**1. Introduction**

In the last decade, we observed a massive upsurge of studies in the field of extracellular vesicles (EVs) [1]. As it is known now, EVs can be loaded with different therapeutic molecules and transport them to recipient cells with little interrogation by the immune system. This property of EVs prompts new possibilities for treatment in various clinical settings [2–4]. In this chapter, we review the biology of EVs as a universal cellular component from a broader perspective, and afterward provide an updated view on red blood cell extracellular vesicles (RBCEVs), their merits and potential applications in therapeutics [5].

### **2. Overview of extracellular vesicles**

#### **2.1 History of extracellular vesicles**

Wolf was the first to discover small procoagulant structures derived from activated platelets in human blood and named them "platelet dust" in 1967. He separated the small structures by ultracentrifugation and further characterized them using an

electron microscopy [6]. In 1987, Johnstone further studied the formation of such vesicles in the duration of sheep reticulocytes maturation *in vitro*. He was able to identify more activities and characteristics of the vesicles. However, he did not name the small vesicles or discover how they were generated in detail [7]. Both of these findings were important milestones in the field, which allowed for further studies on the function of these small vesicles. Today, we call these small vesicles as EVs. Valadi and colleagues were the first who discovered the natural delivery of microRNAs and mRNAs in EVs in mast cells. Later on, nucleic acid transport via EVs was also observed in many other cell types as an essential manner of intercellular communication [8–10]. We now have a much more profound understanding in the field of EVs due to the continuous efforts of various scientists throughout many decades.

#### **2.2 Biogenesis and compositions of extracellular vesicles**

EVs are a heterogeneous class of cell-derived structures with a lipid bilayer membrane, which comprise exosomes, microvesicles, and apoptotic bodies. They are either of the endosomal origins or are shed from the plasma membrane under physiological and pathological conditions. Additionally, they are present in almost all biological fluids, such as blood, urine, breast milk, cerebrospinal fluid, saliva, semen, etc. [11–17]. Further characterizations are based on the different sizes and biogenesis of EVs. Exosomes generally range from 50 to 150 nm in diameter and are secreted from endosomal multivesicular bodies, whereas microvesicles are larger vesicles ranging from 100 to 500 nm in diameter and are formed through a budding or exocytosis process of the plasma membrane [11, 18–23]. Apoptotic bodies are much larger, ranging from 800 to 5000 nm in diameter, and are generated by blebbing of plasma membrane from cells undergoing apoptosis. Hence, apoptotic bodies represent the fragments of dying cells and differ from exosomes and microvesicles in property (**Figure 1**) [17–22]. In this chapter, we will collectively term both exosomes and microvesicles as EVs with apoptotic bodies excluded.

The components of EVs are mainly proteins, lipids, and nucleic acids. However, due to different biogenesis mechanisms, the compositions of exosomes and microvesicles do vary slightly [11, 24–26]. Proteins that are associated with endocytic pathways can be usually found in EVs, such as flotillin and annexin. Some of the biogenesis-associated proteins, such as Tsg101 and Alix, and common tetraspanins, such as CD9 and CD81, are commonly used as EVs markers with CD63 which is mostly regarded as a marker of exosomes. However, currently, there lack well-defined protein markers to distinguish exosomes and microvesicles [11, 24–26]. Lipid components of EVs include phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingomyelin, cholesterol, and so on, which can be found in plasma membrane as well. As microvesicles are formed by budding from plasma membrame, the lipid composition of microvesicles resembles that of plasma membrane of the cells more while exosomes are of higher levels in sphingomyelin, cholesterol, and phosphatidylserine [27–29]. It is noteworthy that many nucleic acid species are highly enriched in EVs. The lipid bilayer structure of EVs acts as a natural shelter against degrading nucleases in the extracellular environment and protects the nucleic acid cargo under adverse conditions such as long-term storage and multiple freeze-thaw cycles. In the recent decade, reports have it that many mRNAs, microRNAs, and other non-coding RNAs are discovered in EVs (**Figure 1**) [30–32].

#### **2.3 Intercellular communication mediated by extracellular vesicles**

As EVs are abundant and widely distributed in biological fluids and carry bioactive cargo, they influence various biological processes of the donor and recipient

**123**

**Figure 1.**

*The Biology and Therapeutic Applications of Red Blood Cell Extracellular Vesicles*

cells [33]. The intercellular communication can occur between cells by transferring EVs that act as an exchange mediator of proteins, lipids, and RNAs. Thus, EVs have a fundamental role to play in important biological processes such as the exchange of surface membrane and horizontal RNA transport between neighboring and remote cells [18]. This aspect is being extensively investigated in cancers [34], neurodegenerative diseases [35], autoimmune disorders [36], aging [37], and so on. The bioactive cargo encapsulated by EVs contain valuable information from the source

*Biogenesis and composition of extracellular vesicles. Extracellular vesicles (EVs) are composed of exosomes, microvesicles, and apoptotic bodies. Exosomes are typically of endosomal origins and are the smallest among them with 50 to 150 nm in diameter. Microvesicles are larger in size from 100 to 500 nm in diameter and are generated through an outward budding or exocytosis of the plasma membrane. Apoptotic bodies are usually the largest ranging from 800 to 5,000 nm in diameter and are generated by blebbing of plasma membrane from cells undergoing apoptosis. Major components of EVs are lipids, proteins, and nucleic acids. Due to different* 

*biogenesis mechanisms, the compositions of exosomes and microvesicles do vary.*

*DOI: http://dx.doi.org/10.5772/intechopen.81758*

*The Biology and Therapeutic Applications of Red Blood Cell Extracellular Vesicles DOI: http://dx.doi.org/10.5772/intechopen.81758*

#### **Figure 1.**

*Erythrocyte*

electron microscopy [6]. In 1987, Johnstone further studied the formation of such vesicles in the duration of sheep reticulocytes maturation *in vitro*. He was able to identify more activities and characteristics of the vesicles. However, he did not name the small vesicles or discover how they were generated in detail [7]. Both of these findings were important milestones in the field, which allowed for further studies on the function of these small vesicles. Today, we call these small vesicles as EVs. Valadi and colleagues were the first who discovered the natural delivery of microRNAs and mRNAs in EVs in mast cells. Later on, nucleic acid transport via EVs was also observed in many other cell types as an essential manner of intercellular communication [8–10]. We now have a much more profound understanding in the field of EVs

due to the continuous efforts of various scientists throughout many decades.

EVs are a heterogeneous class of cell-derived structures with a lipid bilayer membrane, which comprise exosomes, microvesicles, and apoptotic bodies. They are either of the endosomal origins or are shed from the plasma membrane under physiological and pathological conditions. Additionally, they are present in almost all biological fluids, such as blood, urine, breast milk, cerebrospinal fluid, saliva, semen, etc. [11–17]. Further characterizations are based on the different sizes and biogenesis of EVs. Exosomes generally range from 50 to 150 nm in diameter and are secreted from endosomal multivesicular bodies, whereas microvesicles are larger vesicles ranging from 100 to 500 nm in diameter and are formed through a budding or exocytosis process of the plasma membrane [11, 18–23]. Apoptotic bodies are much larger, ranging from 800 to 5000 nm in diameter, and are generated by blebbing of plasma membrane from cells undergoing apoptosis. Hence, apoptotic bodies represent the fragments of dying cells and differ from exosomes and microvesicles in property (**Figure 1**) [17–22]. In this chapter, we will collectively term both

**2.2 Biogenesis and compositions of extracellular vesicles**

exosomes and microvesicles as EVs with apoptotic bodies excluded.

**2.3 Intercellular communication mediated by extracellular vesicles**

As EVs are abundant and widely distributed in biological fluids and carry bioactive cargo, they influence various biological processes of the donor and recipient

The components of EVs are mainly proteins, lipids, and nucleic acids. However, due to different biogenesis mechanisms, the compositions of exosomes and microvesicles do vary slightly [11, 24–26]. Proteins that are associated with endocytic pathways can be usually found in EVs, such as flotillin and annexin. Some of the biogenesis-associated proteins, such as Tsg101 and Alix, and common tetraspanins, such as CD9 and CD81, are commonly used as EVs markers with CD63 which is mostly regarded as a marker of exosomes. However, currently, there lack well-defined protein markers to distinguish exosomes and microvesicles [11, 24–26]. Lipid components of EVs include phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingomyelin, cholesterol, and so on, which can be found in plasma membrane as well. As microvesicles are formed by budding from plasma membrame, the lipid composition of microvesicles resembles that of plasma membrane of the cells more while exosomes are of higher levels in sphingomyelin, cholesterol, and phosphatidylserine [27–29]. It is noteworthy that many nucleic acid species are highly enriched in EVs. The lipid bilayer structure of EVs acts as a natural shelter against degrading nucleases in the extracellular environment and protects the nucleic acid cargo under adverse conditions such as long-term storage and multiple freeze-thaw cycles. In the recent decade, reports have it that many mRNAs, microRNAs, and other non-coding RNAs are discovered in EVs (**Figure 1**) [30–32].

**122**

*Biogenesis and composition of extracellular vesicles. Extracellular vesicles (EVs) are composed of exosomes, microvesicles, and apoptotic bodies. Exosomes are typically of endosomal origins and are the smallest among them with 50 to 150 nm in diameter. Microvesicles are larger in size from 100 to 500 nm in diameter and are generated through an outward budding or exocytosis of the plasma membrane. Apoptotic bodies are usually the largest ranging from 800 to 5,000 nm in diameter and are generated by blebbing of plasma membrane from cells undergoing apoptosis. Major components of EVs are lipids, proteins, and nucleic acids. Due to different biogenesis mechanisms, the compositions of exosomes and microvesicles do vary.*

cells [33]. The intercellular communication can occur between cells by transferring EVs that act as an exchange mediator of proteins, lipids, and RNAs. Thus, EVs have a fundamental role to play in important biological processes such as the exchange of surface membrane and horizontal RNA transport between neighboring and remote cells [18]. This aspect is being extensively investigated in cancers [34], neurodegenerative diseases [35], autoimmune disorders [36], aging [37], and so on. The bioactive cargo encapsulated by EVs contain valuable information from the source

of diseases, which can serve as robust biomarkers in diagnostics and status snapshots in treatment monitoring [38, 39]. The endogenous property of transporting molecules by EVs inspires researchers to utilize them as a superb delivery platform of therapeutic agents as well [40].
