**3.1 Red blood cell extracellular vesicles under physiological and pathological conditions**

Similar to EVs released from other cells, EVs in the circulation carry biomarkers originated from the donor cells [41]. Usually EVs contain various markers which indicate their origins, e.g., CD235a (also called GPA) for RBCs, CD41 for platelets, and CD11c for dendritic cells [42–44]. RBCs express classes of CD59 and DAF, known as complement inhibitors, and signaling of CD47 and SHPS-1 molecules on the cell surface to protect themselves against endogenous elimination [45, 46]. For instance, the RBCs membrane protein CD47 inhibits RBCs phagocytosis via macrophages by binding to the inhibitory receptor signal regulatory protein alpha (SIRPα). The presence of such proteins on the surfaces of RBCEVs may help RBCEVs to escape from the clearance by macrophages if they carry CD47 on their surfaces [47–49]. Mature RBCs lack nuclei and most of the intracellular membrane structures; hence, EVs released from mature RBCs are microvesicles derived from the plasma membrane (**Figure 2**). Even with the same cell origins, the protein or lipid compositions of EVs may differ on account of the lateral cell membrane variation. Further proteomic assays have illustrated that to some extent, the proteomic spectrum difference of EVs and the releasing cells can be attributed to the stimulating conditions during EVs biogenesis [50]. Microvesicles derived from RBCs are reported to be different in protein contents when produced naturally *in vivo*, *ex vivo* released during cold storage of RBCs, or *in vitro* by treatment with EVs' release-inducing chemicals such as calcium ionophore, even though they seem homogeneous merely based on their size and/or surface markers. The most distinctively different proteins are stomatin and flotillin-2 [51].

There are many studies of RBCEVs under diseased conditions with malaria being frequently reported. Mantel and colleagues illustrated that EVs from human RBCs infected with *Plasmodium falciparum* parasites contain microRNAs that are able to moderate target genes in recipient cells [52]. The infected RBC-derived EVs in malaria were internalized by endothelial cells and the EVs-encapsulated miRNA-Argonaute 2 complexes repressed miRNA target genes and changed endothelial barrier properties. Furthermore, multiple miRNA species in such EVs were identified [52]. Ankarklev and colleagues reviewed the role of RBCEVs in malaria and found that the development of EVs by *Plasmodium* sp. is associated with clinical outcomes [53]. Studies have pointed out that elevated EVs levels were detected in patients with severe malaria cases, and increased EV excretion to the endothelium has been linked to infected RBCs [53].

### **3.2 Red blood cell extracellular vesicles for therapeutic purposes**

Chang and colleagues demonstrated the ability of RBCEVs to efficiently deliver ultra-small superparamagnetic iron oxide particles into human bone marrow mesenchymal stem cells for cellular magnetic resonance imaging *in vitro* and *in vivo* in order to develop successful stem cell therapies [54]. The novel method overcomes the difficulty of relatively low intracellular labeling efficiency and addresses biosafety issues associated in comparison with traditional approaches. RBCEVs were

**125**

**Figure 2.**

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

shown to be ultra biosafe and can be used as potential delivery vehicles for clinical applications due to their autologous property to human bone marrow mesenchymal stem cells [54]. Usman and colleagues developed a robust delivery system for RNAbased therapeutics using RBCEVs [55]. Using the novel RBCEVs delivery platform, both small RNA, e.g., antisense oligonucleotides (ASOs), and large RNA, such as Cas9 mRNA, can be electroporated into RBCEVs and transported to target cells in both solid and liquid tumors. In the study, microRNA-125b-ASO-loaded RBCEVs significantly dampened breast tumor growth by intratumoral injection and suppressed acute myeloid leukemia (AML) progression by systemic administration. Genome editing effects were also observed when RBCEVs were loaded with Cas9 mRNA with guide RNAs. The delivery efficiency was higher and far less cytotoxicity was observed as compared to other commercial transfection reagents [55].

*Using red blood cell extracellular vesicles (RBCEVs) for therapeutic delivery. Calcium ionophore is added to RBCs which simulates the release of microvesicles, the only type of RBCEVs. Naturally, RBCEVs contain hemoglobin, Alix, TSG101, and some microRNAs in their lumen. They also display stomatin (STOM) and glycophorin A (GPA) on their membrane. RBCEVs can be loaded with therapeutic molecules including RNAs,* 

*proteins, and chemical drugs for delivery of these molecules to other cell types.*

**3.3 Isolation and loading of red blood cell extracellular vesicles** 

Up to now, standardized protocols for EVs isolation for either scientific research or clinical application are lacking [56]. One of the commonly used

**for therapeutic purposes**

*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 2.**

*Erythrocyte*

of therapeutic agents as well [40].

**and pathological conditions**

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

Similar to EVs released from other cells, EVs in the circulation carry biomarkers originated from the donor cells [41]. Usually EVs contain various markers which indicate their origins, e.g., CD235a (also called GPA) for RBCs, CD41 for platelets, and CD11c for dendritic cells [42–44]. RBCs express classes of CD59 and DAF, known as complement inhibitors, and signaling of CD47 and SHPS-1 molecules on the cell surface to protect themselves against endogenous elimination [45, 46]. For instance, the RBCs membrane protein CD47 inhibits RBCs phagocytosis via macrophages by binding to the inhibitory receptor signal regulatory protein alpha (SIRPα). The presence of such proteins on the surfaces of RBCEVs may help RBCEVs to escape from the clearance by macrophages if they carry CD47 on their surfaces [47–49]. Mature RBCs lack nuclei and most of the intracellular membrane structures; hence, EVs released from mature RBCs are microvesicles derived from the plasma membrane (**Figure 2**). Even with the same cell origins, the protein or lipid compositions of EVs may differ on account of the lateral cell membrane variation. Further proteomic assays have illustrated that to some extent, the proteomic spectrum difference of EVs and the releasing cells can be attributed to the stimulating conditions during EVs biogenesis [50]. Microvesicles derived from RBCs are reported to be different in protein contents when produced naturally *in vivo*, *ex vivo* released during cold storage of RBCs, or *in vitro* by treatment with EVs' release-inducing chemicals such as calcium ionophore, even though they seem homogeneous merely based on their size and/or surface markers. The most distinctively different proteins are stomatin and flotillin-2 [51]. There are many studies of RBCEVs under diseased conditions with malaria being frequently reported. Mantel and colleagues illustrated that EVs from human RBCs infected with *Plasmodium falciparum* parasites contain microRNAs that are able to moderate target genes in recipient cells [52]. The infected RBC-derived EVs in malaria were internalized by endothelial cells and the EVs-encapsulated miRNA-Argonaute 2 complexes repressed miRNA target genes and changed endothelial barrier properties. Furthermore, multiple miRNA species in such EVs were identified [52]. Ankarklev and colleagues reviewed the role of RBCEVs in malaria and found that the development of EVs by *Plasmodium* sp. is associated with clinical outcomes [53]. Studies have pointed out that elevated EVs levels were detected in patients with severe malaria cases, and increased EV excretion to the endothelium has been linked to infected RBCs [53].

**3. Concentrations on red blood cell extracellular vesicles**

**3.1 Red blood cell extracellular vesicles under physiological** 

**3.2 Red blood cell extracellular vesicles for therapeutic purposes**

Chang and colleagues demonstrated the ability of RBCEVs to efficiently deliver

ultra-small superparamagnetic iron oxide particles into human bone marrow mesenchymal stem cells for cellular magnetic resonance imaging *in vitro* and *in vivo* in order to develop successful stem cell therapies [54]. The novel method overcomes the difficulty of relatively low intracellular labeling efficiency and addresses biosafety issues associated in comparison with traditional approaches. RBCEVs were

**124**

*Using red blood cell extracellular vesicles (RBCEVs) for therapeutic delivery. Calcium ionophore is added to RBCs which simulates the release of microvesicles, the only type of RBCEVs. Naturally, RBCEVs contain hemoglobin, Alix, TSG101, and some microRNAs in their lumen. They also display stomatin (STOM) and glycophorin A (GPA) on their membrane. RBCEVs can be loaded with therapeutic molecules including RNAs, proteins, and chemical drugs for delivery of these molecules to other cell types.*

shown to be ultra biosafe and can be used as potential delivery vehicles for clinical applications due to their autologous property to human bone marrow mesenchymal stem cells [54]. Usman and colleagues developed a robust delivery system for RNAbased therapeutics using RBCEVs [55]. Using the novel RBCEVs delivery platform, both small RNA, e.g., antisense oligonucleotides (ASOs), and large RNA, such as Cas9 mRNA, can be electroporated into RBCEVs and transported to target cells in both solid and liquid tumors. In the study, microRNA-125b-ASO-loaded RBCEVs significantly dampened breast tumor growth by intratumoral injection and suppressed acute myeloid leukemia (AML) progression by systemic administration. Genome editing effects were also observed when RBCEVs were loaded with Cas9 mRNA with guide RNAs. The delivery efficiency was higher and far less cytotoxicity was observed as compared to other commercial transfection reagents [55].

#### **3.3 Isolation and loading of red blood cell extracellular vesicles for therapeutic purposes**

Up to now, standardized protocols for EVs isolation for either scientific research or clinical application are lacking [56]. One of the commonly used methods to obtain EVs is ultrafiltration with subsequent differential ultracentrifugation. Ultrafiltration followed by liquid size exclusion chromatography suits the large-scale demand of isolating EV for therapeutics as the method results in, on the one hand, a significantly higher EV yield and, on the other hand, the wellpreserved biophysical properties of the purified EVs [57]. Usman and colleagues provided a lab-based approach to purify RBCEVs using ultracentrifugation with sucrose cushion. To begin with, RBCs in whole blood were separated from white blood cells and plasma by low centrifugation and using leukodepletion filters. Then, the isolated RBCs were diluted in PBS and treated with 10 mM calcium ionophore overnight which can stimulate the release of RBCEVs and significantly increase the yield. In order to purify RBCEVs, RBCs and cell debris were removed by several rounds of low-speed centrifugation. Later, the resulting supernatants were passed through 0.45 μm syringe filters. Afterward, the RBCEVs were concentrated using ultracentrifugation at 100,000 × *g* for 70 min. Subsequently, RBCEVs were resuspended in cold PBS and layered above frozen 60% sucrose cushion and centrifuged at 100,000 × *g* for 16 h. The red layer of RBCEVs above the sucrose was collected and washed again with cold PBS by ultracentrifugation [55]. The approach is cost-effective, features high RBCEVs yield, and can be adopted in most laboratory settings. The use of sucrose cushion is also a highlight as it helps remove the protein contaminants outside RBCEVs, which might trigger unnecessary immune response and protects the integrity and biophysical properties of RBCEVs.

For therapeutic agents to be loaded into EVs, two major strategies currently have been applied. The first option is to load the therapeutic molecules, such as RNAs, into the EVs after EVs isolation, while the second one is conducted during EV biogenesis. These methods are also known as post-loading and pre-loading, respectively. The pre-loading encapsulation approach is also referred to as the endogenous method as it uses the cellular machinery in order to load small RNA into EVs. The pre-loading approach has been shown to work for the packaging of both siRNA and miRNA into EVs. The post-loading method artificially introduces RNAs into EVs, whereas pre-loading is performed in the EVs biogenesis. Post-loading can be subdivided into passive loading, such as by physical incubation, and active loading with instances of electroporation or sonication. Furthermore, the functional small RNAs delivery using electroporated EVs has been shown to be a success in several reports but it depends on the small RNA species [58–62]. Usman and colleagues used the electroporation method for post-loading of RNAs into RBCEVs [55]. Ideally, various therapeutic molecules including ASOs, siRNAs, gRNAs, mRNAs, plasmid DNA, proteins, peptides, and chemical drug compounds can be loaded into RBCEVs using electroporation (**Figure 2**). Other post-loading methods such as mild sonication and physical incubation may be applicable to RBCEVs as tested for other types of EVs. Labeling of EVs is then required to examine the efficiency of delivery to target cells. Various methods and techniques have been applied to label EVs, with most common methods being incubation with biotinylated radioisotope, substrate of luciferase, fluorescence lipophilic dye, streptavidin-conjugated fluorescence dyes, or the use of other modified proteins [55, 64–66].

### **3.4 Advantages of red blood cell extracellular vesicles in therapeutics**

Due to their innate function on cell-cell communication, EVs can be used effectively for drug delivery [12, 67–69]. The biggest advantage of EVs drug delivery is probably that EVs can be taken from an organism and returned to the same organism *in vivo* after being loaded with therapeutic agents, which are thought to be nonimmunogenic. Another advantage to deliver nucleic acids with the help of EVs is

**127**

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

that EVs can carry molecules through physiological barriers, such as the blood-brain barrier, which are hard to cross using conventional delivery methods. Normally, when exogenous RNAs are introduced into the body directly, they will be cleared before reaching the target tissues or cells of interest through degradation by nucleases, or they will be filtered in the kidneys. Both coding and noncoding RNAs were shown to be transferred by EVs intercellular crosstalks. Additionally, it has been shown that microRNAs can be transported in EVs to various cell types. Thus, EVs can be used as a promising vehicle for delivery of RNA-based drugs. Potential fields for therapeutic use include gene therapy, targeted therapy, vaccination, improvement of pregnancy outcome, newborn medicine, kidney disease, and treatment of

It has been reported that diversified types of cells, including RBCs, endothelial cells, monocytes, granulocytes, and platelets, release EVs. Additionally, EVs can be isolated by various methods from cell culture media, plasma, and other biofluids [23, 41, 70, 71]. Although several research groups have demonstrated the advantages of using EVs for RNA delivery, there are still issues with EVs generated from fibroblasts and dendritic cells being not permanently available from all subjects [69, 72]. RBCs are readily obtainable from any human subject and easy to store, and blood transfusion has been a relatively safe, well-established, and routine medical procedure for decades which makes RBCEVs easy to obtain and safe to use. Thus, EVs from whole plasma are easily accessible and substantially present, but these EVs are derived from various cell types, e.g., nucleated cells which represent a risk for horizontal gene transfer [63]. Therefore, obtaining ultrapure RBCEVs solely derived from RBCs is highly preferred as RBCs lack both nuclear and mitochondrial DNA, which means that RBCEVs for pharmaceutical purpose avoid the risk of horizontal gene transfer. RBCEVs formation has been extensively investigated and described in the recent years. Therefore, with such knowledge, RBCEVs are safer and less

Consequently, RBCEVs possess several features which make them better suitable for clinical applications than EVs from other cell types. First of all, blood units are easily accessible from existing blood banks. A large scale of RBCEVs can be produced at low cost as RBCs are the most abundant cell type in the body (84% of all cells) and, during their 120-day lifespan, RBCs continue to release RBCEVs, leading to an approximate 20% loss in RBCs volume and an increase of around 14% in hemoglobin concentration [23, 77–79]. Additionally, RBCEVs are obtainable for allogeneic and autologous transfusion from the patients' own blood. A large number of RBCs (~1012 cells/L) are obtainable from each blood unit. Thus, there exists no need to expand cells in culture and no risk of the emergence of mutations *in vitro*. In addition, no cGMP-qualified media or supplements are required, which are financially desirable. Large-scale amounts (1013–1014) of RBCEVs can be isolated from RBCs (per unit) when treated with calcium ionophore, which is a scalable strategy to obtain EVs. Secondly, RBCEVs are safer compared to EVs from other cell types, because the enucleated RBCs contain no DNA, unlike EVs from nucleated cell types which represent a potential risk for horizontal gene transfer. As plasma EVs are heterogeneous with unpredictable contents, RBCEVs are safer than plasma EVs for allogeneic treatments of cancer because cancer cells and immune cells are known to release large amounts of cancer-promoting EVs into their environment [80, 81]. Thirdly, RBCEVs are nontoxic; hence, they are safer as compared to the toxic synthetic transfection reagents which are typically used. As mentioned before, RNAs in RBCEVs are stable and completely functional *in vitro* and in *vivo* for both liquid and solid cancers. Fourthly, RBCEVs are presumably nonimmunogenic via blood type matching, unlike adenoviruses, adeno-associated viruses, lentiviruses, nanoparticles, and various synthetic

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

autoimmune disease [12, 67–69].

complicated to use [73–76].

that EVs can carry molecules through physiological barriers, such as the blood-brain barrier, which are hard to cross using conventional delivery methods. Normally, when exogenous RNAs are introduced into the body directly, they will be cleared before reaching the target tissues or cells of interest through degradation by nucleases, or they will be filtered in the kidneys. Both coding and noncoding RNAs were shown to be transferred by EVs intercellular crosstalks. Additionally, it has been shown that microRNAs can be transported in EVs to various cell types. Thus, EVs can be used as a promising vehicle for delivery of RNA-based drugs. Potential fields for therapeutic use include gene therapy, targeted therapy, vaccination, improvement of pregnancy outcome, newborn medicine, kidney disease, and treatment of autoimmune disease [12, 67–69].

It has been reported that diversified types of cells, including RBCs, endothelial cells, monocytes, granulocytes, and platelets, release EVs. Additionally, EVs can be isolated by various methods from cell culture media, plasma, and other biofluids [23, 41, 70, 71]. Although several research groups have demonstrated the advantages of using EVs for RNA delivery, there are still issues with EVs generated from fibroblasts and dendritic cells being not permanently available from all subjects [69, 72]. RBCs are readily obtainable from any human subject and easy to store, and blood transfusion has been a relatively safe, well-established, and routine medical procedure for decades which makes RBCEVs easy to obtain and safe to use. Thus, EVs from whole plasma are easily accessible and substantially present, but these EVs are derived from various cell types, e.g., nucleated cells which represent a risk for horizontal gene transfer [63]. Therefore, obtaining ultrapure RBCEVs solely derived from RBCs is highly preferred as RBCs lack both nuclear and mitochondrial DNA, which means that RBCEVs for pharmaceutical purpose avoid the risk of horizontal gene transfer. RBCEVs formation has been extensively investigated and described in the recent years. Therefore, with such knowledge, RBCEVs are safer and less complicated to use [73–76].

Consequently, RBCEVs possess several features which make them better suitable for clinical applications than EVs from other cell types. First of all, blood units are easily accessible from existing blood banks. A large scale of RBCEVs can be produced at low cost as RBCs are the most abundant cell type in the body (84% of all cells) and, during their 120-day lifespan, RBCs continue to release RBCEVs, leading to an approximate 20% loss in RBCs volume and an increase of around 14% in hemoglobin concentration [23, 77–79]. Additionally, RBCEVs are obtainable for allogeneic and autologous transfusion from the patients' own blood. A large number of RBCs (~1012 cells/L) are obtainable from each blood unit. Thus, there exists no need to expand cells in culture and no risk of the emergence of mutations *in vitro*. In addition, no cGMP-qualified media or supplements are required, which are financially desirable. Large-scale amounts (1013–1014) of RBCEVs can be isolated from RBCs (per unit) when treated with calcium ionophore, which is a scalable strategy to obtain EVs. Secondly, RBCEVs are safer compared to EVs from other cell types, because the enucleated RBCs contain no DNA, unlike EVs from nucleated cell types which represent a potential risk for horizontal gene transfer. As plasma EVs are heterogeneous with unpredictable contents, RBCEVs are safer than plasma EVs for allogeneic treatments of cancer because cancer cells and immune cells are known to release large amounts of cancer-promoting EVs into their environment [80, 81]. Thirdly, RBCEVs are nontoxic; hence, they are safer as compared to the toxic synthetic transfection reagents which are typically used. As mentioned before, RNAs in RBCEVs are stable and completely functional *in vitro* and in *vivo* for both liquid and solid cancers. Fourthly, RBCEVs are presumably nonimmunogenic via blood type matching, unlike adenoviruses, adeno-associated viruses, lentiviruses, nanoparticles, and various synthetic

*Erythrocyte*

RBCEVs.

methods to obtain EVs is ultrafiltration with subsequent differential ultracentrifugation. Ultrafiltration followed by liquid size exclusion chromatography suits the large-scale demand of isolating EV for therapeutics as the method results in, on the one hand, a significantly higher EV yield and, on the other hand, the wellpreserved biophysical properties of the purified EVs [57]. Usman and colleagues provided a lab-based approach to purify RBCEVs using ultracentrifugation with sucrose cushion. To begin with, RBCs in whole blood were separated from white blood cells and plasma by low centrifugation and using leukodepletion filters. Then, the isolated RBCs were diluted in PBS and treated with 10 mM calcium ionophore overnight which can stimulate the release of RBCEVs and significantly increase the yield. In order to purify RBCEVs, RBCs and cell debris were removed by several rounds of low-speed centrifugation. Later, the resulting supernatants were passed through 0.45 μm syringe filters. Afterward, the RBCEVs were concentrated using ultracentrifugation at 100,000 × *g* for 70 min. Subsequently, RBCEVs were resuspended in cold PBS and layered above frozen 60% sucrose cushion and centrifuged at 100,000 × *g* for 16 h. The red layer of RBCEVs above the sucrose was collected and washed again with cold PBS by ultracentrifugation [55]. The approach is cost-effective, features high RBCEVs yield, and can be adopted in most laboratory settings. The use of sucrose cushion is also a highlight as it helps remove the protein contaminants outside RBCEVs, which might trigger unnecessary immune response and protects the integrity and biophysical properties of

For therapeutic agents to be loaded into EVs, two major strategies currently have been applied. The first option is to load the therapeutic molecules, such as RNAs, into the EVs after EVs isolation, while the second one is conducted during EV biogenesis. These methods are also known as post-loading and pre-loading, respectively. The pre-loading encapsulation approach is also referred to as the endogenous method as it uses the cellular machinery in order to load small RNA into EVs. The pre-loading approach has been shown to work for the packaging of both siRNA and miRNA into EVs. The post-loading method artificially introduces RNAs into EVs, whereas pre-loading is performed in the EVs biogenesis. Post-loading can be subdivided into passive loading, such as by physical incubation, and active loading with instances of electroporation or sonication. Furthermore, the functional small RNAs delivery using electroporated EVs has been shown to be a success in several reports but it depends on the small RNA species [58–62]. Usman and colleagues used the electroporation method for post-loading of RNAs into RBCEVs [55]. Ideally, various therapeutic molecules including ASOs, siRNAs, gRNAs, mRNAs, plasmid DNA, proteins, peptides, and chemical drug compounds can be loaded into RBCEVs using electroporation (**Figure 2**). Other post-loading methods such as mild sonication and physical incubation may be applicable to RBCEVs as tested for other types of EVs. Labeling of EVs is then required to examine the efficiency of delivery to target cells. Various methods and techniques have been applied to label EVs, with most common methods being incubation with biotinylated radioisotope, substrate of luciferase, fluorescence lipophilic dye, streptavidin-conjugated fluorescence dyes,

or the use of other modified proteins [55, 64–66].

**3.4 Advantages of red blood cell extracellular vesicles in therapeutics**

Due to their innate function on cell-cell communication, EVs can be used effectively for drug delivery [12, 67–69]. The biggest advantage of EVs drug delivery is probably that EVs can be taken from an organism and returned to the same organism *in vivo* after being loaded with therapeutic agents, which are thought to be nonimmunogenic. Another advantage to deliver nucleic acids with the help of EVs is

**126**

transfection reagents. Last but not least, RBCEVs can be frozen and thawed many cycles without affecting their integrity or efficacy. This fact suggests that RBCEVS can be developed into stable pharmaceutical products in the future, but further research needs to be done. Compared to most other current methods for programmable RNA drug therapies, which are unsuitable for the clinical use because of the low uptake efficiency and high cytotoxicity, RBCEVs show promising future prospects [55].
