Mikhail D. Smirnov

Red blood cell targeting to collagen-coated surfaces

The interaction of human red blood cells carrying antihuman collagen antibody with collagen‐coated surfaces was studied. Avidin was used as bifunctional crosslinking agent for the attachment of antibody to the red blood cell surface. Antibody‐carrying red blood cells efficiently and specifically bound to collagen‐coated surface covering a significant part of the surface. The components of normal blood had an insignificant effect on red blood cell binding. A model of drug targeting to the injured sites(s) of blood vessel wall is proposed.

Targeting of enzyme immobilized on erythrocyte membrane to collagen-coated surface

Drug targeting Collagen Immobilized enzyme Antibody attachment Peroxidase Specific binding

Type I and III collagens as a possible target for drug delivery to the injured sites of vascular bed

Interaction of anti-human collagen types I and III antibodies, as well as human red blood cells conjugated with these antibodies, with the surface of denuded intima of human aorta has been studied. Data on the accessibility of antigenic determinants of collagen types I and III for antibodies and red blood cells conjugated with these antibodies have been obtained in ex vivo experiments in an original model. On the basis of the obtained results it is concluded that antigenic determinants of collagen types I and III exposed as a result of blood vessel wall injury can serve as a target for drug delivery to the injured site(s).

Avidin attachment to red blood cells via a phoshpolipid derivative of biotin provides complement-resistant immunoerythrocytes

Preincubation of red blood cells (RBC) in an aqueous dispersion of biotin-phosphatidylethanolamine (biotin-PE) provides binding sites for avidin on the surface of these cells (up to 5 x 10(5) avidin molecules per cell). Previously we have shown that biotin covalently attached to the surface of RBC by a chemical reaction with biotin N-hydroxysuccinimide ester permits attachment of avidin to these cells, resulting in the activation of the alternative pathway of complement with subsequent cell lysis. However, avidin attached to RBC via biotin-PE did not cause complement activation. This is not due to the stabilizing action of biotin-PE. In contrast, various phospholipids, including biotin-PE, enhance the lysis of RBC induced by hemolytic antibodies via the classical complement pathway. The potential of avidin-coated RBC to act as activators of the complement alternative pathway depends on the method of biotin attachment to RBC. Complement-resistant avidin-coated RBC can specifically bind biotinylated antibodies. These immunoerythrocytes effectively and specifically bind to the antigen-coated surface and are not lysed by complement even in the presence of soluble antigen. These data extend the possible applications of immunoerythrocytes in drug targeting.

Avidin attachment to biotinylated amino groups of the erythrocyte membrane eliminates homologous restriction of both classical and alternative pathways of the complement

Lysis of avidin‐coated biotinylated sheep red blood cells (RBC) via the classical pathway of homologous (sheep) and heterologous (guinea pig) complement has been studied. The minimal surface density of avidin inducing antibody‐dependent lysis via the classical pathway is smaller than that inducing antibody‐independent lysis via the alternative pathway. Heterologous lysis via the classical pathway does not depend on the mode of avidin attachment: both biotinylation of membrane amino groups and insertion of biotinyl‐lipid into the membrane provide the same lysis of avidin‐coated RBCs by guinea pig serum in the presence of anti‐avidin antibody. Avidin‐free sheep RBC sensitized with hemolytic anti‐RBC antibody were lysed by guinea pig, but not by sheep serum, confirming high efficiency of homologous restriction of the complement. However, avidin‐coated RBCs were lysed by homologous serum in the presence of anti‐avidin antibody at low surface density of avidin attached. The elimination of the homologous restriction depends on the mode of avidin attachment: biotinylation of membrane amino groups provides antibodymediated lysis via the classical pathway of homologous complement, while insertion of biotinyl‐lipid does not provide lysis.

Interaction of avidin-carrying red blood cells with nucleated cells

In vivo application of red blood cells (RBC) modified with avidin-biotin complex has been suggested recently for various purposes. However, avidin attachment to RBC alters their biocompatibility. Thus, it has been described that avidin-carrying biotinylated RBC were lysed by the complement. In the present work interaction between avidin-carrying RBC and nucleated cells has been examined. It was found that attachment of avidin, but not streptavidin, to RBC led to binding of avidin-carrying RBC to nucleated cells. Adhesiveness of nucleated cells for avidin-carrying RBC varied for different types of nucleated cells. The strongest adhesion was observed with human fibroblasts and rat Kupffer cells, while rat liver endothelial cells were practically non-adhesive for avidin-carrying RBC of corresponding species. In contrast with avidin (streptavidin)-induced lysis by the complement, avidin-induced adhesion was independent of temperature, the presence of divalent ions and mode of avidin attachment. Polyanions (dextran sulphate and heparin) efficiently inhibited the adhesion presumably due to interaction with the membrane-bound avidin. Polyanions to a much lesser extent inhibited lysis of avidin-carrying RBC, which might be a result of their interaction with the complement components. Polycations also blocked adhesion of avidin-carrying RBC to nucleated cells, presumably due to interaction with negatively charged cell-surface components. Therefore, attachment of avidin to RBC alters their biocompatibility, due to both high positive charge of avidin and the cross-linking of biotinylated membrane proteins.