 and anti‐EFGR to improve tumor therapy. Particle size assay using NTA showed that the two types of exosomes— Exo‐OVA with a diameter of about 93 nm and Exo‐OVA‐aCD3/aEGFR with a particle size of 102 nm (Figure 2.5b) [142]—can flexibly cross the tissue barrier and reach the target tissue more easily. Both animals and plants can secrete EVs; but plant‐derived EVs are widely available compared with animal‐derived EVs, and can be extracted and isolated in large quantities, which is more advantageous for tumor nanomedicine development and application. Liu et al. extracted EVs from plant leaves and evaluated the size distribution of leaf nano‐EVs and small extracellular vesicles (sEVs) by NTA. The results showed that the size of most leaf nanovesicles was 218 nm, while the particle size of sEVs was 135 nm (Figure 2.5c) [143].
Figure 2.5 The size of EVs was identified by NTA. (a) Nanotracking analysis of EVs derived from three donor sources [141]; (b) NTA results of Exo‐OVA and Exo‐OVA‐aCD3/aEGFR [142]
