Liposomal delivery of antisense oligonucleotides for efficient downregulation of Bcl-2 and induction of apoptosis.

Aim: The aim of this study was to enhance the delivery and thus anti-tumoral efficiency of antisense bcl-2 oligonucleotides (ODN's).

Methods: Bcl-2 overexpressing DoHH2 lymphoma and HeLa-cells were transfected with ODN's using a polycationic liposome preparation. Specific hybridization of antisense ODN's was demonstrated by gel-shift assays and in vitro transcription/translation studies. Cellular uptake of oligonucleotides was evaluated by fluorescence microscopy. Inhibition of bcl-2 translation was demonstrated by quantitative RT-PCR and Western Blot. TUNEL assay, ANNEXIN V-binding and Apo-2.7 expression were performed to evaluate induction of apoptosis.

Results: Using polycationic liposomes, a ODN transfection rate of 95% in HeLa and 45% in DoHH2 cells were demonstrated by fluorescence microscopy. 24 hours after transfection quantitative RT-PCR detected a 56% decrease of bcl-2 mRNA in antisense and a 7% decrease in sense transfected DoHH2 cells (p < 0.05). In HeLa-cells, bcl-2 expression was almost completely inhibited 72 hours after antisense ODN transfection. Antisense treated cells also showed significant induction of apoptosis.

Conclusions: Polycationic liposome-mediated transfection of bcl-2 antisense ODN's causes enhanced cellular uptake and efficient bcl-2 downregulation in bcl-2 overexpressing cell lines. This delivery strategy may explain why significant induction of apoptosis was achieved at low oligonucleotide concentrations (approximately 200 pmol/5 x 10(5) tumor cells).