α-Cyclodextrin Conjugates as Hepatocyte-Specific siRNA Carriers for

Apr 17, 2012 - and Hidetoshi Arima*. ,†. †. Department of Physical Pharmaceutics, Graduate School of Pharmaceutical Sciences, Kumamoto University,...
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Potential Use of Lactosylated Dendrimer (G3)/α-Cyclodextrin Conjugates as Hepatocyte-Specific siRNA Carriers for the Treatment of Familial Amyloidotic Polyneuropathy Yuya Hayashi,† Yoshimasa Mori,† Shogo Yamashita,† Keiichi Motoyama,† Taishi Higashi,† Hirofumi Jono,‡ Yukio Ando,‡ and Hidetoshi Arima*,† †

Department of Physical Pharmaceutics, Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Kumamoto 862-0973, Japan ‡ Department of Diagnostic Medicine, Graduate School of Medical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-8556, Japan ABSTRACT: To reveal the potential use of lactosylateddendrimer (G3) conjugates with α-cyclodextrin (Lac-α-CDE (G3)) as novel hepatocyte-specific siRNA carriers in order to treat transthyretin (TTR)-related familial amyloidotic polyneuropathy (FAP), we evaluated the RNAi effect of siRNA complexes with Lac-α-CDE (G3) both in vitro and in vivo. Herein, we targeted TTR gene expression because TTRrelated FAP was often caused by amyloidogenic TTR (ATTR), which mainly expresses in hepatocytes. Lac-α-CDE (G3, average degree of substitution of lactose (DSL) 1.2)/siRNA complex had a potent RNAi effect against TTR gene expression through adequate physicochemical properties, asialoglycoprotein receptor (ASGP-R)-mediated cellular uptake, efficient endosomal escape and the delivery of the siRNA complex to cytoplasm, but not nucleus, with negligible cytotoxicity. Lac-α-CDE (G3, DSL 1.2)/siRNA complex had the potential to induce the in vivo RNAi effect after intravenous administration in the liver of mice. The blood chemistry values in the α-CDE (G3) and Lac-α-CDE (G3, DSL 1.2) systems were almost equivalent to those in the control system (5% mannitol solution). Taken together, these results suggest that Lac-α-CDE (G3, DSL 1.2) has the potential for a novel hepatocyte-selective siRNA carrier in vitro and in vivo, and has a possibility as a therapeutic tool for FAP to the liver transplantation. KEYWORDS: dendrimer, cyclodextrin, siRNA, familial amyloidotic polyneuropathy, hepatocyte-specific delivery

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INTRODUCTION Transthyretin (TTR)-related familial amyloidotic polyneuropathy (FAP), which is induced by amyloidogenic transthyretin (ATTR), is an autosomal dominant form of fatal hereditary amyloidosis characterized by systemic accumulation of amyloid fibrils in peripheral nerves and other organs.1,2 To date, more than 100 different point mutations in the TTR gene have been reported, most of which are amyloidogenic.1,3−5 Of the different types of ATTR-related amyloidosis, ATTR Val30Met (V30M), found worldwide, is the most common.2 Because the liver predominantly synthesizes TTR, liver transplantation has been thought to be a promising therapy for halting the progression of clinical FAP symptoms.6−8 However, because no truly effective therapy is available as of this moment, development of alternative therapeutic strategies is urgently needed. RNA interference (RNAi) denotes the sequence-specific cleavage of mRNA after the cellular introduction of complementary, small interfering RNA (siRNA) duplexes 21 to 27 nucleotides in length.9 The development of siRNA-based therapeutics has progressed rapidly because of their specific and potent RNAi triggering activity.10,11 Examples are the use of © 2012 American Chemical Society

RNAi to reduce hepatic TTR expression through antisense nucleotides12 or siRNAs encapsulated with stable nucleic acid lipid particle (SNALP);13 clinical trials are planned using these methodologies. Although siRNAs offer several advantages as potential new biodrugs to treat various diseases, the efficient delivery of siRNAs in vivo remains a crucial challenge for achieving the desired RNAi effect in clinical development.14,15 We previously reported that the starburst polyamidoamine (PAMAM) dendrimer (dendrimer, generation 2, G2) conjugate with α-cyclodextrin (α-CyD) bearing lactose (Lac-α-CDE (G2)) provided remarkable aspects as a gene delivery carrier in vitro and in vivo.16 Lac-α-CDE (G2, degree of substitution of lactose (DSL) 2.6) possesses various advantages for plasmid DNA (pDNA) delivery: (1) simple chemical structure, (2) efficient pDNA transfer activity into cells, (3) negligible cytotoxicity, and (4) hepatocyte-selective pDNA delivery in vivo. However, the gene transfer activity was not sufficient. Received: Revised: Accepted: Published: 1645

December 18, 2011 March 27, 2012 April 17, 2012 April 17, 2012 dx.doi.org/10.1021/mp200654g | Mol. Pharmaceutics 2012, 9, 1645−1653

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Most recently, we newly prepared Lac-α-CDE (G3) and demonstrated that Lac-α-CDE (G3, DSL 1.2) showed much higher gene transfer activity than Lac-α-CDE (G2, DSL 2.6) owing to increase in the number of free primary amino group of the dendrimer.17 However, the use of Lac-α-CDE (G3) as siRNA carriers was not evaluated yet. In the present study, we first evaluated the complexation of siRNA with carriers and physicochemical properties of the siRNA complex with carriers. We next investigated the in vitro RNAi effects of the siRNA complexes with Lac-α-CDE (G3) on TTR, and compared to those of commercial transfection reagents such as jetPEI-Hepatocyte. We also evaluated the cellular uptake, intracellular distribution and cytotoxicity of the siRNA complex with carriers. Finally, we investigated the in vivo RNAi effects and blood chemistry values after intravenous injection of the siRNA complex with Lac-α-CDE (G3) in mice.

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EXPERIMENTAL SECTION Materials. α-CyD was donated by Nihon Shokuhin Kako (Tokyo, Japan) and recrystallized from water. Starburst PAMAM dendrimers (ethylenediamine core, G3, terminal amino groups = 32, molecular weight = 6,909) and asialofetuin (AF, type 1), a ligand of ASGP-R,18 were purchased from Aldrich Chemical (Tokyo, Japan). p-Toluenesulfonyl chloride and lactose monohydrate were purchased from Nakalai Tesque (Kyoto, Japan). Sodium cyanotrihydroborate was obtained from Wako Pure Chemical Industries (Osaka, Japan). Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Nissui Pharmaceuticals (Tokyo, Japan). Opti-MEM Reduced-Serum Medium, BLOCK-iT Alexa Fluor Red Fluorescent Oligo, TrypLE Express with Phenol Red, TRIzol Reagent, fatal bovine serum (FBS) and Lipofectamine 2000 were purchased from Invitrogen (Tokyo, Japan). siTTR and scramble siRNA (control siRNA) were provided by Alnylam Pharmaceuticals (Cambridge, MA). The siTTR sequences used in the present studies are as follows: human TTR sense, 5′GGAUUUCAUGUAACCAAGAdTdT-3′; human TTR antisense, 5′-UCUUGGUUACAUGAAAUCCdTdT-3′; murine TTR sense, 5′-CAGUGUUCUUGCUCUAUAAdTdT-3′; and murine TTR antisense, 5′-UUAUAGAGCAAGAACACUGdTdT-3′. JetPEI-Hepatocyte, a commercially available hepatocyte-selective PEI-based transfection reagent, was obtained from Polyplus transfection (Bioparc, France). Other chemicals and solvents were of analytical reagent grade. Preparation of Lac-α-CDE. α-CDE (G3, DS 2.4) and Lacα-CDE (G2, DSL 2.6) were prepared as previously reported.16,19 Lactose residues were attached to primary amino residues of α-CDE (Figure 1), i.e., 1 mL of borate buffer (pH 7.5) containing α-CDE (G3, DSL 1.2, 19.8 mg; DSL 2.6, 20.0 mg; DSL 4.1, 21.7 mg; DSL 6.1, 23.4 mg) and lactose monohydrate (DSL 1.2, 1.9 mg; DSL 2.6, 4.1 mg; DSL 4.1, 7.3 mg; DSL 6.1, 11.3 mg) and sodium cyanotrihydroborate (DSL 1.2, 3.5 mg; DSL 2.6, 7.1 mg; DSL 4.1, 12.8 mg; DSL 6.1, 19.7 mg) were mixed at 25 °C for 3 h. The DSL values of the conjugates were determined from the integral values of anomeric protons of α-CyD and lactose in 1H NMR spectra. Lac-α-CDEs (G3) (Figure 1) were purified by gel filtration (TOSOH TSKGel HW-40S, Tokyo, Japan) and ethanol precipitation. Cell Culture. HepG2 cells, a human hepatocellular carcinoma cell line, were obtained from Riken Bioresource Center (Tsukuba, Japan). HepG2 cells were grown in DMEM, containing 1 × 105 mU/mL of penicillin, 0.1 mg/mL of

Figure 1. Preparation pathway of Lac-α-CDE (G3).

streptomycin supplemented with 10% FBS at 37 °C in a humidified 5% CO2 and 95% air atmosphere. In Vitro RNAi Effect. The complexes of Lac-α-CDEs (G3)/ siRNA and jetPEI-Hepatocyte/siRNA were prepared at various charge ratios (carriers/siRNA). The concentrations of siRNA for the complex preparation were 100, 200, 400, and 1000 nM. The siRNA complex with Lac-α-CDEs (G3) was then allowed to stand for 15 min at room temperature. The cells (1 × 105/ wells of a 24-well plate) were seeded 24 h before transfection, and then washed twice with serum-free medium. Fifty microliters of Opti-MEM containing the siRNA complex and 244 μL of Opti-MEM in the absence and presence of AF as a competitor were added to each well, and then incubated at 37 °C for 3 h. Six microliters of FBS was add to each well (final FBS concentration was 2%), and then incubated at 37 °C for 21 h. After incubation, total RNA was isolated from the cells using TRIzol Reagent according to the manufacturer’s protocol. The RNA (0.5 μg) was subsequently reverse-transcribed using PrimeScript RT reagent kit (Takara Bio, Shiga, Japan). Real Time Polymerase Chain Reaction (Real-Time PCR). Real-time PCR was performed on a Light Cycler 480 (Roshe, Penzberg, Germany) using 1 μL of cDNA for each sample. SYBR Premix Dimer Eraser (Takara Bio, Shiga, Japan) was used to detect products, and 10 μM concentrations of the following primers were used: human TTR forward, 5′1646

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Figure 2. In vitro RNAi effects of siRNA/Lac-α-CDEs (G3) complexes. (A) Inhibitory effects of complexes of various carriers/siTTR on human TTR mRNA expression in HepG2 cells. The siRNA concentration was 100 nM. The charge ratio of Lac-α-CDEs (G3, DSL 1.2, 2.6, 4.1 and 6.1)/siRNA was 50. The charge ratio of jetPEI-Hepatocyte/siRNA was 8. The level of TTR mRNA expression in scrambled RNA (control siRNA) sample was set at 100%. Each value represents the mean ± SEM of 3 experiments. *p < 0.05, compared with siRNA (control)/Lac-α-CDE. (B) Inhibitory effects of complexes of Lac-α-CDE (G3, DSL 1.2)/siTTR on human TTR mRNA expression in HepG2 cells. The siRNA concentrations were 100, 200, 400, and 1000 nM. The charge ratio of carrier/siRNA was 50. Each value represents the mean ± SEM of 3 experiments. *p < 0.05, compared with control siRNA/Lac-α-CDE (G3, DSL 1.2). (C) Effect of charge ratio of Lac-α-CDE (G3, DSL 1.2)/siTTR complexes on human TTR mRNA expression in HepG2 cells. The siRNA concentration was 400 nM. The charge ratios of carrier/siRNA were 2, 5, 10, 20, 50, and 100. Each value represents the mean ± SEM of 3 experiments. *p < 0.05, compared with siRNA (control)/Lac-α-CDE (G3, DSL 1.2). (D) Western blot analysis of human TTR from HepG2 cells in the supernatant. Human TTR protein expression in the supernatant was determined 24 h after transfection with Lac-α-CDE (G3, DSL 1.2)/siTTR complex. The siRNA concentration was 400 nM. The charge ratio of carriers/siRNA was 50. The TTR-silencing effects of siTTR were determined by Western blotting. Intensity was calculated by ImageJ. Each value represents the mean ± SEM of 5 experiments. *p < 0.05, compared with control siRNA.

amount of cDNA in each sample was normalized using GAPDH, and the melt curve was used to verify specificity. Western Blotting. After transfection, the supernatant was collected and added into Laemmli sample buffer (Bio-Rad Laboratories, Tokyo, Japan), subjected to sodium dodecyl sulfate−polyacrylamide gel electrophoresis, and transferred to nitrocellulose membranes (Bio-Rad Laboratories, Tokyo, Japan). The membranes were blocked by incubation with 2.5% skim milk in PBS containing 0.1% Tween-20. A rabbit polyclonal anti-human prealbumin (Dako, Glostrup, Denmark) diluted 1:1000 was used as primary antibodies. Secondary antibody used was polyclonal goat anti-rabbit immunoglobulin

CATTCTTGGCAGGATGGCTTC-3′; human TTR reverse, 5′-CTCCCAGGTGTCATCAGCAG-3′; human GAPDH forward, 5′-GCACCGTCAAGGCTGAGAAC-3′; human GAPDH reverse, 5′-ATGGTGGTGAAGACGCCAGT-3′; human TNFα forward, 5′-CCAGGCAGTCAGATCATCTTC-3′; human TNFα reverse, 5′-TGAGGTACAGGCCCTCTGAT-3′; murine TTR forward, 5′-CATGAATTCGCGGATGTG-3′; murine TTR reverse, 5′-GATGGTGTAGTGGCGATGG-3′; murine β-actin forward, 5′-TTGGCATAGAGGTCTTTACGGA-3′; and murine β-actin reverse, 5′-GCACCACACCTTCTACAATGAG-3′. PCR was set at 95 °C initially for 30 s, followed by 40 cycles of 95 °C × 5 s, 55 °C × 30 s, 72 °C × 30 s. The relative 1647

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injected from tail vain with 500 μL of a 5% mannitol solution containing the siRNA complex of Lac-α-CDE (G3, DSL 1.2) at the charge ratios of 2 and 20 (carrier/siRNA) at the amount of 20 μg (1 mg/kg) or 180 μg (9 mg/kg) of siRNA in 30 s under anesthesia with ether. Fourty-eight hours after intravenous administration, the mice were sacrificed, and liver was isolated and snap frozen in liquid nitrogen. One milliliter of TRIzol Reagent was added to the Lysing Matrix D (MP Biomedicals Japan, Tokyo, Japan), and then the tissues were frozen. The sample was homogenized using a FastPrep instrument for 20 s at speed setting of 6.0 and then was centrifuged at 12,000 rpm for 5 min at 4 °C. The upper phase of the sample was transferred to a new tube in order to avoid the matrix and debris. Total RNA was isolated from the cells using TRIzol Reagent according to the manufacturer’s protocol, and RNA (0.5 μg) was subsequently reverse-transcribed using PrimeScript RT reagent kit (Takara Bio, Shiga, Japan). Blood samples were taken from the vital artery 48 h after intravenous injection of siRNA complex with Lac-α-CDE (G3, DSL 1.2) at a charge ratio of 20 (carrier/siRNA) at the amount of 20 μg of siRNA. After centrifugation, serum was collected, and creatinine (CRE), blood urea nitrogen (BUN), aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) values were determined by a clinical chemistry analyzer, JCA-BM2250 (JEOL, Tokyo, Japan). Data Analysis. Data are given as the mean ± SEM. Statistical significance of mean coefficients for the studies was performed by analysis of variance followed by Scheffe’s test. p values for significance were set at 0.05.

horseradish peroxidase (Dako, Glostrup, Denmark) diluted 1:1000. Western blotting was visualized by using ECL Plus Western Blotting Detection Reagents (Amersham plc, Amersham, U.K.) according to the manufacturer’s instructions. Cellular Association. Cellular association of the complex was determined by flow cytometry. Four micrograms of BLOCK-iT Alexa Fluor Red Fluorescent Oligo was mixed with Lac-α-CDE (G3, DSL 1.2) or α-CDE (G3) at a charge ratio of 50 (carrier/siRNA). After transfection with the complex of BLOCK-iT Alexa Fluor Red Fluorescent Oligo/Lac-α-CDE (G3, DSL 1.2), the complex of BLOCK-iT Alexa Fluor Red Fluorescent Oligo/α-CDE (G3) or the complex of BLOCK-iT Alexa Fluor Red Fluorescent Oligo/Lipofectamine 2000 for 1 h in HepG2 cells, the cells were washed with PBS (pH 7.4) twice and immediately scraped with 1 mL of PBS (pH 7.4). The cells were collected and filtered through nylon mesh. Data were collected for 1 × 104 cells on a FACSCalibur flow cytometer using CellQuest software (Becton-Dickinson, Mountain View, CA). Fluorescence Microscopy. To observe the cellular association of BLOCK-iT Alexa Fluor Red Fluorescent Oligo and Lac-α-CDE (G3, DSL 1.2), HepG2 cells (1 × 105 cells/ dish) were incubated with the BLOCK-iT Alexa Fluor Red Fluorescent Oligo complex with Lac-α-CDE (G3, DSL 1.2) for 24 h. After incubation, 1.5 μL of Hoechst33342 (DOJINDO, Kumamoto, Japan) was added to each well and incubated at 37 °C for 10 min. The cells were rinsed with PBS (pH 7.4) twice, and added 500 μL of PBS (pH 7.4). Cells were observed by a fluorescence microscope (KEYENCE Biozero BZ-8000, Tokyo, Japan). Cytotoxicity. The effect of the siRNA complex with Lac-αCDE (G3, DSL 1.2) or jetPEI-Hepatocyte on cell viability was measured as reported previously.19 The transfection was performed as described in the transfection section. After washing twice with Hanks balanced salt solutions (HBSS, pH 7.4) to remove siRNA and/or various carriers, 270 μL of fresh HBSS and 30 μL of the WST-1 reagent were added to each well and incubated at 37 °C for 30 min. The absorbance of the solution was measured at 450 nm, with referring absorbance at 655 nm, with a Bio-Rad model 550 microplate reader (Bio-Rad Laboratories, Tokyo, Japan). Interaction between siRNA and Lac-α-CDE (G3, DSL 1.2). Electrophoretic mobility of the siRNA complexes with Lac-α-CDE (G3, DSL 1.2) was measured using a gel electrophoresis system. Various amounts of Lac-α-CDE (G3, DSL 1.2) were mixed with 0.5 μg of siRNA in HBSS (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% agarose gel (include 0.1 μg/mL ethidium bromide) using the Mupid system (Cosmo Bio, Tokyo, Japan) at 100 V for 40 min. The siRNA bands were visualized using an UV illuminator. Physicochemical Properties of siRNA Complexes. The solutions containing Lac-α-CDE (G3, DSL 1.2) at various charge ratios were added to Tris-HCl buffer (10 mM, pH 7.4) containing 5 μg of siRNA. The solution was then incubated for 15 min. The ζ-potential value and particle size of the polyplex of Lac-α-CDE (G3, DSL 1.2) were determined by dynamic light scattering using a Zetasizer Nano (Malvern Instruments, Worcestershire, U.K.). The dynamic light scattering was analyzed by the general purpose mode. The measurements were carried out at least in triplicate. In Vivo RNAi Effects and Blood Chemistry Data. Fourweek-old BALB/c male mice (ca. 20 g) were intravenously

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RESULTS RNAi Effect of Lac-α-CDEs (G3)/siRNA Complexes. To investigate the effect of the number of the lactose moiety in Lac-α-CDEs (G3, Figure 1) on the RNAi effect, TTR mRNA expression after transfection of the complex in HepG2 cells was determined (Figure 2A). We used two siRNAs, such as siTTR, a target siRNA against TTR mRNA, and control siRNA, a scramble RNA. When siTTR/jetPEI-Hepatocyte complex was transfected to cells, the RNAi effect was not observed. An additional attachment of the lactose residue to α-CDE (G3) with DSL value of 1.2 (Lac-α-CDE (G3, DSL 1.2)) elicited much more RNAi effect than the other Lac-α-CDEs (DSL 2.6, 4.1 and 6.1) in HepG2 cells, indicating that Lac-α-CDE (G3, DSL 1.2) had the greatest RNAi effect among all of the Lac-αCDEs (G3). The expression of TTR mRNA levels was suppressed in a siTTR-concentration dependent manner, and approximately 50% of the control siRNA level at the concentration of 400 nM siTTR was shown (Figure 2B). Of various charge ratios, the siTTR complex provided the highest RNAi effect at a charge ratio of 50/1 (Lac-α-CDE (G3, DSL 1.2)/siTTR) (Figure 2C). Likewise, the expression of the TTR protein level in the cell culture supernatant was downregulated about 50% by transfection with the Lac-α-CDE (G3, DSL 1.2)/ siTTR complex, compared to that with control siRNA (Figure 2D), suggesting that Lac-α-CDE (G3, DSL 1.2)/siTTR complex had the RNAi effect against TTR gene expression in vitro. To investigate the asialoglycoprotein receptor (ASGP-R)mediated siTTR transfer of Lac-α-CDE (G3, DSL 1.2), we examined the effect of competitor on TTR mRNA expression in HepG2 cells. Here, AF was used as an ASGP-R competitor.18 As shown in Figure 3, the RNAi effect was inhibited by the 1648

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Alexa Fluor Red Fluorescent Oligo complex with α-CDE (G3), which is lacking lactose moieties, was transfected to cells, cellular uptake of Alexa Fluor Red Fluorescent Oligo did not change in the absence and presence of AF (Figure 4B). Meanwhile, cellular uptake of Alexa Fluor Red Fluorescent Oligo in the Lac-α-CDE (G3, DSL 1.2) complex system decreased in a AF-dose dependent manner (Figure 4A). The RNA-induced silencing complex (RISC) is acknowledged to localize in cytoplasm.20 We next investigated the intracellular localization of Alexa Fluor Red Fluorescent Oligo in HepG2 cells using a fluorescence microscope (Figure 5). Most recently, Figure 3. Effect of asialofetuin (AF) of Lac-α-CDE (G3, DSL 1.2)/ siTTR complex on human TTR mRNA expression in HepG2 cells. The siRNA concentration was 400 nM. The charge ratio of carrier/ siRNA was 50. The concentration of asialofetuin was 0.5 mg/mL. The level of TTR expression in control siRNA sample was set at 100%. Each value represents the mean ± SEM of 3 experiments. *p < 0.05, compared with control siRNA/Lac-α-CDE (G3, DSL 1.2).

addition of AF, indicating that Lac-α-CDE (G3, DSL 1.2) had the ASGP-R-mediated siRNA transfer activity. Cellular Uptake and Intracellular Distribution of Lacα-CDE (G3, DSL 1.2)/siRNA Complex. To gain insight into the mechanism for the ASGP-R-mediated sufficient RNAi effects caused by the complex of Lac-α-CDE (G3, DSL 1.2)/ siRNA, we examined the cellular uptake of Alexa Fluor Red Fluorescent Oligo 1 h after transfection of the complex of Lacα-CDE (G3, DSL 1.2)/Alexa Fluor Red Fluorescent Oligo in HepG2 cells by a flow cytometric analysis (Figure 4). When

Figure 5. Intracellular localization of Alexa Fluor Red Fluorescent Oligo complexes with α-CDE (G3) (A) and Lac-α-CDE (G3, DSL 1.2) (B) in HepG2 cells. The cells were determined by a fluorescence microscope. Incubation time was 24 h. The amount of Alexa Fluor Red Fluorescent Oligo was 0.4 μg. The charge ratio (carrier/Alexa Fluor Red Fluorescent Oligo) was 50. Final Alexa Fluor Red Fluorescent Oligo concentration was 0.1 μM. Scale bar = 20 μm.

we reported that both α-CDE (G3) and siRNA were uniformly observed in cytoplasm 1 h after transfection, and then were gradually accumulated in the periphery of the nucleus until 24 h after transfection.21 Consistent with the previous report, Alexa Fluor Red Fluorescent Oligo was localized in cytoplasm by using α-CDE (G3) (Figure 5A). A similar result was observed in the Lac-α-CDE (G3, DSL 1.2) system (Figure 5B). Cytotoxicity of Lac-α-CDE (G3, DSL 1.2)/siTTR Complex. The drawbacks of siRNA complexes with cationic carriers are known to be cytotoxicity and interferon response.22 Therefore, we evaluated cytotoxicity of the siRNA/carrier complexes in HepG2 cells by the WST-1 method. The complex of α-CDE (G3)/siTTR or Lac-α-CDE (G3, DSL 1.2)/siTTR showed negligible cytotoxicity in HepG2 cells up to a charge ratio of 100/1 (carriers/siTTR) (Figure 6). In sharp contrast, the siTTR complex with jetPEI-Hepatocyte elicited cytotoxicity in a charge ratio-dependent manner (Figure 6), evidently indicating that Lac-α-CDE (G3, DSL 1.2) was a safe carrier for in vitro siRNA delivery, compared with the commercial transfection reagents used in this study. Physicochemical Properties of Lac-α-CDE (G3, DSL 1.2)/siRNA Complex. To clarify physicochemical properties of their siRNA complexes, we examined the complex formation between Lac-α-CDE (G3, DSL 1.2) and siRNA using an agarose gel electrophoresis. As shown in Figure 7, the band intensity derived from siRNA decreased as the charge ratio of Lac-α-CDE (G3, DSL 1.2)/siRNA increased, and the band disappeared at a charge ratio of 2 (carrier/siRNA). In the case of α-CDE (G3, DSL 2.4), the bands vanished at a charge ratio of 2 (carrier/siRNA) (data not shown). At lower charge ratios,

Figure 4. Effect of asialofetuin (AF) on cellular uptake of Alexa Fluor Red Fluorescent Oligo complexes with Lac-α-CDE (G3, DSL 1.2) (A) and α-CDE (G3) (B) in HepG2 cells. The fluorescence intensities of Alexa Fluor Red Fluorescent Oligo in HepG2 cells 1 h after incubation were determined by a flow cytometer. The charge ratio (carrier/Alexa Fluor Red Fluorescent Oligo) was 50. Final Alexa Fluor Red Fluorescent Oligo concentration was 100 nM. The concentrations of AF were 0.5, 1.0, and 2.0 mg/mL. Each figure shows the representative of three independent experiments. 1649

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polydispersity index values of the siRNA complex with Lac-αCDE (G3, DSL 1.2) were somewhat high, compared to a charge ratio of 2 (Table 1). The ζ-potential value of the siRNA complexes with Lac-α-CDE (G3, DSL 1.2) increased at a charge ratio of 2 and was approximately 5.2 mV. Meanwhile, the ζ-potential values of the siRNA complexes increased at the charge ratios of 20, 50 and 100, compared to that at a charge ratio of 2 (Table 1). In Vivo RNAi Effects of Lac-α-CDE (G3, DSL 1.2)/siTTR Complex. We examined the in vivo RNAi effects of siRNA complex in mice. Figure 8 shows the inhibitory effects of the Figure 6. Cytotoxicity of the siTTR complexes with various carriers in HepG2 cells. Cells were incubated with carriers/siTTR complexes for 24 h. Cell viability was assayed by the WST-1 method. The siRNA concentration was 100 nM. Culture medium was supplemented with Opti-MEM (2% FBS). Each point represents the mean ± SEM of 3−4 experiments. *p < 0.05, compared with α-CDE (G3)/siTTR and Lacα-CDE (G3, DSL 1.2)/siTTR .

Figure 7. Effect of Lac-α-CDE (G3, DSL 1.2) on electrophoretic mobility of siTTR. The solutions containing the siTTR complexes with carriers were incubated for 15 min at room temperature after slight agitation. The electrophoresis was performed at 100 V for about 40 min. The charge ratios of Lac-α-CDE (G3, DSL 1.2)/siTTR were 0.1, 0.5, 1, 2, and 5. The amount of siTTR was 0.5 μg.

Figure 8. In vivo inhibitory effects of complexes of Lac-α-CDE (G3, DSL 1.2)/siTTR on murine TTR mRNA expression in BALB/c mice. Murine TTR mRNA expression level was determined 48 h after intravenous injections of siRNA complexes with carriers. The charge ratios of carrier/siRNA were 2 and 20. The amounts of siRNA were 20 and 180 μg. The TTR-silencing effects of siTTR were determined by quantitative real-time PCR. The results of real-time PCR for TTR were normalized by β-actin expression levels. The level of TTR expression in control siRNA sample was set at 100%. Each value represents the mean ± SEM of 3−4 experiments. *p < 0.05, compared with control siRNA (180 μg, 9 mg/kg).

the bands derived from siRNA were observed, because a net charge was negative. As the charge ratio increased, the fraction of free siRNA to carriers decreased due to the complex formation with cationic carriers, and then the band disappeared, when siRNA completely formed complexes with cationic carriers (no free siRNA), because the net charge was shifted from negative to positive. Next, we determined the particle sizes and ζ-potential values of the siRNA complex with Lac-α-CDE (G3, DSL 1.2) (Table 1). The mean diameters of the complexes with Lac-α-CDE (G3, DSL 1.2) decreased at the charge ratios of 20, 50 and 100, compared to that at a charge ratio of 2. The mean diameters of the siRNA complex with Lac-α-CDE (G3, DSL 1.2) were 21.3 nm at a charge ratio of 50 (carrier/siRNA), although the

complexes of Lac-α-CDE (G3, DSL 1.2)/siTTR on TTR mRNA expression in BALB/c mice 48 h after intravenous administration. From the safety point of view, we selected the relatively low charge ratios of 2 and 20 for in vivo experiments because of concern that the Lac-α-CDE (G3, DSL 1.2)/siRNA complex administered intravenously at the charge ratio of 40 shows the off-target effects and cytotoxicity. The Lac-α-CDE (G3, DSL 1.2)/siTTR complexes at a charge ratio of 2 or 20 at the dose of 1 mg/kg siRNA lowered TTR mRNA expression to 93% and 89% of Lac-α-CDE (G3, DSL 1.2)/control siRNA complexes, respectively (Figure 8). The Lac-α-CDE (G3, DSL 1.2)/siRNA complex at a charge ratio of 2 (carrier/siRNA) at a dose of 9 mg/kg siRNA significantly impaired TTR mRNA expression in the liver, compared to Lac-α-CDE (G3, DSL 1.2)/control siRNA complex. These results strongly suggest that the Lac-α-CDE (G3, DSL 1.2)/siRNA complex has the potential to induce the in vivo RNAi effect after intravenous administration in the liver of mice. From the viewpoint of safety, we measured blood chemistry values after intravenous administration of the siRNA complex with Lac-α-CDE (G3, DSL 1.2) (Table 2). In the present study, blood chemistry values such as CRE, BUN, AST, ALT and LDH in the α-CDE

Table 1. Particle Sizes and ζ-Potentials of siRNA Complexes with Lac-α-CDE (G3, DSL 1.2)a carrier Lac-α-CDE (G3, DSL 1.2)

charge ratio (carrier/ siRNA) 2 20 50 100

mean diam (nm) 187.0 29.2 21.3 38.5

± ± ± ±

12.0 4.6 4.8 3.4

PDIb 0.28 0.64 0.44 0.40

± ± ± ±

0.009 0.059 0.200 0.300

ζ-potential (mV) 5.2 14.0 7.2 15.2

± ± ± ±

2.4 5.9 2.7 1.3

a The particle sizes and the ζ-potentials were measured by a Zetasizer Nano. The siTTR complexes with carriers were added to Tris-HCl buffer (10 mM, pH 7.4). The concentration of siRNA was 5 μg/mL. Each value represents the mean ± SEM of 3 experiments. b Polydispersity index.

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Table 2. Blood Chemistry Data after Intravenous Injection of the siTTR Complexes with α-CDE (G3) and Lac-α-CDE (G3, DSL 1.2) in BALB/c Mice

a

carrier

CREa (mg/dL)

BUNb (mg/dL)

ASTc (U/L)

ALTd (U/L)

LDHe (U/L)

control α-CDE (G3) Lac-α-CDE (G3, DSL1.2)

0.11 ± 0.01 0.10 ± 0.01 0.12 ± 0.01

22.8 ± 2.08 21.3 ± 2.29 32.5 ± 5.16

65.8 ± 13.2 39.8 ± 1.69 59.5 ± 8.15

34.0 ± 8.61 23.2 ± 2.28 21.3 ± 1.96

388 ± 79.7 228 ± 18.6 300 ± 44.5

Creatinine. bBlood urea nitrogen. cAspartate aminotransferase. dAlanine aminotransferase. eLactate dehydrogenase.

prepared Lac-dendrimer (G3, DSL 1.2), which is lacking an αCyD molecule, and evaluated the RNAi effect. The RNAi effect of Lac-dendrimer (G3, DSL 1.2) was significantly lower than that of Lac-α-CDE (G3, DSL 1.2) in HepG2 cells (data not shown), suggesting a crucial role of α-CyD for the RNAi effect in vitro. The remarkable RNAi effects of Lac-α-CDE (G3, DSL 1.2)/ siRNA complex may be due to the efficient ASGP-R mediated cellular uptake (Figure 4), the endosomal escaping effect of Lac-α-CDE (G3, DSL 1.2) (Figure 5), cytoplasmic distribution of siRNA (Figure 5) and negligible cytotoxicity (Figure 6). In particular, the enhancing effect of Lac-α-CDE (G3, DSL 1.2) on endosomal escape may result from the cooperative effect of a proton sponge effect of dendrimer (G3) and the inclusion ability of α-CyD with phospholipids in endosomal membrane as reported previously.28 In turn, this high endosomal escaping effect of Lac-α-CDE (G3, DSL 1.2) may increase transfer of the siRNA complex into cytoplasm. In fact, this presumption may be supported by the fact that the siTTR complex with Lac-αCDE (G3, DSL 1.2) induced higher RNAi effect rather than the siTTR complex with Lac-dendrimer (G3, DSL 1.2), which is the lack of an α-CyD molecule (data not shown). Meanwhile, we previously reported that Lac-α-CDE (G2, DSL 2.6)/pDNA complex entered the nucleus by recognition of nuclear lectins.16 In the present study, however, siRNA complex with Lac-α-CDE (G3, DSL 1.2) was localized in cytoplasm. Here, both DNA and siRNA are double stranded nucleic acids, but there are several fundamental structural differences between them (e.g., base pair, length, conformation).24 Therefore, this difference in intracellular distribution between pDNA and siRNA complexes with Lac-α-CDE (G2, DSL 2.6) may be attributed to a lack of binding to nuclear lectin. However, there is no evidence on this speculation. Thereafter, a detailed study should be performed. Meanwhile, the RNAi effects of Lac-α-CDE (G3, DSL 1.2)/ siRNA complex depend on an siRNA concentration and a charge ratio (Figure 2). In fact, the RNAi effect was maximal at a charge ratio of 50, when the concentration of siRNA reached 1000 nM (Figure 2B, C). The existence of this optimal value in the RNAi effects could be attributed to the stable complex formation,29 efficient cellular uptake (Figure 4), marked endosome escaping ability and negligible cytotoxicity. However, the concentrations of 400 and 1000 nM siRNA used the in vitro study are too high for siRNA based gene silencing. Thereafter, it is necessary that the RNAi effects should be increased by a further optimization of the chemical structure of Lac-α-CDE (G3, DSL 1.2). It is well-known that siRNA was easy to degradate after intravenous administration by RNase in serum. Therefore, we investigated the stability of the Lac-α-CDE (G3, DSL 1.2)/ siRNA complex in the presence of serum. The siRNA without complexation with Lac-α-CDE (G3, DSL 1.2) was degraded in the presence of 50% of FCS at 37 °C for 5 h (data not shown). Meanwhile, the siRNA which forms a complex with Lac-α-CDE

(G3) and Lac-α-CDE (G3, DSL 1.2) systems were almost equivalent to those in the control system (5% mannitol solution). These results suggest that the siRNA complex with Lac-α-CDE (G3, DSL 1.2) has a good safe profile in vivo, too.

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DISCUSSION We previously demonstrated that Lac-α-CDE (G2, G3) is likely to be useful for hepatocyte-selective pDNA carrier in the pDNA/carrier system, because the pDNA complex with Lac-αCDE (G3, DSL 1.2) has efficient gene transfer activity in the hepatocyte with negligible cytotoxicity.16,17 However, it is known that an optimized siRNA formulation procedure is quite different from the pDNA formulation, and parameters such as toxicity and efficacy can be controlled by the development of specific protocols for carrier/siRNA complex formation.23,24 Thereby, in the present study, we investigated the potential use of Lac-α-CDEs (G3, DSL 1.2, 2.6, 4.1, 6.1) as an siRNA carrier in the siRNA/carrier complex system in vitro and in vivo. We confirmed that the RNAi effects of Lac-α-CDE (G3, DSL 1.2)/ siRNA complex on endogenous genes such as TTR had emerged, and the effects of Lac-α-CDE (G3, DSL 1.2)/siRNA complex were superior to those of siRNA complexes with jetPEI-Hepatocyte, a commercially available hepatocyte-selective transfection reagent (Figure 2A−C). In the present study, the RNAi effect of Lac-α-CDE (G3, DSL 1.2) was much higher than that of the other Lac-α-CDEs (DSL 2.6, 4.1 and 6.1) in HepG2 cells, suggesting that the low siRNA transfer activity of Lac-α-CDEs with higher DSL values could be ascribed to a weak interaction with siRNA through decrease in the number of a positive charge of the carrier molecule. In fact, we confirmed that the cellular uptake of Lacα-CDE (G3, DSL 1.2)/Alexa Fluor Red Fluorescent Oligo was higher than that of other Lac-α-CDEs (DSL 2.6, 4.1 and 6.1) with a flow cytometry and a fluorescence microscope (data not shown). Furthermore, Western blotting analysis revealed that Lac-α-CDE (G3, DSL 1.2)/siRNA complex suppressed TTR protein expression (Figure 2D). In the current study, there are several reports on successful siRNA delivery in vivo by using cholesterol-functionalized siRNAs and lipid-based nanoparticles, some of which are being evaluated in clinical trials.25,26 However, some of them have their limitations, e.g. activation of toll-like receptors (TLRs), lack of tissue-specific delivery and tissue toxicity. Our findings suggest that Lac-α-CDE (G3, DSL 1.2)/siRNA complex can inhibit TTR gene expressions and selectively deliver siRNA to hepatocytes. Thus, siTTR/Lac-αCDE (G3, DSL 1.2) complex is expected to be the potential new application to siRNA-based FAP therapy. We previously reported that the superior gene transfer activity of α-CDE (G2) to dendrimer (G2) could be attributed to the enhancing endosomal escape of the pDNA complex through the endosomal membrane-disrupting ability of αCyD.27 To examine the role of α-CyD moiety in the Lac-αCDE (G3, DSL 1.2) molecule on siRNA transfer activity, we 1651

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(G3, DSL 1.2) was significantly stabilized as the charge ratio (carrier/siRNA) was increased (data not shown), probably due to the protection of siRNA against RNase through its complex formation with Lac-α-CDE (G3, DSL 1.2). These results suggest that Lac-α-CDE (G3, DSL 1.2) may improve the stability of siRNA after intravenous administration. Lac-α-CDE (G3, DSL 1.2) could be useful as an siRNA carrier for systemic application in vivo. The Lac-α-CDE (G3, DSL 1.2)/siRNA complex exerted the in vivo RNAi effect in hepatocyte after intravenous administrations (Figure 8). In the present in vivo experiments, we selected the relatively low charge ratios of 2 and 20 from the safety point of view, because it was supposed that the Lac-α-CDE (G3, DSL 1.2)/siRNA complex administered intravenously at the charge ratio of 40 shows off-target effects and cytotoxicity. Thereafter, further investigation of the in vivo RNAi effects of the complex at higher charge ratios which show no cytotoxicity is required. Regarding the potent in vivo silencing effect at charge ratio, it is estimated that the difference in the RNAi effects between in vitro and in vivo was caused by the difference in the amount of siRNA (Figures 2 and 8), because the amounts of siRNA used in the in vitro and in vivo studies were 1.6 μg at a concentration of 400 nM and 180 μg at a dose of 9 mg/kg, respectively. Importantly, the blood chemistry values did not change after intravenous injection of Lac-α-CDE (G3, DSL 1.2)/siRNA complex (Table 2). This in vivo safe profile of Lac-α-CDE (G3, DSL 1.2)/siRNA complex is highly likely to be consistent with the in vitro negligible cytotoxicity (Figure 6). Roberts et al. reported that cytotoxicity of dendrimers (G3, G5 and G7) to Chinese hamster lung fibroblasts increased as the generation of dendrimer increased.30 However, in the Lac-α-CDE system, we used low generation (G3) dendrimer, and the number of positively charged amino groups was decreased by an introduction of α-CyD and lactose to the dendrimer molecule. For this reason, the Lac-α-CDE (G3, DSL 1.2)/siRNA complex exhibited safe profiles in vitro and in vivo. However, it should be noted that the RNAi effect of Lac-αCDE (G3, DSL 1.2)/siRNA complex was not sufficient, although the RNAi effect was statistically significant compared to the effect of control (Figure 8). Thereby, further investigations regarding more efficient siRNA delivery and the RNAi effect after intravenous administration of Lac-α-CDE (G3, DSL 1.2)/siTTR complex should be necessary. Now, we have been preparing and evaluating the potential of pegylated Lac-α-CDE (G3) in order to increase the complex stability and half-life in blood circulation. Our findings provided that the Lac-α-CDE (G3, DSL 1.2)/ siRNA complex elicited prominent RNAi effects against TTR gene with efficient cellular uptake, enhanced endosomal escape, distribution in cytoplasm and negligible cytotoxicity of the Lacα-CDE (G3, DSL 1.2)/siRNA complex. Importantly, the preferable RNAi effects and the negligible side effects of Lacα-CDE (G3, DSL 1.2)/siRNA complex were observed after intravenous administration of the siRNA/complex in mice. Taken together, these results suggest that Lac-α-CDE (G3, DSL 1.2) has the potential for a novel hepatocyte-selective siRNA carrier in vitro and in vivo. Therefore, Lac-α-CDE (G3, DSL 1.2)/siTTR complex has possibility as a therapeutic tool for FAP to the liver transplantation.

Article

AUTHOR INFORMATION

Corresponding Author

*Department of Physical Pharmaceutics, Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oehonmachi, Kumamoto 862-0973, Japan. Tel: +81 96 371 4160. Fax: +81 96 371 4420. E-mail: [email protected]. ac.jp. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

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REFERENCES

The authors thank Alnylam Pharmaceuticals for providing the siRNAs. The work described in this paper presents additional results from a Joint Research Project by Kumamoto University and its collaboration partner Alnylam Pharmaceuticals. This work was partially supported by Grant-in-Aid for Scientific Research (C) from Japan Society for the Promotion of Science (16590114, 18590144, 20590037).

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