Folate-PEG-Appended Dendrimer Conjugate with α-Cyclodextrin as a

Jul 26, 2012 - Department of Physical Pharmaceutics, Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamo...
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Folate-PEG-Appended Dendrimer Conjugate with α‑Cyclodextrin as a Novel Cancer Cell-Selective siRNA Delivery Carrier Hidetoshi Arima,*,† Ayumi Yoshimatsu,† Haruna Ikeda,† Ayumu Ohyama,† Keiichi Motoyama,† Taishi Higashi,† Akira Tsuchiya,‡ Takuro Niidome,‡ Yoshiki Katayama,‡ Kenjiro Hattori,§ and Tomoko Takeuchi§ †

Department of Physical Pharmaceutics, Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, Japan ‡ Department of Applied Chemistry, Faculty of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan § Department of Applied Chemistry, Faculty of Engineering, Tokyo Institute of Polytechnics, 1583 Iiyama, Atsugi 243-0297, Japan S Supporting Information *

ABSTRACT: We previously reported that of the various polyamidoamine (PAMAM) STARBURST dendrimer (generation 3, G3) (dendrimer) conjugates with cyclodextrins (CyDs), the dendrimer (G3) conjugate with α-CyD having an average degree of substitution of 2.4 (α-CDE (G3)) has the greatest potential for a novel carrier for siRNA in vitro and in vivo. To improve the siRNA transfer activity and the lack of target specificity of α-CDE (G3), we prepared folate-polyethylene glycol (PEG)-appended α-CDEs (G3) (Fol-PαCs) with various degrees of substitution of folate (DSF) and evaluated their siRNA transfer activity to folate receptor (FR)-overexpressing cancer cells in vitro and in vivo. Of the three Fol-PαCs (G3, DSF 2, 4 and 7), Fol-PαC (G3, DSF 4) had the highest siRNA transfer activity in KB cells (FR-positive). Fol-PαC (G3, DSF 4) was endocytosed into KB cells through FR. No cytotoxicity of the siRNA complex with Fol-PαC (G3, DSF 4) was observed in KB cells (FR-positive) or A549 cells (FR-negative) up to the charge ratio of 100/1 (carrier/siRNA). In addition, the siRNA complex with Fol-PαC (G3, DSF 4) showed neither interferon response nor inflammatory response. Importantly, the siRNA complex with Fol-PαC (G3, DSF 4) tended to show the in vivo RNAi effects after intratumoral injection and intravenous injection in tumor cells-bearing mice. The FITC-labeled siRNA and TRITC-labeled Fol-PαC (G3, DSF 4) were actually accumulated in tumor tissues after intravenous injection in the mice. In conclusion, the present results suggest that Fol-PαC (G3, DSF 4) could potentially be used as a FR-overexpressing cancer cell-selective siRNA delivery carrier in vitro and in vivo. KEYWORDS: PAMAM dendrimer, α-cyclodextrin, folate, PEG, conjugate, siRNA

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INTRODUCTION RNA interference (RNAi) is mRNA degradation mediated by double stranded RNA molecules (small interfering RNAs, siRNAs) 21−27 nucleotides in length, which are intracellularly generated from long endogenous or exogenous doublestranded RNAs (dsRNAs) or directly transfected into cells.1,2 Powerful tools for a gene function study and RNAi therapy are emerging as the most highly effective strategy. In fact, some successful reports on the therapy for intractable disease such as cancer and virus infection have been published.3−6 However, the efficient and safe siRNA delivery systems are required to achieve the desired RNAi effect. The siRNA delivery carriers can be classified into viral and nonviral vectors, and the latter has been widely used due to easy preparation of carrier/nucleic acid complexes, low cytotoxicity, and lack of immunogenicity. Generally, chemically unmodified siRNAs are rapidly degraded in serum, and it is difficult for them to enter mammalian cells. Thus, the strategies to deliver siRNA to target cells include physical or chemical transfection.7,8 © 2012 American Chemical Society

Chemical modification by tumor targeting ligands is wellknown to give an active targeting-ability to drug carriers, for example, antibody,9 sugar,10 folic acid (FA),11,12 transferrin,13,14 epidermal growth factor,15 and Arg-Gly-Asp-Ala-Pro-Arg-ProGly peptide.16 Of these ligands, FA is widely used because of its several advantages;17,18 that is, (1) folate receptor (FR) is upregulated in many human tumor cells, including malignancies of the ovary, brain, kidney, breast, myeloid cells, and lung, (2) FA has a potent binding affinity to FR (Kd ∼10−10 M), (3) low immunogenicity, (4) low molecular weight (Mw 441.4), (5) compatibility with a variety of organic and aqueous solvents, and (6) low cost. We previously reported that the polyamidoamine (PAMAM) STARBURST dendrimer (dendrimer, generation 3, G3) Received: Revised: Accepted: Published: 2591

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conjugate with α-cyclodextrin (α-CyD) having an average degree of substitution of 2.4 (α-CDE (G3)) provided remarkable aspects as a gene delivery carrier.19 α-CDE (G3) has a number of advantages for gene delivery: (1) efficient gene transfer activity into mammalian cells and (2) extremely low cytotoxicity even at high charge ratios of α-CDE (G3)/plasmid DNA (pDNA). Therefore, these preferable properties of αCDE (G3) recommend it as a new candidate of carrier for siRNA transfer. In fact, we previously demonstrated that αCDE (G3) may be utilized as a novel carrier for siRNA transfer in the ternary system of pDNA/siRNA/carrier20 and the binary system of siRNA/carrier.21 In the subsequent study, to improve the siRNA delivery activity and target specificity of α-CDE (G3), we prepared folate-polyethylene glycol (PEG)-appended α-CDEs (Fol-PαCs (G3), Figure 1) with various degrees of substitution of folate (DSF) as novel carriers for siRNA transfer and first evaluated their complexation and physicochemical properties of the siRNA complex with carriers. Second, we examined the in vitro RNAi effects of the siRNA complexes with Fol-PαCs (G3) on luciferase gene to the FR-overexpressing cancer cells. Third, to gain insight into the mechanism for efficient siRNA transfer activity of Fol-PαC (G3) to tumor cells, cellular uptake and intracellular distribution of the siRNA complex with carriers were investigated. Fourth, we evaluated cytotoxicity of these siRNA complexes in vitro. Fifth, we investigated the in vivo RNAi effects after intratumoral and intravenous injections of the complex of siRNA with Fol-PαC (G3) in Colon-26 cellsbearing mice. Finally, we evaluated pharmacokinetics of fluorescein isothiocyanate (FITC)-labeled siRNA and tetramethylrhodamine isothiocyanate (TRITC)-labeled Fol-PαC (G3, DSF 4) after intravenous injection of their complex in the tumor cells-bearing mice.

CyD = 2.4).22 In brief, Fol-PαCs (G3) were prepared as follows: FA in dimethyl sulfoxide (DMSO) containing N,Ndicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS) were mixed at room temperature for 30 min. Then, PEG (MW = 2170) and pyridine were added into the solution and incubated at room temperature for 2 h. After removal of intact FA by a precipitation method with water and purification by gel filtration (TOSOH TSKGelHW-40S, Tokyo, Japan), Fol-PEG-COOH was activated with 0.2 M borate buffer (pH 9) containing EDC and NHS, and then mixed at room temperature for 2 h. Then, α-CDE (G3) was added into the solution and incubated for 48 h. Fol-PαCs (G3) were dialyzed by using a dialysis membrane and purified by gel-filtration and ethanol precipitation methods. To prepare TRITC-Fol-PαC (G3), Fol-PαC (G3) dissolved in sodium chloride (0.9% w/v) and TRITC dissolved in DMSO were added into the flask, and then the mixture was stirred at room temperature for 24 h. The resulting TRITC-Fol-PαC (G3) was purified by dialysis and then precipitated with methanol. Cell Culture. KB cells, a human carcinoma of the nasopharynx, were obtained from the Institute of Development, Aging and Cancer, Tohoku University (Sendai, Japan). A549 cells, a human lung carcinoma, were purchased from American Type Culture Collection (Rockville, MD). Colon-26 cells, a mouse colon adenocarcinoma cell line, were obtained from Riken Bioresource Center (Tsukuba, Japan). Colon-26 cells stably expressing luciferase gene (Colon-26-luc cells) were prepared by hygromycin selection following the transfection with the pGL3 complex with α-CDE (G3). KB cells and Colon26-luc cells were grown in a RPMI-1640 (FA-free) culture medium containing penicillin (1 × 105 mU/mL) and streptomycin (0.1 mg/mL) supplemented with 10% FCS at 37 °C in a humidified 5% CO2 and 95% air atmosphere. A549 cells were cultured as reported previously.23,24 In Vitro Transfection. The protocol described below is based on a published procedure.20 In the transfection system of siRNA, cells were transfected with the complexes of pDNA/ dendrimer (G3) and/or siRNA/carriers. The cells (5 × 104/ wells) were transfected with 200 μL of serum-free medium containing pDNA/dendrimer (G3) complex at 37 °C for 1 h. After being washed with serum-free medium, 120 μL of serumfree medium containing the complex of siRNA/carriers were added to each well and then incubated at 37 °C for 1 h. Thirty microliters of FCS were added to each well (24 well) and then incubated at 37 °C for 21 h. The cell lysates were centrifuged, and then the resulting supernatant was assayed for firefly luciferase activity using a luciferase assay system (Promega, Tokyo, Japan) on a luminometer (Lumat LB9506, Tokyo, Japan) and expressed in relative light units (RLU). Protein concentrations were determined using a bicinchoninic acid assay (BCA protein assay kit, Pierce, IL). Cytotoxicity. The cell viability was assayed using a cell counting kit (WST-1 method) from Wako Pure Chemical Industries (Osaka, Japan).25 In brief, cells (5 × 104/well) were incubated for 1 h with 270 μL of serum-free medium containing the siRNA/carrier complex at the concentration of siRNA (100 nM). The 30 μL of FCS was added to each well, and the cells were further incubated at 37 °C for 23 h. After washing with Hanks’ balanced salt solution (HBSS, pH 7.4), 270 μL of fresh HBSS (pH 7.4) and 30 μL of WST-1 reagent were added. The absorbance of the solution was measured with a microplate reader (Bio-Rad model 550, Tokyo, Japan).

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EXPERIMENTAL SECTION Materials. α-CyD was donated by Nihon Shokuhin Kako (Tokyo, Japan). Dendrimer (G3, ethylenediamine core, molecular weight = 6909) was obtained from Aldrich Chemical (Tokyo, Japan). Linear polyethyleneimine (10 kDa, PEI) was obtained from Wako Pure Chemical Industries (Osaka, Japan). p-Toluenesulfonyl chloride and FA were purchased from Nacalai Tesque (Kyoto, Japan). ω-Amino-α-carboxyl polyethylene glycol (PEG, MW = 2170) was purchased from NOF corporation (Tokyo, Japan). RPMI-1640 (FA-containing) and RPMI-1640 (FA-free) media were purchased from Nissui Pharmaceuticals (Tokyo, Japan) and GIBCO (Tokyo, Japan), respectively. Fetal calf serum (FCS) was purchased from Nichirei (Tokyo, Japan). Lipofectamine 2000 reagent was obtained from Invitrogen (Tokyo, Japan). LysoTracker was purchased from Molecular Probes (Tokyo, Japan). pGL2 and pGL3 were obtained from Promega (Tokyo, Japan). The pGL2 and pGL3 include both SV40 promoter and enhancer elements to produce high levels of firefly luciferase expression. siRNAs and FITC-labeled siGL3 (FITC-siGL3) were obtained from BBridge (Tokyo, Japan). Three mismatches between siGL2 and s i G L 3 se q u e n c e s a r e as f o l l o w s . si G L 2 : se ns e, dTdTGCAUGCGCCUUAUGAAGCU; antisense, dTdTAGCUUCAUAAGGCGCAUGC. siGL3: sense, dTdTGAAUGCGACUCAUGAAGCU; antisense, dTdTAGCUUCAUGAGUCGCAUUC. Other chemicals and solvents were of analytical reagent grade. Preparation of Fol-PαCs (G3) and TRITC-Fol-PαC (G3). Fol-PαCs (G3) were prepared using an α-CDE (G3, DS of α2592

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In Vivo RNAi Effects. Four-week-old BALB/c male mice (ca. 20 g) were subcutaneously injected the suspension containing Colon-26-luc cells (5 × 105 cells/100 μL). About 10 days later, the tumor-bearing mice were intratumorally or intravenously injected with a 5% mannitol solution (100 μL for intratumoral injection or 500 μL for intravenous injection) containing the Fol-PαC (G3)/siRNA complex at a charge ratio of 20/1 or 5/1 (carrier/siRNA), respectively. The doses of siRNA for intratumoral and intravenous injections were 10 μg and 50 μg, respectively. At 24 h after intratumoral or intravenous injection, the mice were sacrificed, and tumor tissues were isolated. The isolated tumor was washed twice with ice-cold saline, and was added to 2 mL of the lysis buffer containing the Roche protease inhibitor, Complete (Tokyo, Japan). The isolated tumor was homogenized at 24 000 rpm. After three cycles of freezing and thawing, the homogenate was centrifuged for 5 min at 10 000 rpm (4 °C), and 20 μL of the supernatant was added to 100 μL of the firefly luciferase assay buffer (Promega, Tokyo, Japan). Luminescence was immediately measured for 10 s (Lumat LB9506, EG&G Berthhold Japan, Tokyo, Japan). Blood samples were taken from the vital artery 24 h after intravenous injection of siRNA complex with Fol-PαC (G3) at a charge ratio of 20 (carrier/siRNA). After centrifugation of blood, serum was collected, and then creatinine (CRE), blood urea nitrogen (BUN), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH) values were determined by using a clinical chemistry analyzer, JCA-BM2250 (JEOL, Tokyo, Japan). Determination of Blood, Various Organs, and Tumor Tissue Levels of FITC-siRNA and Fol-PαC (G3). Blood was collected after intravenous injection with a 5% mannitol solution (500 μL) containing the Fol-PαC (G3)/siRNA complexes at a charge ratio of 20/1 or 5/1 (carrier/siRNA). After centrifugation at 5000 rpm for 10 min, 100 μL of the supernatant was recovered. Various organs and tumor tissues were collected after intravenous injection and homogenized by using a ULTRA-TURRAX T-25 apparatus with lysis buffers (20 mM Tris-HCl, 0.05% Triton X-100, 2 mM EDTA, pH 7.5). After centrifugation at 5000 rpm for 10 min, 100 μL of the supernatant was recovered. The fluorescent intensity of the supernatant was determined via a fluorescent microplate reader 1420 ARVOSX (Wallac, Turke, Finland). Pharmacokinetic Parameters. Pharmacokinetic parameters were calculated by the compartment model analysis using MULTI, a nonlinear least-squares fitting program. The elimination half-life (t1/2), area under the concentration−time curve (AUC), mean resident time (MRT), steady-state distribution volume (Vss), total clearance (CL), maximal blood concentration (Cmax), and time to reach Cmax (Tmax) were estimated. In Vivo Imaging. The fluorescent intensity in various organs and tumor tissues were visualized by using a Maestro EX imaging system (Cambridge Research &. Instrumentation, Inc.). Data Analysis. Data given are presented as the mean ± SEM for each group. Statistical significance of mean coefficients for the studies was determined by analysis of variance followed by Scheffe’s test. p-Values for significance were set at 0.05.

Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) Analysis. The mRNA levels of INF-α, INF-β, and TNF-α in KB cells were assayed via a semiquantitative RT-PCR method. Total RNA was isolated from KB cells (3 × 106 cells/ dish) following manufacturer’s instructions. cDNA was synthesized using a reverse primer and SuperScript III. The sequences of the forward and reverse primer pairs used were the following: 5′-TTGGCATAGAGGTCTTTACGGA-3′ and 5′-GCACCACACCTTCTACAATGAG-3′ for human β-actin; 5′-TCCTGTGGCATCCACGAAACT-3′ and 5′-GAAGCATTTGCGGTGGACGAT-3′ for mouse TNF-α; 5′GCCTTGACACTCCTGGTACAAATGAG-3′ and 5′-CAGCACATTGGCAGAGGAAGACAG-3′ for human INF-α; 5′TGGGTGGAATGAGACTATTGTTG-3′ and 5′CTCCCACGTCAATCTTTCCTC-3′ for human INF-β; 5′CCGAGTGACAAGCCTGTAGC-3′ and 5′-GAAGGAGACGGTAAAGTTGTTGA-3′ for human TNF-α. Interaction between siRNA and Carrier. Electrophoretic mobility of the siRNA/carrier complex was performed using an agarose gel electrophoresis system. The siRNA/carrier complexes having various charge ratios or the volume to amount ratios were prepared with 0.5 μg of siRNA in Tris-HCl buffer (10 mM, pH 7.4). Gel electrophoresis was carried out at room temperature in TBE buffer (45 mM Tris-borate, 1 mM EDTA, pH 8.0) in 2% (w/v) agarose gel including 0.1 μg/mL of ethidium bromide using Mupid system (Advance, Tokyo, Japan) at 100 V for 30 min. The siRNA bands were visualized using an UV illuminator (NLMS-20E, UVP, CA). Determination of Particle Sizes and ζ-Potential Values of Complexes. The particle sizes and ζ-potential values of the complexes of siGL3/carrier were determined by dynamic light scattering using a Zetasizer Nano ZS apparatus (Malvern Instruments, Worcestershire, U.K.). The measurement range of this apparatus for particle sizes is 0.6−6000 nm. Stability Assay of siRNA in FCS. The complexes of siRNA/carriers were incubated in the presence of 50% FCS at 37 °C for 5 h. The solutions were extracted with phenol and phenol/chloroform/isoamyl alcohol (25:24:1 v/v/v). Thereafter, siRNA was precipitated with ethanol. After dissolving the pellet with an RNase-free water, the samples were analyzed by an agarose gel electrophoresis (3.5%) according to the method reported by Minakuchi et al.26 and visualized by an ethidium bromide staining using an UV illuminator (NLMS-20E, UVP, CA). Cellular Uptake of siRNA. The KB cells (5 × 104/wells) in FA-free medium without FCS were incubated in the presence of 1.5, 3, or 6 μL of FA (200 mM) at 37 °C for 1 h. Then, 30 μL of the complex of Alexa555-siRNA (100 nM) with Fol-PαC (G3) at a charge ratio of 20/1 was transfected at 37 °C for 1 h. After transfection, the cells were washed with PBS (pH 7.4) and immediately scraped with 0.5 mL of PBS (pH 7.4). Data were collected for 1 × 104 cells on a FACSCalibur flow cytometer using CellQuest software (Becton-Dickinson, Mountain View, CA). Confocal Laser Scanning Microscopy (CLSM). The KB cells (1 × 104/35 mm glass bottom dish) were transfected with 200 μL of the complex of FITC-siRNA (100 nM) with TRITCFol-PαC (G3) in the presence or absence of LysoTracker (50 nM) at a charge ratio of 20/1 (Fol-PαC (G3)/siRNA) at 37 °C for 24 h. After incubation, the cells were rinsed with RPMI1640 medium (FA-free) twice. Cells were observed using a CLS microscope (Olympus FULUOVIEW FV500BX, Tokyo, Japan) with an argon laser of 350−550 nm.

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RESULTS Preparation of Fol-PαCs (G3). In an attempt to develop FR-overexpressing cancer cell-specific siRNA transfer carriers, 2593

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Figure 1. Preparation pathway of Fol-PαC (G3).

we prepared folate-PEG-appended α-CDEs (Fol-PαCs (G3)) (Figure 1). The degree of substitution of folate-PEG (DSF) values of the conjugates were determined as 2, 4, and 7 by the calculation from the integral values of anomeric protons of αCyD and the benzene ring of FA in 1H NMR spectra (Supporting Information Figure S1). Fol-PαC: 1H NMR (500 MHz, D2O) δ from TMS 8.75 (H7, folic acid), 7.75 (H13/15, folic acid), 6.74 (H12/16, folic acid), 5.03−5.04 (H1, α-CyD), 4.59−4.44 (H9, H19, folic acid), 3.2−3.8 (PEG ethylene), 4.02−3.61 (H3, H5, H6, α-CyD), 3.61−3.35 (H2, H4, α-CyD), 3.35−3.12 (dendrimer methylene), 3.12−2.81 (dendrimer methylene), 2.81−2.63 (dendrimer methylene), and 2.44− 2.01 (dendrimer methylene, H22, H21, folic acid). The yields

of Fol-PαCs (G3, DSL 2, 4, and 7) were approximately 72%, 75%, and 79%, respectively. RNAi Effects of the Fol-PαCs (G3)/siRNA Complexes. To investigate the RNAi effects of the complexes of Fol-PαCs (G3)/siRNA, luciferase activity after transfection of the complex was determined. We used siGL3, a target siRNA, and siGL2, a control siRNA.27 To investigate the effects of the number of the folate-PEG in Fol-PαCs (G3) on the RNAi effects, Renilla luciferase activity after transfection of siRNA complexes with Fol-PαCs (G3, DSF 2, 4, and 7) at a charge ratio of 20 (carrier/siRNA) at a concentration of 100 nM siRNA in KB cells was determined (Figure 2A). An additional attachment of the folate-PEG to α-CDE with DSF value of 4 2594

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Figure 2. (A) Inhibitory effects of complexes of Fol-PαCs (G3) with various DSF/siGL3 on luciferase activity in KB cells transiently expressing pGL3 luciferase gene. The charge ratio of Fol-PαCs/siRNA was 20/1. *p < 0.05, compared with control. †p < 0.05, compared with siGL2. #p < 0.05, compared with Fol-PαC (G3, DSF 2). (B) Inhibitory effects of complexes of Fol-PαC (G3, DSF 4)/siGL3 on luciferase activity in KB cells transiently expressing pGL3 luciferase gene. *p < 0.05, compared with control. †p < 0.05, compared with Fol-PαC (G3, DSF 4)/siGL2. (C) Concentration-dependency of FCS on RNAi effects of complex of Fol-PαC (G3, DSF 4)/siGL3 in KB cells transiently expressing pGL3 luciferase gene. *p < 0.05, compared with siGL2. (D) Inhibitory effects of complex of carriers/siGL3 in KB cells and A549 cells transiently expressing pGL3 luciferase gene. *p < 0.05, compared with A549 cells. Each value represents the mean ± SEM of 3−4 experiments.

(Fol-PαC (G3, DSF 4)) elicited much more RNAi effect than the other Fol-PαCs (G3, DSF 2 and 7) in KB cells. Therefore, it is evident that of all of the Fol-PαCs (G3), Fol-PαC (G3, DSF 4) has the greatest RNAi effect. To examine the effects of the charge ratios of the complexes with Fol-PαC (G3, DSF 4) on the RNAi effect, the luciferase

activity after transfection was determined in KB cells. As shown in Figure 2B, Fol-PαC (G3, DSF 4)/siGL3 complex at a charge ratio of 20 (carrier/siRNA) tended to have the most potent RNAi effect among the charge ratios of 5, 10, and 20, though there was no significant difference in the RNAi effects. The RNAi effect of the complex at a charge ratio of 50 was low, 2595

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compared to that at a charge ratio of 20. Furthermore, the nonsequence specific inhibitory effect of Fol-PαC (G3, DSF 4)/siGL2 complex was observed at a charge ratio of 100. These results suggest an optimal charge ratio of Fol-PαC (G3, DSF 4)/siGL3 complex of 20. Therefore, we mainly used Fol-PαC (G3, DSF 4) at a charge ratio of 20 (carrier/siRNA) in the subsequent in vitro studies. Next, we examined the effect of the FCS concentration on the RNAi effect of Fol-PαC (G3, DSF 4) in KB cells, because the FCS concentration may affect transfection efficiency of siRNA. As a result, the complex maintained the RNAi effect in the presence of 10% FCS (Figure 2C), suggesting that Fol-PαC (G3, DSF 4) was partially resistant to serum in transfection of siRNA. To test the FR-overexpressing cancer cell-selective siRNA transfer activity of Fol-PαC (G3, DSF 4), the RNAi effects of the siRNA complexes with α-CDE (G3) and Fol-PαC (G3, DSF 4) were examined in A549 cells, compared to that in KB cells (Figure 2D). Herein, we confirmed that FR expresses in KB cells, but not in A549 cells, using a RT-PCR method (Supporting Information Figure S2A), which is consistent with the results previously reported.28 The RNAi effect of Fol-PαC (G3, DSF 4)/siRNA complex in KB cells was significantly higher than that in A549 cells. On the other hand, there was no significant difference in the RNAi effect of the α-CDE (G3)/ siRNA complex between KB cells and A549 cells. These results strongly suggest the FR-overexpressing cancer-cell-selective siRNA transfer activity of Fol-PαC (G3, DSF 4). We previously reported that the superior siRNA transfer activity of α-CDE (G3) to dendrimer could be attributed to the enhancing endosomal escape of the siRNA complex through the endosomal membrane-disrupting ability of α-CyD.20,22 Next, to examine the role of α-CyD in the Fol-PαC (G3, DSF 4) molecule on siRNA transfer activity, we prepared Fol-PEGdendrimer (DSF 4), which is lacking an α-CyD molecule, and evaluated its siRNA complex’s RNAi effect. As shown in Figure 3, the RNAi effect of Fol-PEG-dendrimer (DSF 4)/siRNA complex was significantly lower than that of Fol-PαC (G3, DSF 4)/siRNA complex in KB cells, suggesting that α-CyD in the

Fol-PαC (G3, DSF 4) molecule plays an important role in improving siRNA transfer activity. The duration and the extent of the RNAi effect of carrier/ siRNA complex are very important. Therefore, we examined the duration of gene silencing induced by Fol-PαC (G3, DSF 4)/siRNA complex in KB cells, and evaluated the effect of repeated transfection on duration of the silencing (Figure 4).

Figure 4. Inhibitory effects of boost transfection of complex of FolPαC (G3, DSF 4)/siGL3 in KB cells transiently expressing pGL3 luciferase gene. Cells were transfected with complex of Fol-PαC (G3, DSF 4)/siRNA, and incubated for 24 h. Afterward, the cells were transfected again. Each point represents the mean ± SEM of 3−4 experiments. *p < 0.05, compared with first transfection.

The RNAi effects of the complex were increased until 16 h after transfection, and were sustained up to 48 h after transfection (Figure 4). Furthermore, when the second transfection of the siRNA complex was conducted 24 h after first transfection, the RNAi effect was prolonged until 72 h after first transfection. There was no significant cytotoxicity under these experimental conditions (data not shown). Therefore, these results suggest that Fol-PαC (G3, DSF 4) could be applicable for repeated transfection without cytotoxicity. Cytotoxicity, Interferon Response, and Inflammatory Response of the Fol-PαC (G3)/siRNA Complex. The drawbacks of siRNA complexes with cationic carriers are known to be cytotoxicity, interferon response, and inflammatory response. Therefore, we first evaluated cytotoxicity of the carriers/siRNA complexes in KB cells (FR-positive) and A549 cells (FR-negative) by the WST-1 method. The complex of FolPαC (G3, DSF 4)/siRNA showed negligible cytotoxicity in KB cells and A549 cells up to the charge ratio of 100/1 (carrier/ siRNA) (Figure 5). The complex of α-CDE (G3)/siRNA showed slight cytotoxicity in KB cells and A549 cells at a charge ratio of 100/1 (carrier/siRNA) (Figure 5). In sharp contrast, the siRNA complexes with PEI and Lipofectamine 2000 elicited severe cytotoxicity at a charge ratio of 20/1 (PEI/siRNA) and the volume to amount ratio of 37.5/1 (Lipofectamine 2000/ siRNA) according to the manufacture’s recommended conditions, respectively, in KB cells and A549 cells (Figure 5). These results evidently indicate that Fol-PαC (G3, DSF 4) is a safe carrier for siRNA delivery, compared with the commercial transfection reagents used in this study. Second, to examine whether the Fol-PαC (G3, DSF 4)/ siRNA complex induces interferon response and inflammatory response, we evaluated the IFN-α, IFN-β, and TNF-α gene expression 24 h after transfection with the various siRNA complexes by RT-PCR method in KB cells (Figure 6). These experiments were performed under the condition of no cytotoxicity. As shown in Figure 6, polyinosinic-polycytidylic

Figure 3. Inhibitory effects of complexes of Fol-PαC (G3, DSF 4)/ siGL3 and Fol-PEG-dendrimer (G3, DSF 4)/siGL3 in KB cells transiently expressing pGL3 luciferase gene. Each value represents the mean ± SEM of 3−4 experiments. *p < 0.05, compared with Fol-PEGdendrimer (G3, DSF 4). 2596

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Figure 7. (A) Agarose gel electrophoretic analysis of siRNA complexes with carriers. (B) Stability of siRNA in complexes of carriers/siRNA after treated with 50% FCS for 5 h at 37 °C. The complexes of carriers/siRNA were treated with 50% FCS for 5 h at 37 °C.

Figure 5. Cytotoxicity of siRNA complexes with various carriers in KB cells (A) and A549 cells (B). Each value represents the mean ± SEM of 3−4 experiments. *p < 0.05, compared with Fol-PαC (DSF 4). †p < 0.05, compared with control.

respectively. These results suggest that Fol-PαC (G3, DSF 4) formed the complex with siRNA, although the interaction of Fol-PαC (G3, DSF 4) with siRNA was relatively low, compared to that of α-CDE (G3). Next, we determined the particle sizes and ζ-potential values of the complexes (Table 1). The mean diameters of the siRNA Table 1. Particle Sizes and ζ-Potentials of Complexes of αCDE (G3)/siRNA and Fol-PαC (G3, DSF 4)/siRNA in TrisHCl Buffer (10 mM, pH 7.4)a charge ratio (carrier/siRNA)

carrier α-CDE(G3) Fol-PαC (G3,DSF 4)

Figure 6. Effects of various carriers on expression of IFN-α, IFN-β, and TNF-α mRNA in KB cells after transfection of complexes.

1 20 1 20

mean diameter (nm) 219 232 120 111

± ± ± ±

22 43 17bc 15bc

ζ-potential (mV) −6.4 13.0 −8.5 9.7

± ± ± ±

0.9 2.3bd 4.3 1.9bd

a

The siRNA complexes with carriers were added to Tris-HCl buffer (10 mM, pH 7.4). The siRNA concentration was 100 nM. Each value represents the mean ± SEM of 3−6 experiments. bp < 0.05, compared with α-CDE (G3) (charge ratio: 1). cp < 0.05, compared with α-CDE (G3) (charge ratio: 20). dp < 0.05, compared with Fol-PαC (G3, DSF 4) (charge ratio: 1).

acid (Poly I:C), a positive control, clearly induced IFN-α, IFNβ, and TNF-α gene expression. On the other hand, Fol-PαC (G3, DSF 4)/siRNA and α-CDE (G3)/siRNA complexes did not elicit IFN-α, IFN-β, or TNF-α gene expression. In contrast, PEI (10 kDa)/siRNA complex induced IFN-α gene expression, and Lipofectamine 2000/siRNA complex activated IFN-α and TNF-α gene expression. These results suggest that neither interferon response nor inflammatory response of Fol-PαC (G3, DSF 4)/siRNA complex was induced at least under the present experimental conditions. Physicochemical Properties of the Fol-PαC (G3)/siRNA Complex. Physicochemical properties of the siRNA complex with Fol-PαC (G3, DSF 4) could be related to the RNAi effect. To clarify complexation of siRNA with Fol-PαC (G3, DSF 4), we performed the gel mobility assay. As shown in Figure 7A, the band derived from siRNA vanished at the charge ratios of 1 (α-CDE (G3)/siRNA) and 5 (Fol-PαC (G3, DSF 4)/siRNA),

complexes with α-CDE (G3) and Fol-PαC (G3, DSF 4) showed about 220−230 nm and 110−120 nm, respectively, at the charge ratios (carrier/siRNA) of 1/1 and 20/1 (Table 1). The mean diameter of Fol-PαC (G3, DSF 4)/siRNA complex did not show any changes up to the charge ratio (carrier/ siRNA) of 100 (Supporting Information Figure S3A). Meanwhile, the ζ-potential values of the α-CDE (G3)/siRNA and Fol-PαC (G3, DSF 4)/siRNA complexes increased as the charge ratio (carriers/siRNA) increased, and the values showed positive at the charge ratio of 20/1 (carrier/siRNA) (Table 1). The ζ-potential value of the Fol-PαC (G3, DSF 4)/siRNA 2597

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Figure 8. Effects of folic acid on cellular uptake of Alexa555-siRNA complexes with α-CDE (G3) (A) and Fol-PαC (G3, DSF 4) (B) into KB cells.

amount of FITC-siRNA did not colocalize with lysosome/ endosome stained by LysoTracker (Figure 9C). Collectively, these results suggest that Fol-PαC (G3, DSF 4) allowed distribution of siRNA in cytoplasm after the cellular uptake into KB cells, and showed the potent RNAi effects in FRoverexpressing cancer cells. In Vivo RNAi Effects of the Fol-PαC (G3)/siRNA Complex. Next, we examined the in vivo RNAi effects of the siRNA complex with Fol-PαC (G3, DSF 4) in mice. Figure 10 shows the inhibitory effects of the complexes of Fol-PαC (G3, DSF 4)/siGL3 on luciferase activity in BALB/c mice bearing Colon-26-luc cells 24 h after intratumoral injection. Here, we confirmed FR expression in Colon-26 cells using a RT-PCR method (Supporting Information Figure S2B). The Fol-PαC (G3, DSF 4)/siGL3 complex, not the Fol-PαC (G3, DSF 4)/ siGL2 complex, reduced luciferase activity to 40% of control (5% mannitol solution) (Figure 10A). This RNAi effect of FolPαC (G3, DSF 4)/siGL3 complex was higher than that of αCDE (G3)/siGL3 complex, suggesting that folate ligands have an important role to elevate the RNAi effects. Moreover, we evaluated the in vivo RNAi effects of siRNA complexes 24 h after intravenous injection of the solution containing the FolPαC (G3, DSF 4)/siRNA complex to tail vein of mice bearing Colon-26-luc cells. As shown in Figure 10B, the Fol-PαC (G3, DSF 4)/siGL3 complex tended to impair luciferase gene expression, compared to control (5% mannitol solution). In addition, the Fol-PαC (G3, DSF 4)/siGL2 complex did not change luciferase activity, which strongly suggests that (1) the carrier did not have silencing activity per se and (2) the control siRNA was biologically inert. However, there was no significant difference in the RNAi effects between Fol-PαC (G3, DSF 4)/ siGL3 and α-CDE (G3)/siGL3 complexes. Furthermore, the RNAi effect (ca. 20% knockdown) elicited by Fol-PαC (G3, DSF 4)/siGL3 by intravenous injection was slightly weaker than that (ca. 60% knockdown) by intratumoral injection. These results suggest that the Fol-PαC (G3, DSF 4)/siRNA complex had the potential to induce the in vivo RNAi effect after not only intratumoral but also intravenous administration in tumor-bearing mice. From the viewpoint of safety, we measured blood chemistry values after intravenous injection of the siRNA complex with Fol-PαC (G3, DSF 4)/siGL3 in mice bearing Colon-26-luc

complex did not show any further increase at charge ratios (carrier/siRNA) over 20/1 (Supporting Information Figure S3B). These results suggest that α-CDE (G3) and Fol-PαC (G3, DSF 4) formed the complexes with siRNA, but the physicochemical properties were different, possibly leading to the distinct RNAi effects. Next, we examined the effects of Fol-PαC (G3, DSF 4) on the enzymatic degradation of siRNA by FCS. As shown in Figure 7B, the intensities of band derived from siRNA increased as the charge ratios of α-CDE (G3)/siRNA and Fol-PαC (G3, DSF 4)/siRNA increased. Fol-PαC (G3, DSF 4)/siRNA complex showed clear bands derived from siRNA even at the low charge ratio, compared to α-CDE (G3)/siRNA complex. These results suggest that Fol-PαC (G3, DSF 4) formed the complex with siRNA even in the presence of FCS and inhibited siRNA degradation against RNase in FCS, rather than α-CDE (G3). Cellular Association and Intracellular Distribution of the Complex of Fol-PαC (G3)/siRNA. To gain insight into the mechanism for the FR-overexpressing cancer cell-selective siRNA transfer activity of Fol-PαC (G3, DSF 4), we examined the cellular association of Alexa-siRNA 1 h after transfection of the complex of Fol-PαC (G3, DSF 4)/Alexa-siRNA in KB cells (FR-positive) in the presence and absence of FA as a competitor of FR by a flow cytometric analysis (Figure 8). The cellular association of Alexa-siRNA in the Fol-PαC (G3, DSF 4) system, but not α-CDE (G3) system, was lowered by the addition of FA in a concentration-dependent manner. These results suggest that Fol-PαC (G3, DSF 4) had a FRoverexpressing cancer cell-selective siRNA transfer activity. The RNA-induced silencing complex (RISC) is acknowledged to localize in cytoplasm and form the complex with siRNA to elicit the RNAi effect.29 Thus, we investigated the cellular association and intracellular distribution of FITCsiRNA and TRITC-Fol-PαC (G3, DSF 4) in KB cells using a CLS microscope (Figure 9). It should be noted that uptake of FITC-siRNA in KB cells was observed 24 h after transfection with α-CDE (G3) and Fol-PαC (G3, DSF 4) (Figure 9A). Furthermore, TRITC-α-CDE (G3) and FITC-siRNA were uniformly observed in cells 24 h after transfection (Figure 9B). A similar result was observed in the TRITC-Fol-PαC (G3, DSF 4)/FITC-siRNA system (Figure 9B). In addition, the large 2598

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Figure 9. (A) Cellular association of FITC-siRNA complexes with α-CDE and Fol-PαC (G3, DSF 4) in KB cells. (B) Intracellular distribution of FITC-siRNA complexes with TRITC-α-CDE (G3) and TRITC-Fol-PαC (G3, DSF 4) in KB cells. (C) Intracellular distribution of FITC-siRNA and LysoTracker in KB cells.

tumor-bearing mice, we determined the blood concentrations of FITC-siRNA and TRITC-Fol-PαC (G3, DSF 4) and calculated their pharmacokinetic parameters. As shown in Figure 11A, the FITC-siRNA levels in blood rapidly decreased to 40% of the total concentration in 5−10 min after intravenous administration, and completely disappeared within 1 h from blood. Meanwhile, TRITC-Fol-PαC (G3, DSF 4) was slowly eliminated from blood, compared to FITC-siRNA, after intravenous injection of the complex to tumor-bearing mice (Figure 11B). From the compartment model analysis using

cells (Table 2). In the present study, blood chemistry values such as CRE, BUN, AST, ALT, and LDH in the Fol-PαC (G3, DSF 4)/siRNA complex system were almost equivalent to those in the control system (5% mannitol solution) and α-CDE (G3)/siRNA complex system. These results suggest that the siRNA complex with Fol-PαC (G3, DSF 4) has negligible acute organ injury in vivo. Pharmacokinetic Analysis of the Fol-PαC (G3)/siRNA Complex. To investigate the pharmacokinetics of the Fol-PαC (G3, DSF 4)/siRNA complex after intravenous injection to 2599

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Figure 10. In vivo RNAi effect after intratumoral (A) and intravenous (B) injections of siRNA complex with α-CDE (G3) or Fol-PαC (G3, DSF 4) in BALB/c mice bearing Colon-26-luc tumor cells. Each value represents the mean ± SEM of 4−5 experiments. *p < 0.05, compared with control. †p < 0.05, compared with siGL2.

Figure 11. Time courses of blood levels of FITC-siRNA (A) and TRITC-Fol-PαC (G3, DSF 4) (B) after intravenous injection of the solution containing TRITC-Fol-PαC (G3, DSF 4)/FITC-siRNA complex to tail vein of BALB/c mice bearing Colon-26 tumor cells. Each point represents the mean ± SEM of 3−6 experiments.

MULTI, the pharmacokinetics of FITC-siRNA and TRITCFol-PαC (G3, DSF 4) fitted with the one-compartment model and two-compartment model, respectively. From the in vivo pharmacokinetic parameters of Vd and Vd2, the amount of FITC-siRNA transferred into tissues was found to be low compared to that of TRITC-Fol-PαC (G3, DSF 4) (Table 3). Also, judging from the values of elimination constant (ke), halflife (t1/2), total clearance (CLtot), and mean residence time (MRT), FITC-siRNA may be eliminated more rapidly than TRITC-Fol-PαC (G3, DSF 4). These results suggest that the biodistribution behaviors of FITC-siRNA and TRITC-Fol-PαC (G3, DSF 4) were different, probably due to the partial dissociation after intravenous injection of the complex. In Vivo Imaging after Intravenous Injection of the TRITC-Fol-PαC (G3)/FITC-siRNA Complex. To reveal the biodistribution of FITC-siRNA and TRITC-Fol-PαC (G3, DSF 4) after intravenous injection to tumor-bearing mice, we determined their fluorescence in various organs and tumors by using a Maestro EX imaging system (Figure 12). The fluorescence derived from FITC-siRNA was mainly observed

in kidney at 10 min after intravenous injection, and was strongly detected in tumors 30 min after administration. On the other hand, the fluorescence derived from TRITC-Fol-PαC (G3, DSF 4) showed up mainly in liver and kidney until 1 h, and then it gradually increased in tumor tissues. These results suggest that FITC-siRNA and TRITC-Fol-PαC (G3, DSF 4) are accumulated in tumor tissues after intravenous administration of the complex to tumor-bearing mice. Tumor Tissue Levels of FITC-siRNA and TRITC-Fol-PαC (G3). Finally, we examined the tumor tissue levels of FITCsiRNA and TRITC-Fol-PαC (G3, DSF 4) after intravenous injection to tumor-bearing mice. As shown in Figure 13A, tumor tissue level of FITC-siRNA after intravenous injection of the complex was significantly higher than that of FITC-siRNA alone. The tumor tissue levels of FITC-siRNA and TRITC-FolPαC (G3, DSF 4) increased until 1 h after administration, and then gradually decreased. The tumor tissue levels of FITC-

Table 2. Blood Chemistry Data after Intravenous Injection of the siRNA Complexes with α-CDE (G3) and Fol-PαC (G3, DSF 4) in BALB/c Mice Bearing Colon-26-luc Tumor Cellsa carrier/siRNA

CRE (mg/dL)b

BUN (mg/dL)c

AST (U/L)d

ALT (U/L)e

LDH (U/L)f

control α-CDE (G3)/siRNA Fol-PαC(G3, DSF4)/siRNA

0.10 ± 0.01 0.10 ± 0.01 0.10 ± 0.01

22.9 ± 1.69 23.9 ± 1.16 22.1 ± 1.45

66.8 ± 27.8 43.3 ± 3.5 53.6 ± 3.2

20.1 ± 2.70 25.6 ± 4.08 33.1 ± 5.44

396 ± 184 231 ± 20 377 ± 87

a

The charge ratio of carrier/siRNA was 20/1. Each value represents the mean±S.E.M. of 3-5 mice. bCreatinine. cBlood urea nitrogen. dAsparate aminotransferase. eAlanine aminotransferase. fLactate dehydrogenase. 2600

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Table 3. In Vivo Pharmacokinetic Parameters of FITCsiRNA and TRITC-Fol-PαC (G3, DSF 4) after Intravenous Injection of TRITC-Fol-PαC (G3, DSF 4)/FITC-siRNA Complex to Tail Vein of BALB/c Mice Bearing Colon-26 Tumor Cells parameter C0 (μg/mL) ke (min−1) t1/2 (min) Vd (mL) CLtot(mL/min) AUC (μg·min/mL) MRT (min) parameter C0 (μg/mL) k12 (min−1) k21 (min−1) ke (min−1) t1/2,α (min) t1/2,β (min) Vd1 (mL) Vd2 (mL) Vss (mL) CLtot (mL/min) AUC (μg·min/mL) MRT (min)

CDE (Fol-α-CDE (G3)) in order to deliver pDNA to FRoverexpressing cancer cells through FR-mediated endocytosis. Unfortunately, this Fol-α-CDE (G3) failed to deliver pDNA in a tumor cell-selective manner, probably due to low interaction between FA and FR, resulting from the direct linkage of FA to dendrimer without any spacers. Therefore, we attempted to insert the PEG chain as a spacer between FA and α-CDE (G3) (Figure 1) to enhance the FR recognition ability to FRoverexpressing cancer cells. As a result, it is highly likely that Fol-PαC (G3, DSF 4) was recognized by KB cells (FRpositive) (Figure 8). In addition, to estimate the extent of interaction between Fol-PαC (G3, DSF 4) and FR, we determined the association constant of Fol-PαC (G3, DSF 4) and folate-binding protein (FBP) instead of FR by surface plasmon resonance analysis. The association constant of FolPαC (G3, DSF 4) with FBP was ca. 3.8 × 107 M−1 and was significantly higher than that of folic acid alone with FBP (1.1 × 103 M−1). The enhancement of the association constant may be due to the multipoint recognition of Fol-PαC (G3, DSF 4) with FBP. These results suggest that Fol-PαC (G3, DSF 4) can strongly interact with FR. The interaction of siRNA with carriers, particle size, and ζpotential value of the complex could be related to the siRNA transfer activity. As the results of agarose gel electrophoretic analysis show, Fol-PαC (G3, DSF 4) forms the complex with siRNA; however, the interaction of Fol-PαC (G3, DSF 4) with siRNA was relatively low, compared to that of α-CDE (G3) (Figure 7A). This low interaction of Fol-PαC (G3, DSF 4) with siRNA may be caused by the reduction of the number of primary amino groups of dendrimer by the addition of FolPEG, or steric hindrance of PEG chains. To elicit the RNAi effect, siRNA should be released and incorporated into RISC in cytoplasm. The low electrostatic interaction of Fol-PαC (G3, DSF 4) and siRNA may reduce the stability of siRNA against enzymatic degradation, while that may accelerate the release of siRNA after endosomal escaping of the complex. Therefore, it is thought that the latter effect was superior to the former one under the present experimental conditions. The mean diameter of the siRNA complexes with Fol-PαC (G3, DSF 4) was about 110−120 nm at the charge ratios (carrier/siRNA) of 1/1 and 20/1 (Table 1) and was smaller than that with α-CDE (G3) (220−230 nm). This may be due to the steric hindrance by the addition of Fol-PEG, resulting in the inhibition of the aggregation. Peer et al. demonstrated that the tumor vessels have increased permeability due to aberrant angiogenesis, thus allowing nanoparticles with diameters less than 200 nm to passively extravasate into the tumor sites through the EPR effect.32 In addition, Reddy et al. reported that the particle size of folate-appended carrier/siRNA complex through FR-mediated endocytosis was less than ca. 150 nm.33 Therefore, the particle sizes of Fol-PαC (G3, DSF 4)/siRNA complex were of great advantage in the EPR effect and FRmediated endocytosis. Meanwhile, the ζ-potential value of FolPαC (G3, DSF 4)/siRNA complex were slightly positive at a charge ratio (carrier/siRNA) of 20/1 (Table 1). Leamon et al. reported that folate-appended carrier/siRNA complex with neutral ζ-potential value enhanced the FR recognition, probably due to the inhibition of nonspecific adsorption onto cell surface.31 Therefore, the reason for the highest RNAi effect of Fol-PαC (G3, DSF 4)/siRNA complex at the charge ratio (carrier/siRNA) of 20/1 (Figure 2B) was may be due to its low ζ-potential value.

FITC-siRNA 5.9 0.103 7 1.7 0.18 57 14 TRITC-Fol-PαC (G3, DSF4) 23.6 0.092 0.037 0.023 3 30 9.4 23.7 33.1 0.22 1015 61

siRNA and TRITC-Fol-PαC (G3, DSF 4) after 5 h intravenous injection were about 6 μg/tissue (10 ng/mg of tissue) and 0.4 μg/tissue (0.75 ng/mg of tissue), respectively. These results suggest that even though the FITC-siRNA complex with TRITC-Fol-PαC (G3, DSF 4) may dissociate partially in part after intravenous injection to tumor-bearing mice, FITC-siRNA and TRITC-Fol-PαC (G3, DSF 4) can accumulate in tumors.

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DISCUSSION In this study, we clarified that Fol-PαC (G3, DSF 4) has FRoverexpressing cancer-cell-selective siRNA transfer activity and negligible cytotoxicity, compared to α-CDE (G3) and the other Fol-PαCs (G3, DSF 2 and 7) through FR-mediated endocytosis. Cationic polymer-mediated siRNA transfer should overcome several major barriers for transfection: cellular association, endosomal escape, and translocation into cytosol.30 Leamon et al. reported that when the polyplexes with Fol-PEG-appended poly-L-lysine were transfected in FR-overexpressing cells, the optimal DSF value existed.31 In the present study, siRNA transfer activity of Fol-PαC (G3) was found to be strongly dependent on the DSF value. Fol-PαCs (G3, DSF 4) elicited much more efficient siRNA transfer activity than the other FolPαCs (G3, DSF 2 and 7) in KB cells (Figure 2A), suggesting that the low siRNA transfer activity of Fol-PαC (G3, DSF 7) with higher DSF value could be ascribed to a weak interaction with siRNA owing to the low number of free primary amino groups in the molecule. Meanwhile, the low siRNA transfer activity of Fol-PαC (G3, DSF 2) could be due to slight receptor recognition with FR. Therefore, these results suggest that the DSF value of 4 is optimal in Fol-PαC (G3) as a siRNA transfer carrier to FR-overexpressing cancer cells. It should be noted that the spacers between dendrimer and ligands are very important for cell-selective nucleic acids transfer activity. Previously, we prepared folate-appended α2601

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Figure 12. In vivo imaging after intravenous injection of TRITC-Fol-PαC (G3, DSF 4)/FITC-siRNA complex to tail vein of BALB/c mice bearing Colon-26 tumor cells.

To reveal the role of α-CyD in the Fol-PαC (G3, DSF 4) molecule on siRNA transfer activity, we prepared Fol-PEGdendrimer (G3, DSF 4) and evaluated its siRNA complex’s RNAi effect (Figure 3). The RNAi effect of Fol-PEG-dendrimer (G3, DSF 4), which is lacking an α-CyD molecule, was significantly lower than that of Fol-PαC (G3, DSF 4) in KB cells. So far, we have reported that the enhancing effect of αCDE (G3) on gene and siRNA transfer activity could be ascribed to high endosomal escaping ability through the cooperative effect of a proton sponge effect of dendrimer and the inclusion ability of α-CyD with phospholipids in endosomes.22,34,35 Generally, glycosylphosphatidylinositol (GPI)anchored proteins including FR are known to accumulate into GPI-anchored protein-enriched early endosomal compartments (GEECs), where the pH is not low, compared to that in general

late-endosome (ca. pH 5.5) and lysosomes (ca. pH 5.0).36 This fact suggests that the proton-sponge effect of the dendrimer in the Fol-PαC (G3) molecule is very slight. Actually, the RNAi effect of Fol-PαC (G3, DSF 4)/siRNA was significantly higher than that of Fol-PEG-dendrimer (G3, DSF 4)/siRNA complex (Figure 3). Taken together, α-CyD in the Fol-PαC (G3, DSF 4) molecule may play an important role for its endosomal escaping in KB cells. Fol-PαC (G3, DSF 4)/siRNA complex showed the potential to induce the in vivo RNAi effects after intratumoral injection to tumor cells-bearing mice (Figure 10). In the present study, to enhance the FR-expression in tumor tissues, Colon-26 cells were cultured with folic acid-free medium before inoculation, and the mice were concomitantly grown with folic acid-free food. Intratumoral injection of Fol-PαC (G3, DSF 4)/siRNA 2602

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or 11−14 ng/g tissue, suggesting that enough siRNA was delivered into tumor tissues to obtain the 50% RNAi effect. The reasons for the weak RNAi effect after intravenous injection of Fol-PαC (G3, DSF 4)/siRNA complex, even though enough siRNA was delivered into tumor tissues, were thought to be (1) the interstitial space with high pressure in tumor tissues may prohibit the penetration of the siRNA complex, (2) the large amount of siRNA delivered to tumor tissues was free siRNA (already dissociated from carrier) and could not enter the tumor cells, and (3) the enzymatic degradation of siRNA may occur in tumor cells. Actually, the pharmacokinetics of FITCsiRNA and TRITC-Fol-PαC (G3, DSF 4) were different, probably due to the partial dissociation after intravenous injection of the solution containing the TRITC-Fol-PαC (G3, DSF 4)/FITC-siRNA complex (Figures 11 and 12). Therefore, further investigations on the reduction of a dissociation of siRNA from Fol-PαC (G3, DSF 4) and the delivery of Fol-PαC (G3, DSF 4)/siRNA complex to tumor cells to obtain the higher RNAi effect after intravenous injection are required. In conclusion, our findings provided that the Fol-PαC (G3, DSF 4)/siRNA complex elicited the potent RNAi effects in KB cells (FR-positive) with negligible cytotoxicity, efficient cellular uptake, enhanced endosomal escape, and distribution in cytoplasm. Importantly, the Fol-PαC (G3, DSF 4)/siRNA complex showed the preferable RNAi effects in tumor-bearing mice. Taken together, Fol-PαC (G3, DSF 4) could be potentially used as a FR-overexpressing cancer cell-selective siRNA delivery carrier in vitro and in vivo, and these data may be useful for design of α-CyD and Fol-PEG conjugates with nonviral vectors.

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ASSOCIATED CONTENT

S Supporting Information *

Additional figures showing 1H NMR spectra, RT-PCR analysis results, particle sizes, and ζ-potentials. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Mailing address: Department of Physical Pharmaceutics, Graduate School of Pharmaceutical Sciences, and Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, Japan. Telephone: +81 96 371 4160. Fax: +81 96 371 4420. Email: [email protected].

Figure 13. Time courses of tumor tissue levels of FITC-siRNA (A) and TRITC-Fol-PαC (G3, DSF 4) (B) after intravenous injection of TRITC-Fol-PαC (G3, DSF 4)/FITC-siRNA complex to tail vein of BALB/c mice bearing Colon-26 tumor cells. Each point represents the mean ± SEM of 3−4 experiments. *p < 0.05, compared with FITCsiRNA alone.

Notes

Additional figures The authors declare no competing financial interest.

complex showed 50% reduction of luciferase gene expression (Figure 10A). The reason for the induction of the potent RNAi effects after intratumoral injection of Fol-PαC (G3, DSF 4)/ siRNA complex could be related to the enhancement of not only FR-mediated endocytosis in tumor cells but also the stability of siRNA against enzymatic degradation by the complex formation with Fol-PαC (G3, DSF 4). These results suggest that local administration of Fol-PαC (G3, DSF 4)/ siRNA complex to tumor tissues could be applicable. On the other hand, the RNAi effect in intravenous injection of Fol-PαC (G3, DSF 4)/siRNA complex system was relatively low (Figure 10B). Landesman et al. reported that the delivery of 300−500 molecules of siRNA/cell or 1 ng of siRNA/g tissue is required for the acquisition of the 50% RNAi effects.37 In the present study, the amount of siRNA accumulated in tumor tissue after intravenous injection of the Fol-PαC (G3, DSF 4)/siRNA complex was calculated as 3.8 × 106−1.1 × 107 molecules/cell

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ACKNOWLEDGMENTS This work was partially supported by a Grant-in-Aid for Scientific Research (C) from Japan Society for the Promotion of Science (14572158).

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REFERENCES

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dx.doi.org/10.1021/mp300188f | Mol. Pharmaceutics 2012, 9, 2591−2604