Enhanced intrapulmonary delivery of anticancer siRNA for lung cancer therapy using cationic ethylphosphocholine-based nanolipoplexes.

Here, we report a cationic nanolipoplex as a pulmonary cellular delivery system for small-interfering RNA (siRNA). Six nanoliposomes differing in cationic lipids were formulated and screened in vitro and in vivo for cellular delivery functions in lung cells/tissues. Although the six nanoliposomes showed similar siRNA delivery efficiency in vitro, they exhibited significant differences in pulmonary cellular delivery functions in vivo. Among the various nanoliposomes, cationic dioleoyl-sn-glycero-3-ethylphosphocholine and cholesterol (ECL)-based nanoliposomes showed the highest pulmonary cellular delivery in vivo and the lowest cytotoxicity in vitro. The delivery efficiency of fluorescent siRNA in ECL nanoliposomes was 26.2-fold higher than that of naked siRNA in vivo. Treatment with Mcl1 (myeloid cell leukemia sequence 1)-specific siRNA (siMcl1) using ECL nanolipoplexes reduced target expression in B16F10 cell lines, whereas control, luciferase-specific siGL2 in ECL nanolipoplexes did not. In metastatic lung cancer mouse models induced by B16F10 or Lewis lung carcinoma (LLC) cells, intratracheal administration of siMcl1 in ECL nanolipoplexes significantly silenced Mcl1 mRNA and protein levels in lung tissue. Reduced formation of melanoma tumor nodules was observed in the lung. These results demonstrate the utility of ECL nanoliposomes for pulmonary delivery of therapeutic siRNA for the treatment of lung cancers and potentially for other respiratory diseases.

Cationic solid lipid nanoparticles for co-delivery of paclitaxel and siRNA.

In this study, we formulated cationic solid lipid nanoparticles (cSLN) for co-delivery of paclitaxel (PTX) and siRNA. 1,2-Dioleoyl-sn-glycero-3-ethylphosphocholine-based cSLN were prepared by emulsification solidification methods. PTX-loaded cSLN (PcSLN) were characterized by zeta potential and gel retardation of complexes with small interfering RNA (siRNA). The sizes of PcSLN did not significantly differ from those of empty cSLN without PTX (EcSLN). The use of cSLN increased the cellular uptake of fluorescent dsRNA in human epithelial carcinoma KB cells, with PcSLN complexed to fluorescence-labeled dsRNA promoting the greatest uptake. For co-delivery of therapeutic siRNA, human MCL1-specific siRNA (siMCL1) was complexed with PcSLN; luciferase-specific siRNA (siGL2) complexed to EcSLN or PcSLN was used as a control. MCL1 mRNA levels were significantly reduced in KB cells treated with siMCL1 complexed to PcSLN, but not in groups treated with siMCL alone or siGL2 complexed to PcSLN. siMCL1 complexed to PcSLN exerted the greatest in vitro anticancer effects in KB cells, followed by siMCL1 complexed to EcSLN, siGL2 complexed to PcSLN, PTX alone, and siMCL1 alone. In KB cell-xenografted mice, intratumoral injection of PcSLN complexed to siMCL1 significantly reduced the growth of tumors. Taken together, our results demonstrate the potential of cSLN for the development of co-delivery systems of various lipophilic anticancer drugs and therapeutic siRNAs.

Trilysinoyl oleylamide-based cationic liposomes for systemic co-delivery of siRNA and an anticancer drug.

Oligolysine-based cationic lipid derivatives were synthesized for delivery of siRNA, and formulated into cationic liposomes. Among various oligolysine-based lipid derivatives differing in lysine residue number and lipid moiety, trilysinoyl oleylamide (TLO)-based liposomes (TLOL) showed the highest delivery efficiency combined with minimal cytotoxicity. Delivery of siRNA using TLOL silenced target genes both in vitro and in vivo. In green fluorescent protein (GFP)-expressing tumor tissue, a significant reduction of fluorescence was observed after intratumoral administration of siGFP using TLOL compared with control siGL2. Intravenous administration of siMcl1 employing pegylated TLOL (pTLOL) reduced the expression of human Mcl1 protein in KB-xenografted tumor tissue. Despite the reduction in target protein Mcl1 expression following such systemic delivery, tumor growth was only slightly reduced compared to a siGL2-treated control group. To potentiate the anticancer activity of siMcl1, the anticancer drug suberoylanilide hydroxamic acid (SAHA) was additionally encapsulated in pTLOL. After intravenous administration of siMcl1 using SAHA-loaded pTLOL (pSTLOL), a significant reduction in tumor growth was observed compared to that seen in animals treated with free SAHA or siGL2 complexed with pSTLOL. The results indicate that pTLOL could be further developed as a systemic delivery system for synergistic anticancer siRNA and a drug.

Anionic amino acid-derived cationic lipid for siRNA delivery.

Viable siRNA therapeutic strategies require the concurrent development of effective and safe delivery systems. Here, we described the synthesis of a new cationic lipid, N,N''-dioleylglutamide (DG), and evaluated DG-based liposomes as an siRNA delivery system. DG, an amino acid derivative, was synthesized by peptide bond linkage of oleylamine to each carboxylic acid group of glutamic acid. Gel retardation assays showed that DG-based cationic liposomes and siRNA began to form complexes from the N/P ratio of 1.8. The viability of A549, HeLa and WM266.4 cells was significantly higher after treatment with DG-based liposomes than with Lipofectamine 2000 and cationic 3beta-[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol)-based liposomes. The DG-based cationic liposomes could effectively deliver a fluorescent model siRNA into A549, HeLa, and WM266.4 human cancer cell lines, showing at least 2-fold higher fluorescence mean intensity values than did Lipofectamine 2000. When survivin-specific siRNA was delivered to cells in lipoplexes, survivin mRNA levels were reduced by DG-based liposomes to the higher extent than Lipofectamine 2000 and DC-Chol-based liposomes. When red fluorescent protein (RFP)-expressing cells were treated with RFP-specific siRNA (siRFP), RFP expression significantly decreased in cells treated with DG-based liposomes. Molecular imaging revealed that intratumoral injection of siRFP and DG-based liposome complexes significantly reduced fluorescence in RFP-expressing tumor tissues in mice. These results suggest that DG-based cationic liposomes would be of value for cellular delivery and in vivo local delivery of siRNA.