Selective transfer of a lipophilic prodrug of 5-fluorodeoxyuridine from immunoliposomes to colon cancer cells

Abstract

A monoclonal antibody against the rat colon carcinoma CC531 was covalently coupled to liposomes containing a dipalmitoylated derivative of the anticancer drug FUdR as a prodrug in their bilayers. We investigated the in vitro interaction of these liposomes with CC531 target cells and the mechanism by which they deliver the active drug FUdR intracellularly to the cells by monitoring the fate of the liposomal bilayer markers cholesterol-[(14)C]oleate and [(3)H]cholesteryloleylether as well as the (3)H-labeled prodrug and colloidal gold as an encapsulated liposome marker. After binding of the immunoliposomes to the cell surface, only limited amounts were internalized as demonstrated by a low level of hydrolysis of liposomal cholesterol ester and by morphological studies employing colloidal gold-labeled immunoliposomes. By contrast, already within 24 h immunoliposome-incorporated FUdR-dP was hydrolyzed virtually completely to the parent drug FUdR intracellularly. This process was inhibited by a variety of endocytosis inhibitors, indicating that the prodrug enters and is processed by the cells by a mechanism involving an endocytic process, resulting in intracellular FUdR concentrations up to 3000-fold higher than those in the medium. Immunoliposomes containing poly(ethyleneglycol) (PEG) chains on their surface, with the antibody coupled either directly to the bilayer or at the distal end of the PEG chains were able to deliver the prodrug into the tumor cells at the same rate as immunoliposomes without PEG. Based on these observations, we tentatively conclude that during the interaction of the immunoliposomes with the tumor cells the lipophilic prodrug FUdR-dP is selectively transferred to the cell surface and subsequently internalized by constitutive endocytic or pinocytic invaginations of the plasma membrane, thus ultimately delivering the prodrug to a lysosomal compartment where hydrolysis and release of parent drug takes place. This concept allows for an efficient delivery of a liposome-associated drug without the need for the liposome as such to be internalized by the cells.

Topology of Surface Ligands on Liposomes: Characterization Based on the Terms, Incorporation Ratio, Surface Anchor Density, and Reaction Yield

“…The values for incorporation ratios in parenthesis were calculated by us based on following equation: maleimide lipid molar ratio (of the total lipids in the liposomes)×0.55 (ratio facing outward from the membrane) (Eq. 1 in Chart 1) with the assumption of a 100% reaction yield, which means that all of the anchors contained an attached ligand [33][34][35][36][37][38][39][40][41][42][43][44][45][46][47] . mAb: monoclonal antibody, r.t.: room temperature, n.d.; no data.…”

“…33) Most literature reports do not provide these two values or provide sufficiently accurate values to permit the surface topologies to be evaluated. Instead, bioactivities are evaluated with reference to differences in the composition of liposomal lipids [33][34][35][36][37][38][39][40][41][42][43][44][45][46][47] (Table 1). The affinity of a liposome toward binding and its pharmacokinetics are related to its surface topology, 29,30,[48][49][50] which is defined as the ratio of the density of the surface ligand and PEG that are located on the liposomal surface.…”

Targeting of angiogenic endothelial cells at sites of inflammation by dexamethasone phosphate–containing RGD peptide liposomes inhibits experimental arthritis

“…30) were synthesized at a purity of 95% by Ansynth Service BV (Roosendaal, The Netherlands). The thioacetyl group was used for thioether linkage to the liposomes (32). Briefly, peptide was deacetylated in an aqueous solution of 0.05M HEPES/0.05M hydroxylamine HCl/0.03 mM EDTA (pH 7.0) for 30 minutes at room temperature.…”

Cytosolic Delivery of Liposomally Targeted Proteins Induced by Photochemical Internalization

“…The monoclonal antibody mAb425 and the irrelevant isotype antibody were modified with SATA to introduce sulfhydryl groups randomly as described previously (14). In short, an 8-fold molar excess of SATA dissolved in DMF was added to the antibody solution (1/10 of volume) and incubated for 45 min at room temperature.…”