In order to use the LDL receptor pathway to target radionuclides to cancer sites for imaging and diagnostic purposes, a labeling procedure of LDL with 111In using the DTPA-bis(stearylamide) (L) has been developed. This bifunctional ligand is intended to be incorporated into the phospholipid monolayer of LDL and to specifically chelate the In3+ cation at the surface. The ligand was incorporated into LDL in buffered medium with a 65-80% yield. The L-LDL samples are stable over a 24 h period when examined by dialysis, allowing their storage before indium-111 radiolabeling. In vitro studies of In-L-LDL particles show that indium labeling is rapidly achieved (1 h). More than 85% of the indium atoms are bound to the chelating functions of the incorporated DTPA derivatives and less than 10% to the nonspecific complexation sites of LDL (e.g., protein residues). After incubation in human serum, the indium activity recovered in the LDL fraction of In-L-LDL samples (95%) is much higher than in In-LDL samples (35%), pointing out the strong stabilizing chelating effect of the ligand. Competitive binding studies show that In-L-LDL are recognized by LDL receptors of A549 cells like native LDL when the In-L/LDL ratio varies from 5 to 30. All these in vitro experiments demonstrate that the In-L-LDL conjugates possess properties suitable for further work with in vivo experiments.

ABSTRACT:The aim of this study was to evaluate the role of low-density lipoproteins (LDLs) as a vehicle for aluminum sulfophthalocyanine (AlPcS) to target tumor cells for photodynamic therapy (PDT). LDLs are biological particles containing an apolipoprotein which recognizes with high affinity specific receptors on many cell types, including cancer cells. LDL was loaded with AlPcS in two different manners. In the first procedure, the tetrasulfonated AlPcS 4 was covalently bound to the protein part of the LDL via amide bonds to 6-carboxypentylaminosulfonyl spacer chains attached to two of the four sulfonate groups, i.e. AlPcS 4 A 2 . In the second procedure, the AlPcS 4 was substituted with a linear dodecylaminosulfonyl chain, i.e. AlPcS 4 (C 12 ) and noncovalently inserted into the phospholipid monolayer of the LDL. Both preparations contained over 50 moles AlPcS/ mole LDL. They were tested in vitro for their phototoxic properties against the EMT-6 mouse mammary tumor cell line and the A-549 human lung adenocarcinoma cell line. Cell survival was assessed using the MTT colorimetric assay. Association of the free AlPcS 4 (C 12 ) with LDL increased the in vitro phototoxicity of the dye substantially against both cell lines while the covalently loaded AlPcS 4 A 2 -LDL preparation showed little cytotoxicity, even at a 10-fold-higher light or drug doses. It was postulated that the covalent labeling of the protein moiety with the Pc greatly reduced LDL receptor recognition, rendering this derivative photo-inactive. Photodynamic therapy of EMT-6 tumor-bearing mice showed that the free and LDL-associated AlPcS 4 (C 12 ) exhibited similar activities, with 100% cure one week post-PDT at 0.2 mmol kg À1. We conclude that the attached aliphatic chain of the AlPcS 4 (C 12 ) greatly enhances the phototoxicity of the parent AlPcS 4 but that pre-association of the AlPcS 4 (C 12 ) with LDL does not further augment its in vivo photodynamic efficacy.

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