Preparation of immunoliposomes bearing poly(ethylene glycol)-coupled monoclonal antibody linked via a cleavable disulfide bond for ex vivo applications.

Several methods for the preparation of sterically stabilized immunoliposomes (SIL) have recently been described. This report examines an established method for coupling anti-CD34 My10 mAb to poly(ethylene glycol)-liposomes (PEG-liposomes) containing the anchor pyridyldithiopropionylamino-PEG-phosphatidylethanolamine (PDP-PEG-PE) via a cleavable disulfide bond. Efficient attachment of pyridyldithio-derivatized mAb took place (equivalent to coupling ca. 70% of total input protein) at 2 mol percent of the functionalized PEG-lipid. The My10-SIL bound specifically to CD34+ cells (human leukemic KG-1a and hematopoietic progenitor cells) and the extent of binding was a function of liposomal lipid concentration, the mAb density in the liposome surface and the CD34 cell expression. In mixtures with CD34- cells (CHO or Jurkat), CD34+KG-1a cells were determined by flow cytometry at percentages (1-4%) similar to those reported in clinical samples (such as cord blood, mobilized peripheral blood and bone marrow) using a direct immunostaining with My10-SIL. The disulfide bond was stable in cell culture medium (10% of fetal calf serum) during 8 h and cell-bound SIL can be released from cells by treatment with dithiothreitol as reducing agent under mild conditions (1 h of incubation with 50 mM DTT at 20 degrees C). SIL binding and subsequent dithiothreitol treatment did not influence the cell viability. Our approach should contribute to the development of targetable liposomal vehicles to CD34+ cells for use in ex vivo conditions as sorting of hematopoietic stem cells.