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Liposomes containing alkylated methotrexate analogues for phospholipase A2 mediated tumor targeted drug delivery |
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| Authors: | Thomas Kaasgaard Thomas L Andresen Simon S Jensen Lotte T Jensen |
| Institution: | a LiPlasome Pharma A/S, Department of Chemistry, Technical University of Denmark, DK-2800 Lyngby, Denmark b MEMPHYS - Center for Biomembrane Physics, Physics Department, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark c Danish Technological Institute, Holbergsvej 10, DK-6000 Kolding, Denmark d DTU Nanotech, Technical University of Denmark, 4000 Roskilde, Denmark e Bioneer A/S, Kogle Allé 2, DK-2970 Hørsholm, Denmark |
| Abstract: | Two lipophilic methotrexate analogues have been synthesized and evaluated for cytotoxicity against KATO III and HT-29 human colon cancer cells. Both analogues contained a C16-alkyl chain attached to the γ-carboxylic acid and one of the analogues had an additional benzyl group attached to the α-carboxylic acid. The cytotoxicity of the γ-alkylated compound towards KATO III (IC50 = 55 nM) and HT-29 (IC50 = 400 nM) cell lines, was unaffected by the alkylation, whereas the additional benzyl group on the α-carboxyl group made the compound nontoxic. The γ-derivative with promising cytotoxicity was incorporated into liposomes that were designed to be particularly susceptible to a liposome degrading enzyme, secretory phospholipase A2 (sPLA2), which is found in high concentrations in tumors of several different cancer types. Liposome incorporation was investigated by differential scanning calorimetry (DSC), and sPLA2 hydrolysis was examined by fluorescence spectroscopy and high performance liquid chromatography (HPLC). The results showed that the methotrexate (MTX)-analogue could be incorporated into liposomes that were degradable by sPLA2. However, the in vitro cytotoxicity of the MTX-liposomes against KATO III and HT-29 cancer cells was found to be independent of sPLA2 hydrolysis, indicating that the alkylated MTX-analogue was available for cancer cell uptake even in the absence of liposome hydrolysis. Using a DSC based method for assessing the anchoring stability of alkylated compounds in liposomes, it was demonstrated that the MTX-analogue partitioned into the water phase and thereby became available for cell uptake. It was concluded that liposomes containing alkylated MTX-analogues show promise as a drug delivery system, although the MTX-analogue needs to be more tightly anchored to the liposomal carrier. Also, the developed DSC-assay for studying the anchoring stability of alkylated drugs will be a useful tool in the development of liposomal drug delivery systems. |
| Keywords: | Methotrexate analogue Phospholipase A2 Drug delivery Liposome Tumor targeting Synthesis |
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