Cytoskeleton-specific immunoliposomes: sealing of hypoxic cells and intracellular delivery of DNA

Abstract

Various pathological conditions, including hypoxia and inflammation, provoke cell membrane lesions. These lesions represent microscopic holes in the sarcolemma through which the components of the cytoskeleton become exposed to the surroundings. Labeled antibodies against intracellular cytoskeletal antigens, such as antimyosin antibody, may be used to reveal cell membrane lesions. Being coupled to liposomes, such antibodies can deliver phospholipid vesicles to the affected cell surface and ‘plug’ them directly into the holes. The in vitro antimyosin antibody-mediated liposome transport to cytoskeletal antigen of hypoxic cardiomyocytes was used in order: (1) to prevent cell deaths by sealing membrane lesions; and (2) to achieve intracellular DNA delivery. A hypoxic model of injury in H9C2 rat embryonic cardiocytes was used in these experiments. Under hypoxic culture conditions, cells were incubated with 150–200 nm antimyosin-immunoliposomes (IL), plain liposomes (PL), and non-specific IgG-liposomes (IgL). After hypoxia (which lasted in different experiments from 1 to 5 days), cell viability was assessed following [ 3H]thymidine incorporation, Trypan Blue exclusion test and by fluorescent microscopy. All tests demonstrated highly improved survival of hypoxic cells in the presence of IL (up to 95% survival after 24 h of hypoxia), whereas, cell survival in the presence of control PL and IgL never exceeded 40%. The presence of IL maintained the survival of hypoxic cells for approximately 5 days, while all control cultures never survived for more than 24–36 h. In addition, salvaged cells maintained normal proliferation after hypoxia in the presence of IL. Electron microscopy experiments with silver grains containing IL demonstrated that after incubation, silver grains can be identified in the cytoplasm, which we see as evidence of possible fusion of ‘plugging’ liposomes with cell membrane. Based on this phenomenon, it is hypothesized that if target cells are under artificial stress, cytoskeleton-specific IL can deliver their contents into the cytoplasm, which provides a good opportunity for the intracellular delivery of drugs and DNA. This hypothesis was confirmed by successful delivery of a plasmid pEScFv 2G42D7 (antimyosin antibody) vector in hypoxic H9C2 cardiocytes by IL.