Chitosan-alginate Modified Red Blood Cell Used For Xenotransfusion

As a kind of special resources, blood has very important application in the clinical emergency treatment. However, countries around the world have different degrees of blood shortage because of the blood itself characteristics and preservation limitation. People have been trying to find an adequate, safe and effective blood substitute as the supplement of current blood bank system. Blood substitute research mainly includes two development approaches of hemoglobin-based oxygen carries(HBOCs) and heterogeneous modified red blood cells(RBCs). The main impediment of xenotransfusion is immune rejection problem between animals and humans. In recent years, advances in the field of xenotransplantation raise the intriguing possibility of using modified heterogeneous red blood cell as an alternative source for blood transfusion. This study is an attempt of modified heterogeneous RBCs as potential blood substitutes, using the micro capsule immune isolation technology.Objective On the basis of the principle of polyelectrolyte complexation, chitosan-alginate modifying conditions were studied to prepare single-celled modified heterogeneous RBCs. Analyzing the influence of chitosan-alginate modification on the properties and functions of RBCs and studying the immunogenicity and circulation time in vivo of modified RBCs to investigate the immune isolation effect of chitosan-alginate coating. The effect and influence of modified RBCs transfusion to animals were study to explore the potential possibility of chitosan-alginate modified RBCs used for xenotransfusion.Methods Using different concentrations of chitosan and alginate to incubate red blood cells, red blood cell count was measured by blood cell analyzer. Microscopic(cells) electrophoresis experiment was carried out to determine the surface charge of RBCs in modification process. Using the fluorescent tagged sodium alginate to modify erythrocyte and identify the structure of chitosan-alginate modified RBCs. The morphology and integrity of RBCs were observed by optical microscope and scanning electron microscope. Determining the size and osmotic fragility of RBCs before and after modification, and the red blood cell deformability, aggregation and oxygen affinity were also tested to analyze the mechanical properties and oxygen carrying function of modified RBCs. The cross matching test was used to determine the response of chitosan-alginate modified RBCs reacting with human standard serum, plasma and the rat plasma. The cycle time of red blood cell in animals was measured by tail vein injection of RBCs in mice experiment to study the immune isolation effect of chitosan-alginate modification. The oxygen carrying ability, improvement of microcirculation, blood flow dynamics characteristics and comprehensive anti-shock curative effect of modified RBCs in vivo were investigated by using the rat model of blood exchange, hemorrhagic shock-microcirculation observation model and severe uncontrolled hemorrhagic shock model in rats to study the potential possibility of chitosan-alginate modified RBCs as blood substitutes.Results Based on polyelectrolyte layers of self-assembly technology, chitosan and alginate form micro capsule membrane outside of the RBCs, preparing single-celled modified RBCs with yield close to 90%. The surface charge of RBCs changed twice during the modification process. Chitosan-alginate modified RBCs have similar surface negative charge with natural red blood cell. The diameter of modified RBCs increases slightly, and modification has no effect on red blood cell morphology and osmotic fragility. Chitosan-alginate modification decreased deformability of RBCs, and the aggregation had no significantly alteration. The oxygen affinity of RBCs increased after chitosan-alginate modification(P50 value from 29.8 down to 25.4 mmHg), more similar to natural human erythrocyte. The responses of modified porcine RBCs reacting with human standard serum, frozen plasma and rat plasma reduced from strong positive to approximate negative or negative in the cross matching tests. The results showed that the 24 h survival rate of modified RBCs in vivo was close to 50% and the half-life was 20.7h, about 9 times that of the RBCs without chitosan-alginate modification. Compared with dextran solution, transfusion with modified RBCs maintained higher mean arterial pressure(MAP) and hematocrit(HCT), improving the survival time of rats. Chitosan-alginate modified RBCs also can effectively improve and restore microcirculation disorder caused by hemorrhagic shock. Transfusion with chitosan-alginate modified RBCs improved metabolic acidosis caused by hypoxia in hemorrhagic shock. Similar to allogeneic RBCs, chitosan-alginate modified RBCs can develop the function in vivo, having significant resistance to hemorrhagic shock.Conclusions This study achieved the single-celled RBCs modification by chitosan and alginate, based on the layers of polyelectrolyte self-assembly technology. Chitosan-alginate modification had little effects on the structure and function of RBCs, while the mechanical properties and oxygen carrying function of modified RBCs were still similar to natural human erythrocytes. The immune isolation effect of chitosan-alginate modification significantly reduced the immunogenicity of heterogeneous RBCs and prolonged circulation time in vivo. Heterogeneous RBCs with chitosan-alginate modification can play function as normal RBCs in circulation in a certain time, having the potential for xenotransfusion.