Preparation and characterization of biotinylated red blood cells

Abstract

Biotinylation of intact mammalian red blood cells (RBC) was performed either by attachment to the amino groups by means of biotin N-hydrosuccinimide ester (NHS-biotin) or by oxidation of the induced aldehyde groups of the RBC membrane by biotin hydrazide. Comparison of these different procedures showed that biotinylation by NHS-biotin provided the highest cell recovery (> 90%), the binding of approximately 1000 biotin molecules/cell (on mouse RBC) and the 24 h survival in circulation was unaffected. In contrast, biotin hydrazide produced cell recovery in the 5-30% range (depending on the extent of oxidation), an undetectable number of molecules of biotin/cell and negligible 24 h survival. Among the NHS derivatives of biotin, further studies were performed on those containing a spacer arm of 2.2 nm (22 A) [sulphosuccinimidyl-6-(biotinamido)-hexanoate]. In vitro this derivative was similar to, or better than, the NHS-biotin in terms of cell recovery and the number of molecules/cell. In vivo this derivative showed a 24 h circulation survival similar to that of NHS-biotin. Unfortunately, biotin bound with such a spacer arm is lost after a few days of RBC circulation, probably due to plasma biotinidase. Possible applications of biotinylated RBCs include the in vivo measurement of RBC volume, the RBC survival and the delivery of enzyme and antigens.

Homing of annexin-labeled stem cells to apoptotic cells

“…1 B, D), showing the safeguard of the physiological membrane protein turnover. Consistently with this last observation, several investigators reported that the biotinylation reagents do not seem cytotoxic in in vitro and in vivo experimental models [18,20,21].…”

“…Avidin-biotin interaction has been largely exploited for "protein detection" techniques (in immunology, histochemistry, in situ hybridization, affinity chromatography and other areas) as well as in pioneer techniques aimed at improving cell adhesion or drug delivery. Of these, biotinylation of TNF-α was proposed as an approach to direct the cytokine to the membrane of melanoma cells [17], and biotinylation of red blood cells as a potential antigen delivery system via the biotin-avidin-biotin bridge [18]. An interesting report demonstrates the safety and specificity of red blood cell delivery of HIV-Tat as a step in the design of a new anti-HIV vaccine [19].…”

Regulation of the Complement-Mediated Elimination of Red Blood Cells Modified with Biotin and Streptavidin

“…This result indicates that detachment of b-IgG may occur in the pulmonary vasculature, enriched by capillaries. In contrast, splenic this section document that b-IgG/SA/b 23 -RBC circulate amined for the possibility of selective delivery of antigens to immunocompetent cells (10) and for the elimination of circulating molecules and particles from the bloodstream (11). Therefore, SA-mediated attachment of biotinylated antibodies is a key aspect in the design of immunoerythrocytes for drug targeting, targeted immunization, and selective blood clearance.…”

“…Iodogen was obtained from Pierce (Rockford, IL). immunization (10)) and specific elimination of circulating pathogens from blood (11).…”

Immunization with HIV protease peptides linked to syngeneic erythrocytes

“…A human leukocyte antigen (HLA) A0201 restricted epitope, deriving from HIV-1 protease (PR 75–84 wt , VLVGPTPVNI) and a mutant variant (PR 75–84 d.mut ) harboring two drug resistance mutations, V82F and I84V were resuspended in phosphate buffered saline (PBS), biotinylated and coupled to syngeneic erythrocytes by avidin-biotin bridges [5,9-12]. The amount of peptide bound to the red blood cells was estimated to 25 ng/mL blood [7,9].…”