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In vivo targeting of liposomal drug carriers

University of British Columbia

Studies in this thesis were aimed at optimizing the in vivo targeting properties of liposomal drug carriers. A major obstacle to targeted delivery of liposomal carriers concerns access to the target site and subsequent binding to the target cell population in vivo. This thesis presents strategies to enhance circulation lifetimes of liposomes with surface-associated targeting proteins as a means to improve access of these carriers to a target site. Further, a model approach to investigate binding of liposomal drugs to targets in vivo is developed. This research effort was initiated following an investigation focused on demonstrating in vivo targeting of drug (doxorubicin)-loaded large unilamellar liposomes with covalently attached streptavidin (SA-LUV5) to a murine lymphocytic leukaemia cell line (P388) residing in the peritoneal cavity of mice. Streptavidin, a protein derived from Streptomyces avidinii, has 4 high-affinity binding sites for biotin. A two-step targeting procedure was used in which target cells were pre-labeled with a biotinylated monoclonal antibody, specific for the Thy 1.2 antigen expressed on P388 cells, prior to systemic administration of SA-LUVs. In vitro studies based on this two-step procedure resulted in a 30-fold and 20-fold increase in cell-associated lipid and doxorubicin, respectively, for doxorubicin-loaded SA-LUVs compared to protein-free liposomes or to conditions where the target cells were not pre-labeled with biotinylated antibody. Under optimal conditions approximately 6,000 SA-LUVs were specifically bound per cell using this procedure. In vivo targeting of drug-loaded SA-LUVs (injected intraperitoneally or intravenously) to P388 target cells was also achieved. However, the specificity and the efficiency of targeting was significantly less than expected on the basis of in vitro results. One explanation for the reduced efficiency concerns the fact that only a small amount (<3%) of the injected lipid dose reached the peritoneal cavity following intravenous administration. Surprisingly, however, under conditions where high local concentration of liposomes was achieved (i.p. injection of SA-LUVs), the specificity of the targeting was also less than that achieved in vitro. Non-specific binding of liposomes was largely a consequence of liposome uptake by tumor-associated macrophages. Observations suggesting that SA-LUVs have limited access to the target site led to studies designed to improve passive targeting to the peritoneal cavity. These studies were based on the hypothesis that procedures that increased circulating blood levels of injected (i.v.) liposomes would increase the total amount of lipid that accessed an extravascular site. Two procedures were developed to increase the circulation lifetimes of intravenously injected SA-LUVs. The first procedure involved blockade of liposome uptake by phagocytic cells in the liver with a low pre-dose (2 mg/kg drug) of liposomal doxorubicin. The second involved the incorporation of a polyethylene glycol-modified phospholipid (2-DP0SE0PGE0) in SA-LUVs. It was shown that incorporation of up to 2 mol% PEG-PE in liposomes resulted in an improved protein-coated liposome that exhibited optimal size characteristics as well as efficient binding to target cells in vitro. It was established that each of these procedures prolonged circulation lifetimes, decreased uptake in liver and increased accumulation in the peritoneal cavity following intravenously administration of SA-LUVs. Combining the strategies of liver blockade and incorporation of PEG-PE further increased circulation lifetimes and decreased liver uptake of SA-LUVs, however there was no further increase in passive targeting to the peritoneal cavity. The presence of an established P388 tumor in the peritoneal cavity markedly increased the passive targeting of SA-LUVs to that location. These results are of interest in terms of developing an understanding of the mechanism by which liposomes leave the vascular compartment. In order to investigate why there was reduced in vivo binding of SA-LUVs to target cells, a model approach was developed based on using biotin-labeled multilamellar vesicles as a target. SA-LUVs incorporating 2 mol% PEG-PE were found to bind optimally to multilamellar vesicles that incorporated biotinoylaniinohexanoyl DSPE (BAH-MLV). This binding was not reduced in the presence of normal mouse serum and SA-LUVs isolated from the blood of mice previously injected (i.v.) with the liposomes, exhibit no change in binding characteristics. In vivo studies based on SA-LUVs injected intraperitoneafly demonstrated a 17-fold and 8-fold increase in binding to BAH-MLVs in the peritoneal cavity of tumor-free and tumor-bearing animals, respectively, compared to non-targeted systems. The extent of targeting achieved under these conditions was comparable to that observed in vitro. SA-LUVs injected intravenously demonstrated a 5-fold increase in binding compared to both tumor-free and tumor-bearing animals. These studies were extended to a solid tumor model where it was shown that the presence of intratumorally injected BAH-MLVs promoted the accumulation of i.v. administered SA-LLJVs. SA-LUVs injected intravenously into mice bearing subcutaneous Lewis lung tumors accumulated 2- fold greater in tumors that had been injected with BAH-MLVs than tumors injected with MLVs.

Thesis/Dissertation

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2009-04-14

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http://hdl.handle.net/2429/7044

Biochemistry and Molecular Biology

University of British Columbia

UBCV

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