Chemical modification of red blood cells for development of blood substitute

Abstract

Chemical modifications of red blood cells (RBC) were performed to develop blood substitute which was universially transfusible, stable and active in oxygen transport. As a chemical modification reagents,we used methoxy-polyethylene glycol(mPEG),glutaraldehyde(GA),divinylsulfone(DVS) and bezafibrate(BZF).To evaluate the chemically modified RBCs as a blood substitute,it was performed that membrane protein analysis by SDS-polyacrylamide gel electrophoresis, blood group typing with anti-A,-B and-D antiserum, hemagglutination test, antibody binding test,oxygen equilibrium curve analysis and osmotic fragility curve analysis. Human Rh(+) AB type RBCs were modified with methoxy-polyethylene glycol(mPEG) in order to decrease agglutinabilities toward clinically important anti- A,anti-B and anti-D reagents. Attachment of mPEG to membrane proteins of the RBC was identified by the shift of well-known blood group active glycoprotein bands such as band 3, 4.5 and PAS-1 on the SDS-polyacrylamide gel electrophoresis. When 4 mM concentration of mPEG was added to RBCs, agglutination was minimum by a blood group typing and microwell agglutination tests. Antibody binding tests showed decreased antibody binding was the result of mPEG attachment to blood group active glycoprotein of the cell membrane. The morphology of RBCs after mPEG attachment was the usual discocytic cell. Oxygen equilibrium curves of the mPEG attached RBCs were similar to unmodified RBCs. This approach to decrease agglutinability of the RBCs toward blood group antibodies may be used to develop a universially transfusible blood substitute. However, this mPEG attached RBCs were less stable in osmotic fragility against NaCl concentrations than unmodified RBCs. This unstability would be overcomed by crosslinking of the RBCs. RBCs were crosslinked with glutaraldehyde by using a hemodialyzer which is used as artificial kidney. Human RBC,which was flowed in a flow of 2 ml/min flow rate, was extensively crosslinked ...