Accumulation of protein-coated liposomes in an extravascular site: influence of increasing carrier circulation lifetimes.

The primary objective of this work was to test whether increased blood levels and circulation lifetimes result in increased passive targeting of protein-coated liposomal drug carriers. The system used to evaluate this was based on i.v. injection of 100 nm of distearoyl phosphatidylcholine/cholesterol liposomes with covalently bound streptavidin. The circulation lifetime of these liposomes was increased by procedures that involved blockade of liposome uptake by phagocytic cells in the liver and/or the incorporation of a poly(ethylene glycol)-modified phospholipid [poly(ethylene glycol)2000-modified distearoyl phosphatidylethanolamine]. Blockade of liver phagocytic cells with a low predose (2 mg/kg of drug) of liposomal doxorubicin increased the circulation half-life of the streptavidin liposomes from less than 1 hr to greater than 3 hr. A further 2-fold increase in circulating half-life (to approximately 7.5 hr) was achieved by using liposomes with 2 mole % of poly(ethylene glycol)2000-modified phosphatidylethanolamine. In combination with RES blockade, the circulation lifetimes of poly(ethylene glycol)phosphatidylethanolamine containing streptavidin liposomes could be increased to greater than 12 hr. The ability of these liposomes to move from the plasma compartment to an extravascular compartment was measured by using the peritoneal cavity as a convenient, accessible, extravascular site. The tendency for liposomes to accumulate in this site was not, however, clearly dependent on circulating blood levels. Comparable levels of liposomes in the peritoneal cavity were achieved when using systems that exhibited significantly different circulation lifetimes.

Poly(ethylene glycol)-modified phospholipids prevent aggregation during covalent conjugation of proteins to liposomes.

Liposome aggregation is a major problem associated with the covalent attachment of proteins to liposomes. This report describes a procedure for coupling proteins to liposomes that results in little or no change in liposome size. This is achieved by incorporating appropriate levels of poly(ethylene glycol)-modified lipids into the liposomes. The studies employed thiolated avidin-D coupled to liposomes containing the thio-reactive lipid N-(4-(p-maleimidophenyl)butyryl)dipalmitoyl phosphatidylethanolamine (1 mol % of total lipid) and various amounts of MePEG-S-POPE (monomethoxypoly(ethylene glycol) linked to phosphatidylethanolamine via a succinate linkage). The influence of PEG chain length and density was also assessed. The presence of PEG on the surface of liposomes is shown to provide an effective method of inhibiting aggregation and the corresponding increase in liposome size during the covalent coupling of avidin-D. A balance between the size of the PEG used and the amount of PEG-lipid incorporated into the liposome had to be achieved in order to maintain efficient coupling. Optimal coupling efficiencies in combination with minimal aggregation effects were achieved using 2 mol % MePEG2000-S-POPE (PEG of 2000 MW) or 0.8 mol % MePEG5000-S-POPE (PEG of 5000 MW). At these levels, the presence of PEG did not affect the biotin binding activity of the covalently attached avidin. The ability of the resulting liposomes to specifically target to biotinylated cells is demonstrated.

A two-step targeting approach for delivery of doxorubicin-loaded liposomes to tumour cells in vivo.

A two-step targeting approach was used to deliver doxorubicin-loaded liposomes to a murine tumour cell (P388 leukaemia) grown in culture and, more importantly, in vivo. Targeting was mediated through the use of an antibody specific for the Thy 1.2 antigen that is highly expressed on P388 cells. Briefly, the approach consists of prelabeling target cells with biotinylated anti-Thy 1.2 antibody prior to administration of drug-loaded liposomes that have streptavidin covalently attached to their surface. Results from in vitro studies demonstrate that a 30-fold increase in cell-associated lipid and a 20-fold increase in cell-associated doxorubicin can be achieved over control liposomes using this two-step procedure. Flow-cytometry and fluorescent-microscopy data were used to confirm that P388 cells can be stably labeled with the biotinylated anti-Thy 1.2 antibody in vivo. Subsequently, liposome-targeting studies were initiated in vivo, where target cell binding was assessed following i.p. or i.v. injection of doxorubicin-loaded liposomes into animals bearing P388 tumours prelabeled with biotinylated antibody. A streptavidin-mediated 3.7-fold increase in cell-associated lipid and drug was achieved when the liposomes were given i.p. When doxorubicin-loaded streptavidin liposomes were injected i.v., P388 cells located in the peritoneal cavity were specifically labeled, although the efficiency of this targeting reaction was low. Less than a 2-fold increase in cell-associated lipid was achieved through the use of target-specific (streptavidin-coated) liposomes. These studies demonstrate that the presence of a well-labeled target cell population within the peritoneal cavity will not promote accumulation of an i.v. injected, targeted liposomal drug. Furthermore, the importance of separating target-cell-specific binding from non-specific uptake by tumour-associated macrophages is discussed.