Biophysical consequences of linker chemistry and polymer size on stealth erythrocytes: size does matter

Immunocamouflaged red blood cells (RBC) are produced by cell surface derivatization with methoxypolyethylene glycol (mPEG). These immunologically attenuated cells may reduce the risk of allosensitization in chronically transfused patients. To characterize the effects of differing linker chemistries and polymer lengths, RBC were modified with cyanuric chloride activated mPEG (C-mPEG 5 kDa), benzotriazole carbonate methoxyPEG (BTC-mPEG; 5 or 20 kDa) or N-hydroxysuccinimidyl ester of mPEG propionic acid (SPA-mPEG; 2, 5 or 20 kDa). Biophysical methods including particle electrophoresis and aqueous two-phase polymer partitioning were employed to compare the PEG derivatives. While C-mPEG was faster reacting, both BTC-mPEG and SPA-mPEG gave comparable findings after 1 h. Both PEG surface density and molecular mass had a large effect on RBC surface properties. Proportional changes in electrophoretic mobility and preferential phase partitioning were achieved by increasing either the quantity of surface PEG or the PEG molecular mass. In addition, two-phase partitioning may provide a means for efficiently removing unmodified or lightly modified (hence potentially immunogenic) RBC in the clinical setting. Furthermore, mPEG modification significantly inhibits cell-cell interaction as evidenced by loss of Rouleaux formation and, consequently, sedimentation rate. Importantly, BTC-mPEG 20 kDa RBC showed normal in vivo survival in mice at immunoprotective concentrations (up to 2 mM).