**2.2 Transporters**

*Erythrocyte*

**2.1 Membrane receptors**

**Membrane receptor**

CD55 (a

**Transporter**

**Cell adhesion molecule**

*DAF, decay-accelerating factor.*

*MAC, membrane attack complex.*

*SIRPα, signal regulatory protein alpha.*

*Various membrane proteins in RCs [5].*

researches were conducted together. For example, just like RC does, RCM-coated nanoparticles showed long circulation in the blood by evading immune responses such as MPS and CS [7, 8]. Also, RCM-coated glucose sensors showed high permselectivity to glucose [9]. As a result, the sensor was barely affected by interfering molecules such as saccharides and antioxidants. Likewise, the utilization of the functionalities of membrane protein of RC advances nanobiotechnology in the field of drug delivery system and biosensor. In this chapter, we investigate the various membrane proteins expressed on RC and its functionalities. The techniques for extraction and functionalization of cell membrane have been researched. Also, we

discuss about the application of RCM functionalization for the last decade.

**2. Membrane proteins of red cell membrane and its functionalities**

major membrane proteins on RCM classified by their function [5].

**Protein Gene Function**

AE1 (band 3) SLC4A1 Anion transporter RhAG RhAG Ammonia transporter Nucleoside transporter SLC29A1 Nucleoside transporter Urea transporter SLC14A1 Urea transporter Glucose transporter SLC2A Glucose transporter

*transporter, and glucose transporter; cell adhesion molecule consists of CD47.*

CD59 CD59 <sup>b</sup>

Membrane proteins are essential components allowing specific functionalities for cells. There are three categories classified by its function. Membrane proteins perform as receptors, transporters, and cell adhesion molecules. **Table 1** represents

Membrane receptors are one of integral membrane proteins. They mediate cell signaling via binding extracellular molecules. Specifically, membrane receptors allow communication between the cell and external environment. Hormones, cytokines, cell adhesion molecules, and immunoproteins are examples of the extracellular molecules. The ligand bound of the membrane receptor may induce changes in the metabolism or activity of the cell. In RC, CD55 (decay-accelerating factor) and

> DAF) CD55 Decay-accelerating factor that prevents the activation of complement system

> > attack complex

CD47 CD47 "do not eat me" signal protein that interacts with c

*Membrane receptors consist of CD55 and CD59; transporters consist of AE1, RhAG, nucleoside transporter, urea* 

inhibit phagocytosis

MAC-inhibitory protein that prevents complement membrane

SIRPα to

**138**

**Table 1.**

*a*

*b*

*c*

Transporters are involved in the movement of specific molecules or ions across cell membrane. The proteins are involved in the movement of molecules by active transport or facilitated diffusion. It is revealed that anion, gas, nucleoside, urea, and glucose transporters are expressed on the RC. In detail, (AE1, also called band 3) is responsible for mediating the exchange of chloride ion with bicarbonate (HCO3ˉ) across RCM [13]. Rh-associated glycoprotein (RhAG) is a gas transporter which permeates carbon dioxide [1]. Nucleoside transporter mediates the transport of nucleoside substrates like adenosine [14]. Urea transporter is specialized in urea transportation, which is activated by antidiuretic hormone (vasopressin) [15]. Glucose transporter (GLUT) is a uniporter that transports glucose toward intracellular orientation (**Figure 3**) [16]. GLUT is an essential protein for glucose uptake of the cell by catalyzing facilitative diffusion. Especially, RC expresses a large number of GLUT compared to other cells because the cell lacks mitochondria and the energy is produced by glycolysis of glucose [17].

#### **Figure 1.**

*Hemolysis mechanism of paroxysmal nocturnal hemoglobinuria (PNH) via the complement system [4]. (a) Normal RBC possesses CD55 and CD59 which are glycosylphosphatidylinositol (GPI)-anchored self-protective complement regulatory factors. CD55 is a widely expressed membrane protein that accelerates the decay of C3 convertases. CD59 is the major inhibitor of terminal complement, which blocks the generation of the membrane attack complex (MAC). (b) Intravascular hemolysis of PNH RBC through C3 convertase and MAC. (c) Extravascular hemolysis of PNH RBC via macrophage. Eculizumab inhibits the complement activation by compensating CD59. \*PNH, a life-threatening disease characterized by destruction of RBC by complement system; eculizumab, a monoclonal antibody complement inhibitor which is highly effective for PNH; C3 con, C3 convertase; C5b-8, complex of C5b, C6, C7, and C8 proteins; C3dg, a fragment of C3 protein, which is ligand of integrin (CR3) on macrophage; iC3b, inactivated C3b; Hb, hemoglobin; CR3, complementary 3.*

#### **Figure 2.**

*Complement system signal cascade [4]. Proximal complement consists of three pathways. The lectin, classical, and alternative pathways initiate and converge at the step of complement component 3 (C3) activation. Terminal complement is initiated by C5 convertases, leading to cleavage of C5 to C5a and C5b. C5b oligomerizes with C6, C7, C8, and multiple C9 molecules to form the membrane attack complex (MAC). The CD55 inhibits proximal complement activation by accelerating the decay of C3 convertases preventing the incorporation of C9 into the MAC.*

#### **Figure 3.**

*(a) Overall structure of human glucose transporter-1 (GLUT1) and (b) working model for GLUT1 [16]. The working model is predicted to have four conformations (outward open, ligand-bound and occluded, inwardopen, and ligand-free and occluded) required for a complete glucose transport cycle. Intracellular helix (ICH) domain of GLUT1 has two critical roles. First, the ICH domain appears to maintain a defined conformation with respect to the N domain. Second, the ICH domain is pulled toward the C domain during the inter-domain rotation from occluded to inward-open conformation.*

**141**

[7, 8, 23].

*Application of Red Cell Membrane in Nanobiotechnology DOI: http://dx.doi.org/10.5772/intechopen.84274*

Cell adhesion molecules interact with membrane receptors of various cells. RC has CD47 as a cell adhesion molecule [18]. CD47 belongs to the immunoglobulin superfamily and sends a "*don't eat me"* signal to MPS such as monocyte and macrophage (**Figure 4**) [6]. This intriguing signal is derived from the interaction between CD47 and signal regulatory protein alpha (SIRPα) which is expressed on monocytes and most of subpopulations such as macrophages. Indeed, CD47-eliminated RCs

*The cocrystal structure shows human CD47 (hCD47) with human signal regulatory protein α (hSIRPα). The left panel depicts that the occurrence of phagocytosis depends on the reaction between hCD47 and hSIRPα [6].*

were easily phagocytosed by macrophages unlike RC with CD47 [3, 19].

**3.1 Red cell membrane extraction procedure**

**3. Red cell membrane extraction and functionalization techniques**

RC can easily be extracted from whole blood of mice or human. Normally, the whole blood is extracted at vacutainer tube (evacuated tube) containing anticoagulant such as heparin, citrate, or ethylenediaminetetraacetic acid (EDTA) according to the purpose (**Table 2**). Heparin collection tubes are preferred for peripheral blood in cytogenic studies. Heparin activates antithrombin III which deactivates thrombin and serine endopeptidase, which are essential enzymes for coagulation [20]. Citrate collection tubes are employed for blood transfusion and coagulation assays because citrate reversibly binds to calcium which is an essential molecule in many steps of coagulation cascade [21]. EDTA collection tubes are usually used for complete blood count (CBC) test because EDTA is a strong anticoagulant that irreversibly binds to calcium [22]. Since coagulation requires RC to form blood clot, EDTA collection tubes are mostly used for RC extraction

The procedure for extracting RC from whole blood is as follows [23]. Whole blood withdrawn from mice or human is centrifuged at 800–1000 g for 5 min at 4°C in order to remove the plasma and the buffy coat. The resulting sediment is washed three times with ice-cold 1× PBS to remove blood proteins adsorbed on RCs. To extract cell membrane from RCs, hypotonic treatment, homogenize, or sonication is conducted for hemolysis. Hypotonic treatment is the most convenient hemolysis procedure without disruption of membrane and membrane proteins. Washed RCs are suspended in 0.25× PBS for 20 min at 4°C. As hemolysis progresses, hemoglobin is released from RCs, and RC ghosts (empty cell membrane without cytoplasmic contents) are formed [24]. As a result, RC ghost can be verified with phase contrast

**2.3 Cell adhesion molecules**

**Figure 4.**

*Application of Red Cell Membrane in Nanobiotechnology DOI: http://dx.doi.org/10.5772/intechopen.84274*

#### **Figure 4.**

*Erythrocyte*

**140**

**Figure 3.**

*rotation from occluded to inward-open conformation.*

**Figure 2.**

*into the MAC.*

*Complement system signal cascade [4]. Proximal complement consists of three pathways. The lectin, classical, and alternative pathways initiate and converge at the step of complement component 3 (C3) activation. Terminal complement is initiated by C5 convertases, leading to cleavage of C5 to C5a and C5b. C5b oligomerizes with C6, C7, C8, and multiple C9 molecules to form the membrane attack complex (MAC). The CD55 inhibits proximal complement activation by accelerating the decay of C3 convertases preventing the incorporation of C9* 

*(a) Overall structure of human glucose transporter-1 (GLUT1) and (b) working model for GLUT1 [16]. The working model is predicted to have four conformations (outward open, ligand-bound and occluded, inwardopen, and ligand-free and occluded) required for a complete glucose transport cycle. Intracellular helix (ICH) domain of GLUT1 has two critical roles. First, the ICH domain appears to maintain a defined conformation with respect to the N domain. Second, the ICH domain is pulled toward the C domain during the inter-domain* 

*The cocrystal structure shows human CD47 (hCD47) with human signal regulatory protein α (hSIRPα). The left panel depicts that the occurrence of phagocytosis depends on the reaction between hCD47 and hSIRPα [6].*

#### **2.3 Cell adhesion molecules**

Cell adhesion molecules interact with membrane receptors of various cells. RC has CD47 as a cell adhesion molecule [18]. CD47 belongs to the immunoglobulin superfamily and sends a "*don't eat me"* signal to MPS such as monocyte and macrophage (**Figure 4**) [6]. This intriguing signal is derived from the interaction between CD47 and signal regulatory protein alpha (SIRPα) which is expressed on monocytes and most of subpopulations such as macrophages. Indeed, CD47-eliminated RCs were easily phagocytosed by macrophages unlike RC with CD47 [3, 19].

#### **3. Red cell membrane extraction and functionalization techniques**

#### **3.1 Red cell membrane extraction procedure**

RC can easily be extracted from whole blood of mice or human. Normally, the whole blood is extracted at vacutainer tube (evacuated tube) containing anticoagulant such as heparin, citrate, or ethylenediaminetetraacetic acid (EDTA) according to the purpose (**Table 2**). Heparin collection tubes are preferred for peripheral blood in cytogenic studies. Heparin activates antithrombin III which deactivates thrombin and serine endopeptidase, which are essential enzymes for coagulation [20]. Citrate collection tubes are employed for blood transfusion and coagulation assays because citrate reversibly binds to calcium which is an essential molecule in many steps of coagulation cascade [21]. EDTA collection tubes are usually used for complete blood count (CBC) test because EDTA is a strong anticoagulant that irreversibly binds to calcium [22]. Since coagulation requires RC to form blood clot, EDTA collection tubes are mostly used for RC extraction [7, 8, 23].

The procedure for extracting RC from whole blood is as follows [23]. Whole blood withdrawn from mice or human is centrifuged at 800–1000 g for 5 min at 4°C in order to remove the plasma and the buffy coat. The resulting sediment is washed three times with ice-cold 1× PBS to remove blood proteins adsorbed on RCs. To extract cell membrane from RCs, hypotonic treatment, homogenize, or sonication is conducted for hemolysis. Hypotonic treatment is the most convenient hemolysis procedure without disruption of membrane and membrane proteins. Washed RCs are suspended in 0.25× PBS for 20 min at 4°C. As hemolysis progresses, hemoglobin is released from RCs, and RC ghosts (empty cell membrane without cytoplasmic contents) are formed [24]. As a result, RC ghost can be verified with phase contrast

