# Erythrocytes and Nanoparticles: New Therapeutic Systems
## 5 RBCs Loaded with Biologically Active Compounds and NPs: New Perspective  
Exploiting the advantages of biocompatibility, biodegradability, long-life in bloodstream, decreased side effects of therapeutic agents, immune evasion and the possibility of targeting specific organs such as liver, kidney and spleen, RBCs can be employed not only for targeted drug delivery, but also as bioreactors, as carriers with smart release, and also in diagnostics (Figure 5) [84; 85]. Figure 5: Different application of RBCs loaded with therapeutic agents and/or NPs. The employment of RBCs as bioreactors has also found several applications in anticancer therapy. This employment has focused mainly on l-asparaginase, methioninease and arginine deaminase, which are able to decrease the blood level of amino acids (asparagine, methionine or arginine, respectively) necessary for biosynthesis during cell division. These are essential for cancer cells that are not able to synthetize asparagine or arginine on their own, since they do not contain asparagine synthetase [86]. Erythrocytes (as bioreactors) represent a new and interesting subfield for biomedical applications, giving researchers the opportunity to improve the treatment of specific diseases caused by the absence or decrease in the activity of certain enzymes that, if administrated alone, could cause an immune response or the rapid removal from the bloodstream. Despite these opportunities, the RBC membranes' impermeability represents a limit for their effectiveness in application as bioreactors. To overcome this barrier, specific membrane transporters can be exploited to allow the loading of the specific substrate of interest through an active transport. Otherwise, RBCs as bioreactors with a specific enzyme could allow entrance of cargo of interest. It is possible to utilize a non-diffusible form of the therapeutic agent, thanks to the creation of transient pores. Then the enzymatic conversion of the drug into the diffusible form is released in the blood, thanks the bioreactor's RBCs. An example of this strategy would be the prodrug dexamethasone 21-phosphate which, once inside erythrocytes, is subjected to conversion in the diffusible drug by the enzyme loaded inside [87]. Recently, the above-mentioned therapeutic strategies have found good results in the treatment of acute lymphoblastic leukemia, acute myeloid leukemia [88] and metastatic pancreatic cancer by loading RBCs with asparaginase. In the last case, a high effectiveness was successfully achieved for pancreatic cancer, with a reduction of the mortality rate of 40%, compared with chemotherapy treatment alone [89]. Nevertheless, the engineering of RBCs as carriers for the gradual release of anticancer drugs resulted in good performance in clinical trials. The administration of pharmacological agents such as anthracycline antibiotics (e.g., doxorubicin) can lead to cardiotoxicity and other undesired side effects on healthy tissues due to the production of reactive oxygen species [90]. These drawbacks have been overcome through the encapsulation of doxorubicin into erythrocytes, which were then employed for the treatment of acute lymphoblastic and myeloid leukemia. This engineered erythrocyte-based carrier led to a decrease in the peak plasma concentration of antibiotics compared with the administration of the free drug. Additionally, a targeted delivery to the liver, better effectiveness and a prolonged half-life in the bloodstream were reported, as were decreases in cardiotoxicity and the number of adverse reactions [91]. As is well known, senescent and damaged RBCs are removed upon passage through the spleen trabeculae and by the macrophages of the RES, including peritoneal macrophages, hepatic Kupffer cells and alveolar macrophages of the lung. These properties can be exploited for targeted drug delivery by a suitable modification of the RBC's surface in order to impart the damaged features.