The long term objective of this research is to utilize liposomes to microinject exogeneous molecules into encaryotic cells. To accomplish this liposomes with a novel pH sensitive composition will be made that can fuse with the endosomal compartment thereby deliver their encapsulated contents into the cytoplasm. This idea is based upon observations that many types of viruses deliver their contents into cells subsequent to endocytosis. Transfer of the viral contents involves a pH induced conformational change of a viral membrane protein. This results in fusion of the viral membrane with the endosomal membrane and transfer of the viral DNA into the cytoplasm. A liposome can be designed to accomplish such a pH dependent microinjection by incorporating novel pH sensitive trigger lipids with phosphatidyl ethanolamine. At pH 7.0 the lipids assume a bilayer structure but as the pH is lowered to 4.5, that found in the endosome, the liposome changes its structure. The efficiency of this pH sensitive endocytotically mediated liposomal system for microinjecting exogeneous molecules into cells will be determined. The parameters that influence the collapse and fusion of such liposomes will be studied by sensitive fluorescence techniques both in cell culture and in liposome-liposome model systems in order to elucidate the mechanism. The consequences of the pH mediated microinjection on cellular metabolism protein, DNA and adenine nucleotide synthesis will be examined and the fate of microinjected molecules including plasmid DNA will be studied. Knowledge resulting from this project could be used to devise systems to deliver a wide variety of membrane impermeable agents into the cytoplasm. Such a tool would be useful in cellular physiology and would also lead to increase knowledge of how viral DNA escapes from the endosomal compartment. If a targeting ligand is attached to the liposome enhanced delivery of contents into designated cell types could ensue. Such site-specific delivery of nucleic acids to cells in vitro or in vivo could have therapeutic applications for currently non-curable genetic diseases.
