Drug delivery using natural biological carriers, especially erythrocytes, is a rapidly developing field. Such erythrocytes can act as carriers that prolong the drug’s action due to its gradual release from the carrier; as bioreactors with encapsulated enzymes performing the necessary reactions, while remaining inaccessible to the immune system and plasma proteases; or as a tool for targeted drug delivery to target organs, primarily to cells of the reticuloendothelial system, liver and spleen. To date, erythrocytes have been studied as carriers for a wide range of drugs, such as enzymes, antibiotics, anti-inflammatory, antiviral drugs, etc., and for diagnostic purposes (e.g., magnetic resonance imaging). The review focuses only on drugs loaded inside erythrocytes, defines the main lines of research for erythrocytes with bioactive substances, as well as the advantages and limitations of their application. Particular attention is paid to in vivo studies, opening-up the potential for the clinical use of drugs encapsulated into erythrocytes.

Background. L-asparaginase is an enzyme, widely used in the therapy of acute lymphoblastic leukemia in children and adults, but its use is limited due to a wide range of side effects and anaphylactic reactions. L-asparaginase loaded into erythrocytes can solve these problems. This enzyme is protected from the immune system and plasma proteases due to erythrocyte membrane, but continues to work inside the cell because its membrane is permeable to L-asparagine. Thus, the half-life of the drug increases and anaphylactic reactions reduce. The encapsulation of L-asparaginase into erythrocytes can be performed by various osmotic methods. Each of them is characterized by the amount of encapsulated enzyme, the cell yield, as well as by the quality indices of the survived erythrocytes. An important parameter of each method is the possibility to provide sterility of this dosage form for the clinical use.The aim of the study was the comparing of three osmotic methods of L-asparaginase encapsulation into erythrocytes (hypo-osmotic lysis, dialysis and flow dialysis) to select the most promising method for clinical use.Materials and methods. A suspension of erythrocytes of healthy donors (hematocrit 60–70%) was mixed with L-asparaginase from E. сoli. The procedures of hypotonic reversible lysis, dialysis in dialysis bags, or flow dialysis using pediatric dialyzers were performed. The physiological osmolality was restored in suspensions after the procedure by the addition of a hypertonic solution, and they were incubated for 30 min at 37 °C. Then the cells were washed in isotonic phosphate-buffered saline with pH 7.4. Activity of L-asparaginase, volume, hematocrit, hematological indices and osmotic cell fragility of erythrocytes were measured in the suspensions of erythrocytes before and after the enzyme encapsulation procedure.Results. An optimal osmolality of the hypotonic buffer for each method was selected and was equal to 90–110 mOsm/kg. The yields of encapsulation were 4.2 ± 2.0, 6.0 ± 2.3 and 16.2 ± 2.2 % for hypotonic lysis, dialysis and flow dialysis, respectively. The hematological indices of the obtained erythrocyte-carriers differed from the corresponding parameters of the initial erythrocytes, but did not differ significantly for different methods.Conclusion. Comparative investigation of mentioned above parameters allowed choosing the method of flow dialysis as the most promising for clinical use.

The limitations of the efficiency of ammonium-neutralizing erythrocyte-bioreactors based on glutamate dehydrogenase and alanine aminotransferase reactions were analyzed using a mathematical model. At low pyruvate concentrations in the external medium (below about 0.3 mM), the main limiting factor is the rate of pyruvate influx into the erythrocyte from the outside, and at higher concentrations, it is the disappearance of a steady state in glycolysis if the rate of ammonium processing is higher than the critical value (about 12 mM/h). This rate corresponds to different values of glutamate dehydrogenase activity at different concentrations of pyruvate in plasma. Oxidation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) by glutamate dehydrogenase decreases the fraction of NADPH in the constant pool of nicotinamide adenine dinucleotide phosphates (NADP + NADPH). This, in turn, activates the pentose phosphate pathway, where NADP reduces to NADPH. Due to the increase in flux through the pentose phosphate pathway, stabilization of the ATP concentration becomes impossible; its value increases until almost the entire pool of adenylates transforms into the ATP form. As the pool of adenylates is constant, the ADP concentration decreases dramatically. This slows the pyruvate kinase reaction, leading to the disappearance of the steady state in glycolysis.

The review is devoted to one of the main regulatory enzymes of glycolysis in erythrocytes – pyruvate kinase, a deficiency of which is often the cause of hereditary nonspherocytic hemolytic anemia. The article presents data on the structure and function of pyruvate kinase and the currently known mutations of coding this enzyme gene. Authors analyzed associations between various genetic types and impaired enzyme function and the severity of the hemoly sis.

The differential diagnosis of hereditary spherocytosis is a great challenge because of the similar clinical and laboratory signs it shares with other hereditary hemolytic anemias as well as due to the limited availability of molecular genetic testing. The development of easy-to-perform laboratory tests for the differential diagnosis of hereditary hemolytic anemias remains as relevant as ever. Here, a method of measuring red blood cell filterability for the diagnosis of hereditary spherocytosis is proposed for the first time. The aim of our study was to compare red blood cell filterability measurement with other diagnostic tests for hereditary spherocytosis as well as to assess its specificity and sensitivity. We included 30 patients (18 girls and 12 boys, with the median age of 8.6 years) with hereditary spherocytosis and 15 patients (9 girls and 6 boys, with the median age of 10 years) with other hereditary hemolytic anemias (pyruvate kinase deficiency (n = 14) and stomatocytosis (n = 1)). The diagnostic work-up for hereditary hemolytic anemia included a complete blood count test using an automated hematology analyzer, an osmotic resistance analysis before and after 24 hours of incubation at 37°С, erythrocytometry with sphericity index calculation, EMA test (eosin-5-maleimide binding assay) and red blood cell filterability measurement using artificial filters with cylindrical pores 3-5 μm in diameter. The study was approved by the Independent Ethics Committee and the Scientific Council of the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology. The parents of all of the enrolled children signed a voluntary informed consent form for peripheral blood collection and diagnostic testing. In all the cases of hereditary spherocytosis diagnosed in accordance with the relevant clinical recommendations, red blood cell filterability was very low (0–0.31 units). It was higher only in 3 instances, reaching 0.47, 0.64 and 0.82 units, but in two of these cases there were no genetic data available, and the remaining patient was found to harbor the SPTA1 c.433999CT mutation which was characterized both as spherocytosis and elliptocytosis. Red blood cell filterability in the group of the patients with other hemolytic anemias equalled 0.55 to 0.86 units (with the median of 0.77 units). The sensitivity of the RBC filterability measurement method in diagnosing hereditary spherocytosis was 93% (with 100% specificity), while the EMA test had a sensitivity of 89% and specificity of 95%. Our comparative study showed that red blood cell filterability measurement and the EMA test have similar sensitivity and specificity in diagnosing hereditary spherocytosis but the former is much cheaper and easier to perform since it does not require expensive equipment and can be carried out at any laboratory.

Drug delivery using natural biological carriers, especially erythrocytes, is a rapidly developing field. Erythrocytes can act as carriers with the gradual release of a pharmacological agent, as bioreactors with encapsulated enzymes, or as a tool for targeted delivery of drugs to target organs especially tissue macrophages, liver and spleen. To date, red blood cells have been studied as carriers for a wide range of drug compounds, such as enzymes, antibiotics, anti-inflammatory, antiviral drugs, etc. The review is devoted to the advantages of erythrocytes as carriers for the delivery of drugs loaded into the erythrocyte, or related to its surface, and defines the main directions of research on erythrocytes carriers of biologically active substances. Particular attention is paid to in vivo studies that reveal the potential of carrier erythrocytes for clinical use.