Surface Coating of Red Blood Cells with Monomethoxy poly(ethylene glycol) Activated with Two Different Reagents

Document Type: Research Article


Biotechnology Group, Department of Chemical Engineering, Tarbiat Modares University, P.O. Box 14115-143 Tehran, I.R. IRAN


Methoxy poly(ethylene glycol) (mPEG) with molecular mass of 5 kDa activated with succinimidyl carbonate and cyanuric chloride, separately was covalently attached to human red blood cells (RBCs). Inhibition of agglutination by blood-type specific antisera (anti-D) was employed to evaluate the effect of the polymer coating. The remaining single cells after incubation with anti-D sera were counted using a simple hemocytometer (Improved Neubauer Ruling). The extent of surface coating was evaluated by addition of FITC labeled-anti-D to the cells and recording the fluorescence intensity ratio of FITC-anti-D bound cells of the PEG-RBCs versus control (uncoated) RBCs. The morphology of RBCs was evaluated by scanning electron microscopy (SEM). The effect of polymer coating, based on the immunological response of RBCs, using two kinds of activated mPEG, at optimum conditions of PEGylation was compared. It was found that succinimidyl carbonate at its optimum condition (pH=8.7, temperature =14°C and reaction time =60 min) is more effective than cyanuric chloride at its optimum condition (pH=8.7, temperature =14 °C and reaction time =30 min) for RBC coating with mPEG.  


Main Subjects

[1] Scott, M. D. and Murad, K. L., Cellular Camouflage: Fooling the Immune System with Polymers, Curr. Pharmaceut. Des., 4, 423 (1998).

[2] Blackall, D. P., Armstrong, J. K., Meiselman, H. J. and Fisher, T. C., Polyethylene Glycol-Coated Red Blood Cells Fail to Bind Glycophorin A-Specific Antibodies and are Impervious to Invasion by the Plasmodium falciparummalaria parasite, Blood, 97, 551 (2001).

[3] Murad, K. L., Mahany, K. L., Brugnara, C., Kuypers, F. A., Eaton, J. W. and Scott, M. D., Structural and Functional Consequences of Antigenic Modulation of Red Blood Cells with Methoxypoly(Ethylene Glycol), Blood ,93, 2121 (1999).

[4] Castro, O., Sandler, S. G., Houston-Yu, P. and Rana, S.,   Predicting    the   Effect   of    Transfusing only Phenotype-Matched RBCs to Patients with Sickle Cell Disease: Theoretical and Practical Implications, Transfusion, 42, 684 (2002).

[5] Vichinsky, E. P., Earles, A., Johnson, R. A., Hoag, M.S., Williams, A. and Lublin, B., Alloimmunization in Sickle Cell Anemia and Transfusion of Racially Unmatched Blood, New Engl. J. Med., 322, 1617 (1990).

[6] Hashemi Najafabadi, S., Vasheghani Farahani, E., Shojaosadati, S. A., Rasaee, M. J., Armstrong, J. K., Moin, M. and Pourpak, Z., A Method to Optimize PEG-Coating of Red Blood Cells, Bioconjug. Chem., 17, 1288 (2006).

[7] Hashemi Najafafabadi, S., Vasheghani Farahani, E., Shojaosadati, S. A. and Rasai, M. J., Evaluation of the Surface Treatment of Red Blood Cells by Polymer Coating, Iran. J. Polym. Sci. Technol., 18, 311 (2006).

[8] Gundersen, S. I. and Palmer, A. F., Conjugation of Methoxy Polyethylene Glycol to the Surface of Bovine Red Blood Cells, Biotech. Bioeng., 96 (6), 1199 (2007).

[9] Bradley, A. J. and Scott, M. D., Immune Complex Binding by Immunocamouflaged [Poly(ethylene glycol) grafted] Erythrocytes, Am. J. Hematol., 82, 970 (2007).

[10] Roberts, M. J., Bentley, M. D. and Arris, J. M., Chemistry for Peptide and Protein PEGylation, Adv. Drug Deliv. Rev., 54, 459 (2002).

[11] Kodera, Y., Matsushima, A., Hiroto, M., Nishimura, H., Ishii, A., Ueno, T. and Inada, Y., Pegylation of Proteins and Bioactive Substances for Edical and Technological Applications, Prog. Polym. Sci., 23, 1233 (1998).

[12] Armstrong, J. K., Meiselman, H. J. and Fisher, T. C., Covalent Binding of Poly(ethyleneglycol) to the Surface of Red Blood Cells Inhibits Aggregation and Reduces Low-Shear Blood Viscosity, Am. J. Hematol., 56, 26 (1997).

[13] Scott, M. D. and Chen, A. M., Beyond the Red Cell: PEGylation of Other Blood Cells and Tissues, Trans. Clin. Biol., 11, 40 (2004).

[14] Sarvi,  F., Vasheghani-Farahani, E., Shojaosadati, S. A., Hashemi Najafabadi, S., Moin, M. and Pourpak, Z., Surface Treatment of Red Blood Cells with  Monomethoxy poly (ethylene glycol) Activated by Succinimidyl Carbonate, Iran. Polym. J., 6, 525 (2006).

[15] Miron, T. and Wilchek, M., A Simplified Method for the Preparation of Succinimidyl Carbonate Poly-ethylene Glycol for Coupling to Proteins, Bioconjug. Chem., 4, 568 (1993). 

[16] Bradley,  A. J.,  Murad,  K. L., Regan, K. L. and Scott, M. D., Biophysical Consequences of Linker Chemistry and Polymer Size on Stealth Erythrocytes: Size dose Matter, Biochim. Biophys. Acta, 1561, 147 (2002).

[17] Coligan, J. E.,  Kruisbeek, A. M, Margulies, D. H., Shevach, E. M. and Strober, W., “Current Protocols in Immunology”,  John Wiley & Sons, Inc., (1991). 

[18] Kayden,  H. J. and  Bessis,  M.,  Morphology of Normal Erythrocyte and Acanthocyte Using Nomarski Optics and the Scanning Electron Microscope, J. Hematol., 35, 427 (1970).

[19] Davies,  L., “Efficiency  in  Research, Development and Production: The Statistical Design and Analysis of Chemical Experiments”, The Royal Society of Chemistry, Cambridge, p. 1 (1993).

[20] Roy, R. K., “A Primer on the Taguchi Method”, Van Nostrand Reinhold, New York, p. 1 (1990).

[21] Jackson, C. C., Charlton, J. L., Kuzminski, K., Lang, G. M. and Sehon, A. H., Synthesis, Isolation and Characterization of Conjugates of Ovalbumin with Monomethoxy Polyethylene Glycol Using Cyanuric Chloride as the Coupling Agent, Anal. Biochem., 165, 114 (1987).

[22] Scott, M. D., Murad, K. L., Koumpouras, F., Talbot, M. and Eaton, J. W., Chemical Camouflage of Antigenic Determinants: Stealth Erythrocytes, Proc. Natl. Acad. Sci. USA, 94, 7566 (1997).

[23] Fisher, T. C., PEG-Coated Red Blood Cells-Simplifying Blood Transfusion in the New Millennium?, Immunohematol., 16, 37 (2000).

[24] Hortin, G. L., Lok, H. T. and Huang, S. T., Progress Toward Preparation of Universal Donor Red Cells, Artif. Cells, Blood Substitutes, Immobilization Biotechnol., 25, 487 (1997).

[25] Poraicu, D., Sandor, S. and Menessy, I., Decrease of Red Blood Cell Filterability Seen in  Intensive Care II. Red Blood Cell Crenation in-vivo as Morphological Evidence of Increased Red Cell Viscosity in Low Flow States, Resuscitation, 10, 305 (1983).

[26] Miltin, H. J., Martin,N. E. and Modi, M., Pegylation a Novel Process for Modifying Pharmacokinetics, Clin. Pharmacokinet., 40 (7), 539 (2001).