No 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.

Novel polycation liposomes decorated with cyclic(Cys-Arg-Gly-Asp-d-Phe) peptide (cyclicRGD)-polyethylene glycol (PEG) (RGD-PEG-polycation liposomes (PCL)) were previously developed for cancer therapy based on RNA interference. Here, we demonstrate the in vivo delivery of small interfering RNA (siRNA) to tumors by use of RGD-PEG-PCL in B16F10 melanoma-bearing mice. Pharmacokinetic data obtained by positron emission tomography showed that cholesterol-conjugated siRNA formulated in RGD-PEG-PCL markedly accumulated in the tumors. Delivered by RGD-PEG-PCL, a therapeutic cocktail of siRNAs composed of cholesterol-conjugated siRNAs for c-myc, MDM2, and vascular endothelial growth factor (VEGF) were able to significantly inhibit the growth of B16F10 melanoma both in vitro and in vivo. These data suggest that targeted delivery of siRNAs by use of RGD-PEG-PCL has considerable potential for cancer treatment.

No abstract

In the development of nucleic acid medicines such as small interfering RNA (siRNA) drugs, one problem is how to study the pharmacokinetics and pharmacodynamics, since the precise in vivo behavior of siRNA is hard to detect. In this research, to establish a highly sensitive detection system of siRNA biodistribution in the whole body, the technology of positron imaging was applied. First, a one-step synthetic method in which double-stranded siRNA was directly labeled by a positron emitter, 18 F, was developed. By using [ 18 F]-labeled siRNA ([ 18 F]-siRNA), the complex of siRNA and polycation liposomes (PCL) containing dicetylphosphate tetraethylenepentamine (TEPA-PCL) was prepared. Then, the biodistribution of the siRNA after intravenous administration to mice was analyzed by planar positron imaging system (PPIS). As a result, whereas naked [ 18 F]-siRNA was immediately excreted in mouse bladder after administration, the complex with cationic liposome (CL) was trapped in the lungs. Furthermore, [ 18 F]-siRNA carried with PEGylated CL (PL) was distributed throughout the body, suggesting that it circulated in the bloodstream for an extended period of time. Additionally, PET imaging revealed more detailed biodistribution of the siRNA than in vivo imaging system (IVIS) because PET imaging is not aŠected by the depth variation of target tissues. On the other hand, to induce high accumulation of siRNAs against c-myc, MDM2, and VEGF in tumor tissue, a tumor-targeting probe, RGD peptide, was grafted at the top of PEG chain in PEGylated TEPA-PCL and the eŠect of the complex on experimental lung metastasis of B16 melanoma was examined. The complex suppressed the progression of tumor. We believe that the positron imaging data would support the development of siRNA agent for clinical use.