# Erythrocytes and Nanoparticles: New Therapeutic Systems
## 1 Introduction
Human blood groups depend on the presence of specific surface antigens, such as sugars. This aspect must be taken into consideration during the administration of modified RBCs. If the receiving patient presents a different blood group from the donor, problems of agglutination or hemolysis may arise. For example, the 0-blood group (also called universal donor due to the absence of surface antigens) is characterized by the production of antibodies against the A and B blood group antigens. Additionally, an RHD-negative patient can suffer an immunogenic response if given RBCs from an RHD-positive subject. To avoid such problems, red blood cells should be sourced from healthy donors with the same blood group for which they are intended. In addition, where possible, autologous red blood cells should be removed, loaded and reinfused into the same subject, ensuring absolute biocompatibility. Another strategy to overcome these drawbacks is based on the conversion of one blood group to another through the action of specific enzymes able to apply specific modification of the surface markers. In this regard, specific exoglycosadases have been used to convert blood group B to 0. In those cases, no clearance problems were detected for these modified RBCs once injected in the receiving patient. On the other hand, the same cannot be said for the conversion of blood group A to 0, because of very low conversion efficacy [12]. Alternatively, the hyperosmotic method [13] allows for the creation of temporary pores in RBCs. Via contemporary exposure to the cargo of interest, RBCs are loaded after the osmotic gradient. After that, by washing the cells, the external concentration of substance decreases, the pores close and the loaded molecules remain inside the RBCs [11]. Another method is electroporation, which is the encapsulation of therapeutic molecules in RBCs through a high-intensity electric field applied to the cell suspensions [12]. Despite the potential of employing RBCs as carriers for therapeutic agents, their use can be hampered by different critical points during their manipulation. These should not be underestimated, and can include: damage to RBCs membranes during drug encapsulation, changes in osmotic pressure, contamination during manipulation, etc. [14]. A pivotal role in overcoming these drawbacks is played by the physicochemical properties of the drug. This is because loading molecules with high affinity toward RBCs leads to a reduction of the steps of cell manipulation that could compromise the biocompatibility of the RBCs [15]. The other principal approach to produce RBC vectors for therapeutic purposes is loading therapeutic molecules onto the cell surface. One possibility is the direct attachment of specific ligands onto the RBC surface by exploiting physical interactions such as van der Waals forces or hydrogen bonds [16]. Another option is coupling therapeutic molecules to ligands specific for the RBC membrane [9]. However, surface loading can compromise the biocompatibility of RBCs, leading to lysis and elimination through activation of complement pathways (and others). To overcome this drawback, one developed solution could utilize the employment of biotherapeutic agents (e.g., fibrinolytics) conjugated with antibodies for complement receptor type 1 (CR1), present on erythrocytes' surfaces [17]. Furthermore, there are other targets on RBCs which can be used for this purpose. One example is human glycoporin A (hGPA), which can interact with the single-chain variable fragment (scFv) fragment of the monoclonal antibody TER119, conjugated with different recombinant biotherapeutics [18]. For example, this antibody--when fused to complement-regulating proteins, including CR1--can enhance RBC resistance to lysis [19]. Similarly, by employing the endothelial anticoagulant and anti-inflammatory glycoprotein thrombomodulin, conjugated with TER119 scFv, a longer half-life in the bloodstream can be obtained compared with soluble thrombomodulin, improving the prevention of arterial and venous thrombosis [20].