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

Center for Immunology and Microbial Disease, Albany Medical College, Albany, NY, USA.
Biochimica et Biophysica Acta (Impact Factor: 4.66). 05/2002; 1561(2):147-58. DOI: 10.1016/S0005-2736(02)00339-5
Source: PubMed

ABSTRACT 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).

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    ABSTRACT: The grafting of low-immunogenic polymers to cells dramatically reduces antigenic recognition and immunogenicity of allogeneic donor cells consequent to steric and charge camouflage (i.e., immunocamouflage). While methoxypoly(ethylene glycol) [mPEG] has historically been utilized for the immunocamouflage of cells, other low-immunogenic polymers such as polyethyloxazoline propionic acid (PEOZ) may also be capable of conferring immunoprotection. Moreover, PEOZ may have attributes that could have enhanced pharmacological and biological utility relative to mPEG. To evaluate the immunocamouflage efficacy of PEOZ relative to mPEG, human red blood cells (RBC) and leukocytes were modified with mPEG or PEOZ. The differential effects of mPEG and PEOZ was assessed via grafting efficacy, cell morphology and viability, immunocamouflage of surface antigens, and the prevention of in vitro immune recognition (RhD and HLA). Although membrane grafting of mPEG and PEOZ were similar, mPEG demonstrated superior immunocamouflage efficacy as measured by antibody binding and phagocytosis of opsonized RBC while PEOZ showed improved RBC morphology. While mPEG appears to be superior to PEOZ in the immunocamouflage of cells, PEOZ may still be a valuable addition to our repertoire of immunomodulatory polymers. Moreover, our results demonstrate the importance of indirect immunocamouflage of antigens found in membrane protein complexes.
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