In vivo investigations into the drug delivery potential of liposomes

The drug delivery potential of liposomes has been well documented in vitro. Liposomes composed primarily of phosphatidylethanolamine (PE) have been developed which release drug to the target cell cytoplasm. These liposomes are triggered to release drug in response to multivalent cell surface antigen binding or to the acidic pH of the endosome. PE is essential to the liposome destabilization/fusion mechanisms involved. For therapeutic liposome applications, considerations regarding liposome biodistribution must be addressed. Rapid uptake of injected liposomes by the reticuloendothelial system (RES) has posed limitations for drug delivery to other tissues. Recently, a new generation of long-circulating liposomes has been developed which include the monosialoganglioside G_M1 or amphipathic poly(ethylene glycol) (PEG) in the lipid composition. The development of targetable PE liposomes in vivo is described here. These liposomes contain G_M1 in the lipid composition and are targeted by coupled monoclonal antibody to mouse lung. The results are compared to more conventional liposomes with phosphatidylcholine (PC) as the major matrix lipid, and it is demonstrated that for cholesterol-containing liposomes, targetability is positively correlated with reduced clearance from the circulation. In further attempts to reduce RES uptake of PE liposomes, dioleoyl-N-(monomethoxypolyethyleneglycolsuccinyl)-phosphatidylethanol- amine (PEG-PE) was used in the liposome composition. These liposomes accumulated in spleen, unlike PC liposomes with PEG-PE which showed more prolonged circulation in the blood. The PE liposomes with PEG-PE are plasma stable. Furthermore, spleen uptake is dependent on the injection dose, PEG-PE concentration, and the PEG chain length. These results reveal the significance of the matrix lipid in determining the circulation time of PEG-PE-containing liposomes. Circulation of PEG-PE-containing liposomes is also dependent on the liposome size. Using PC as the matrix lipid in combination with PEG-PE, very small (d<70nm) and very large (d>300nm) liposomes accumulate to elevated levels in liver and spleen, respectively compared to intermediate size liposomes (d~150-200nm) which show a more prolonged circulation time. Fluorescence microscopy revealed that liposomes accumulated in liver are localized to Kupffer cells regardless of liposome size. These findings disagree with the hypothesis that the reduced circulation time of small liposomes with PEG-PE results from an ability to pass through the fenestrated liver endothelium and thereby reach the parenchymal cells. Results from a serum protein binding assay and binding of streptavidin to biotinylated liposomes demonstrate a decreased steric barrier activity of the PEG-PE with the small liposomes. More avid adsorption of plasma protein, possibly including opsonin(s), to small PEG-PE- containing liposomes may account for their more rapid clearance from the circulation. For the very large liposomes, fluorescence microscopy revealed localization to the red pulp and marginal zone in spleen. The long-circulating intermediate size liposomes revealed an increased tumor accumulation compared to liposomes without PEG-PE. Fluorescence microscopy revealed these liposomes are localized to areas surrounding blood vessels with a significant degree of extravasation. These investigations demonstrate the ability to target liposomes in vivo, and establish parameters and limitations to be considered in future liposome development.