# Erythrocytes and Nanoparticles: New Therapeutic Systems
## 5 RBCs Loaded with Biologically Active Compounds and NPs: New Perspective
The exploited modification concerns the opsonization with antibodies of their membrane, the binding of complement component C3b or, alternatively, treatment with calcium ionophore [92]. In this regard, a targeted delivery of methotrexate to the liver was successfully carried out [93]. For the loading of the drug into RBCs, researchers have experimented with the electroporation, hypoosmotic and hyperosmotic methods. The prepared RBC carriers were administered, leading to decreased peak plasma concentration (which is important for a gradual release of the drug), along with increasing accumulation in the liver, higher methotrexate concentration in the bloodstream, and a longer half-life that treatment with the free drug. In a similar way, RBC-based carriers envisaged a new contribution in the treatment of retroviral infections caused by the human immunodeficiency virus (HIV). The loading of antiretroviral drugs enabled inhibition of the activity of reverse transcriptase. Additionally, encapsulation of reduced glutathione (GSH) was shown to block viral replication and interfere with the protein envelope folding. These carriers were further modified in order to target macrophages, preventing the transmission of HIV from macrophages to lymphocytes and reducing typical symptoms of the disease within 3 months [94]. Furthermore, the importance of targeted drug delivery is shown in other studies concerning immunization, cancer immunotherapy and immune tolerance [95]. The possibility of delivering antigens directly to macrophages or dendritic cells (DCs) can guarantee an improvement in the immunological response when compared to the response obtained using classical adjuvant. A classical study demonstrated these properties by the conjugation of glycoproteins B of Herpes Simplex virus type 1 onto the surface of red blood cells [96]. The ultimate goal of immunotherapy is to stimulate the immune system against cancer. With this in mind, loading specific tumor-associated antigens into RBCs could offer the opportunity to stimulate the activation of specific T-lymphocytes which act against tumoral growth. In a recent study, ovalbumin antigen was encapsulated into erythrocytes whose surface had been modified with specific antibodies in order to induce direct phagocytosis by macrophages and DCs. In this way, an immune response toward cell expressing ovalbumin was obtained, with the observed cell lysis induced by the activation of CD4+ and CD8+ T cells [97]. In other conditions, an immune tolerance towards a specific antigen is needed in order to prevent the attack of the immune system against its own antigens, a condition which can occur in autoimmune diseases or during a transplant. In this case, RBCs employed as a carrier can offer great opportunities. One potential application concerns enzyme replacement therapy--loading the enzyme alglucosidase alfa (AGA) to treat glycogen storage disease caused by alfa-glucosidase deficiency. The targeted delivery of the therapeutic agent to the antigen-presenting cells of the liver and spleen has led to an elimination of the humoral response toward AGA--which is otherwise caused by administration of the free drug. This occurs because liver DCs have an immature phenotype and there are not able to elicit an Ag-specific T-cell response; however, they can induce the development of T-cell tolerance [98].