RGD-containing ligands for targeting liposomal nanoparticles

The use of a targeting ligand to enhance the delivery of liposomal nanoparticles (LNs) to specific cells in diseased tissue is an attractive strategy that has progressed very slowly since the idea originated over 25 years ago. The slow progress can be attributed, in part, to the difficulties involved in producing well-defined targeted LN systems. This work concerns the development of peptide-based ligands for targeting LNs to cells that overexpress the α[sub v]β₃ integrin, which is considered a unique marker of the tumor-associated endothelium. Initial work focused on using Fab' fragments of monoclonal antibodies as targeting ligands and employing literature techniques for coupling these proteins to lipids that can then be inserted in preformed LNs. However, it was found that the coupling procedures resulted in low yields of poorly defined lipid-ligand conjugates that were difficult to quantitate when incorporated into LN. Attention was then given to the development of a peptide-targeting agent that could be incorporated into an LN at the time that the LN is made. The RGD-containing cyclic peptide, eRGDfK, was employed as the targeting ligand. In order to characterize its binding to the α[sub v]β₃ integrin, a fluorescently labeled analogue, cRGDfK-488, was synthesized and capillary electrophoresis was employed for analysis. This procedure proved advantageous for studying receptor ligand interactions, since it allowed for the binding to be characterized in solution without the need for covalent modification of receptor or ligand. A 2:1 RGD ligand to integrin specific binding stoichiometry was revealed with the second binding event having a similar affinity as the first binding event. The next phase of these studies investigated the ability of cRGDfK-488 to bind to integrins on human umbilical vascular endothelial cells (HUVEC) and subsequently undergo endocytosis. This was compared to the binding and uptake properties of a fluorescently labeled monoclonal antibody, LM609X, which specifically binds α[sub v]β₃ integrin. Using flow cytometry and fluorescence microscopy, it is shown that the RGD ligand exhibited considerably greater uptake following incubation at endocytosis permitting temperatures (37°C) as compared to endocytosis inhibiting temperatures (4°C). A 7.4-fold increase in uptake of the RGD-lipid was observed following a one-hour incubation with HUVEC at 37°C, as compared to 4°C. In contrast, only a 1.9 fold increase in cell-associated fluorescence was observed on incubation with LM609X at 37°C as compared to 4°C. It is suggested that this ability of RGD ligands to stimulate endocytosis may be of utility for achieving enhanced intracellular delivery of ligand-associated drugs in anti-angiogenic applications. The cyclic peptide was then used to construct a fluorescently labeled RGD-spacer-lipid construct of defined molecular weight that could be incorporated into LN at the time of manufacture. It is shown that the resulting RGD-LNs bind to HUVEC with increasing avidity as the amount of RGD-spacer-lipid incorporated is increased. Further, these RGD-LNs are straightforward to make and can load and retain anti-cancer drugs such as doxorubicin. It is shown that RGD-LNs loaded with doxorubicin and incubated with HUVEC selectively deliver the drug to the cytosol while non-targeted LNs are not internalized by the cell, suggesting potential utility as targeted drug delivery systems in vivo.Medicine, Faculty ofBiochemistry and Molecular Biology, Department ofGraduat