Dicetyl Phosphate-Tetraethylenepentamine-Based Liposomes for Systemic siRNA Delivery

Abstract

Dicetyl phosphate-tetraethylenepentamine (DCP-TEPA) conjugate was newly synthesized and formed into liposomes for efficient siRNA delivery. Formulation of DCP-TEPA-based polycation liposomes (TEPA-PCL) complexed with siRNA was examined by performing knockdown experiments using stable EGFP-transfected HT1080 human fibrosarcoma cells and siRNA for GFP. An adequate amount of DCP-TEPA in TEPA-PCL and N/P ratio of TEPA-PCL/siRNA complexes were determined based on the knockdown efficiency. Then, the biodistribution of TEPA-PCL modified with poly(ethylene glycol) (PEG) was examined in BALB/c mice. As a result, TEPA-PCL modified with PEG6000 avoided reticuloendothelial system uptake and showed long circulation in the bloodstream. On the other hand, PEGylation of TEPA-PCL/siRNA complexes caused dissociation of a portion of the siRNA from the liposomes. However, we found that the use of cholesterol-conjugated siRNA improved the interaction between TEPA-PCL and siRNA, which allowed PEGylation of TEPA-PCL/siRNA complexes without siRNA dissociation. In addition, TEPA-PCL complexed with cholesterol-conjugated siRNA showed potent knockdown efficiency in stable luciferase-transfected B16-F10 murine melanoma cells. Finally, the biodistribution of cholesterol-conjugated siRNA formulated in PEGylated TEPA-PCL was examined by performing near-infrared fluorescence imaging in Colon26 NL-17 murine carcinoma-bearing mice. Our results showed that tumor targeting with siRNA via systemic administration was achieved by using PEGylated TEPA-PCL combined with active targeting with Ala-Pro-Arg-Pro-Gly, a peptide used for targeting angiogenic endothelium.

Systemic Delivery of Small Interfering RNA by Use of Targeted Polycation Liposomes for Cancer Therapy

“…We previously developed TEPA-PCL, liposomes containing DCP-conjugated synthetic polycation, for the purpose of systemic administration of siRNA. 8) In the present study, we used siRNA with cholesterol conjugated to it at the 3′-end of its sense strand, which modification is known to have no effect on the knockdown of specific protein expression, 9) for the stabilization of siRNA complexed with carrier PCL in the bloodstream. Thus, siRNA-C is considered to be internalized by cells as a complexed form.…”

“…8) Briefly, dimerized dicetylphosphate anhydride in anhydrous chloroform was added to a solution of tetraethylenepentamine (SigmaAldrich, St. Louis, MO, U.S.A.) in anhydrous pyridine, and the reaction mixture was stirred for 4 h. After the removal of the solvent by evaporation, the residue was resuspended in distilled water and filtered to remove any unreacted tetraethylenepentamine. DCP-TEPA was purified by column chromatography using aminated silica gel (Chromatorex NH, Fuji Silisia Chemical Ltd., Aichi, Japan).…”

“…[5][6][7][8] Among various PCL composed of different kinds of polycations, PCL containing dicetylphosphate-tetraethylenepentamine (DCP-TEPA) as a main component have been found to be quite efficient for siRNA delivery and for gene silencing.…”

“…Although PEGylation of siRNA/TEPA-PCL complexes causes partial dissociation of the siRNA from the complex during the PEGylation step, this dissociation was found to be avoidable by conjugating cholesterol to the 3′-end of the sense strand of the siRNA (siRNA-C). 8) For active targeted delivery of siRNA to tumor tissues, we earlier investigated the usefulness of modification of these PEGylated TEPA-PCL (PEG-PCL) with cyclic(Cys-Arg-Gly-Asp-d-Phe) peptide (cyclicRGD) and termed them RGD-PEG-PCL.…”

Nanoparticle-Mediated Delivery of Anticancer Agents to Tumor Angiogenic Vessels

“…On the other hand, we have explored a DDS targeting angiogenic vessels in which cytotoxic drugs, [12][13][14][15][16][17][18][19][20] angiogenesis inhibitors, 21,22) and nucleic acids such as siRNA 23,24) have been delivered to angiogenic endothelial cells using targeted liposomes. Since proliferating cells are killed by cytotoxic drugs nonspecifically, not only tumor cells but also angiogenic endothelial cells should be attacked by these drugs.…”