title: Targeting Her-2+ breast cancer cells with bleomycin immunoliposomes linked to LLO.
abstract: Bleomycin is a membrane impermeable chemotherapeutic agent that is relatively innocuous extracellularly but highly cytotoxic when delivered directly to the cytoplasm. We report on the development of a liposome delivery system that targets Her-2 overexpressing breast cancer cells and breaches the endosomal barrier, delivering bleomycin to the cytoplasm. The liposomes are conjugated to the antibody trastuzumab, which results in specific binding and internalization of liposomes into Her-2 overexpressing cells. In addition, the liposomes are disulfide bonded to a pore-forming protein listeriolysin O (LLO) which forms pores in the endosome and allows the liposomal cargo to pass into the cytoplasm. We demonstrate specific delivery to Her-2 positive MCF-7/Her18 cells relative to Her-2 negative MCF-7 cells using a fluorescent probe calcein within the immunoliposomes. When calcein is replaced by bleomycin, the liposomes effectively reduce viability of five different Her-2 overexpressing cell lines (BT-474, SKBR-3, MCF-7/Her18, HCC-1954 and MDA-453) while harming to a much lesser extent Her-2 negative breast cell lines (MCF-7, MCF-12a and MCF-10a). The liposomes also affect trastuzumab-resistant cells, reducing MDA-453 cell number by 97% compared to untreated cells. Importantly, the concentration of drug needed to reduce tumor cell growth and viability using this liposome therapy is approximately 57,000-fold less than the concentration needed if drug is delivered extracellularly, raising the possibility of increased therapeutic specificity with decreased side effects.
authors.affiliation: Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Denver, 12850 Montview Boulevard, Aurora, Colorado 80045, United States.
authors.forename: Max
authors.initials: M
authors.lastname: Kullberg
authors.forename: Kristine
authors.initials: K
authors.lastname: Mann
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Anchordoquy
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology / Q000494:pharmacology; D061067:Antibodies, Monoclonal, Humanized / Q000276:immunology / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000276:immunology / Q000494:pharmacology; D001427:Bacterial Toxins / Q000276:immunology / Q000378:metabolism*; D001761:Bleomycin / Q000276:immunology / Q000494:pharmacology*; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000276:immunology / Q000378:metabolism; D045744:Cell Line, Tumor; D003593:Cytoplasm / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D011992:Endosomes / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D005260:Female; D005452:Fluoresceins / Q000494:pharmacology; D006360:Heat-Shock Proteins / Q000276:immunology / Q000378:metabolism*; D006460:Hemolysin Proteins / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D008081:Liposomes / Q000276:immunology* / Q000494:pharmacology*; D061986:MCF-7 Cells; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000276:immunology / Q000378:metabolism*; D000068878:Trastuzumab
chemical_list: D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D000970:Antineoplastic Agents; D001427:Bacterial Toxins; D001761:Bleomycin; D005452:Fluoresceins; D006360:Heat-Shock Proteins; D006460:Hemolysin Proteins; D008081:Liposomes; D018719:Erb-b2 Receptor Tyrosine Kinases; D000068878:Trastuzumab; C038705:hlyA protein, Listeria monocytogenes; C007740:fluorexon