Delivery of siRNA Using Cationic Liposomes Incorporating Stearic Acid-modified Octa-Arginine

Materials and Methods

Materials. 2-Chlorotrityl chloride resins and Fmoc-L-arginine were obtained from JiEr Biochemical Company (Shanghai, China). Stearic acid ((StA), 98.5%), cholesterol (Chol), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Egg phosphatidylcholine (ePC) was purchased from Avanti Polar Lipids (Alabaster, AL, USA). Fetal bovine serum (FBS) and Dulbecco's modified Eagle's medium (DMEM) were from HyClone (Logan, UT, USA). Anti-survivin siRNA, FAM and Cy3 fluorescence-labeled siRNA were synthesized by Ribo Biochemistry (Guangzhou, China). 4’,6-Diamidino-2-phenylindole (DAPI) was purchased from Invitrogen Molecular Probes (Eugene, OR, USA). All other reagents were commercially purchased in reagent grade.

Synthesis of StA-R8. StA-R8 was prepared by Fmoc solid-phase peptide synthesis on a 2-chlorotrityl chloride resin as reported previously (22). Fmoc-L-arginine was loaded onto the resin one by one to synthesize R8 (23) and, then, stearic acid was coupled to R8 via an amide bond using the same approach (24). Next, the target peptide was purified and structurally confirmed by reverse-phase high-performance liquid chromatography (RP-HPLC) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). The scheme of StA-R8 synthesis is shown in Figure 1A.

Preparation of StA-R8 liposomes and transfection complexes. An ethanol injection method was used to prepare StA-R8 liposomes. StA-R8, ePC and Chol were dissolved in ethyl alcohol at different molar ratios. This solution was injected at a constant speed into 20 mM HEPES buffer (pH 7.4) with vortex mixing and at an ethanol/water ratio of 1:3 v/v. Then, the resulting liposomes were sonicated for 30 sec. siRNA complexes were synthesized by combining StA-R8 liposomes with anti-survivin siRNA. The composition of the complexes was optimized by varying the charge ratios of StA-R8-to-siRNA from 0.5:1 to 8:1. A schematic diagram of the preparation of StA-R8 liposomes and transfection complexes is shown in Figure 1B.

Particle size and zeta potential measurements. The particle size and zeta potential of StA-R8-modified liposomes were determined on a Zetasizer Nano ZS 90 (Malvern Instruments, Ltd., Malvern, UK). The measurement of each complex was repeated three times.

Agarose gel retardation assay. StA-R8 liposomes are capable of forming electrostatic complexes with siRNA, which was measured by gel retardation. The siRNA/StA-R8 complexes at varying charge ratios ranging from 0.5:1 to 8:1 were analyzed by electrophoresis on a 2% (w/v) agarose gel in TAE buffer, containing 0.5 μg/ml ethidium bromide (Sigma, St. Louis, MO, USA). The gel was run for 20 min at 80 V and visualized with a UV lamp using a Vilber Lourmat imaging system (Marne La Vallée, France). The pictures were digitized and analyzed using the Image J software (National Institutes of Health, Bethesda, MD, USA) to compute the mean density of siRNA band.

Cell culture. HeLa and HepG2 cells were cultured in DMEM with 10% fetal bovine serum (FBS) and 1% antibiotics/antimycotics in a humidified atmosphere containing 5% CO₂ at 37°C.

Cytotoxicity assay. A549, HepG2 and 293T cells were seeded in a 96-well plate at 1×10⁴ cells/well and cultured for 24 h. Then, siRNA complexes of StA-R8 or StA-R8 liposomes at different concentrations were added into the plate. The serum-free medium was replaced with fresh medium with FBS after 4 h incubation and the cells were incubated for another 20 h. Next, 15 μl MTT stock solution (5 mg/ml) was added into each well and the cells were incubated for 4h at 37°C. Finally, the culture medium was removed and 100 μl/well DMSO was added to dissolve the formazan converted from MTT. Cell viability data were obtained by measuring OD490 on a BioTek Synergy™ 4 Hybrid Microplate Reader (Winooski, VT, USA).

Cellular uptake analysis. Cells (1×10⁵/well) were seeded onto a 24-well plate. The StA-R8 liposomes/siRNA^FAM or StA-R8/siRNA^FAM complexes diluted in 1 ml of medium without FBS were added to the cells and the cells were incubated for 4 h in dark at 37°C. Subsequently, cells were washed three times with 1×PBS, trypsinized and fixed in 4% paraformaldehyde at 4°C overnight. Fluorescence intensity of the cells was measured on a flow cytometer (BD Biosciences, San Jose, CA, USA).

Confocal microscopy. Cells (1×10⁵/well) were incubated with StA-R8 liposomes/siRNA^Cy3 or StA-R8/ siRNA^Cy3 complexes for 4 h in the dark, washed three times with 1×PBS and fixed with 4% paraformaldehyde for 20 min. DAPI, 2μg/ml, was used to stain the cellular nuclei for 5 min at room temperature. Then, the cells were observed on a Zeiss 710 LSMNLO Confocal Microscope (Carl Zeiss, Jena, Germany).

Bioactivity of the siRNA complexes. HepG2 cells (2×10⁵ cells/well) seeded in a 6-well plate were treated with various transfection complexes as described above. The incubation medium was remove after 4 h and fresh medium with 10% FBS was added. The cells were cultured for another 44 h, washed and harvested. Quantitative real-time polymerase chain reaction (qRT-PCR) and western blot were used to determine survivin mRNA and protein expression.