Optimal combination of cationic lipid and phospholipid in cationic liposomes for gene knockdown in breast cancer cells and mouse lung using siRNA lipoplexes

Formulation of cationic liposomes is a key factor that determine the gene knockdown efficiency by cationic liposomes/siRNA complexes (siRNA lipoplexes). Here, to determine the optimal combination of cationic lipid and phospholipid in cationic liposomes for in vitro and in vivo gene knockdown using siRNA lipoplexes, three types of cationic lipid were used, namely 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), dimethyldioctadecylammonium bromide (DDAB) and 11-[(1,3-bis(dodecanoyloxy)-2-((dodecanoyloxy)methyl)propan-2-yl)amino]-N,N,N-trimethyl-11-oxoundecan-1-aminium bromide (TC-1-12). Thereafter, 30 types of cationic liposome composed of each cationic lipid with phosphatidylcholine or phosphatidylethanolamine containing saturated or unsaturated dialkyl chains (C14, C16, or C18) were prepared. The inclusion of phosphatidylethanolamine containing unsaturated and long dialkyl chains with DOTAP- or DDAB-based cationic liposomes induced strong luciferase gene knockdown in human breast cancer MCF-7-Luc cells stably expressing luciferase gene. Furthermore, the inclusion of phosphatidylcholine or phosphatidylethanolamine containing saturated and short dialkyl chains or unsaturated and long dialkyl chains into TC-1-12-based cationic liposomes resulted in high gene knockdown efficacy. When cationic liposomes composed of DDAB/1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), TC-1-12/DOPE and TC-1-12/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine were used, significant gene knockdown occurred in the lungs of mice following systemic injection of siRNA lipoplexes. Overall, the present findings indicated that optimal phospholipids in cationic liposome for in vitro and in vivo siRNA transfection were affected by the types of cationic lipid used.


Introduction
Small interfering (si)rna is a promising tool for inhibiting specific gene expression (1). Cationic liposomes have attracted attention for sirna delivery to cells (2,3) as sirna/cationic liposome complexes (siRNA lipoplexes) are efficiently delivered into cells and notably suppress target gene expression (2). To prepare cationic liposomes, liposomal formulations are used to combine cationic and neutral helper lipids, such as phospholipid or cholesterol (chol), to increase transfection efficiency and stability (4). However, the structures of cationic lipid and phospholipid, such as head group and alkyl chains, affect the transfection efficiency of siRNA lipoplexes (5,6). Therefore, discovering an optimal combination of cationic and neutral helper lipid for liposomal formulations is key for achieving efficient siRNA transfection.
To determine the effect of phospholipids in cationic liposomes on gene knockdown activity of sirna lipoplexes, three types of cationic lipid (doTaP, ddaB and Tc-1-12) were selected to prepare 30 types of cationic liposome composed of cationic lipid with phosphatidylcholine or phosphatidylethanolamine containing saturated or unsaturated dialkyl chains (c14, c16 or c18) and in vitro and in vivo gene knockdown efficacy was evaluated to find optimal phospholipids in cationic liposome for sirna transfection.
Preparation of cationic liposomes and siRNA lipoplexes. The cationic liposomes were prepared from cationic lipid and phospholipid at a molar ratio of 1:1. To prepare cationic liposomes, cationic lipid and phospholipid were dissolved in chloroform or chloroform/methanol (9:1, v/v). chloroform and methanol were evaporated under vacuum on a rotary evaporator at 60˚C to obtain a thin film. The thin film was hydrated with water at 60˚C by vortex mixing for 30 sec at 3,000 rpm and sonicated in a bath-type sonicator (Bransonic ® 2510J-MTH, 42 kHz; Branson UL Trasonics Co.) for 5-10 min at 60˚C.
To prepare sirna lipoplexes, liposome was added to 50 pmol sirna at a charge ratio (+:-) of 4:1, vortexed for 10 sec at 3,000 rpm and room temperature and left at room temperature for 15 min, as previously described (9,20). The charge ratio represents molar ratio of cationic lipid in cationic liposomes to sirna phosphate. The particle size, particle size distribution [polydispersity index (Pdi)] and ζ-potential of cationic liposomes and sirna lipoplexes were measured using a light-scattering photometer (cat. no. elS-Z2; otsuka electronics co., ltd.), as previously reported (21).
Free siRNA levels in siRNA lipoplexes. sirna lipoplexes were prepared at charge ratios (+:-) of 1:1-4:1. The amount of free sirna in sirna lipoplexes were measured using exclusion assay with SYBr ® Green i nucleic acid Gel Stain (Takara Bio inc.) and calculated based on the standard curves of free sirna as previously reported (21).
Gene knockdown in vitro using siRNA lipoplexes. McF-7-luc cells were seeded in 6-well culture plates at a density of 3x10 5 cells/well at 37˚C. After 24 h, each siRNA lipoplex with 50 pmol cont sirna or luc sirna was diluted in 1 ml rPMi-1640 medium supplemented with 10% FBS and added to the cells (final concentration, 50 nM). At 48 h post-transfection, luciferase activity was measured as counts per second (cps)/µg protein using PicaGene MelioraStar-lT luminescence reagent (Toyo ink co. ltd.) and Bca reagent (Pierce™ BCA Protein Assay kit; Thermo Fisher Scientific, inc.) as reported previously (21). luciferase activity (%) was calculated relative to that of untransfected cells.
Biodistribution of siRNA following intravenous injection of siRNA lipoplexes into mice. ethical approval for this study was obtained from the institutional animal care and use committee of Hoshi university (approval no. P21-039). a total of 8 female BalB/c mice (weight, 18-20 g; age, 8 weeks; Sankyo Labo Service Corporation, Inc.) were housed at 24˚C and 55% humidity under 12/12-h light/dark cycle (lights on at 8:00 a.m.) with food and water ad libitum. sirna lipoplexes with 20 µg cy5-sirna were administered intravenously to mice via the lateral tail vein (n=1/sirna lipoplex). at 1 h post-injection of siRNA lipoplexes, mice were sacrificed via cervical dislocation; death was confirmed by cessation of heartbeat. Tissue (lung, heart, liver, spleen, and kidney) was analyzed by cy5 fluorescence imaging using nightoWl lB981 nc100 system (Berthold Technologies GmbH & co. KG), as previously described (21). The images were analyzed using indiGo2 software (version 2.0.1.0) provided with the in vivo imaging system (Berthold Technologies). Following fluorescence imaging, tissue samples were frozen on dry ice and sliced into 16 µm sections. The localization of Cy5-siRNA was examined using a fluorescent microscope (Eclipse TS100-F; Nikon Corporation) with optical filter Cy5 HQ (excitation, 620/60 nm; dichroic mirror, 660 nm; emission, 700/75 nm; nikon corporation).
Agglutination assay. erythrocyte suspension was prepared from whole blood of female BalB/c mice (age, 8 weeks; Sankyo labo Service corporation, inc.), as previously described (13). sirna lipoplexes with 2 µg sirna were added to 100 µl 2% (v/v) erythrocyte suspension. Following incubation for 15 min at 37˚C, the sample was placed on a glass plate and agglutination was observed with light microscope at 100x magnification.
Expression of Tie2 mRNA in the lung following systemic injection of siRNA lipoplexes. sirna lipoplexes with 20 µg cont 2 or Tie2 sirna were administered intravenously to 8-week-old female BalB/c mice via the lateral tail veins (n=3-4/sirna lipoplex). no sirna lipoplexes caused mouse death following systemic injection. The lung was excised at 48 h post-injection of sirna lipoplexes and total rna was isolated using isogen ii (nippon Gene co., ltd.). cdna was synthesized from total rna using PrimeScript™ rT Master Mix (Takara Bio inc.) according to the manufacturer's protocol and quantitative Pcr was performed using a roche light cycler 96 system with FastStart essential dna Probes Master (roche diagnostics GmbH) and TaqMan Gene expression assays [Tie2, cat. no. Mm00443243_m1 and phosphatase and tensin homolog (PTen), cat. no. Mm00477208_m1; both applied Biosystems; Thermo Fisher Scientific, Inc.; primer sequences not available]. The thermocycling conditions were as follows: Initial denaturation at 95˚C for 600 sec, followed by 45 cycles of denaturation at 95˚C for 10 sec and primer annealing and extension at 60˚C for 30 sec (two-step amplification). The expression levels of Tie2 mrna were normalized to PTen in each sample as reported previously (19) and analyzed using the comparative cq (2 -ΔΔcq ) method (22).
Statistical analysis. data are presented as the mean + Sd of triplicate assessments. The statistical significance was determined by unpaired Student's t test or one-way anoVa followed by Tukey's post hoc test using GraphPad Prism version 4.0 (GraphPad Software, inc.). P<0.05 was considered to indicate a statistically significant difference.

Characterization of cationic liposomes and siRNA lipoplexes.
The present study aimed to determine whether alkyl chain length, saturation and the head group of phospholipids in cationic liposomes affect gene knockdown efficacy following treatment with sirna lipoplexes. doTaP and ddaB were used as dialkyl cationic lipids, Tc-1-12 was used as a trialkyl cationic lipid and dSPe, dPPe, dMPe, PoPe, doPe, dSPc, dPPc, dMPc, PoPc and doPc were used as phospholipids ( Fig. 1). cationic liposomes were prepared from cationic lipid/phospholipid at a molar ratio of 1:1 (Tables i-iii).
Binding of siRNA to cationic liposomes. The binding of sirna to each cationic liposome was assessed by exclusion assay with SYBr Green i. The addition of cationic liposomes to sirna beyond charge ratios of 2:1-3:1 was found to markedly decrease the fluorescence of SYBR ® Green i in the ddaB-, doTaP-, and Tc-1-12-based liposomes (Fig. 2). These results suggested that most ddaB-, doTaP-and Tc-1-12-based cationic liposomes exhibited greatest binding to sirna at a charge ratio of 4:1.

Discussion
Phosphatidylcholine and phosphatidylethanolamine are phospholipids in the cell membrane of most prokaryotes that are used as neutral helper lipids to prepare liposomes (15). cationic liposomes composed of dialkyl cationic lipids with neutral helper lipids, such as doPe, have been evaluated as carriers for sirna delivery (5,6). The present study determined the effect of phospholipids in cationic liposomes on gene knockdown using sirna lipoplexes. Following in vitro transfection, inclusion of phosphatidylcholines in doTaP or ddaB-based cationic liposomes did not induce high gene knockdown by sirna lipoplexes, although lP-ddaB/doPc lipoplexes exhibited moderate gene knockdown activity. This may be because phosphatidylcholine has a larger head group than phosphatidylethanolamine (15). du et al (27) reported that doPc promotes stable laminar structure, limiting escape of cationic liposomes composed of doTaP and doPc from the endosome. However, doPe promotes formation of inverted hexagonal lipid structures and cationic liposomes composed of DOTAP and DOPE exhibit improved transfection efficiency by destabilizing the endosomal membrane compared with those composed of DOTAP and DOPC (27). Such finding suggests that difference in headgroup structure between phosphatidylethanolamine and phosphatidylcholine may affect  Phospholipid  ddaB  doTaP  Tc-1-12 dSPe   In vivo, inclusion of phosphatidylethanolamine containing unsaturated and long dialkyl chains into ddaB or Tc-1-12based liposomes induced significant gene knockdown in the lung. Here, combination of doTaP with phospholipid did not induce high in vivo gene knockdown. However, inclusion of chol in doTaP-based cationic liposomes was previously reported to increase gene knockdown in the lung (6), indicating that combination of doTaP and chol may be suitable for in vivo transfection. By contrast, ddaB-based cationic liposomes showed high in vivo gene knockdown efficacy when ddaB was combined with doPe. our recent study reported that cationic liposomes composed of ddaB and chol induce high gene knockdown efficiency of Tie2 mRNA (>70%) when lipoplexes with 20 µg Tie2 sirna are systemically injected into mice (13). doTaP has unsaturated dialkyl chains (Tm, -12˚C) (30) while DDAB has saturated dialkyl chains (Tm, 43˚C) (31), indicating that combination of DOTAP and phospholipid may be unstable in blood circulation (37˚C) following systemic injection, resulting in poor gene knockdown. although Tm value of Tc-1-12 is unknown, it was hypothesized that this value is low because of the short alkyl chains. nonetheless, Tc-1-12-based liposomes induced significant gene knockdown when combined with doPe or PoPe. it is unclear why Tc-1-12 exhibited high in vivo transfection efficiency when combined with phosphatidylethanolamine containing unsaturated and long dialkyl chains. Tc-1-12 may affect liposome membrane stability via its trialkyl chains (29). Further study is needed to evaluate the mechanism underlying in vivo gene knockdown by Tc-1-12-based cationic liposomes. among the present liposomal formulations, lP-ddaB/doPe, lP-Tc-1-12/doPe and lP-Tc-1-12/PoPe are potential vectors for delivering sirna into the lung. The present study evaluated mrna levels in the lung following injection of sirna lipoplexes; however, future experiments should also evaluate changes of protein levels in the lung.