*2.12. Antiproliferative Activity*

The KB cells were seeded in 96-well culture plate, 24 h prior to transfection. Each siRNA lipoplex with 50 pmol Cont siRNA or PLK1 siRNA was diluted in 1 mL of folate-deficient RPMI-1640 medium supplemented with 10% FBS, and then the mixture (100 μL) was added to the cells at 20–30% confluency in the well (final 50 nM siRNA concentration). After the 48 h incubation period, cell viability was measured by a WST-8 assay as described above.

### *2.13. Measurement of Expression Level of PLK1 mRNA*

For the knockdown of PLK1 mRNA by transfection with PLK1 siRNA, KB cells were plated into 6-well culture plate at a density of 3 <sup>×</sup> <sup>10</sup><sup>5</sup> cells/well. The siRNA lipoplexes with 50 pmol Cont siRNA or PLK1 siRNA were diluted in 1 mL of folate-deficient RPMI-1640 medium (50 nM siRNA) supplemented with 10% FBS and then added to the cells. At 24 h after transfection, total RNA was isolated using NucleoSpin RNA (Macherey-Nagel, GmbH, Düren, Germany). First strand cDNA was synthesized from 1 μg of total RNA using PrimeScript RTase (Takara Bio, Inc., Otsu, Japan). Quantitative (q)PCR was performed using a Roche Light Cycler 96 system (Roche Diagnostics, Basel, Switzerland) and TaqMan Gene expression assays (PLK1, Hs00983227\_m1 and GAPDH, Hs02786624\_g1, Applied Biosystems, Thermo Fisher Scientific Inc.). Samples were run in triplicate, and the expression levels of PLK1 mRNA were normalized by the amount of GAPDH mRNA in the same sample, and analyzed using the comparative Cq (2−ΔΔCq) method [31].

#### *2.14. In Vivo Anti-Tumor E*ff*ect*

All animal experiments were conducted in accordance with the "Guide for the Care and Use of Laboratory Animals" published by the U.S. National Institutes of Health and the "Guide for the Care and Use of Laboratory Animals" adopted by the Institutional Animal Care and the Use Committee of Hoshi University (Tokyo, Japan) (which is accredited by the Ministry of Education, Culture, Sports, Science, and Technology, Japan). Ethical approval for this study was obtained from the Institutional Animal Care and the Use Committee of Hoshi University (permission number: 30-074).

To generate KB tumor xenografts, 1 <sup>×</sup> 107 cells suspended in 50 <sup>μ</sup>L of PBS were inoculated subcutaneously in the flank region of female BALB/c nu/nu mice (8 weeks of age, CLEA Japan Inc., Tokyo, Japan). The tumor volume was calculated using the formula, tumor volume = 0.5 <sup>×</sup> a <sup>×</sup> b2, where a and b are the larger and smaller diameters, respectively. When the average volume of the xenograft tumors reached 80 mm3 (day 0), LP-HAPC-2mol%PEG2000 or LP-HAPC-2mol%FA-PEG2000 lipoplexes of 10 μg of Cont siRNA or PLK1 siRNA per tumor were directly injected into xenografts on days 0, 2, and 4. Tumor volume was measured on days 0, 2, 4, 6, and 8. Tumor volume (%) was calculated as relative to each tumor volume at the day 0. At day 8, mice were sacrificed by cervical dislocation, and then the excised tumors were weighed.

#### *2.15. Statistical Analysis*

The statistical significance of differences between mean values was determined using Student's *t*-test. A *p*-value of 0.05 or less was considered significant.

#### **3. Results**

#### *3.1. Characterization of FA-PEG-Modified Cationic Liposomes and siRNA Lipoplexes*

Firstly, we examined whether the cationic lipid type in FA-PEG-modified cationic liposomes affected gene silencing after transfection of FA-PEG-modified siRNA lipoplexes into KB cells, which overexpressed FR-α [32]. Here, we used HAPC-Chol, OH-Chol, and OH-C-Chol (Figure 1) as cationic cholesterol derivatives for preparation of cationic liposomes. LP-HAPC, LP-OH, and LP-OH-C were prepared from HAPC-Chol/DOPE, OH-Chol/DOPE, and OH-C-Chol/DOPE, respectively, at a molar ratio of 3:2 (Table 1). For PEGylated cationic liposomes, 1, 2, or 3 mol% PEG2000-DSPE was added to the formulation of LP-HAPC, LP-OH, and LP-OH-C (LP-HAPC-1–3mol%PEG2000, LP-OH-1– 3mol%PEG2000, and LP-OH-C-1–3mol%PEG2000, respectively). For FR-targeted cationic liposomes, 1, 2, or 3 mol% FA-PEG2000-DSPE was added to the formulation of LP-HAPC, LP-OH, and LP-OH-C (LP-HAPC-1–3mol%FA-PEG2000, LP-OH-1–3mol%FA-PEG2000, and LP-OH-C-1–3mol%FA-PEG2000, respectively). In addition, as PEG- or FA-PEG-modified cationic liposomes with a longer PEG chain, 1 mol% PEG5000-DSPE and FA-PEG5000-DSPE, respectively, were added to the formulation of LP-HAPC (LP-HAPC-1mol%PEG5000, and LP-HAPC-1mol%FA-PEG5000, respectively).

In HAPC-Chol-based liposomes, the sizes of LP-HAPC, LP-HAPC-1–3mol%PEG2000, LP-HAPC-1–3mol%FA-PEG2000, LP-HAPC-1mol%PEG5000, and LP-HAPC-1mol%FA-PEG5000 were approximately 90–110 nm, polydispersity index (PDI) 0.21–0.26, and the ζ-potentials were approximately +35–47 mV (Table 2). In OH-Chol-based liposomes, the sizes of LP-OH, LP-OH-1– 3mol%PEG2000, and LP-OH-1–3mol%FA-PEG2000 were approximately 84–110 nm (PDI 0.17–0.31), and the ζ-potentials were approximately +34–47 mV (Table 2). In OH-C-Chol-based liposomes, the sizes of LP-OH-C, LP-OH-C-1–3mol%PEG2000 and LP-OH-C-1–3mol%FA-PEG2000 were approximately 90–110 nm (PDI 0.12–0.26), and the ζ-potentials were approximately +43–52 mV (Table 2).

**Figure 1.** Structure of cationic cholesterol derivatives, neutral helper lipid, and FA-PEG-DSPE: HAPC-Chol; cholesteryl (3-((2-hydroxyethyl)amino)propyl)carbamate hydroiodide, OH-Chol; *N*-(2-(2-hydroxyethylamino)ethyl)cholesteryl-3-carboxamide, OH-C-Chol; cholesteryl (2-((2 hydroxyethyl)amino)ethyl)carbamate, DOPE; 1,2-dioleoyl-*sn*-glycero-3-phosphoethanolamine, FA-PEG- DSPE; FA-PEG-distearoylphosphatidylethanolamine.



*Pharmaceutics* **2019** , *11*, 181

For the preparation of siRNA lipoplexes, their liposomes were mixed with siRNA at a charge ratio (+:−) of 7:1 as reported previously [13], because siRNA lipoplexes at this charge ratio (+:−) exhibited high gene silencing efficacy (Supplemental Figure S1). In HAPC-Chol-based lipoplexes, the sizes of LP-HAPC, LP-HAPC-1–3mol%PEG2000, LP-HAPC-1–3mol%FA-PEG2000, LP-HAPC-1mol%PEG5000, and LP-HAPC-1mol%FA-PEG5000 lipoplexes were approximately 170–210 nm (PDI 0.24–0.28), and the ζ-potentials were approximately 21–40 mV (Table 2). In OH-Chol-based lipoplexes, the sizes of LP-OH, LP-OH-1–3mol%PEG2000, and LP-OH-1–3mol%FA-PEG2000 lipoplexes were approximately 170–300 nm (PDI 0.22–0.28), and the ζ-potentials were approximately 21–45 mV (Table 2). In OH-C-Chol-based lipoplexes, the sizes of LP-OH-C, LP-OH-C-1–3mol%PEG2000, and LP-OH-C-1–3mol%FA-PEG2000 lipoplexes were approximately 170–420 nm (PDI 0.12–0.26), and the ζ-potentials were approximately 22–39 mV (Table 2). Regardless the cationic lipid type in cationic liposomes, PEG-modification, or FA-PEG-modification of cationic liposomes trended to decrease the ζ-potentials of cationic liposomes and siRNA lipoplexes. However, the type of cationic cholesterol derivatives in cationic liposomes did not largely affect size and ζ-potential of cationic liposomes and siRNA lipoplexes.

#### *3.2. E*ff*ect of Cationic Lipid of FA-PEG-Modified Cationic Liposomes on In Vitro Gene Knockdown E*ffi*cacy*

To examine the gene silencing effect in FR-expressing cells by FA-PEG-modified siRNA lipoplexes, KB-Luc cells were incubated with siRNA lipoplexes modified with 1–3 mol% PEG2000-DSPE or FA-PEG2000-DSPE, and the gene silencing activity was assessed by assaying luciferase activity. LP-HAPC, LP-OH, and LP-OH-C lipoplexes with Luc siRNA strongly suppressed luciferase activity (>80% knockdown, compared with Cont siRNA) (Figure 2A,C,D). In HAPC-Chol- and OH-C-Chol-based liposomes, above 2 mol% PEG-modification of LP-HAPC and LP-OH-C lipoplexes with PEG2000-DSPE completely abolished the gene silencing effect; however, LP-HAPC-2mol%FA-PEG2000 and LP-OH-C-2mol%FA-PEG2000 lipoplexes with Luc siRNA exhibited strong suppression of luciferase activity (Figure 2A,D). In contrast, in OH-Chol-based liposomes, above 1 mol% PEG-modification of LP-OH lipoplexes with PEG2000-DSPE abolished the gene silencing effect; however, LP-OH-1mol%FA-PEG2000 lipoplexes with Luc siRNA exhibited strong suppression of luciferase activity (Figure 2C). From these results, in FR-mediated gene silencing, the optimal amount of PEG2000-DSPE or FA-PEG2000-DSPE in the liposomal formulation may be affected by the cationic lipid type in FA-PEG-modified liposomes. Regarding the length of the PEG chain between FA and lipid, both LP-HAPC-1mol%PEG5000 and LP-HAPC-1mol%FA-PEG5000 lipoplexes did not exhibit gene silencing activity (Figure 2B), indicating that a longer PEG chain inhibited FR-mediated uptake by cells. From these results, optimal modifications of PEG2000-DSPE or FA-PEG2000-DSPE in formulations of LP-HAPC, LP-OH, and LP-OH-C were 2 mol%, 1 mol%, and 2 mol%, respectively, for selective FR-mediated gene silencing in tumor cells.

**Figure 2.** Effect of FA-PEG modification of cationic liposomes on suppression of luciferase expression in KB-Luc cells after transfection with FA-PEG-modified siRNA lipoplexes. (**A**) LP-HAPC-1–3mol%PEG2000 and LP-HAPC-1–3mol%FA-PEG2000, (**B**) LP-HAPC-1mol%PEG5000 and LP-HAPC-1mol%FA-PEG5000, (**C**) LP-OH-1–3mol%PEG2000 and LP-OH-1–3mol%FA-PEG2000, (**D**) LP-OH-C-1–3mol%PEG2000 and LP-OH-C-1–3mol%FA-PEG2000 were used. The siRNA lipoplexes with Cont siRNA or Luc siRNA were added to KB-Luc cells at 50 nM siRNA, and the luciferase assay was carried out 48 h after incubation. Each column represents the mean + SD (*n* = 3). \*\* *p* < 0.01, compared with Cont siRNA.

#### *3.3. Cytotoxicity by FA-PEG-Modified siRNA Lipoplexes*

To examine whether FA-PEG-modification of cationic liposomes affected the cytotoxicity, we investigated cell viability at 24 h after transfection into KB cells with FA-PEG-modified siRNA lipoplexes. LP-HAPC, LP-OH, and LP-OH-C lipoplexes did not exhibit marked cytotoxicity (80–90% cell viability), and PEG-modification of their lipoplexes also did not affect cytotoxicity (approximately 90% cell viability) (Figure 3). However, FA-PEG-modification of LP-HAPC, LP-OH, and LP-OH-C slightly increased cytotoxicity (70–80% cell viability) with increasing the amounts of FA-PEG2000-DSPE in the cationic liposomes, compared with the PEG-modification. These results indicated that

FA-PEG-modification of cationic liposomes might increase cytotoxicity by increasing the cellular uptake of siRNA lipoplexes.

**Figure 3.** Effect of FA-PEG-modification of cationic liposomes on cell viability 24 h after transfection with FA-PEG-modified siRNA lipoplexes into KB-Luc cells. (**A**) LP-HAPC-1–3mol%PEG2000 and LP-HAPC-1–3mol%FA-PEG2000, (**B**) LP-OH-1–3mol%PEG2000 and LP-OH-1–3mol%FA-PEG2000, (**C**) LP-OH-C-1–3mol%PEG2000 and LP-OH-C-1–3mol%FA-PEG2000 were used. The siRNA lipoplexes were added to KB cells at 50 nM siRNA. Each column represents the mean + SD (*n* = 4). Each column represents the mean + SD (*n* = 3). \* *p* < 0.05, \*\* *p* < 0.01, compared with PEG2000-DSPE.
