Chemistry and Physics of Lipids 157 (2009) 94–103 Contents lists available at ScienceDirect Chemistry and Physics of Lipids journal homepage: www.elsevier.com/locate/chemphyslip Liposomes containing alkylated methotrexate analogues for phospholipase A 2 mediated tumor targeted drug delivery Thomas Kaasgaard a,b,c,∗ , Thomas L. Andresen a,d , Simon S. Jensen e , René O. Holte a , Lotte T. Jensen a , Kent Jørgensen a 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 article info Article history: Received 27 May 2008 Received in revised form 12 September 2008 Accepted 17 November 2008 Available online 27 November 2008 Keywords: Methotrexate analogue Phospholipase A2 Drug delivery Liposome Tumor targeting Synthesis 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 C 16 -alkyl chain attached to the - carboxylic acid and one of the analogues had an additional benzyl group attached to the -carboxylic acid. The cytotoxicity of the -alkylated compound towards KATO III (IC 50 = 55 nM) and HT-29 (IC 50 = 400 nM) cell lines, was unaffected by the alkylation, whereas the additional benzyl group on the -carboxyl group made the compound nontoxic. The -derivative with promising cytotoxicity was incorporated into lipo- somes that were designed to be particularly susceptible to a liposome degrading enzyme, secretory phospholipase A 2 (sPLA 2 ), which is found in high concentrations in tumors of several different cancer types. Liposome incorporation was investigated by differential scanning calorimetry (DSC), and sPLA 2 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 sPLA 2 . However, the in vitro cytotoxicity of the MTX-liposomes against KATO III and HT-29 cancer cells was found to be independent of sPLA 2 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. © 2008 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Methotrexate (MTX) is an anticancer drug that has been used in the treatment of various cancer types for decades (Acute Leukemia Group B, 1965). It is an antimetabolite that inhibits dihydrofolate reductase (DHFR), which is an enzyme involved in the biosynthetic pathway of nucleotides and MTX is therefore highly toxic to rapidly dividing cancer cells (Subramanian and Kaufman, 1978; Bertino et al., 1996). However, MTX has a narrow therapeutic index, and its clinical use is hampered by severe dose-limiting side effects, as well as intrinsic and acquired drug resistance mechanisms of cancer cells ∗ Corresponding author at: Danish Technological Institute, Holbergsvej 10, DK-6000 Kolding, Denmark. Fax: +45 72201919. E-mail address: thomas.kaasgaard@teknologisk.dk (T. Kaasgaard). (Frei et al., 1984; Bertino et al., 1996; Mauritz et al., 2002; Serra et al., 2004). Various approaches have been attempted to overcome drug resistance and improve the therapeutic index of MTX. Synthesis of MTX-analogues having a molecular structure that is slightly different from that of the parent molecule is one approach to over- come drug resistance that has been employed for several years (Montgomery et al., 1979). The main strategy is to make alter- ations to the MTX-molecule that renders the analogue insensitive to, or able to bypass drug resistance mechanisms, and yet retain the cytotoxic activity towards the DHFR target enzyme. An exhaustive number of publications report on the effects that different alter- ations to the MTX-molecule have on DHFR inhibition and cytotoxic potency of the synthesized analogues (Piper et al., 1982a; Rosowsky et al., 1983, 1988a,b). Such studies have shown that particularly the -carboxyl group (Rosowsky et al., 1981, 1986), and to a lesser 0009-3084/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.chemphyslip.2008.11.005