Internalization of indium-labeled LDL through a lipid chelating anchor in human pancreatic-cancer cells as a potential radiopharmaceutical for tumor localization

Abstract

Low-density lipoproteins (LDL) labeled with indium via a lipid-chelating agent, the bis(stearylamide) of diethylenetri-aminepentaacetic acid (L), were evaluated as a potential radiopharmaceutical (111 In-L-LDL) for tumor localization by studying their internalization in human pancreatic cancer cells (Capan-1). Using DiI-LDL (1,18-dioctadecyl-3,3,38,38-tetramethylindodicarbocyanine perchlorate-LDL), this cell line was shown to bind human LDL with a high-affinity saturable component and a low-affinity non-saturable (40%) component. The single saturable high-affinity binding site had a K D of 27.5 6 2.1 mg/ml and a maximal binding of 610 6 7.5 ng/ml protein. Electron-microscopic examination of the In-L-LDL particles revealed the peripheral distribution of the electron-dense indium atoms at the outer surface of LDL. The modified LDL were then shown to be internalized by the cells. After conjugation of In-L-LDL to colloidal gold to follow the different stages of internalization, electron-microscopic examination showed that the In-L-LDL gold conjugates were stuck to the external sheet of the plasma apical and microvilli membrane, into earlier and later endosomes and into multi-vesicular bodies, suggesting the penetration of the In-L-LDL particles into lysosomal vacuoles. The observation of In-L-LDL-gold conjugates in deep-seated cytoplasm suggests that LDL could be employed as a drug-transport vehicle for targeting cytotoxics or radionuclides close to the cell nucleus. Int. J. Cancer, 70:315-322, 1997. r 1997 Wiley-Liss, Inc. Low-density lipoproteins (LDL) have attracted increasing attention as endogenous drug-transport vehicles to cancer cells since reports of higher receptor-mediated LDL uptake in tumor cells than in normal cells (Ho et al., 1978; Hynds et al., 1984; Lombardi et al., 1989; Samadi-Baboli et al., 1993). Various methods have been proposed for labeling LDL with a metal ion in order to use LDL as target imaging agents in diagnostic applications. For the radiomet-als detectable by gamma scintigraphy, the radioelement was affixed directly or indirectly to protein residues of Apoprotein B-100 (ApoB), the binding ligand to the LDL cell receptor (R-LDL) (Rosen et al., 1990; Virgolini et al., 1991; Ponty et al., 1993). In these procedures, the radiometal/LDL ratio was kept low to avoid disrupting the 3-dimensional structure of the recognition protein. To localize the radionuclide preferentially on the lipid layer and to minimize the structural modification of ApoB, we have developed a new labeling method using a lipid-chelating anchor (Jasanada et al., 1996). A lipophilic derivative of diethylenetriamine-pentaacetic acid (DTPA), the N,N9-bis(stearylamide) of DTPA (L), was first incorporated into LDL giving L-LDL conjugates before adding 111-indium to obtain the final tracer 111 In-L-LDL. In vitro experiments demonstrated the stability of the lipophilic anchorage of the chelator, the stability of the indium-111 complexation in plasma, while binding studies showed recognition of the 111 In-L-LDL particles by LDL...

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