Delivery of antisense oligonucleotides to neurons by anionic liposomes: Therapeutic potential and mechanisms of endocytosis

A major drawback for the use of antisense technology to treat neurological diseases is the lack of an efficient delivery vector. Numerous caveats govern the behavior of cationic lipids, limiting their application for neuronal DNA delivery. As the need for a net-positive charge on transfection-competent cationic liposome-DNA complexes is unresolved, we explored the potential of anionic liposomes for oligonucleotide delivery to neurons. Liposomes composed of dioleoyl phosphatidylglycerol and dioleoyl phosphatidylcholine were monodisperse and encapsulated oligonucleotides with 40–60% efficiency. Ionic strength, bilayer charge density and oligonucleotide chemistry influenced encapsulation. To demonstrate the biological efficacy of this vector, antisense oligonucleotides to p53 delivered in anionic liposomes were tested in an in vitro model of excitotoxicity. Treatment of hippocampal neurons with 1 μM p53 antisense oligonucleotides in anionic liposomes prevented glutamate-induced upregulation of p53 and increased neuronal survival to ∼75% compared to 35% in glutamate-treated cells. Encapsulated phosphorothioate p53 antisense oligonucleotides were neuroprotective at 5–10 fold lower concentrations than the unencapsulated oligonucleotides. Replacing the anionic lipid with phosphatidylserine significantly decreased neuroprotection and cationic lipid-oligonucleotide complexes were biologically ineffective. Neuroprotection by p53 antisense oligonucleotides in anionic liposomes was comparable to that by glutamate receptor antagonists or a chemical inhibitor of p53. Next, we studied the mechanisms involved in the uptake and intracellular transport of anionic liposomes. Anionic liposomes encapsulating Cy3-labeled oligonucleotides were internalized in a time- and temperature-dependent manner. Uptake occurred by receptor-mediated endocytosis via the LDL receptor-related protein, independent of cell surface proteoglycans. Liposomal endocytosis required clathrin, dynamin, an intact cytoskeletal network and phosphatidylinositol 3-kinase activity. Nuclear localization of Cy3-labeled oligonucleotides was observed within 1–3 h of incubation, suggesting that this delivery system bypassed the lysosomal pathway and the oligonucleotides were freely available to the neuron. Liposomal lipids were recycled back to the cell surface via transferrin-containing compartments. Uptake of cationic lipid-oligonucleotide complexes and oligonucleotides without lipid vectors was much lower than that of oligonucleotides delivered by anionic liposomes. Thus, the rapid endocytosis of anionic liposomes in hippocampal neurons, their lack of cytotoxicity and enhancement of biological activity of antisense oligonucleotides, make anionic liposomes an attractive vector system for oligonucleotide delivery to neurons.