Successful gene silencing by small interfering RNA (siRNA) requires efficient uptake of siRNA into targeted cells. For in vitro transfection of siRNA using cationic liposomes, two types of transfection method are currently being used: conventional (forward; Fw) and reverse (Rev) transfections. Here, to investigate an efficient siRNA transfection method using cationic liposomes, we compared the transfection efficiency of siRNA between Fw-transfection and Rev-transfection methods with various types of cationic liposomes. In Fw-transfection, siRNA/cationic liposomes complex (siRNA lipoplexes) was added to pre-plated cells. In contrast, Rev-transfection was performed by co-incubation of cells with siRNA lipoplexes in suspension. As a result, Rev-transfection with 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)-based or cationic cholesterol derivative-based liposomes could deliver siRNA into the cells via efficient cellular association, and induce an improved gene silencing effect by siRNA compared with Fw-transfection. Furthermore, Rev-transfection did not show increased cytotoxicity compared with Fw-transfection. These findings suggested that Rev-transfection in suspension has better potential for efficient transfection of siRNA into cells with minimal toxicity.
Synthetic small interfering RNAs (siRNAs), which are small, double-stranded RNAs, are substrates for the RNA-induced silencing complex [
1,2-Dioleoyl-3-trimethylammonium-propane (DOTAP) methyl sulfate salt (
siRNAs targeting nucleotides of firefly pGL3 luciferase (Luc siRNA) and nonsilencing siRNA (control [Cont] siRNA) as a negative control were synthesized by Sigma Genosys (Tokyo, Japan). The siRNA sequences of the Luc siRNA were: sense strand: 5'-GUGGAUUUCGAGUCGUCUUAA-3' and antisense strand: 5'-AAGACGACUCGAAAUCCACAU-3'. The siRNA sequences of the Cont siRNA as a negative control for Luc siRNA were: sense strand: 5'-GUACCGCA CGUCAUUCGUAUC-3' and antisense strand: 5'-UACGAAUGACGUGCGGU ACGU-3'. Alexa Fluor®488-labeled AllStars Negative Control siRNA (AF-siRNA) was obtained from Qiagen (Valencia, CA, USA).
Cationic liposomes were prepared from DOTAP/Chol or DOTAP/DOPE at a molar ratio of 1:1 using a thin-film hydration method, as reported previously [
To prepare complexes of siRNA/cationic liposome (siRNA lipoplexes), each liposome preparation was added to 50 pmol siRNA at a charge ratio (+:−) of 4:1 of cationic lipid to siRNA for DOTAP/Chol and DOTAP/DOPE liposomes or 7:1 for OH-Chol/DOPE and OH-C-Chol/DOPE liposomes with gentle shaking and left at room temperature for 15 min. The charge ratio (+:−) of liposomes: siRNA is expressed as the molar ratio of cationic lipid to siRNA phosphate.
The particle size distributions of cationic liposomes and lipoplexes were measured by the cumulant method using a light-scattering photometer (ELS-Z2, Otsuka Electronics Co., Ltd., Osaka, Japan) at 25˚C after diluting the dispersion with an appropriate volume with water. The ζ-potentials were measured using the ELS-Z2 at 25˚C after diluting the dispersion with an appropriate volume of water.
Human breast cancer MCF-7-Luc (TamR-Luc#1) cells stably expressing firefly luciferase (pGL3) were donated by Dr. Kazuhiro Ikeda (Division of Gene Regulation and Signal Transduction, Research Center for Genomic Medicine, Saitama Medical University, Saitama, Japan). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 μg/mL kanamycin, and 0.5 mg/mL G418 at 37˚C in a 5% CO2 humidified atmosphere.
For Fw-transfection, MCF-7-Luc cells were detached with TrypLETM Express (Invitrogen), plated into 6-well (35 mm) culture dishes at a density of 2.5 × 105 cells per well and then incubated for 1 h at 37˚C in a 5% CO2 humidified atmosphere. Each lipoplex with 50 pmol siRNA was diluted in 1 mL of DMEM (50 nM siRNA) supplemented with 10% FBS, and then added to the cells (
For Rev-transfection, MCF-7-Luc cells were detached with TrypLETM Express, and 2.5 × 105 cells were suspended in 0.5 mL of DMEM supplemented with 10% FBS (
Each lipoplex with 50 pmol Luc siRNA or Cont siRNA was transfected by the Fw-transfection or Rev-transfection methods into MCF-7-Luc cells in 6-well culture dishes (final 50 nM siRNA concentration). Forty-eight hours after transfection, luciferase activity was measured as counts per sec (cps)/μg protein using a luciferase assay system (Pica Gene, Toyo Ink Mfg. Co. Ltd., Tokyo, Japan) and BCA reagent (Pierce, Rockford, IL, USA), as reported previously [
For Fw-transfection, MCF-7-Luc cells were detached with TrypLETM Express, plated into 6-well culture dishes at a density of 5.0 × 105 cells per well and then incubated for 1 h. Each lipoplex with 50 pmol AF-siRNA was diluted in 1 mL of medium (50 nM siRNA) supplemented with 10% FBS, and then added to the cells. For Rev-transfection, each lipoplex with 50 pmol AF-siRNA was diluted in
0.5 mL of medium supplemented with 10% FBS, and then added to 5.0 × 105 cells suspended in 0.5 mL of medium. The mixtures (1 mL) of siRNA lipoplex (50 nM AF-siRNA) and suspended cells were transferred into 6-well culture dishes. After 3 h incubation, the cells were washed twice with 1 mL phosphate- buffered saline (PBS) to remove any unbound lipoplexes. The amount of AF- siRNA in the cells was determined by examining fluorescence intensity using a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA), as described previously [
For Fw-transfection, MCF-7-Luc cells were plated into 96-well culture dishes at a density of 2.5 × 104 cells per well and incubated for 1 h. Each lipoplex with 50 pmol Cont siRNA was diluted in 1 mL of medium supplemented with 10% FBS, and then the mixture (100 μL) was added to the cells (final 50 nM siRNA concentration).
For Rev-transfection, each lipoplex with 50 pmol Cont siRNA was diluted in 0.5 mL of medium supplemented with 10% FBS, and then added to 2.5 × 105 cells suspended in 0.5 mL of the medium. The mixtures (100 μL) of the lipoplexes and 2.5 × 104 cells were transferred into 96-well culture dishes (final 50 nM at siRNA concentration).
After a 24 h incubation period, cell numbers were determined using a Cell Counting Kit-8 (Dojindo Laboratories, Kumamoto, Japan). Cell viability was expressed as relative to the absorbance at 450 nm of untransfected cells.
Data were compared using analysis of variance and evaluated with Student’s t test. A p value of 0.05 or less was considered significant.
First, we prepared DOTAP-based and cationic cholesterol derivative-based liposomes for siRNA delivery by Fw- and Rev-transfections, respectively. In this study, we used DOTAP, OH-Chol, or OH-C-Chol (
Next, we examined the effect of transfection methods on the gene knockdown effect by the lipoplexes with 50 nM siRNA using a luciferase assay system with MCF-7-Luc cells. Conventional transfection (Fw-transfection) is a common technique for delivering siRNA into cells for gene silencing (
Formulation | Molar ratio | Liposomea) | Lipoplex of siRNAa,b) | ||
---|---|---|---|---|---|
Size (nm) | ζ-potential (mV) | Size (nm) | ζ-potential (mV) | ||
DOTAP/Chol DOTAP/DOPE OH-Chol/DOPE OH-C-Chol/DOPE | 1/1 1/1 3/2 3/2 | 101.2 ± 2.5 116.2 ± 2.9 126.0 ± 2.9 120.5 ± 2.7 | 56.0 ± 1.0 49.7 ± 0.6 52.8 ± 0.9 47.5 ± 1.8 | 189.5 ± 5.0 189.0 ± 6.0 220.1 ± 1.9 195.3 ± 5.2 | 41.7 ± 0.4 44.4 ± 0.8 46.7 ± 0.8 46.1 ± 1.8 |
a)In water. b)Charge ratio (+/−) of cationic liposome/siRNA = 4/1 for DOTAP/Chol and DOTAP/DOPE lipoplexes, and 7/1 for OH-Chol/DOPE and OH-C-Chol lipoplexes, respectively. Values represent means ±S.D. (n = 3).
Fw-transfection and Rev-transfection must be uniform. In contrast, in Rev- transfection, siRNA lipoplexes were diluted in culture medium, and mixed with an equal volume of medium diluted with MCF-7 cells (
For DOTAP-based formulations, DOTAP/Chol lipoplexes with Luc siRNA did not suppress luciferase activity by Fw-transfection; however, moderate suppression of luciferase activity was observed by Rev-transfection (
DOTAP/DOPE lipoplexes exhibited higher gene silencing activity than DOTAP/Chol lipoplexes (
To clarify the relationship between the gene silencing effect and cellular uptake in Fw-transfection and Rev-transfection with siRNA lipoplexes, we examined the cellular association of lipoplexes by flow cytometric analysis. In Fw- transfection, DOTAP/Chol lipoplexes exhibited higher cellular association than DOTAP/DOPE lipoplexes (
Finally, we investigated cytotoxicity after Fw- and Rev-transfections with siRNA lipoplexes. As a result, in any siRNA lipoplexes, Rev-transfection did not
strongly increase cytotoxicity compared with Fw-transfection (
In solid-phase Rev-transfection, the siRNA complex must be freeze-dried on a culture plate in small volumes. The one of advantages for Rev-transfection in suspension is the ability to easily prepare a large number of plates in larger volumes. However, the reasons for the improvements in gene silencing (
In conclusion, Rev-transfection with siRNA lipoplexes in suspension could deliver siRNA into the cells via efficient cellular association and induce an improved gene silencing effect by siRNA compared with Fw-transfection. These findings suggested that the Rev-transfection method in suspension has potential for efficient transfection of siRNA into cells with minimal toxicity.
We declare no conflict of interests. This project was supported in part by a Grant-in-Aid for Scientific Research (C) from the Japan Society for the Promotion of Science (KAKENHI Grant Number 26460046).
Hattori, Y., Yoshiike, Y., Honda, M., Ohno, H. and Onishi, H. (2017) Evaluation of Small Interfering RNA Delivery into Cells by Reverse Transfection in Suspension with Cationic Liposomes. Pharmacology & Pharmacy, 8, 129-139. https://doi.org/10.4236/pp.2017.85009