Thiolation Mediated Pegylation Platform to Generate Functional Universal Red Blood Cells

Abstract

The PEGylation that adds an extension arm on protein amino groups with the conservation of their positive charge masks the A and D antigens of erythrocytes efficiently. In the present study, the efficiency of masking the antigens of RBC by PEGylation protocols that do not conserve the charge with and without adding extension arms is compared. The conjugation of PEG-5000 to RBCs through the addition of extension arms masked the D antigen more efficiently than the other protocol. A combination of PEG-5 K and PEG-20 K is needed to mask the A antigen, irrespective of the PEGylation approach. The oxygen affinity of the PEGylated RBCs increased by the extension arm facilitated PEGylation. The protocol involving the conjugation of PEG-chains without adding extension arm did not alter the oxygen affinity of RBCs. A combination of PEGylation protocols is an alternate strategy to generate universal red blood cells with good levels of oxygen affinity.

Drug delivery by red blood cells: vascular carriers designed by mother nature

“…Masking against immune reaction to mismatched ABO antigens is the most challenging goal; in this setting, PEG-coating offered no protection and rather aggravated hemolysis in the initial studies191. Additional studies using alternative PEG-coupling techniques and combinations of PEG with different chain length produced encouraging results in masking ABO and Rh(D) blood group antigens192–194. More recently, even masking of xeno-antigens on RBC has been attempted, yet the outcome was not too encouraging195.…”

“…PEG coupling using sulfo-group chemistry192,194, insertion of lipid derivatives of PEG197 and other methods have also been developed186,198,199. Extent of “stealth” feature or masking increases proportionally to PEG length in the range 5–35 kD, branching and surface density200.…”

Molecular Engineering of Cell and Tissue Surfaces with Polymer Thin Films

“…PEGylation of cell and tissue surfaces has been utilized for a number of applications, some of which will be described later in this chapter. Two approaches to increasing the surface density of chains while minimizing bulk polymer concentration is to perform multiple sequential reactions [18,19] or to utilize reactions that target multiple anchor groups [20,21]. In general, hydrophilic and neutral polymers, such as PEG, associate poorly with the cell surface [17]; the same is also true for negatively charged polymers (polyanions), which are electrostatically repelled by the negatively charged cell surface.…”

Elimination of blood group antigens: hope and reality

“…Ext, external side of red cell membrane; Int, internal side of red cell membrane. in RBC (Jeong & Byun, 1996;Scott et al, 1997;Scott & Murad, 1998;Nacharaju et al, 2007), there is no relevant in vitro or in vivo model available to assess the impact of changes in cell-tocell and cell-to-serum protein interactions or of alterations in the in vivo pharmacodynamic properties of modified blood cells (Jovtchev et al, 2008). Similarly, there is no current data on the long-term fate of PEG-treated red cells and of membranes and proteins coated with PEG derivatives.…”

“…Comparison of the reported data from research on human stealth RBC is difficult due to great differences between study designs involving the type and mode of PEG derivative, methodologies used and expertise of teams in transfusion medicine. Several techniques have been used to assess antigen visibility on the red cell surface including haemagglutination with serum from incompatible donors (Jeong & Byun, 1996; Bradley et al , 2001; Nacharaju et al , 2005, 2007), cytometry to detect cell surface antigens (Nacharaju et al , 2005), or IgM‐type antibodies attached to their antigen target (Bradley et al , 2001). Although all these techniques have shown a significant reduction effect on antigen visibility after PEG treatment, it is more realistic to speak of decreasing the exposure of antigenic determinants than of removing red cell antigenicity.…”