**1. Introduction**

RNA interference (RNAi) is a powerful gene-silencing process that holds great promise in the field of cancer therapy. Small double-stranded RNAs, i.e., synthetic small interfering RNAs (siRNAs) suppress the expression of a target gene by triggering specific degradation of the complementary mRNA sequence [1], and therefore siRNA therapeutics have become an increasingly important strategy for anticancer therapy [2]. For siRNA therapy against tumors, siRNAs are designed for targeting mRNAs transcribed from a tumor-causing gene, and siRNAs are introduced into the cytoplasm of tumor cells to cause cleavage of the target mRNA. For example, polo-like kinase 1 (PLK1) is a key regulator for cell mitosis, and its expression is elevated in many types of human tumors [3,4]. The inhibition of PLK1 expression by PLK1 siRNA can lead to death of tumor cells with multiple stages of mitosis [5], and therefore PLK1 is expected to be one of potential targets for siRNA therapy against tumors. However, the success of siRNA therapy relies on the development of safe and efficacious delivery systems that can introduce siRNAs into target tumor cells [2,6,7].

For effective transfection of siRNAs into tumor cells, siRNA carriers such as cationic liposomes are currently the most widely validated means [8]. Many different cationic lipids have been synthesized for lipid-based gene delivery [9] and shown activity in delivering siRNA into cells [10]. For siRNA delivery by cationic liposomes, cationic cholesterol derivatives have often been used [11]. Recently, we reported that cationic liposomes composed of cholesteryl (3-((2-hydroxyethyl)amino)propyl)carbamate hydroiodide (HAPC-Chol), *N*-(2-(2-hydroxyethylamino)ethyl)cholesteryl-3-carboxamide (OH-Chol), or cholesteryl (2-((2-hydroxyethyl)amino)ethyl)carbamate (OH-C-Chol) could efficiently suppress the expression of target genes by siRNA in cells [12,13]. Regarding the liposomal formulation, we previously reported that cationic liposomes composed of OH-Chol/DOPE and OH-C-Chol/DOPE exhibited high gene silencing efficacies compared with cationic nanoparticles composed of OH-Chol/Tween80 and OH-C-Chol/Tween80, respectively [14]. DOPE is thought to improve transfection efficiency by destabilizing the endosomal membrane [15,16], thereby facilitating the release of siRNAs into the cytoplasm. Therefore, a combination of cationic cholesterol derivative with DOPE in cationic liposomes might be a suitable formulation in siRNA delivery. However, selective delivery of siRNAs into tumor cells by cationic liposomes must be achieved for clinical applications.

Folate receptors (FRs) have been found to be overexpressed in a wide range of tumors, including ovary, uterus, lung, kidney, breast, colon, prostate, and brain cancers [17–19]. The following four isoforms of FR have been identified: folate receptor alpha (FR-α), beta (FR-β), delta (FR-Δ), and gamma (FR-γ); and FR-α and FR-β are attached to the cell by a GPI-anchor [18]. Among these FR isoforms, FR-α is the most widely studied as a biomarker for tumors, because a few normal tissues have been found to express FR-α, although most express the protein at much lower levels than are detected in FR-α-positive carcinoma. Therefore, FR-mediated tumor targeting has emerged as an attractive method of active targeting of siRNAs into tumor cells by cationic liposomes. When folic acid (FA) or its conjugates bind to FRs, they are taken up into the cells via receptor-mediated endocytosis. Therefore, FA-polyethylene glycol (PEG)-modification has been employed in cationic liposomes to facilitate the uptake of siRNA lipoplexes into tumor cells. Generally, PEGylated cationic liposomes can significantly reduce nonspecific gene transfer. However, conjugation of folate to the PEG chain can restore the cellular association with FR-positive tumors. To the best of our knowledge, there have been no reports about the effect of cationic lipids in FA-PEG-modified cationic liposomes on FR-targeting, although several studies have investigated the application of FA-PEG-modified cationic liposomes for siRNA delivery into FR-expressing cells [20–23]. In this study, to examine the effect of cationic lipid type in FA-PEG-modified cationic liposomes on gene-silencing effects, we selected three kinds of cationic cholesterol derivatives, HAPC-Chol, OH-Chol, and OH-C-Chol, and prepared three types of FA-PEG-modified cationic liposomes composed of cationic cholesterol derivatives and DOPE for the evaluation of gene-silencing effects. Here, we found that in FR-selective siRNA delivery, the cationic lipid type in FA-PEG-modified cationic liposomes affected an optimal amount of FA-PEG2000-DSPE in liposomal formulation, and the optimized formulation of FA-PEG-modified cationic liposomes by in vitro transfection was not necessarily correlated with formulation by in vivo transfection.

#### **2. Materials and Methods**

#### *2.1. Materials*

*N*-(2-(2-Hydroxyethylamino)ethyl)cholesteryl-3-carboxamide (OH-Chol) and cholesteryl (2-((2 hydroxyethyl)amino)ethyl)carbamate (OH-C-Chol) were synthesized as described previously [14,24,25]. Cholesteryl (3-((2-hydroxyethyl)amino)propyl)carbamate hydroiodide (HAPC-Chol) was synthesized as described previously [26]. Methoxy-poly(ethyleneglycol)-distearylphosphatidylethanolamine (PEG2000- DSPE/SUNBRIGHT DSPE-020CN, and PEG5000-DSPE/SUNBRIGHT DSPE-050CN, and PEG mean molecular weight, 2000 and 5000, respectively) and 1,2-dioleoyl-*sn*-glycero-3-phosphoethanolamine

(DOPE, COATSOME ME-8181) were obtained from NOF Co. Ltd. (Tokyo, Japan). All other chemicals were of the highest grade available.

#### *2.2. Small Interfering RNAs*

The siRNAs targeting nucleotides of firefly pGL4 luciferase (Luc), enhanced green fluorescent protein (EGFP), human polo-like kinase 1 (PLK1), and non-silencing siRNA control (Cont) as a negative control were synthesized by Sigma Genosys (Tokyo, Japan). Alexa Fluor®488-labeled AllStars Negative Control siRNA (AF-siRNA) was obtained from Qiagen (Valencia, CA, USA). The siRNA sequences for Luc siRNA: sense strand: 5 -GGACGAGGACGAGCACUUCUU-3 and antisense strand: 5 -GAAGUGCUCGUCCUCGUCCUU-3. The siRNA sequences for EGFP siRNA: sense strand: 5 -GGCUACGUCCAGGAGCGCACCTT-3 and antisense strand: 5 -GGUGCGCUCC UGGACGUAGCCTT-3. The siRNA sequences of the PLK1 siRNA were as reported previously [27]. The siRNA sequences of the Cont siRNA were as reported previously [28].

## *2.3. Preparation of Cationic Liposomes and siRNA Lipoplexes*

FA-PEG-distearoylphosphatidylethanolamine (FA-PEG-DSPE) (mean MW of PEG: 2000 and 5000) was synthesized as described previously [29]. Cationic liposomes were prepared from HAPC-Chol/ DOPE (composition designated as LP-HAPC), OH-Chol/DOPE (composition designated as LP-OH), and OH-C-Chol/DOPE (composition designated as LP-OH-C), at a molar ratio of 3:2. PEG2000 and FA-PEG2000-modified cationic liposomes were incorporated at 1, 2, or 3 mol% PEG2000-DSPE and FA-PEG2000-DSPE, respectively, into the cationic liposomal formulation, as shown in Table 1. PEG5000- and FA-PEG5000-modified LP-HAPC (LP-HAPC-PEG5000 and LP-HAPC-FA-PEG5000) were incorporated with 1 mol % PEG5000-DSPE and FA-PEG5000-DSPE, respectively, into the formulation of LP-HAPC.

For the preparation of cationic liposomes by a thin-film hydration method, cationic cholesterol derivative, DOPE, and PEG-DSPE or FA-PEG-DSPE were dissolved in chloroform, and then the chloroform was 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. The liposomes were sonicated in a bath-type sonicator (Bransonic® 2510J-MTH, 100W, Branson UL Trasonics Co., Danbury, CT, USA) to produce liposomes with a final size of approximately 100 nm.

In in vitro transfection, to prepare cationic liposome/siRNA complexes (siRNA lipoplexes), each cationic liposome was added to 50 pmol siRNA at a charge ratio (+:−) of 7:1 with vortex mixing for 10 s and left at room temperature for 15 min. The charge ratio (+:−) of cationic liposome:siRNA is expressed as the molar ratio of cationic lipid to siRNA phosphate.



#### *2.4. Size and* ζ*-Potential of Cationic Liposomes and siRNA Lipoplexes*

The siRNA lipoplexes were formed by addition of cationic liposomes to 5 μg Cont siRNA at a charge ratio (+:−) of 7:1 with vortex mixing for 10 s. The particle size distributions of cationic liposomes and siRNA 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 to an appropriate volume (~1.5 mL) with water. The ζ-potentials of cationic liposomes and siRNA lipoplexes were measured by electrophoresis light-scattering methods using ELS-Z2 at 25 ◦C after diluting the dispersion with an appropriate volume (~1.5 mL) with water.

#### *2.5. Cell Culture*

Human nasopharyngeal tumor KB cells (also known as a subline of human cervix adenocarcinoma HeLa) were obtained from the Cell Resource Center for Biomedical Research, Tohoku University (Miyagi, Japan). KB cells were cultured in RPMI-1640 medium with 10% heat-inactivated fetal bovine serum (FBS) and 100 μg/mL kanamycin in a humidified atmosphere containing 5% CO2 at 37 ◦C.

For the preparation of KB cells stably expressing firefly pGL4 luciferase, KB cells were plated on 35-mm culture dishes. Twenty-four hours later, the cells were transfected with 2 μg of plasmid DNA encoding firefly pGL4 luciferase under the control of a cytomegalovirus (CMV) promoter (pGL4.51[luc2/CMV/Neo] Vector, Promega, Madison, WI, USA) using Lipofectamine 2000 transfection reagent (Invitrogen, Thermo Fisher Scientific, Inc., Carlsbad, CA, USA). The transfected cells were selected in medium with 1200 μg/mL G418 sulfate. G418-resistant colonies were subcultured and established as a permanent cell line expressing firefly pGL4 luciferase (KB-Luc).

For the preparation of KB cells stably expressing EGFP, KB cells were transfected with 2 μg of plasmid DNA encoding EGFP under the control of a CMV promoter (pEGFP-C1, Clontech, Palo Alto, CA, USA) using Lipofectamine 2000 transfection reagent. The transfected cells were selected in medium with 1 mg/mL G418 sulfate. G418-resistant colonies were subcultured and established as a permanent cell line expressing EGFP (KB-EGFP).

#### *2.6. Gene Silencing E*ff*ect by FA-PEG-Modified siRNA Lipoplexes in KB-Luc Cells*

The KB-Luc cells were seeded in 6-well culture plate at a density of 3 <sup>×</sup> 105 cells per well, 24 h prior to transfection. The siRNA lipoplexes were formed by the addition of cationic liposomes into 50 pmol Cont siRNA or Luc siRNA with vortex mixing for 10 s and left at room temperature for 15 min. For siRNA transfection, each siRNA lipoplex was diluted in 1 mL of folate-deficient RPMI-1640 medium (Invitrogen) supplemented with 10% FBS and then the mixture was added to the cells (final 50 nM siRNA concentration). Lipofectamine RNAiMAX lipoplexes (Invitrogen) were prepared in accordance with the manufacturer's protocol and then added to the cells. Forty-eight hours after the transfection, luciferase activity was measured as counts per sec (cps)/μg protein using the luciferase assay system (PicaGene MelioraStar-LT Luminescence Reagent, Toyo Ink Mfg. Co. Ltd., Tokyo, Japan) and BCA reagent (Pierce™ BCA Protein Assay Kit, Pierce, Rockford, IL, USA). Luciferase activity (%) was calculated as relative to the luciferase activity (cps/μg protein) of untransfected cells.

#### *2.7. Gene Silencing E*ff*ect by FA-PEG-Modified siRNA Lipoplexes in KB-EGFP Cells*

The KB-EGFP cells were seeded in 6-well culture plate at a density of 3 <sup>×</sup> 105 cells per well, 24 h prior to transfection. The siRNA lipoplexes were formed by addition of cationic liposomes into 50 pmol Cont siRNA or EGFP siRNA with vortex mixing for 10 s and left at room temperature for 15 min. For siRNA transfection, each siRNA lipoplex was diluted in 1 mL of folate-deficient RPMI-1640 medium supplemented with 10% FBS and then the mixture was added to the cells (final 50 nM siRNA concentration). Lipofectamine RNAiMAX lipoplexes were prepared in accordance with the manufacturer's protocol and then added to the cells. Forty-eight hours after transfection, EGFP expression levels in the cells were determined by examining fluorescence intensity using a

FACSVerseTM flow cytometer (Becton Dickinson, San Jose, CA, USA) equipped with a 488-nm argon ion laser. Data for 10,000 fluorescence events were obtained by recording forward scatter (FSC), side scatter (SSC), and green (530/30 nm) fluorescence. EGFP expression level (%) was calculated as relative to the fluorescence intensity of untransfected cells.
