1. Field of the Invention
This invention relates to liposomes, and more particularly relates to ligand linked liposomes having a surface chemically modified to provide enhanced performance as immunoassay reagents or as site directed carriers of therapeutic agents.
2. Background of the Invention
In recent years, liposomes have been extensively studied as reagents for immunoassay and as carriers of therapeutic agents. When used in an immunoassay, a liposome generally encapsulates a reporter molecule, such as a dye or an enzyme, and is complexed with a ligand, usually an antigen or antibody. The liposome-ligand complex is often referred to as the assay tracer. In a sandwich assay the tracer is a loaded liposome-antibody complex which binds noncompetitively to the assay analyte bound to a capture antibody. In a competitive assay, the tracer is a complex of the loaded liposome with the analyte, and the complex and the free analyte compete for a limited number of binding sites on the capture antibody.
When used as a carrier for a therapeutic agent, the liposome is covalently conjugated to a site-directed antiligand, usually an antibody specific for an antigen associated with the site. In this way, the therapeutic agent-carrier complex is transported to the intended site of action.
Much research has been directed to suitable methods for forming the liposome ligand complex. In one method, the liposomes and ligand are maintained in contact whereby the hydrophobic portion of the ligand nonspecifically associates with hydrophobic components of the liposome. This absorption method has not achieved widespread use because the stability of the complex may be insufficient for the intended purpose leading to dissociation and/or leakage of the dye or therapeutic agent from the liposome.
A generally suitable method for forming a more stable liposome complex is by covalent bonding of the ligand to a reactive constituent of the liposome membrane. The covalent bond may be formed either before or preferably after formation of the liposomes. In the preferred and generally used method, functional groups .on the liposome and the ligand are joined by a heterobifunctional spacer molecule. One of the functional groups forms a covalent bond with a reactive group on the surface of the liposome. The second functional group forms a covalent bond with a reactive group, usually a thiol group, on the ligand.
Martin et al., in U.S. Pat. No. 4,429,008 discloses liposomes conjugated to a variety of spacer moieties terminating in thiol-reactive functional groups projecting outwardly from the liposome surface. The thiol reactive groups react with thiol groups of ligands, such as antigens and antibodies, to covalently attach the ligands to the lipsomes via stable thioether bonds. This disclosure makes no provision for unreacted thiol-reactive groups which may react with other nucleophilic groups in the assay environment.
Published European patent Application No. 248,621 to Hatoh et al. discloses a liposome-ligand complex which includes a group which blocks any thiol reactive groups on the liposome which are not conjugated to the ligand. The blocking group may be a sulfhydryl containing reducing agent such as cysteine, mercaptoethanol or dithiothreitol (DTT), or an amino group containing substance such as glycine, serine or TRIS.
Bredehorst et al., Biochemistry 25, 5693 (1986), discusses the problem of liposome stability during coupling of Fab fragments to liposomes containing N-[4 (p-maleimidophenyl)butyryl]phosphatidylethanolamine (MPB-PE) wherein the maleimido group is thiol reactive. It is reported that stability is high if the mole percentage of MPB PE is 2.5 or lower whereas 5.0% causes release of 95% of an encapsulated fluorescein dye.
A further problem often encountered during or after coupling of thiolated ligands to thiol reactive liposomes is aggregation of the liposomes. Martin et al. in Macromolecules as Drugs and as Carriers for Biologically Active Molecules, New York Academy of Sciences, 446, 443 (1985), discloses that the problem of aggregation of liposomes is significantly reduced when the liposomal membrane contains from 10 to 30% of phosphatidylglycerol. The same authors note that an approach to preventing or reversing liposome aggregation is to lower the ion strength of the coupling medium.
The above disclosures have contributed to overcoming the problems of liposome leakage, loss of ligand and reversible aggregation during storage or use but have not provided a satisfactory solution to the problem of irreversible aggregation due to liposome fusion, protein-protein or protein-liposome interactions. The present invention is directed to solution of this problem.
USPTO Backgrounds
USPTO Backgrounds