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

• Published in: Biochimica et biophysica acta 2000-12, Vol.1509 (1-2), p.299-310
• Main Authors: Mercadal, M, Domingo, J.C, Petriz, J, Garcia, J, de Madariaga, M.A
• Format: Article
• Language: English
• Subjects: Animals; Antibodies, Monoclonal - chemistry; Antibodies, Monoclonal - immunology; Antigen-Antibody Reactions; Antigens, CD34 - immunology; CD34 antigen; Cell Separation - methods; Cell Survival; CHO Cells; Cricetinae; Cross-Linking Reagents; Culture Media; Disulfide bond; Disulfides - chemistry; Dithiothreitol; Flow Cytometry; Fluorescein-5-isothiocyanate; Hematopoietic Stem Cells - immunology; Humans; Liposomes - chemical synthesis; Liposomes - immunology; Microscopy, Confocal; Poly(ethylene glycol); Polyethylene Glycols - chemistry; Stem cell; Sterically stabilized immunoliposome; Succinimides; Tumor Cells, Cultured
• ISSN: 0005-2736; 0006-3002; 1879-2642
• DOI: 10.1016/S0005-2736(00)00305-9

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°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.