title: Targeted liposomes for delivery of protein-based drugs into the cytoplasm of tumor cells.
abstract: Our goal was to deliver therapeutically active macromolecules into the cytosol of target cells. First, attempts were made to prepare virosomes that specifically interact with OVCAR-3 cells (human ovarian cancer cells). Detergent solubilized influenza virus envelopes were reconstituted forming virosomes. Cell specificity was introduced by incorporating PEG-derivatized lipids with mAB 323/A3 (Fab' fragments) connected to their distal PEG end. These cell-specific, modified virosomes maintained their fusogenic activity when lowering the pH. Most importantly, antibody-mediated binding was a prerequisite for low-pH induced membrane fusion. However, basically, there are two problems with this approach: (1) these virosomes are quite leaky and (2) virosomes can be expected to be immunogenic. A solution to tackle leakage and potential immunogenicity of these site-specific liposomal structures is to use immuno-PEG-liposomes with a pH-dependent fusogen inside the liposome. The system that we designed to test this concept consisted of (1) the fusogenic di-peptide dINF-7, (2) the monoclonal antibody 425 connected to the distal end of PEG-PE (for site specific binding and endosomal uptake), (3) diphtheria toxin chain A (DTA, as carrier-dependent active compound) and phosphatidylcholine/cholesterol as 'bilayer backbone'. A series of tests were performed to show that selective binding and pH-dependent destabilization of (endosomal) membranes indeed occurred. To test the cytotoxic activity of these DTA loaded liposomes, OVCAR-3 cells were used for testing. OVCAR-3 cells express the epidermal growth factor receptor, which is the ligand for antibody 425. In vitro, these site specific and fusogenic liposomes showed a remarkable, cell specific cytotoxic effect.
authors.affiliation: Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands.
authors.forename: Enrico
authors.initials: E
authors.lastname: Mastrobattista
authors.forename: Daan J A
authors.initials: DJ
authors.lastname: Crommelin
authors.forename: Jan
authors.initials: J
authors.lastname: Wilschut
authors.forename: Gert
authors.initials: G
authors.lastname: Storm
mesh_terms: D003593:Cytoplasm / Q000378:metabolism*; D016503:Drug Delivery Systems; D011992:Endosomes; D005434:Flow Cytometry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008055:Lipids / Q000737:chemistry; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism*; D008954:Models, Biological; D009975:Orthomyxoviridae / Q000502:physiology; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D014407:Tumor Cells, Cultured
chemical_list: D008055:Lipids; D008081:Liposomes; D010455:Peptides