Dicetyl Phosphate-Tetraethylenepentamine-Based ... - ACS Publications

Nippon Fine Chemical Co. Ltd., 5-1-1 Umei, Takasago, Hyogo 676-0074, Japan. ⊥DDS Research, Formulation Research Laboratories, Eisai Co., Ltd., 5-1-3...
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Dicetyl Phosphate-Tetraethylenepentamine-Based Liposomes for Systemic siRNA Delivery Tomohiro Asai,†,* Saori Matsushita,† Eriya Kenjo,† Takuma Tsuzuku,† Norihito Yonenaga,† Hiroyuki Koide,† Kentaro Hatanaka,† Takehisa Dewa,‡ Mamoru Nango,‡ Noriyuki Maeda,§ Hiroshi Kikuchi,^ and Naoto Oku† †

Department of Medical Biochemistry and Global COE, University of Shizuoka Graduate School of Pharmaceutical Sciences, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan ‡ Department of Life and Materials Engineering, Nagoya Institute of Technology, Gokiso-cho, Showa-ku, Nagoya 466-8555, Japan § Nippon Fine Chemical Co. Ltd., 5-1-1 Umei, Takasago, Hyogo 676-0074, Japan ^ DDS Research, Formulation Research Laboratories, Eisai Co., Ltd., 5-1-3 Tokodai, Tsukuba, 300-2635, Japan

bS Supporting Information ABSTRACT: Dicetyl phosphate-tetraethylenepentamine (DCP-TEPA) conjugate was newly synthesized and formed into liposomes for efficient siRNA delivery. Formulation of DCP-TEPA-based polycation liposomes (TEPA-PCL) complexed with siRNA was examined by performing knockdown experiments using stable EGFP-transfected HT1080 human fibrosarcoma cells and siRNA for GFP. An adequate amount of DCP-TEPA in TEPA-PCL and N/P ratio of TEPA-PCL/ siRNA complexes were determined based on the knockdown efficiency. Then, the biodistribution of TEPA-PCL modified with poly(ethylene glycol) (PEG) was examined in BALB/c mice. As a result, TEPA-PCL modified with PEG6000 avoided reticuloendothelial system uptake and showed long circulation in the bloodstream. On the other hand, PEGylation of TEPA-PCL/ siRNA complexes caused dissociation of a portion of the siRNA from the liposomes. However, we found that the use of cholesterolconjugated siRNA improved the interaction between TEPA-PCL and siRNA, which allowed PEGylation of TEPA-PCL/siRNA complexes without siRNA dissociation. In addition, TEPA-PCL complexed with cholesterol-conjugated siRNA showed potent knockdown efficiency in stable luciferase-transfected B16-F10 murine melanoma cells. Finally, the biodistribution of cholesterolconjugated siRNA formulated in PEGylated TEPA-PCL was examined by performing near-infrared fluorescence imaging in Colon26 NL-17 murine carcinoma-bearing mice. Our results showed that tumor targeting with siRNA via systemic administration was achieved by using PEGylated TEPA-PCL combined with active targeting with Ala-Pro-Arg-Pro-Gly, a peptide used for targeting angiogenic endothelium.

’ INTRODUCTION RNA interference (RNAi) refers to post-transcriptional gene silencing induced by double-stranded RNA.1,2 Small RNAs such as small interfering RNA (siRNA) and microRNA (miRNA) are expected to become novel drug therapeutics for intractable diseases such as cancer.3 Compared with local injection of siRNA drug candidates, systemic injection of them is quite limited at present because of a serious problem of siRNA delivery;4,5 i.e., naked RNA is readily degraded by nucleases and poorly penetrates plasma membrane of target cells. Thus, establishment of an siRNA delivery system is a major challenge for the development of RNAi-based drugs.5 In recent studies, a variety of drug delivery system (DDS) technologies such as liposomes,6 micelles,7 other nanoparticles,8,9 and cholesterol-conjugation10 of siRNA have been investigated for siRNA delivery. Also, useful nonviral r 2011 American Chemical Society

vectors enabling both potent gene silencing in target cells and in vivo delivery to target cells have been awaited to achieve siRNA therapy via systemic administration. We reported earlier that polycation liposomes (PCL), one of the nonviral vectors, possess the advantages of both liposomes and polycations for the delivery of plasmid DNA and siRNA.11-17 Previously, we synthesized cetylated polyethylenimine[1800] (cetyl-PEI[1800]) and polycation (spermidine, spermine or PEI[1800])-dialkyl phosphate conjugates to prepare various types of PCLs.11-13 PCLs are simply prepared with these conjugates through anchoring via the hydrophobic portion of Received: October 25, 2010 Revised: January 26, 2011 Published: March 01, 2011 429

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of cetyl-PEI-PCL complexed with a plasmid DNA encoding luciferase can drive gene expression in the first-pass organs in mice.16 Our previous results also demonstrate that siRNA formulated in cetyl-PEI-PCL can induce efficient gene silencing in vitro.17 However, cetyl-PEI-PCL accumulates in the first-pass organs and reticuloendothelial system (RES) after intravenous injection, which makes it difficult to deliver siRNA to target cells. Our recent study using positron emission tomography (PET) demonstrates that 18F-labeled siRNA formulated in cationic liposomes accumulates in the lungs of mice immediately after intravenous injection via a tail vein.18 Poly(ethylene glycol) (PEG) forms a water shell on the liposomal surface and provides a steric barrier to avoid interactions with plasma proteins, resulting in escape from trapping by the RES.19,20 Therefore, PEGylated liposomes have a property of long circulation in the bloodstream and are useful for drug and gene delivery to inflamed sites such as tumors through the enhanced permeability and retention (EPR) effect.21 An important aspect of PEGylation of liposomes is that it serves for not only RES-avoidance but also improvement of undesirable properties of liposomes.22 In general, PEGylation of cationic liposomes can reduce their nonspecific binding to plasma proteins and improves their biodistribution. However, our previous data indicate that modification with PEG6000 cannot improve the biodistribution of PCL containing PEI[1800]. It is considered that the molecular size of PEI[1800] is too large to improve the undesirable properties of PCL by PEG6000 modification. In the present study, dicetylphosphate-tetraethylenepentaamine (DCP-TEPA, Figure 1A) was newly synthesized and formed into liposomes for efficient siRNA delivery based on our previous results. Furthermore, DCP-TEPA-based PCL (TEPA-PCL) was PEGylated and applied to systemic delivery of siRNA.

’ EXPERIMENTAL PROCEDURES Synthesis of DCP-TEPA. Dicetyl phosphate and tetraethylenepentamine were purchased from Sigma-Aldrich and Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan), respectively. DCPTEPA was synthesized by using a procedure analogous to that reported previously;13,14 i.e., two-step reactions, (i) dimerization of dicetyl phosphate anhydride and (ii) nucleophilic substitution with tetraethylenepentamine, provided the conjugate. Dimerized dicetyl phosphate anhydride (323 mg, 0.3 mmol) in anhydrous pyridine (2.0 mL) was added to a solution of tetraethylenepentamine (351 mg, 1.85 mmol) in 1.0 mL of anhydrous pyridine. The reaction mixture was stirred under nitrogen atmosphere for 4 h at room temperature. After removal of the solvent under reduced pressure, the residue was suspended in distilled water and then filtered to remove any unreacted polyamine. The residue was subjected to column chromatography using aminated silica gel (Chromatorex NH, Fuji Silisia Chemical LTD, Aichi, Japan) with an eluting solution of CHCl3/MeOH (39/1, v/v), giving 129 mg (60%) as a white solid. 1H NMR (600 MHz, CDCl3) δ: 0.88 (t, 6H), 1.25 (s, 52H), 1.34-1.37 (m, 4H), 1.65-1.67 (m, 6H), 2.41-3.24 (bm, 16H), 3.93-4.00 (bm, 4H); MALDI-TOF-MS for (C40H89N5O3P)þ calcd 718.7, found 718.2. Preparation of DCP-TEPA-Based Liposomes for siRNA Delivery. Dipalmitoylphosphatidylcholine (DPPC), cholesterol, PEG2000-distearoylphosphatidylethanolamine (DSPE), PEG6000DSPE, and Ala-Pro-Arg-Pro-Gly (APRPG)-PEG6000-DSPE

Figure 1. Preparation of DCP-TEPA-based liposomes for efficient siRNA delivery. (A) Chemical structure of DCP-TEPA. (B) Effect of DCP-TEPA amount in TEPA-PCL on the knockdown efficiencies. siGFP (25 pmol, N/P = 24 equiv) was formulated in TEPA-PCL composed of DCPTEPA/DOPE/DPPC/cholesterol = X/1/Y/1. DCP-TEPA/DPPC = X/Y is indicated in the figure. (C) Determination of an adequate N/P ratio of TEPA-PCL/siRNA by performing a similar knockdown experiment. (B, C) EGFP/HT1080 cells (4  104 cells/ well) were precultured overnight. TEPA-PCL complexed with siGFP (25 pmol, N/P = 6-24 equiv) was added to these cells and then removed after a 24 h incubation. These cells were subsequently cultured for an additional 24 h. The fluorescence intensities of EGFP were measured and corrected for protein amounts. The control is that of cells not transfected with siGFP. Data are presented as the percentage of control fluorescence intensities with SD bars.

the conjugate. Our previous results indicate that PCLs containing cetyl-PEI[1800] (cetyl-PEI-PCL), dicetyl phosphate (DCP)spermidine or DCP-spermine possess high gene transfer activity without severe cytotoxicity.11,14 A possible mechanism of gene transfer with cetyl-PEI-PCL is internalization of cetyl-PEI-PCL/ plasmid DNA complexes into cells via the endocytic pathway, followed by the release of plasmid DNA from the endosomes through the “proton-sponge” effect of PEI.15 Intravenous injection 430

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were products of Nippon Fine Chemical Co. (Takasago, Hyogo, Japan). Dioleoylphosphatidylethanolamine (DOPE) was purchased from NOF Co. (Tokyo, Japan). Alexa Fluor 750-labeled control siRNA (AF750-siCont) was obtained from Japan Bio Services Co., Ltd. (Saitama, Japan). All other siRNAs including cholesterol-conjugated ones were purchased from Hokkaido System Science Co. (Hokkaido, Japan). The nucleotide sequences of siRNA for GFP (siGFP) with a 2-nucleotide overhang (underline) were 50 -GGCUACGUCCAGGAGCGCACC-30 (sense) and 50 -UGCGCUCCUGGACGUAGCCUU-30 (antisense); for luciferase (siLuc), 50 -CUUACGCUGAGUACUUCGATT-30 (sense) and 50 -UCGAAGUACUCAGCGUAAGTT-30 (antisense); for enhanced luciferase 2 (siLuc2), 50 -GCUAUGGGCUGAAUACAAATT-30 (sense) and 50 -UUUGUAUUCAGCCCAUAGCTT-30 (antisense); and for the control (siCont), 50 CGAUUCGCUAGACCGGCUUCAUUGCAG-30 (sense) and 50 -GCAAUGAAGCCGGUCUAGCGAAUCGAU-30 (antisense). In the case of AF750-siCont, AF750 was conjugated with siCont at the 30 -prime end of the antisense strand. In the case of cholesterol-conjugated siRNA (siRNA-C), cholesterol was conjugated with siRNA at the 30 -prime of the sense strand. DCP-TEPA, DOPE, DPPC, and cholesterol were dissolved in t-butyl alcohol and freeze-dried. TEPA-PCL was produced by hydration of the lipid mixture with RNase-free water. TEPA-PCL was sized by extruding it 10 times through a polycarbonate membrane filter having a pore size of 100 nm (Nuclepore, Maidstone, UK). TEPA-PCL and siRNA (or siRNA-C) were mixed gently and incubated for 20 min at room temperature to form TEPA-PCL/siRNA complexes. The ratio of the nitrogen moiety of TEPA-PCL to the phosphorus one of siRNA (N/P ratio) was varied from 6 to 48 equiv to examine the formulation, and was then fixed at 18 equiv based on the results. For systemic administration, TEPA-PCL (or TEPA-PCL/siRNA-C complexes) was incubated with PEG2000-DSPE, PEG6000-DSPE, or APRPG-PEG6000-DSPE dissolved in RNase-free water at 50 C for 15 min. The percent molar ratio of PEG-conjugates to total lipids was 10% or 20%. Particle size and ζ-potential of TEPAPCL-based siRNA vectors diluted with RNase-free water were measured by using a Zetasizer Nano ZS and MPT-2 (Malvern, Worcs, UK). Free siRNA dissociated from TEPA-PCL was separated by electrophoresis in 15% acrylamide gel and visualized by ethidium bromide staining. TEPA-PCL complexed with siLuc or siLuc-C was used for this gel electrophoresis experiment. Cell Cultures. HT1080 human fibrosarcoma cells constitutively expressing EGFP (EGFP/HT1080 cells) had been previously established23 and were cultured in DME/Ham F12 medium (WAKO, Osaka, Japan) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO), 100 units/ mL penicillin (MP Biomedicals, Irvine, CA), 100 μg/mL streptomycin (MP Biomedicals), and 100 μg/mL Geneticin (SigmaAldrich). A549-luc-C8 Bioware Cell Line, a luciferase-expressing cell line derived from A549 human lung carcinoma cells, and B16-F10-luc2 Bioware Ultra Cell Line, a luciferase-expressing cell line stably transfected with the firefly luciferase gene (Luc2), were purchased from Caliper Life Sciences (Hopkinton, MA). Colon 26 NL-17 colon carcinoma cells, established by Dr. Yamori (Japanese Foundation for Cancer Research, Tokyo, Japan), were kindly provided by Dr. Nakajima (Johnson & Johnson KK, Tokyo, Japan). A549-luc-C8, B16-F10-luc2, and Colon26 NL-17 cells were cultured in DME/Ham F12 medium supplemented with 10% FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin.

Experimental Animals. Five-week-old BALB/c male mice were purchased from Japan SLC Inc. (Shizuoka, Japan). The animals were cared for according to the Animal Facility Guidelines of the University of Shizuoka. All animal experiments were approved by the Animal and Ethics Review Committee of the University of Shizuoka. Evaluation of RNAi Efficiencies in Vitro. EGFP/HT1080 cells were seeded onto 24-well FALCON plates (BD Bioscience, San Jose, CA) at the density of 4  104 cells/well and precultured overnight. Before siRNA transfection, the medium was changed to a fresh one containing 10% FBS but no antibiotics. TEPA-PCL complexed with siGFP (25 pmol, N/P = 6-48 equiv) were added to the medium at a final siRNA concentration of 50 nM and removed after 24 h incubation. These cells were subsequently cultured for an additional 24 h and then lysed with 1% reduced Triton-X100 containing protease inhibitors (2 mM PMSF, 200 μM leupeptin, 50 μg/mL aprotinin, and 100 μM pepstatin A). The fluorescence intensities of EGFP were measured by using a Tecan Infinite M200 microplate reader (Salzburg, Austria) and corrected for protein amounts by using a BCA Protein Assay Reagent Kit (PIERCE Biotechnology, Rockford, IL) according to the manufacturer’s instructions. A549-luc-C8 or B16-F10-luc2 cells were seeded onto 96-well luminunc white plates (Nalge Nunc International, Naperville, IL) at the density of 5  103 cells/well and precultured overnight. Before siRNA transfection, the medium was changed to a fresh one containing 10% FBS but no antibiotics. TEPA-PCL complexed with siLuc, cholesterol-conjugated siLuc (siLuc-C), cholesterol-conjugated siCont (siCont-C), or cholesterolconjugated siLuc2 (siLuc2-C) were added to the medium at a final siRNA concentration of 50-100 nM (10-20 pmol, N/P = 18 equiv) and removed after 24 h incubation. These cells were subsequently cultured for an additional 24 h and used for the luciferase assay. The luminescence intensity and cell viability were measured by using ONE-Glo Luciferase Assay System (Promega, Madison, WI) and CellTiter-Fluor Cell Viability Assay (Promega), respectively, according to the manufacturer’s instructions. B16-F10-luc2 cells were also transfected with siLuc2 mixed with Lipofectamine 2000 (LFA2K, Invitrogen, Rockville, MD) according to the manufacturer’s instructions. Biodistribution of TEPA-PCL in Mice after Intravenous Injection. In order to label TEPA-PCL with a radioisotope, we added a trace amount of [3H] cholesteryl hexadecyl ether (GE Healthcare UK Ltd., Buckinghamshire, England) to the initial tbutyl alcohol solution and freeze-dried it with the other lipids of the TEPA-PCL preparation. Five-week-old BALB/c male mice (n = 5) were injected with [3H]-labeled TEPA-PCL via a tail vein (74 kBq/mouse). Three hours after the injection, these mice were sacrificed under deep anesthesia for collection of the blood. The plasma was obtained by centrifugation (600 g for 5 min). Then, the heart, lungs, liver, spleen, and kidneys were removed, washed with saline, and weighed. The radioactivity in each organ was determined with a liquid scintillation counter (Aloka LSC3100, Tokyo, Japan). The total amount in the plasma was calculated based on the body weight of the mice, where the plasma volume was assumed to be 4.27% of the body weight based on the data for total blood volume.24 Near-Infrared Fluorescence Imaging of siRNA in Vivo. The biodistribution of AF750-siCont-C formulated in TEPA-PCL modified with 10% PEG6000 (PEG-TEPA-PCL) or 10% APRPG-PEG6000 (APRPG-TEPA-PCL) was assessed by near-infrared fluorescence (NIRF) imaging with a Xenogen IVIS 431

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Table 1. Particle Size and ζ-Potential of TEPA-PCL-Based Nanoparticlesa size (nm) PDI ζ-potential (mV) 134

0.172

þ52.5

TEPA-PCL modified with 10% PEG2000 107 TEPA-PCL modified with 20% PEG2000 133

0.154 0.011

-6.92 -13.1

TEPA-PCL

TEPA-PCL modified with 10% PEG6000 104

0.269

-6.01

TEPA-PCL modified with 20% PEG6000 135

0.402

-3.60

TEPA-PCL/siCont-C

0.121

PEG-TEPA-PCL/siCont-C APRPG-TEPA-PCL/siCont-C

139

98.1 0.234 121

0.190

þ46.2 þ2.23 -0.05

Particle size and ζ-potential of TEPA-PCL-based nanoparticles diluted with RNase-free water were measured by using the Zetasizer Nano ZS. Lipid composition of TEPA-PCL is DCP-TEPA/DOPE/DPPC/cholesterol = 0.25/1/0.75/1 as a molar ratio. N/P ratio of TEPA-PCL/ siCont-C is 18 equiv. PDI: polydispersity index.

a

Lumina System coupled to Living Image software for data acquisition (Xenogen Corp., Alameda, CA). Colon26 NL-17 cells (1.0  106 cells/mouse) were implanted subcutaneously into the foot of 5-week-old BALB/c male mice. These mice were fed an alfalfa-free feed (Oriental Yeast Co. Ltd., Tokyo, Japan) to reduce the influence of background fluorescence. Ten days after tumor implantation, the carcinoma-bearing mice (n = 3) were anesthetized continuously via inhalation of 2% escain (Mylan Pharmaceuticals, Morgantown, WV) and intravenously injected with AF750-siCont-C formulated in PEG-TEPA-PCL or APRPG-TEPA-PCL (15 μg, N/P = 18 equiv) via a tail vein. The fluorescence of AF750 was acquired 10 min, 3 h, and 24 h after the injection. These mice were sacrificed under anesthesia 96 h post injection. Then, the organs (heart, lungs, liver, spleen, kidneys, and tumor) were collected and imaged ex vivo with the IVIS. Statistical Analysis. Differences between groups were evaluated by analysis of variance (ANOVA) with the Tukey posthoc test.

’ RESULTS Gene Silencing with DCP-TEPA-Based Liposomes. An adequate lipid composition of TEPA-PCL for siRNA transfection was examined by performing knockdown experiments using EGFP/HT1080 cells and siGFP. As shown in Figure 1B, the amount of DCP-TEPA in the PCLs clearly affected the knockdown efficiency, but did not show simple dose-dependency: The highest knockdown efficiency was obtained when the lipid composition of TEPA-PCL was DCP-TEPA/DOPE/DPPC/ cholesterol = 0.25/1/0.75/1 as a molar ratio. Thus, this composition of TEPA-PCL was adopted for the subsequent experiments. Next, an adequate N/P ratio of TEPA-PCL/siRNA was determined by using the same knockdown experiment. As a result, the highest knockdown efficiency was obtained when the N/P ratio was 18 or 24 equiv (Figure 1C). The knockdown efficiency did not change even when the N/P ratio was increased up to 48 equiv (data not shown). siGFP without TEPA-PCL did not show any knockdown effect. Obvious cytotoxicity was not observed in any of the experiments (data not shown). Particle size and ζ-potential of TEPA-PCL-based nanoparticles are summarized in Table 1. The ζ-potential of TEPA-PCL under various pH conditions (pH = approximately 4.1-7.9) was also determined as an indicator of the buffering effects of TEPA in

Figure 2. Biodistribution of PEGylated TEPA-PCL in mice after intravenous injection. TEPA-PCL labeled with [3H] cholesteryl hexadecyl ether (74 kBq/mouse) was modified with DSPE-PEG2000 or DSPE-PEG6000. The percent molar ratio of DSPE-PEG to total lipids was 10% or 20%. BALB/c mice (n = 5) were injected with TEPA-PCL or PEGylated TEPA-PCL via a tail vein. Three hours after the injection, the radioactivity in each organ was determined with a liquid scintillation counter. Data are presented as % dose per tissue (A) and % dose per 100 mg tissue (B). Significant differences from the TEPA-PCL group are indicated (*p < 0.05, **p < 0.01, ***p < 0.001).

PCL and compared with that of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) liposomes. The ζ-potential of TEPAPCL under low pH conditions was higher than that under the neutral condition, whereas that of DOTAP liposomes did not change at any pH tested (Supporting Information Figure 1). Biodistribution of TEPA-PCL in Mice after Intravenous Injection. The biodistribution of PEGylated TEPA-PCL was determined in 5-week-old BALB/c male mice (Figure 2). The relative amount of DSPE-PEG to total lipids affected the RESavoidance activity in the case of TEPA-PCL modified with PEG2000: Modification of TEPA-PCL with 10 mol % PEG2000 could not avoid RES uptake, resulting in rapid 432

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Figure 3. Stabilization of complexes by use of cholesterol-conjugated siRNA. (A) The portion of siRNA dissociated from TEPA-PCL by PEGylation was separated by gel electrophoresis and stained with ethidium bromide. (B) The interaction between TEPA-PCL and siRNA was improved by modifying siRNA with cholesterol at the 30 -prime of the sense strand. The portion of siRNA or siRNA-C dissociated from TEPA-PCL by PEGylation was visualized by a similar procedure. (C, D) RNAi efficiency of siRNA-C transfected with TEPA-PCL was determined. A549-luc-C8 (C) or B16-F10-luc2 (D) cells (5  103 cells/well) were precultured overnight. TEPA-PCL complexed with siRNA (siLuc, siLuc-C; 10 pmol, N/P = 18 equiv (C), siCont-C, or siLuc2-C, 20 pmol, N/P = 18 equiv (D)) was added to these cells and removed after a 24 h incubation. These cells were subsequently cultured for additional 24 h and used for the luciferase assay. B16-F10-luc2 cells were also transfected with siLuc2 mixed with LFA2K according to the manufacturer’s instructions. The control is that of cells not transfected with siRNA. Data are presented as the percentage of control luminescence intensity.

clearance from the bloodstream. The biodistribution of TEPAPCL modified with 10% PEG2000 was almost similar to that of TEPA-PCL without PEG. On the other hand, TEPA-PCL modified with 20% PEG2000 avoided RES uptake and showed long-term circulation in the bloodstream. The length of PEG on TEPA-PCL also affected the avoidance of PEGylated TEPA-PCL from the RES uptake. TEPA-PCL modified with PEG6000 avoided RES uptake and showed long-term circulation in the bloodstream even when the percent molar ratio of DSPE-PEG to total lipids was 10%. The long-term circulation property of

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TEPA-PCL modified with PEG6000 was superior to that with PEG2000. On the basis of these data, TEPA-PCL modified with 10% PEG6000 was adopted for subsequent experiments. Stabilization of Complexes by Use of Cholesterol-Conjugated siRNA. Complex formation of PEGylated TEPA-PCL/ siRNA was examined by electrophoresis in 15% acrylamide gels. As a result, free siRNA dissociated from TEPA-PCL was detected after PEGylation (Figure 3A). In contrast, siRNA was stably associated to TEPA-PCL by modifying the siRNA with cholesterol at the 30 -prime of the sense strand (Figure 3B). In the case of TEPA-PCL/siRNA-C complexes, free siRNA-C was not detected when they were formed at an N/P ratio of 18-30. In contrast, free siRNA dissociated from TEPA-PCL/siRNA complexes at all N/P ratios tested. The influence of cholesterolconjugation of siRNA on knockdown efficiency was examined by using A549-luc-C8 cells and siLuc-C. TEPA-PCL complexed with siLuc or siLuc-C was used to transfect A549-luc-C8 cells. As a result, cholesterol-conjugation at the 30 -prime end of the sense strand of the siRNA did not affect the knockdown efficiency (Figure 3C). The knockdown efficiency of TEPA-PCL was also examined in another experimental model using B16-F10-luc2 cells and siLuc2-C. The knockdown efficiency of TEPA-PCL/ siLuc-C was higher than that obtained with LFA2K (Figure 3D). NIRF Imaging of siRNA in Tumor-Bearing Mice. The biodistribution of AF750-siCont-C after intravenous injection in Colon26 NL-17 carcinoma-bearing mice was imaged in vivo by use of IVIS (Figure 4A). In vivo imaging data showed that fluorescent signals of AF750 were observed in the whole body of all mice at 10 min and 3 h after the injection. Accumulation of AF750 in the bladder was also observed in all groups up to 24 h after the injection. The total fluorescence of AF750 injected with TEPA-PCL was stronger than that without TEPA-PCL at 24 h after the injection. Importantly, AF750-siCont-C formulated in APRPG-TEPA-PCL accumulated in the tumor 24 h after the injection. Ex vivo images of each organ were obtained 96 h after the injection (Figure 4B). The total fluorescence of AF750 was low in siCont-C without TEPA-PCL. Accumulation of AF750 in the liver was observed with PEG-TEPA-PCL. In addition, strong fluorescent signals of AF750 in the tumor were observed only in the case of APRPG-TEPA-PCL.

’ DISCUSSION Our previous results showed that siRNA formulated in cetylPEI-PCL showed efficient gene silencing in EGFP/HT-1080 cells and human umbilical vein endothelial cells.17 However, cetyl-PEI-PCL modified with PEG6000 accumulated in the RES immediately after intravenous injection into mice. We considered that PEG6000 modification could not negate the undesirable properties of cetyl-PEI-PCL because of the molecular size of PEI[1800]. On the basis of these results, DCP-TEPA was newly synthesized and liposomalized for systemic siRNA delivery. Since TEPA is a relatively short polycation, we expected it to be stably presented on the liposomal membrane and not to interfere with the beneficial effects of PEGylation. In fact, PEG6000 endowed TEPA-PCL with the property of long circulation. On the other hand, we previously reported gene delivery with liposomal polyamine (spermidine, spermine, and PEI[1800])-dialkyl (dilauryl (DLP), dimyristyl (DMP), and DCP) phosphate conjugates.14 Our previous results demonstrated that not only the polyamine but also the dialkyl parts affect their transfection efficiencies and that DCP is favorable for polyamine-mediated 433

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PCL and the N/P ratio of TEPA-PCL/siRNA are important for gene silencing. On the other hand, PEGylation of TEPA-PCL/ siRNA complexes caused dissociation of a part of siRNA from the TEPA-PCL, even though the formation of TEPA-PCL/siRNA complexes is based on electrostatic interactions. However, we could prevent this undesirable phenomenon by adding hydrophobic cholesterol to complexes by using siRNA-C. It is still not presently clear how DSPE-PEG removed siRNA from the TEPAPCL. Modification of siRNA with cholesterol at the 30 -prime of the sense strand did not affect the knockdown efficiency, as previously reported.10 Thus, modification of siRNA with certain compounds to improve the properties of nanoparticles could be a useful strategy and applicable to other situations. In addition, the transfection efficiency of TEPA-PCL/siRNA-C was higher than that of LFA2K. TEPA-PCL/siRNA-C would be internalized into cells by endocytosis and efficiently escape from endosomes possibly through the buffering effects, which have been observed in the case of cetyl-PEI[1800]-based PCL.15 The biodistribution study demonstrates that PEG6000 is suitable for PEGylation of TEPA-PCL. In general, the length of PEG2000 is adequate to endow neutral liposomes with long circulation. In fact, Doxil, which is a liposomal doxorubicin modified with MPEG2000, has such a property and is used worldwide as a cancer drug. In contrast, modification of TEPAPCL with 10% PEG2000 did not alter the biodistribution of it after intravenous administration. This phenomenon might have been due to TEPA exposure on the liposomal surface despite PEGylation. Our data suggest that relatively long PEG chains would be needed to improve the biodistribution of positively charged liposomes in some cases. In addition, we modified PEGylated TEPA-PCL with APRPG for efficient delivery of siRNA into target cells since it is assumed that PEGylation of TEPA-PCL attenuates cellular uptake and gene silencing effects of siRNA formulated in TEPA-PCL. A steric barrier formed on the surface of liposomes by PEGylation is known to inhibit binding of liposomes to cells. In fact, our previous in vitro study using human umbilical vein endothelial cells revealed that PEGylation of cetyl-PEI-PCL attenuates cellular uptake of siRNA formulated in cetyl-PEI-PCL, whereas modification of PEGylated cetylPEI-PCL with APRPG improves this attenuation.25 In the NIRF imaging, we obtained not only in vivo but also ex vivo data, since the fluorescence intensities of in vivo NIRF imaging are strongly affected by tissue depth in animals.18 As a result, both our in vivo and ex vivo data showed that AF750-siContC formulated in APRPG-TEPA-PCL accumulated in the tumor after intravenous injection into Colon26 NL-17 carcinoma-bearing mice. In vivo data also showed that the total fluorescent signals of AF750 observed in the whole body were almost similar between PEG-TEPA-PCL and APRPG-TEPA-PCL, suggesting that the presence of APRPG did not diminish the long-circulating property achieved by PEGylation. These data indicate that the accumulation of AF750-siCont-C formulated in APRPG-TEPA-PCL in the tumor would be achieved by both active targeting to angiogenic vessels and passive targeting by the EPR effect. These results are consistent with our previous results showing that modification of liposomes with APRPG-PEG conjugate should be useful for both drug and gene delivery to angiogenic vessels in tumors.22,26-28 In conclusion, we developed TEPA-PCL for efficient delivery of siRNA into tumor cells and angiogenic endothelial cells. Furthermore, we also developed APRPG-TEPA-PCL for targeting angiogenic vessels and achieved systemic delivery of siRNA to the tumors in in vivo study.

Figure 4. NIRF-imaging of APRPG-TEPA-PCL/AF750-siCont-C in tumor-bearing mice. (A, B) Colon26 NL-17 carcinoma-bearing BALB/c mice (n = 3) were intravenously injected with AF750-siCont-C (15 μg), PEG-TEPA-PCL/AF750-siCont-C or APRPG-TEPA-PCL/AF750-siCont-C (15 μg, N/P = 18 equiv) via the tail vein under anesthesia. The biodistribution of AF750-siCont-C was assessed with a Xenogen IVIS Lumina System coupled to Living Image software for data acquisition. (A) The fluorescence of AF750 in these mice was acquired in vivo 10 min, 3 h, and 24 h after the injection. (B) The fluorescence of AF750 in each organ (heart, lungs, liver, spleen, kidneys, and tumor) was acquired ex vivo at 96 h after the injection.

gene delivery. For these reasons, we designed DCP-TEPA for systemic siRNA delivery. In the present study, we first determined the formulation of TEPA-PCL/siRNA complexes by performing knockdown experiments. Interestingly, the amount of DCP-TEPA in PCL clearly affected the knockdown efficiency, but did not show dose dependency. This phenomenon might be related to the stability of TEPA-PCL, since the hydrophilic portion of DCP-TEPA is relatively large compared with conventional liposomal components. Our data suggest that the amount of DCP-TEPA in TEPA434

dx.doi.org/10.1021/bc1004697 |Bioconjugate Chem. 2011, 22, 429–435

Bioconjugate Chemistry

ARTICLE

’ ASSOCIATED CONTENT

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bS

Supporting Information. The ζ-potential of TEPA-PCL under various pH conditions (pH = approximately 4.1-7.9) was determined as an indicator of the buffering effects of TEPA in PCL and compared with that of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) liposomes. The ζ-potential of TEPAPCL under low pH conditions was higher than that under the neutral condition, whereas that of DOTAP liposomes did not change at any pH tested. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Corresponding author. Tomohiro Asai, Department of Medical Biochemistry and Global COE, University of Shizuoka School of Pharmaceutical Sciences, 52-1 Yada, Suruga-ku, Shizuoka 4228526, Japan. TEL: (81) 54 264 5703, FAX: (81) 54 264 5705. E-mail address: [email protected].

’ ACKNOWLEDGMENT This research was supported by a Grant-in-Aid for Scientific Research (19790131) and the Mochida Memorial Foundation for Medical and Pharmaceutical Research. ’ REFERENCES (1) Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S. E., and Mello, C. C. (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806–811. (2) Elbashir, S. M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., and Tuschl, T. (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494–498. (3) Sibley, C. R., Seow, Y., and Wood, M. J. (2010) Novel RNAbased strategies for therapeutic gene silencing. Mol. Ther. 18, 466–476. (4) Tiemann, K., and Rossi, J. J. (2009) RNAi-based therapeuticscurrent status, challenges and prospects. EMBO Mol. Med. 1, 142–151. (5) Manjunath, N., and Dykxhoorn, D. M. (2010) Advances in synthetic siRNA delivery. Discovery Med. 9, 418–430. (6) Zimmermann, T. S., Lee, A. C., Akinc, A., Bramlage, B., Bumcrot, D., Fedoruk, M. N., Harborth, J., Heyes, J. A., Jeffs, L. B., John, M., Judge, A. D., Lam, K., McClintock, K., Nechev, L. V., Palmer, L. R., Racie, T., Rohl, I., Seiffert, S., Shanmugam, S., Sood, V., Soutschek, J., Toudjarska, I., Wheat, A. J., Yaworski, E., Zedalis, W., Koteliansky, V., Manoharan, M., Vornlocher, H. P., and MacLachlan, I. (2006) RNAi-mediated gene silencing in non-human primates. Nature 441, 111–114. (7) Kim, S. H., Jeong, J. H., Lee, S. H., Kim, S. W., and Park, T. G. (2008) LHRH receptor-mediated delivery of siRNA using polyelectrolyte complex micelles self-assembled from siRNA-PEG-LHRH conjugate and PEI. Bioconjugate Chem. 19, 2156–2162. (8) Chen, Y., Zhu, X., Zhang, X., Liu, B., and Huang, L. (2010) Nanoparticles modified with tumor-targeting scFv deliver siRNA and miRNA for cancer therapy. Mol. Ther. 18, 1650–1656. (9) Li, J., Chen, Y. C., Tseng, Y. C., Mozumdar, S., and Huang, L. (2010) Biodegradable calcium phosphate nanoparticle with lipid coating for systemic siRNA delivery. J. Controlled Release 142, 416–421. (10) Wolfrum, C., Shi, S., Jayaprakash, K. N., Jayaraman, M., Wang, G., Pandey, R. K., Rajeev, K. G., Nakayama, T., Charrise, K., Ndungo, E. M., Zimmermann, T., Koteliansky, V., Manoharan, M., and Stoffel, M. (2007) Mechanisms and optimization of in vivo delivery of lipophilic siRNAs. Nat. Biotechnol. 25, 1149–1157. (11) Yamazaki, Y., Nango, M., Matsuura, M., Hasegawa, Y., Hasegawa, M., and Oku, N. (2000) Polycation liposomes, a novel nonviral 435

dx.doi.org/10.1021/bc1004697 |Bioconjugate Chem. 2011, 22, 429–435