SURFACE TREATMENT OF RED BLOOD CELLS WITH MONOMETHOXYPOLY(ETHYLENE GLYCOL) ACTIVATED BY SUCCINIMIDYL CARBONATE

The immunological response to transplanted allogeneic tissue and cells has been a significant barrier in both organ transplantation and blood transfusion. The most common example of allogeneic cell exposure is the use of red blood cells (RBCs) in transfusion. The red blood cell (RBC) membrane is architecturally complex and is characterized by significant biochemical diversity. Protein, lipoprotein, glycoprotein, and carbohydrate-rich structures play an important role in ensuring the physical integrity of the cell and carrying out its physiologic functions. However, presence of these molecules, on the RBC surface, also has adverse consequences. Several of these structures carry defined polymorphic epitopes, recognized serologically as blood group antigens, which can stimulate alloimmune responses (alloimmunization) after red blood cell transfusion or pregnancy. Alloimmunization is particularly problematic in patient groups who receive multiple transfusions [1]. The first and still the best characterized example of tissue rejection due to antigenic variation of cell surface components is reaction to transfusions of mis-matched red blood cell (RBC) [2]. The transition of blood transfusions from a highly risky surgical procedure to a mundane yet clinically crucial procedure was enabled by the discovery of the ABO/RhD blood groups by Landsteiner (1900) and Landsteiner and Wiener (1940) [2]. In most transfusions, ABO and D (Rhesus) blood typing is sufficient to identify appropriate donors. More often, problems are encountered in individuals who receive multiple transfusions, such as patients with sickle cell anemia and thalassemia. In such patients, alloimmunization against minor RBC antigens can make it nearly impossible to identify appropriate blood donors [3-4]. In the late 1950s, the chemical modification of proteins became common and techniques were developed to facilitate the analysis of the structure–function relationships in the protein molecules. Since the late 1970s, many articles concerning the chemical modification of proteins by conjugation with synthetic macromolecules, that is polyethylene glycol (PEG) derivatives, have been published [17]. The aims of these protein modifications have included the reduction of immunoreactivity or immunogenicity in medical processes [5]. The covalent attachment of PEG is now commonly used to modify a variety of proteins, enzymes, drugs, and artificial surfaces. The most common reactive groups are the alpha or epsilon amino groups of the lysine [6]. Despite these applications, RBCs have only recently been considered for use as substrate for PEG modification [5]. For proteins, typical reactive amino acids include lysine, cysteine, histidine, arginine , aspartic acid, glutamic acid, serine, threonine, tyrosine, N-terminal amino group and the C-terminal carboxylic acid [6]. Several reactive derivatives of methoxy-PEG (mPEG) have been used to covalently attach mPEG to the surface of RBCs [18,19]. One of the reagents for activating mPEG is succinimidyl carbonate. Despite the development of this technology to PEG-coating of RBCs, optimization of the reaction conditions has not been thoroughly investigated. Hashemi-Najafabadi et al. [17] have recently studied the attachment of mPEG activated with cyanuric chloride to RBCs in order to optimize reaction conditions. In the peresent study, RBCs were coated with a reactive succinimidyl carbonate mPEG derivative of 5 kDa molecular mass. The effects of process variables (polymer concentration, temperature, time and pH) were investigated. Full factorial and Taguchi designe methods were employed to identify the optimum reaction conditions for PEGylation of RBCs. The inhibition of agglutination by a blood-type specific antiserum (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 method (Improved Neubauer Ruling). The extent of surface coating was evaluated by the attachment of cells to FITC labeled-anti-D and recording as the fluorescence intensity ratio of FITC-anti-D bound cells of the PEG-RBCs versus control (uncoated) RBCs. Finally, the morphology of PEG-RBCs was studied by scanning electron microscopy (SEM) technique. Materials and Methods Materials Methoxypoly(ethylene glycol) of 5 kDa molecular mass, triethanolamine and fluoresceinisothiocyanate (FITC) were purchased from Sigma. Dioxan was purchased from BDH (England) and N,N′-Disuccinimidyl carbonate was purchased from Aldrich (U.S.A). Anhydrous sodium carbonate, D-glucose, sodium chloride, isopropanol, diethylether, 4-(dimethylamio)pyridine and potassium chloride were obtained from Merck-Schuchardt (Darmstadt, Germany). Cyclohexane was purchased from Roth Iranian Polymer Journal / Volume 15 Number 6 (2006) 526 Sarvi F. et al. Surface Treatment of Red Blood Cells with .