Lipid nanoparticles for hepatic delivery of small interfering RNA

Abstract

Clinical application of small interfering RNA (siRNA) requires safe and efficient delivery in vivo. Here, we report the design and synthesis of lipid nanoparticles (LNPs) for siRNA delivery based on cationic lipids with multiple tertiary amines and hydrophobic linoleyl chains. LNPs incorporating the lipid containing tris(2-aminoethyl)amine (TREN) and 3 linoleyl chains, termed TRENL3, were found to have exceptionally high siRNA transfection efficacy that was markedly superior to lipofectamine, a commercial transfection agent. In addition, inclusion of polyunsaturated fatty acids, such as linoleic acid and linolenic acid in the formulation further enhanced the siRNA delivery efficiency. TRENL3 LNPs were further shown to transport siRNA into the cytosol primarily via macropinocytosis rather than clathrin-mediated endocytosis. The new LNPs have demonstrated preferential uptake by the liver and hepatocellular carcinoma in mice, thereby leading to high siRNA gene-silencing activity. These data suggest potential therapeutic applications of TRENL3 mediated delivery of siRNA for liver diseases.

Figure 1

Chemical structures of three cationic lipid-like materials.

Figure 2. In vitro evaluation of siRNA-LNPs containing TRENL

(A) In vitro luciferase gene silencing by siRNA encapsulated in various TRENL based formulations. SK-HEP-1 cells, stably expressing the luciferase gene, were transfected with luciferase siRNA complexed with LNPs at concentrations of 100nM and 20nM for 24hr. As a positive control, lipofectamine 2000 was used in the experiment. (B) MTS study analysis of cell cytoxicity of various TRENL based LNPs. Values represent the mean ± S.D. (n = 4). (C) Down-regulation of Cdk4 by Cdk4 siRNA in TRENL3 based LNP. SK-HEP-1 cells were treated by Lipofectamine 2000/siRNA complex and TRENL3-LNP-siRNA at the concentration level of 100 nM for 48 h. Cdk4 protein level expression was determined by western blotting (1- untreated; 2-Lipofectamine-NC siRNA; 3-Lipofectamine-Cdk4 siRNA; 4- TRENL3-LNP-NC siRNA; 5-TRENL3-LNP-Cdk4 siRNA). The quantification of Cdk4 protein level was normalized by β-actin.

Figure 3. Enhanced in vitro delivery efficiency by including unsaturated fatty acid in LNPs

(A) Chemical structures of three unsaturated fatty acids. (B) Cryo-TEM image of nanostrucure of siRNA-LNP containing TRENL3/LA. White arrows show the onion-like structure of LNPs. (C) In vitro luciferase gene silencing by TRENL3 based LNPs containing various fatty acids. SK-HEP-1 cells were transfected with luciferase siRNA complexed with LNPs at indicated concentrations for 24hr. The luciferase expression was analyzed by luminance. (D) MTS study analysis of cell cytoxicity of various TRENL3 based LNPs. Values represent the mean ± S.D. (n = 4).

Figure 4. Mechanism study of siRNA delivery by TRENL based LNPs

(A) Measurement of pH-dependent zeta potential of various TRENL based LNPs. (B) Ceullar uptake of LNPs carrying FAM- siRNA by SK-HEP-1 cells. Cells were treated various LNPs formulated FAM-siRNA (100 nM) at 4 h and the mean fluorescence intensity (MFI) was determined by flow cytometry. (C) Internalization mechanism of siRNA delivery by TRENL based LNPs. SK-HEP-1 cells were treated with 100 nM Cy3 labeled siRNA (red) in the presence of various labeled markers for 1 h. Markers for macropinosomes (70 kDa dextran), clathrin pits (transferrin), and caveosomes(CT-B). (D) Co-localization study of LNP-FAM-siRNA (green) with lysotracker (red). White arrows indicate the colocalization. Scale bar=20 μm.

Figure 5. Colloidal stability of siRNA encapsulated LNPs in serum

(A) Colloidal stability of TRENL3-LA-based LNP versus TRENL based LNP. (B) Serum stability of TRENL3-LA-based LNP versus TRENL based LNP.

Figure 6. In vivo biodistribution study of LNP mediated systemic delivery of siRNA

Confocal microscopic imaging of liver sections (A) and other organs (B). Mice were injected with TRENL3-LA based LNPs carrying Cy3-siRNA via tail vein. After 4 hours the liver was harvested and sections were counterstained with Alexa Fluor 488-Phalloidin (for actin) and Hoechst 33342 (for nuclei). Scale bar=20 μm. (C) Tissue distribution of Cy5-ODN containing LNP with TRENL3 and LA. 4 hr after intravenous administration, tissues were harvested and then Cy5 fluorescence signals were measured by IVIS imaging system. Left panel: a typical Cy5 fluorescence images of vital organs. Right panel: biodistribution based on the intensity of Cy5 signal (n=4). (D) A representative IVIS fluorescence image of tissues from a DEN induced mouse treated by Cy5-ODN (2.5 mg/kg) in LNP with TRENL3 and LA for 4 h. (E) Confocal images of liver and liver tumor sections from the DEN mouse treated by TRENL3-LA based LNP carrying Cy3-siRNA. After 4 hours the liver and liver tumor was harvested and sections were counterstained with Alexa Fluor 488-Phalloidin (for actin) and Hoechst 33342 (for nuclei). Scale bar=40 μm.

Figure 7. In vivo down-regulation by siRNA encapsulated in the TRENL3-LA based LNP

A single 1 mg/kg Factor VII siRNA dose was administered via tail vein injection to ICR mice. Livers were harvested at 48 hours postdose and assayed using a chromogenic assay. Data were normalized to PBS-treated mice and represented as group mean ± SD, n = 3/group. TRENL3-LA carrying Factor VII siRNA was that carrying NC siRNA(*P < 0.05).