Conjugation of Methoxypolyethylene Glycol to Bovine Red Blood Cells as a Potential Universal Oxygen Therapeutic

Increasing shortages of human red blood cells (hRBC) donations is making the supply for transfusions scarce and expensive. Investigations are ongoing to find acceptable oxygen therapeutics from hemoglobin modifications, but they have many side affects and are not able to replace red blood cell transfusions. Methoxypolyethylene glycol (mPEG) covalently bound to the surface of hRBCs has been shown to decrease immunological recognition of hRBC surface antigens. The goal of this study is to similarly PEGylate the surface of bovine RBCs (bRBCs) to reduce the demand on human blood donations needed for blood transfusions. This study investigates the feasibility of conjugating the 20 kDa succinimidyl ester of mPEG propionic acid (SPA-mPEG) to the bRBC's surface for use as an oxygen therapeutic. It was found that SPA-mPEG reacts with bRBCs in a dose dependent manner. The overall oxygen binding affinity of PEGylated bRBCs was moderately increased with greater initial SPA-mPEG concentrations when reacted with bRBCs. Control bRBCs exhibited a P50 of ~28 mmHg, while bRBCs reacted at 6 mM SPA-mPEG exhibited a reduced P50 ~21 mmHg. Oxygen transport simulations modeling the O2 distribution within the capillary and tissue space were conducted to insure that PEGylated bRBCs were capable of delivering O2 to tissues. These simulations showed that SPA-mPEG conjugated cells could still transport vital O2 to pancreatic islet tissues and maintain the tissue's O2 tension above hypoxic levels even under extreme conditions. PEGylated bRBCs reconstituted to a physiological hematocrit of 40% exhibited viscosities on the order of ~ 3 cp, similar to hRBCs. Finally, PEGylated bRBCs exhibited similar glass capillary flow characteristics indicating PEGylated bRBCs retain most of their deformability. Altogether, this shows that mPEG-bRBCs are physically capable as an oxygen therapeutic. Unfortunately, PEGylation was not able to attenuate the human immune system response. Flow cytometry experiments demonstrated that IgG and IgM in human serum and GS-IB4 were still able to recognize exposed xenoantigens and aggregate the mPEG-bRBCs. Therefore, this method of PEGylation is not recommended to create an oxygen therapeutic. Future studies to knock out this primary xenoantigen expression with siRNAs are recommended to create a universal oxygen therapeutic from bRBCs.