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

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).

Immunocamouflage of latex surfaces by grafted methoxypoly(ethylene glycol) (mPEG): Proteomic analysis of plasma protein adsorption

“…Previous studies have shown that hydrophilic surfaces have decreased total protein adsorption and are also more biocompatible [12]. Thus, mPEG grafting generates a steric barrier while simultaneously biophysically camouflaging surface charge thus reducing immune recognition of implantable biomaterials, allogeneic red blood cells [5,[13][14][15][16][17][18], lymphocytes [3,4,[19][20][21] and viruses ( Figure 6BD) [22]. As shown in this study, consequent to PEGylation, latex particles demonstrated significant inhibition of protein adsorption in a polymer doseand length-dependent manner, as measured quantitatively with colorimetric protein assay and flow cytometry ( Figure 2A and B) and qualitatively by SDS-PAGE, fluorescent microscopy and iTRAQ-MS analysis (Figures 3-5).…”

Polymer-mediated immunocamouflage of red blood cells: Effects of polymer size on antigenic and immunogenic recognition of allogeneic donor blood cells

“…To quantitate the degree of immunocamouflage induced by the grafted polymers, flow cytometry (FACSCalibur; BD Biosciences San Jose, CA, USA) was performed using previously established erythrocyte parameters [12][13][14]. A minimum of 20000 cells was analyzed per sample.…”

“…To examine the effects of the various mPEG derivatives in vivo, RBC survival studies were performed in Balb/c mice [9,12,13]. All animal studies were institutionally approved and conducted in compliance with all applicable Canadian animal care regulations.…”

“…Ongoing work on the covalent modification of RBC and other blood cells with methoxypoly(ethylene glycol) (mPEG; PEGylation) suggests that polymer grafting may prove to be a practical prophylactic method of decreasing the risk of transfusional alloimmunization and a treatment for patients that have already been alloimmunized [9][10][11][12][13][14][15][16][17][18][19][20]. To further examine the potential clinical application of "immunocamouflaged" erythrocytes, the effects of polymer size on the antigenic recognition of non-ABO blood group antigens expressed by normal human RBCs was investigated.…”

Potent suppression of agglutinability of human A and B type erythrocytes by KMnO4-EDTA concerted action

“…When universal donor is not available, the conversion of A or B type to universal type is an alternative to overcome the life-threatening situation. For this reason multi-dimensional research works have already been initiated to camoufl age the RBCs [4][5][6][7]. However , there remain various drawbacks in new techniques including development of neoantigen, formation of PEG antibodies against mPEGylated erythrocyte, high chances of phagocytic recognition, poor oxygen diffusion, incomplete loss of antigenicity of A, B blood types [8].…”