1:Universitat de Barcelona

There is a need for improving the delivery of oligonucleotides intended for gene therapy. Previously, we synthesized dioleyl pyridinium (DOPY) that was able to transfect cells with great efficiency. Now we describe the synthesis of two new cationic liposomes: trioleyl pyridinium (TROPY) and tetraoleyl pyridinium (SPIDER). Both syntheses were carried out in two synthetics steps. TROPY is a tris-pyridinium salt connected through a mesitylene spacer, SPIDER is a tetrakis pyridinium salt connected through a 1,2,4,5-tetramethylphenyl spacer. The pyridinium moieties in both liposomes bear an oleyl chain.  We studied their biological activity in human prostate cancer PC3 cells. As a model for therapeutic oligonucleotides, we used a Polypurine Reverse Hoogsteen (PPRH) hairpin specifically directed against the promoter region of the antiapoptotic survivin gene, either labelled with fluorescein or unmodified. The DNA binding capability was tested by gel agarose electrophoresis, observing a decrease in the amount of free oligonucleotide in the presence of liposomes. Tropy and Spider/PPRH particles were 142 and 198  nm in size, respectively, as determined by DLS. The internalization of the PPRH was followed by fluorescent microscopy, flow cytometry and confocal microscopy in PC3 cells, showing that PPRHs were delivered into cytosol and nuclei of the cells. Both liposomes had no intrinsic cytotoxicity up to 1.5 µg/ml. The decrease in viability of PC3 cells, due to the PPRH against survivin, transfected with either 1.5 µg/ml of TROPY or SPIDER was 84% and 91%, respectively, as determined by MTT assays.