Delivery of Macromolecules into Cytosol Using Liposomes Containing Hemolysin from Listeria monocytogenes

The cytosolic space of cells is an important but relatively inaccessible target for the delivery of therapeutic macromolecules. Here we describe the efficient delivery of macromolecules into the cytosolic space of macrophages from liposomes that contain listeriolysin O (LLO), the hemolytic protein of Listeria monocytogenes that normally mediates bacterial passage from phagosomes into cytosol. LLO was purified and encapsulated inside pH-sensitive liposomes, along with other molecules to be delivered. When internalized by bone marrow-derived macrophages, these liposomes rapidly released encapsulated fluorescent dye, first into endosomes and then into the cytosol, without measurably harming the cells. Furthermore, these liposomes efficiently delivered encapsulated ovalbumin to the cytosolic pathway of antigen processing and presentation, as measured by the major histocompatibility complex (MHC) class I-restricted presentation of peptides derived from ovalbumin. Delivery was significantly better than that obtained by other currently available liposome formulations. LLO-containing liposomes should therefore provide an efficient vehicle for delivery of antigens or therapeutic molecules in vivo. The cytosolic space of cells is an important but relatively inaccessible target for the delivery of therapeutic macromolecules. Here we describe the efficient delivery of macromolecules into the cytosolic space of macrophages from liposomes that contain listeriolysin O (LLO), the hemolytic protein of Listeria monocytogenes that normally mediates bacterial passage from phagosomes into cytosol. LLO was purified and encapsulated inside pH-sensitive liposomes, along with other molecules to be delivered. When internalized by bone marrow-derived macrophages, these liposomes rapidly released encapsulated fluorescent dye, first into endosomes and then into the cytosol, without measurably harming the cells. Furthermore, these liposomes efficiently delivered encapsulated ovalbumin to the cytosolic pathway of antigen processing and presentation, as measured by the major histocompatibility complex (MHC) class I-restricted presentation of peptides derived from ovalbumin. Delivery was significantly better than that obtained by other currently available liposome formulations. LLO-containing liposomes should therefore provide an efficient vehicle for delivery of antigens or therapeutic molecules in vivo.