Association Of Bioscavengers To Human Red Blood Cells

We are developing technologies to increase the in vivo retention of bioscavengers by attaching them to red blood cells (RBCs). We have examined cell‐penetrating peptides (CPPs) of 5‐40 amino acids that potentially could either anchor the bioscavengers to the cell surface or internalize them by associated transport. In addition, we tested the binding ability of RBC surface antigens (anti‐type A and B blood group) antibody‐bioscavenger conjugates. RBCs obtained from human blood were incubated with CPPs such as the protein transduction domain of Tat‐HIV (Tat‐PTD), calcitonin, and inverse antennapedia (Inverse‐Antp) biotin congeners in complex with Neutravidin‐fluospheres. No significant differences were observed between control RBCs and CPPs‐incubated RBCs. RBC binding of blood group antibodies was initially monitored with secondary antibodies conjugated to fluorescent marker CY2. Bioscavenger BChE was bound to RBCs by a bridge formed of primary antibody, biotinylated secondary antibody, Neutravidin, and finally biotinylated bioscavenger butyrylcholinesterase (BChE). The preparations were washed and the bound BChE was detected by fluorescence microscopy following incubation with CY2 labeled anti‐butyrylcholinesterase antibodies and BChE enzymatic activity. Our results show that there was significant binding of BChE to the RBCs demonstrating that antibodies can be used as an effective means to bind BChE to RBCs surfaces. This work was supported by DTRA#1.D0011_08_WR_C.