Targeted liposomes for delivery of protein-based drugs into the cytoplasm of tumor cells.
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.
Mastrobattista
Enrico
E
Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands.
Crommelin
Daan J A
DJ
Wilschut
Jan
J
Storm
Gert
G
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
D008055:Lipids; D008081:Liposomes; D010455:Peptides