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siRNA complex system
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Journal of Controlled Release j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j c o n r e l
RNA interference in vitro and in vivo using an arginine peptide/siRNA complex system Sung Wook Kim 1, Na Young Kim 1, Yu Bin Choi, Seo Hyun Park, Jai Myung Yang, Sungho Shin ⁎ Department of Life Science, Sogang University, Shinsu-Dong, Mapo, Seoul, 121-742, South Korea
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Article history: Received 14 August 2009 Accepted 5 January 2010 Available online 14 January 2010 Keywords: Arginine peptide siRNA cellular location siRNA delivery system Xenograft tumor model
a b s t r a c t Efficient delivery systems are required to exploit the enormous potential of RNA interference. We introduced an arginine peptide-based small-interference RNA (siRNA) delivery system for in vitro and in vivo RNA interference. Arginine peptides formed stable complexes with siRNA and transduced siRNA into COS-7 cells in vitro, resulting in efficient gene silencing. The intracellular path of the peptide/siRNA complex was investigated in live cells using fluorescent labeling and confocal microscopy. At 24 h after transfection, most of the siRNA signals were observed in the perinuclear region, indicating that siRNA was targeted to the perinuclear region for interactions with RNA-induced silencing complex (RISC). Effective in vivo RNA interference was achieved in a mouse model bearing a subcutaneous tumor. Intratumoral administration of HER-2-specific siRNA/peptide complexes resulted in a marked reduction of tumor growth. Body weight monitoring during treatment showed that our delivery system was nontoxic. Our approach offers the potential for siRNA delivery in various in vitro and in vivo applications. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Post-transcriptional gene silencing by RNA interference (RNAi) is an attractive approach for the targeted inhibition of gene expression both in vitro and in vivo [1–3]. One of main advantages of using RNAi is its ability to specifically and potently knock down the expression of target genes. Gene silencing can be induced by small-interference RNA (siRNA) through sequence-specific cleavage of perfectly complementary mRNAs [4,5]. Introduction of siRNA into the mammalian cell cytoplasm triggers mRNA cleavage by RNA-induced silencing complexes (RISC) [6]. RISC activation is thought to initially involve endonuclease Argonaute 2 (AGO2)-mediated cleavage of the passenger or sense strand of the double-stranded siRNA, whereby the single-stranded antisense strand produced serves to guide the RISC to complementary sequences in target mRNAs [7,8]. The activated AGO2-RISC complex then binds to an mRNA strand and cleaves the mRNA strand between the bases that are complementary to bases 10 and 11 relative to the 5′ end of the siRNA guide strand, thereby leading to subsequent degradation of the cleaved mRNA transcript by cellular exonucleases [9,10]. Although their endogenous mechanism is not yet fully understood, the potential of siRNA molecules for treating a wide range of diseases has become the focus of the pharmaceutical industry in recent years [5,11]. Since the biological functions of the siRNA molecules take place in the cytoplasm, intracellular delivery of siRNA is a key factor in RNAi ⁎ Corresponding author. Tel.: + 82 2 701 8550; fax: + 82 2 704 3601. E-mail address: [email protected] (S. Shin). 1 These two authors contributed equally to this work. 0168-3659/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2010.01.009
technology. Because of the high molecular weight and net-negative charge, naked siRNA cannot freely access the intracellular environment [12]. Thus, delivery systems are required to facilitate cellular accumulation of siRNA molecules. Currently, various siRNA-delivery systems have been developed including liposomes, polymers, and peptides [13]. In recent times, membrane penetrating peptides (MPPs) have been widely used as cellular delivery vectors. These peptide-mediated delivery systems are advantageous because of the low costs incurred for reagents, nontoxic effects, and the ease of preparation. A common characteristic of MPPs is the presence of highly basic amino acids, such as arginine and lysine, thus suggesting that basic amino acids are critical motifs for the efficient delivery of exogenous biomolecules into cells [14]. Further investigation demonstrated that peptides composed only of arginine residues were able to translocate efficiently through the cell membrane [15]. Thus, the transduction properties of arginine peptides are not limited only to protein delivery, because they can deliver DNA as well. In a previous study, we demonstrated that short arginine peptides capable of forming complexes with DNA, promote efficient transfection in various mammalian cells types [16]. Despite the high proficiency of arginine peptide as a carrier for protein and DNA transfer, the molecular mechanism for the siRNA delivery process is poorly understood. In the present work, we report that functional siRNA can be delivered into cells by using a short arginine peptide (R15), and the siRNA can be localized to specific cytoplasmic compartments in the perinuclear region. In addition, arginine peptide-mediated delivery of HER-2 siRNA was found to downregulate HER-2 mRNA efficiently; inducing inhibition of tumor growth in vivo. Taken together, these
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results support the potential for an arginine peptide-mediated siRNA delivery system for therapeutic application. 2. Materials and methods 2.1. Materials GenePorter2 was obtained from Invitrogen (Carlsbad, CA, USA). Heparin was provided by Amersham/USB (Piscataway, NJ, USA). A luciferase assay kit was purchased from Promega (Madison, WI, USA). All chemicals used in the study were provided by Sigma-Aldrich (St. Louis, MO, USA), unless otherwise mentioned.
2.7. Arginine peptide-mediated siRNA delivery COS-7 cells stably expressing luciferase were seeded into the 24well plates at a density of 5 × 104 cells in 1 ml of culture medium. After overnight incubation, the cells were treated with the peptide/siRNA complexes. The solution containing these peptide/siRNA complexes (300 μl) was gently added to the cells, followed by incubation at 37 °C for 2 h in a 5% CO2 atmosphere. The cultures were then washed three times with serum-free medium and transferred to the complete medium for growth. Twenty-four hours after treatment with peptide/ siRNA complexes, cells were lysed and analyzed for luciferase activity. 2.8. Luciferase activity measurement
2.2. Peptide and siRNA synthesis Arginine peptide (R15) was prepared by solid-phase peptide synthesis using standard Fmoc (9-fluorenylmethyloxycarbonyl) chemistry. High performance liquid chromatography (HPLC) analysis indicated that the synthetic peptide was at least 95% pure. The peptide was dissolved in phosphate-buffered saline (PBS, pH 7.4) to appropriate concentrations. The siRNA used for luciferase gene silencing (sense-5′ CUU ACG CUG AGU ACU UCG ATT 3′, antisense5′ UCG AAG UAC UCA GCG UAA GTT 3′) and 5′ Cy3-labeled siRNAs were ordered from Samchully Pharm (Seoul, South Korea).
Cells were lysed by the addition of 150 μl of passive lysis buffer per well. The cell lysate (100 μl) was then recovered for the luciferase activity assay. Light emission was measured by integration over 10 s at 25 °C using a luminometer (MicroLumat Plus LB96V, Berthold, Germany). The total protein concentration in the cell lysate was determined using the BCA assay (Pierce Chemical, Rockford, IL, USA). Luciferase activity in each sample was normalized to relative light units (RLU) per mg of cell lysate proteins. 2.9. Observation of labeled peptide/siRNA complexes by confocal microscopy
2.3. Cell culture The COS-7 cells were cultured in Dulbecco's Modified Eagle Media (DMEM) (Invitrogen) with 10% heat-inactivated fetal bovine serum and 1% antibiotics (streptomycin + penicillin). For RNAi assay, cells were grown in 24-well plates and incubated at 37 °C in an incubator containing 5% CO2 atmosphere. For confocal laser scanning microscopy, cells were grown in glass bottom cell culture dishes. 2.4. Establishment of stable luciferase-expressing cells To obtain a stable luciferase-expressing cell line, COS-7 cells were transfected with luciferase gene by pcDNA 3.1/zeo-expression vector and maintained in DMEM in the presence of zeocin. Stable COS-7 cell clones were selected with zeocin (200 μg/ml) as described previously [17]. 2.5. Preparation of peptide/siRNA complexes The siRNA was diluted with 50 µl of PBS to a concentration of 10 µg/ml, and arginine peptides were dissolved in 250 µl of serumfree medium. To form the peptide/siRNA complex, the siRNA solution was pipetted into the peptide solution and mixed vigorously by vortexing. The complexes were incubated at room temperature (25 °C) for l h. The particle size of complexes was measured by dynamic light scattering (DLS) method and zeta-potential was determined by laser Doppler electrophoresis (LDE) method with Zetasizer Nano ZS (Malvern, UK). 2.6. Gel shift assay To determine the optimal relative concentration at which peptide/ siRNA complex formation would be facilitated, 0.5 µg of siRNA was incubated with various amounts of arginine peptide for 1 h at 25 °C in serum-free media, with a peptide/siRNA charge ratio ranging from 0 − to 20. The ratio between peptide (NH+ 3 ) and siRNA (PO4 ) was defined as the nitrogen/phosphate (N/P) ratio [18]. The peptide/siRNA complexes were then analyzed by electrophoresis on a 20% polyacrylamide gel in TAE (40 mM Tris acetate/2 mM EDTA) buffer at 100 V for 70 min, which was followed by staining with ethidium bromide (EtBr) for 20 min.
To investigate the distribution of the peptide/siRNA complexes in live cells, Cy3-labeled siRNA and fluorescein isothiocyanate (FITC)tagged arginine peptides were used. Double-labeled complexes were gently added to cells and incubated for various time periods. Following incubation, the cells were washed three times with PBS; they were again extensively washed with heparin-containing PBS (1 mg/ml) to completely remove the cell surface-bound complexes as described previously [19]. Distribution of the fluorescently labeled peptide/DNA complexes was analyzed using a confocal laser scanning microscope (LSM 510; Carl Zeiss, Germany) without fixing the cells. 2.10. Cytotoxicity assay The MTT assay was conducted essentially according to the manufacturer's protocol (Boehringer Mannheim, Mannheim, Germany). Briefly, cells were plated on to 96-well plates (4× 104/well) in DMEM medium with 10% heat-inactivated fetal bovine serum in the presence of peptide/siRNA complexes (3:1). After various incubation periods, MTT (200 μg/ml) was added to each well. Cells were further incubated with MTT for 4 h. The insoluble formazan was dissolved in DMSO solution. Cell viability was assessed by measuring the absorbance at 570 nm and expressed as the ratio of the A570 of cells treated with arginine peptide/ siRNA over the control samples. LDH assay was performed according to the manufacturer's protocol (BioVision, USA). 2.11. In vivo experiments In order to generate tumors, Balb/c mice (5 weeks of age; Samtako, Osan, South Korea) were injected subcutaneously in the left flank with 100 μl of cell suspension containing 5 × 106 SKOV-3 cells. Tumor size was measured using a Vernier caliper and the volume of tumor was calculated using the formula V = 0.523 × longest diameter× shortest diameter2. Treatment of the tumors was started after 10–15 days, based on when the subcutaneous tumors reached a size of 100 mm3. Argininecomplexed HER-2-specific siRNA (N/P ratio of 3.0), arginine-complexed mismatched siRNA, or naked siRNA were prepared in 5% glucose solution, and the complexes were injected directly into the tumors every 3 days at a dose of 4-μg siRNA per mouse. For HER-2 targeting, three designed siRNAs (CCUGGAACUCACCUACCUGdTdT/CAGGUAGG UGAGUUCCAGGdTdT, CUACCUUUCUACGGACGUGdTdT/CACGUCCGUA
GAAAGGUAGdTdT, and GAU CCGGAAGUACACGAUGdTdT/CAUCGUGUACUUCCG GAUCdTdT) were synthesized, and the three duplexes were mixed at equimolar ratios as described previously [20]. Mouse body weight was also monitored. 2.12. Western blot The protein (30 μg) from nude mouse xenograft for each sample was loaded on 8% SDS-PAGE gel and electrophoresed. Protein bands in the gel were then transferred to a nitrocellulose membrane. Membranes were blocked for 1 h in 5% nonfat dry milk in TBST (10 mM Tris–HCl, pH 7.5; 150 mM NaCl, and 0.1% Tween 20) and probed for 1.5 h with primary HER-2 antibodies diluted in TBST/5% nonfat dry milk (1:8000) as per the supplier's recommendations (Thermo SCIENTIFIC, Fremont, CA, USA). Membranes were then washed with TBST for 20 min × 3 and incubated for 1 h with horseradish peroxidase-labeled secondary antibodies in TBST. After an additional four washes with TBST, bound antibody was visualized by X-ray film. Membranes were tested for GAPDH (0.1 µg/ml antiGAPDH primary antibody) to confirm equal loading.
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Table 1 Characteristics of siRNA complex formation with arginine peptide at various N/P ratios. N/P ratio
Complex size (nm)
Zeta-potential (mV)
0.5 1.0 3.0 10.0
113.65 ± 0.78 182 ± 0.1 224.85 ± 4.17 270.45 ± 31.04
−10.24 ± 2.91 −1.46 ± 1.00 7.06 ± 0.10 15.63 ± 0.06
Experiments were performed at least in triplicate each time; data shown are mean value and S.D. for three different measurements.
Table 1, at N/P ratio 3.0, arginine peptide was able to condense the siRNA into small positively charged particles suitable for intracellular trafficking and biodistribution. In order to use arginine peptide as a siRNA delivery agent, the degree of cytotoxicity of peptides/siRNA complex is critical. We measured the cytotoxicity of peptide/siRNA complexes by MTT assay. To check the cell membrane integrity, LDH assay was also carried out (Fig. 2). The cell viability of peptide/siRNA complex-treated cells was almost same as that in nontreated cells. We observed no significant impairment of cell survival at a 3:1 arginine peptide/siRNA ratio. After 48 h, COS-7 cells displayed greater than a 98% survival rate.
2.13. Statistical analysis The quantitative data collected were expressed as means ± S.D. Statistical significance was analyzed by a paired, two-sample Student's t test. Statistical significance was inferred at a value of P b 0.05. 3. Results 3.1. Complex formation of arginine peptide with siRNA To assess formation of peptide/RNA complexes, a gel shift assay was employed to measure the electrostatic interaction in the complexes as a function of the positive peptide (NH+ 3 )/negative RNA (PO− 4 ) charge ratios. Luciferase siRNA (0.5 μg) was mixed with arginine peptides at various N/P ratios (0.5–20). The results showed that the electrophoretic migration of RNA was retarded with an increasing ratio of peptides/RNA (Fig. 1). The intensity of the siRNA band decreased as the N/P ratio increased. No migration RNA band was observed at N/P ratio 3.0 or higher. This absence of migration was probably due to the neutralization of nucleic acid by the arginine peptide and/or formation of a large complex between the arginine and the RNA [16]. These results suggest that an arginine peptide is sufficient to form a complex with siRNA. We next evaluated physical properties such as size and zetapotential of peptide/siRNA complexes at various N/P ratios. As seen in
Fig. 1. Gel shift assay of arginine/siRNA complex formation. Luciferase siRNA was incubated for 1 h at 25 °C with different concentrations of arginine peptides, corresponding to peptide (N)/siRNA (P) charge ratios in a range between 0 and 20.0, as indicated above each lane. The complexes were analyzed by electrophoresis on 20% polyacrylamide gel stained with ethidium bromide.
3.2. Silencing effect of arginine/siRNA complexes Since arginine peptides are known to bind siRNA, and the complexes show no cytotoxicity, we tested the biological effectiveness of this system. We investigated the inhibitory activity of siRNA against the expression of luciferase. COS-7 cells that stably express luciferase were used as a gene knockdown assay system [17]. A wide range of N/P ratios were used for transfection to explore the effect of charge parameters on delivery efficiency. As indicated in Fig. 3A, reduction of luciferase expression was observed in COS-7 cells treated with arginine/siRNA complexes when the siRNA concentration was kept constant at 0.5 μg /ml and N/P ratio was varied from 0.5 to 10.0. The expression of luciferase was decreased with increasing N/P ratio up to 3.0. At an N/P ratio of 3.0, the complex suppressed luciferase expression by 50% compared with the untreated control. The delivery of siRNA using a commercially available nonviral transfection reagent, liposome (GenePORTER2), resulted in a smaller decrease in luciferase expression compared with arginine peptide. A mismatched siRNA complexed with peptide did not result in a change in luciferase expression levels, demonstrating the selective inhibition by siRNA. Once the N/P ratio parameter was set, the time-course of inhibition produced by arginine/siRNA complexes was studied. Inhibition of
Fig. 2. Cytotoxicity of peptide/siRNA complexes at N/P ratio 3:1 for COS-7 cells was evaluated by MTT and LDH assay. Experiments were performed at least in triplicate each time; data shown are mean value and S.D. for three different measurements.
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3.3. Cellular localization of arginine/siRNA complexes Despite the current widespread use of MPPs as siRNA delivery vectors, the cellular localization of peptide/siRNA complexes has remained controversial. Previous reports have suggested that siRNA was localized in the cytoplasm around the periphery of the nucleus [21,22]. In contrast, other reports have indicated that siRNA was detected in the nucleus [14,23]. Therefore, we investigated the cellular location of siRNA in live cells. We double-labeled the complexes with Cy3 and FITC and evaluated the cellular locations of the complexes using confocal microscopy. Fig. 4A shows a representative confocal image of live COS-7 cells treated with FITC-tagged arginine peptide and Cy3-labeled siRNA complexes. Since RNAi efficiency was maximized at 24 h after transfection, microscopic observations were performed 24 h after initial treatment with peptide/siRNA complexes. Red (siRNA) and yellow fluorescence (peptide/siRNA complexes) was found in the cytoplasm in the form of a clustered-dot pattern. Notably, the complexes appeared to be heterogeneously distributed in the perinuclear region of the cells, and no signals were observed inside of the nucleus in a Z-stack image (Fig. 4B), indicating that siRNA was localized in the cytoplasm. Our results are consistent with previous findings that suggested that TAT peptide-mediated delivery of siRNA resulted in cytoplasm location of siRNA [20]. 3.4. Intracellular trafficking of arginine/siRNA complexes
Fig. 3. Luciferase activity after transfection of arginine/siRNA complexes in COS-7 cells stably expressing luciferase. Cells were incubated in the presence of preformed arginine/ luciferase siRNA complexes for 2 h, after which they were washed with serum-free medium and replaced in complete medium. Twenty-four hours later, cell extracts were prepared, and their luciferase activities were determined as described in Materials and methods. (A) Charge (N/P)-dependent silencing effect of arginine/siRNA complexes. (B) Time-dependent silencing effect of arginine/siRNA complexes. (C) Dosedependent silencing effect of arginine/siRNA complexes. Experiments were performed at least in triplicate each time; data shown are mean value and S.D. for each measurement. *, P b 0.05 compared with control.
luciferase expression remained essentially constant between 24 h and 36 h (Fig. 3B). Arginine/siRNA complexes showed maximum inhibition effect at 24 h after treatment. However, 48 h later, the luciferase activity returned to normal levels. To further identify the optimal experimental conditions, we then assessed the optimal concentration of siRNA necessary to maximize luciferase gene silencing. As shown in Fig. 3C, the silencing effect was dose-dependent. Inhibition of luciferase expression gradually increased from 20% in the presence of 15 nM siRNA to over 50% inhibition with 150 nM siRNA.
Next, to improve understanding of the cellular location of siRNA, we investigated the intracellular pathway taken by the arginine/siRNA complexes from the margin of the cell surface until their entry into the perinuclear region of live cells. When cells were incubated with the complexes for 1 h, various sized fluorescent spots were detected predominantly on the extracellular surface of the plasma membrane (Fig. 5A). After 3 h of incubation, punctuated fluorescence was observed in the cytoplasm, indicating the entry of the complexes into the cells (Fig. 5B). However, some portions of the complexes were still on the cell surface. In the cytoplasm, labeled complexes appeared as vesicles rather than as a diffuse distribution, suggesting an endocytotic uptake mechanism. With increasing incubation time, fluorescence appeared to aggregate to more discrete areas in the cytoplasm. Following 6–24 h of incubation, we noticed the appearance of fluorescence in a perinuclear location (Fig. 5C–E). The fluorescence revealed round structures restricted primarily to the external surface of the nuclear membrane. Finally, 48 h after incubation, most of the FITC signals of the arginine/ siRNA complexes disappeared, and only faint Cy3 signals were observed (Fig. 5F). All these observations suggest that the arginine/siRNA complexes make their way into the cytoplasm of cells. In addition, we assessed whether the cellular location of siRNA in the perinuclear region of the cytoplasm was affected by the presence or absence of the mRNA target. As shown in our results, siRNA localization in cells was similar without (Fig. 6) or with luciferase reporter plasmid (Fig. 4A), indicating that siRNA cellular localization was independent of the endogenous mRNA target. 3.5. Inhibition of tumor growth by arginine/siRNA complexes Although a number of MPP-mediated siRNA delivery systems have been shown to be powerful methods for inhibiting gene expression in vitro, little is known about in vivo application of these systems for therapeutic purposes. To examine the in vivo therapeutic efficiency of the arginine peptide-mediated siRNA delivery system, we investigated the effect of arginine/siRNA complexes on growth inhibition in tumors. A previous report demonstrated that the stable reduction of HER-2 expression in SKOV-3 cells inhibited subcutaneous tumor growth in a mouse model, indicating that expression of HER-2 is rate-limiting for tumor growth in vivo [20]. Therefore, we used this model to test
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Fig. 4. Cellular localization of double-labeled arginine/siRNA complexes. COS-7 cells stably expressing luciferase (5 × 104) were incubated in the presence of arginine/siRNA complexes for 24 h. After incubation, samples were prepared as described in Materials and methods and examined by confocal laser scanning microscopy. Nuclei (blue) were stained with Hoechst 33258 dye. (A) a, FITC-labeled arginine (green); b, Cy3-labeled siRNA (red); c, differential interference contrast (DIC); and d, merged image (yellow fluorescence indicates colocalization of arginine and siRNA). (B) Z-stack image of the cells. The green and red lines indicate the position within the image of the projections given in the upper and right, respectively.
whether application of arginine/HER-2-specific siRNA complexes resulted in the growth inhibition of subcutaneous tumors. SKOV-3 cells (5× 106) were subcutaneously injected into the flanks of mice. When the tumors reached a size of approximately 100 mm3, we started intratumoral treatment with arginine/HER-2-specific siRNA, arginine/ HER-2-mismatched RNA complexes, or naked siRNA and repeated treatment every 3 days for a total of five treatments. As shown in Figs. 7 and 8, arginine/HER-2-specific siRNA complex significantly reduced the tumor growth compared with arginine/HER-2-mismatched siRNA
complex, naked siRNA, or 5% glucose solution used as control. Differences were obvious as soon as 1 week after onset of treatment (Pb 0.05 at day 7). These findings were confirmed by western blot analysis of tumor sections. Measurement of HER-2 by western blot of tumor lysate showed that tumor collected 17 days following administration of arginine/siRNA complexes showed significantly decreased HER-2 expression compared with treatment with mismatched siRNA, naked siRNA, or control (Fig. 9).
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Fig. 5. Intracellular tracking of double-labeled arginine/siRNA complexes in COS-7 cells. Cells were incubated with FITC-labeled peptide and Cy3-labeled siRNA complexes at 37 °C for the indicated time periods. After incubation, the cells were washed as described in Materials and methods to remove surface-bound materials. The live cells were examined by confocal laser scanning microscopy. (A) 1 h, (B) 3 h, (C) 6 h, (D) 12 h, (E) 24 h, and (F) 48 h.
Finally, the in vivo toxicity of arginine/siRNA complexes was studied. As shown in Fig. 10, mouse body weight did not change significantly during the arginine/siRNA complex treatment. From these results, we inferred that the arginine peptide-mediated siRNA delivery system was safe. 4. Discussion Interest in siRNA as a tool for genome-wide analysis of mammalian cells is growing. As a consequence, siRNA holds great potential as a therapeutic agent against genes causing a wide range of diseases. In the current study, we introduced the arginine peptide-mediated delivery system for RNA interference in vitro and in vivo. Furthermore, to obtain the best delivery efficiency, we optimized the various technical factors such as charge ratio of arginine peptide/siRNA complex, cell/complex incubation period, and concentration of siRNA.
Using gel shift assay, we showed the ability of arginine peptides to form complexes with siRNA, as reported previously for condensing DNA [16]. Thus, siRNA may be protected from cellular enzymatic degradation and is suitable for intracellular transport. To investigate whether this complex formation ability was sufficient to induce RNAi, a COS-7 cell line stably expressing luciferase was used as a gene knockdown assay system. As much as a 50% reduction of luciferasespecific expression was obtained in cells transfected for 24 h with arginine/siRNA complex at a charge ratio of 3:1. However, further increase of the peptide/siRNA charge ratio showed decreased RNAi efficiency. One possible explanation for this result is that an overly strong peptide-siRNA interaction may prevent spatially appropriate release of siRNA to the cytoplasm, as in peptide-DNA interaction [24]. The silencing effect was also dose-dependent (Fig. 3C). A significant reduction of luciferase expression was observed when peptide was complexed with luciferase siRNA at a concentration of
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Fig. 6. Cellular localization of arginine/siRNA complexes in wild COS-7 cells. Cells (5× 104) were incubated in the presence of arginine/siRNA complexes for 24 h. After incubation, samples were prepared as described in Materials and methods and examined by confocal laser scanning microscopy. Nuclei (blue) were stained with Hoechst 33258 dye. A, FITC-labeled arginine (green); B, Cy3-labeled siRNA (red); C, differential interference contrast (DIC); and D, merged image.
150 nM. A similar effective siRNA concentration range was observed in a previous MPG peptide-mediated siRNA delivery study [25]. Prior to this study, Kim et al. investigated the potency of cholesteryl-R9 conjugates as a siRNA delivery vehicle in vitro and in vivo model [26]. Adding to hydrophobic moieties to oligoarginine was shown by others to improve the plasmid gene transfection efficiency [27], presumably due to the hydrophobic character to the arginine oligopeptides would enhance their ability to cross the plasma membrane, and hence, facilitate intracellular delivery of the plasmid DNA cargo. Comparing the R9 with cholesteryl, the maximal
Fig. 7. Arginine/siRNA complex inhibited tumor growth in nude mice. SKOV-3 cancer cells (5 × 106 cells/mouse) were implanted in the mice. Once the tumors reached approximately 100 mm3, the mice were treated intratumorally with arginine/HER-2specific siRNA, arginine/HER-2-mismatched siRNA, or naked siRNA. As a control, 5% glucose solution was injected. Data = mean, n = 6. S.D. of the data points is not shown for clarity.
achievable levels of siRNA-mediated target protein down-regulation are roughly same as arginine peptide (R15) only in our study. However, the observed RNAi effects between our study and theirs must reflect the experimental conditions, such as number of arginine residues, target genes and cells utilized for siRNA delivery. The present study was not intended to investigate this issue, and future studies will be needed to elucidate RNAi effect of hydrophobic molecule-conjugated R15 peptides.
Fig. 8. SKOV-3 xenograft tumor growth inhibition by arginine/siRNA complexes. Representative example of tumors in the four treatment groups. On day 17 tumors were taken. A, control; B, naked siRNA; C, arginine/HER-2-mismatched siRNA; and D, arginine/ HER-2-specific siRNA.
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Fig. 9. Western blot analysis of HER-2 in the SKOV-3 xenograft tumor after treatment with various formulations. HER-2 levels in tumors at 17 days after treatment initiation. A, control; B, naked siRNA; C, arginine/HER-2-mismatched siRNA; and D, arginine/HER2-specific siRNA.
Identifying the cellular location of siRNA is crucial to developing efficient peptide-mediated siRNA delivery systems. Although a considerable body of data has accumulated, the cellular location of siRNA is still controversial. Previous studies have demonstrated that siRNA delivery mediated by TAT peptide resulted in a cytoplasmic location of siRNA [21]. Further investigations have reported that part of siRNA was identified in the nucleus with the R-8 MEND carrier [14]. In addition, Veldhoen et al. suggested that the cellular location of siRNA was dependent on the type of carrier peptide used [25]. As shown in a Z-stack scanning confocal microscopy image (Fig. 4B), in our study, siRNAs were localized in the cytoplasm, especially in the perinuclear region. Furthermore, previous experiments have shown that Dicer and RNAi activity was located in cytoplasm [28,29]. Combining all these results, the present report supports the view that subcellular compartmentalization may play an important role in the function of the RNAi machinery. Since siRNA was localized in the cytoplasm in our system, we next investigated whether this cellular location of siRNA was target mRNAspecific. The luciferase siRNA was localized in the peripheral region of the nucleus in wild COS-7 cells (Fig. 6). These results suggest that the subcellular location of siRNA is not specific to the targeted mRNA. Similar results have shown that siRNA localization of both functional and nonfunctional siRNA sequences was in the perinuclear region of cytoplasm [21]. To examine further the cellular location siRNA, we investigated the intracellular path of double-labeled arginine/siRNA complexes. To avoid any artifacts due to cell fixation, our experiments were performed with living cells. The course of the intracellular pathway of complexes in our experiments is summarized as follows. At 1 h after incubation, arginine/siRNA complexes began to attach to the cell surface. At 3 h after incubation, arginine/siRNA complexes were observed in the cytoplasm. However, some of the complexes were still on the cell surface. With time, complexes gradually migrated closer to the nucleus and, 6 h after incubation, a significant fraction of the
arginine/siRNA complexes reached the cytoplasm and appeared as vesicles. The localization of complexes peripheral to the nucleus was relatively prevalent after 12–24 h following treatment of the cells. Finally, 48 h after incubation, most of the FITC signals from the arginine/siRNA complexes disappeared and only red signals (siRNA) were observed in the perinuclear region, indicating that the siRNA was released from the arginine peptides and was in a stable condition. Therefore, these results suggest that after uptake via endocytosis, peptide/siRNA complexes can escape from endosome and finally move to the perinuclear region of cytoplasm. Recently, El-Sayed et al. [30] reported that modification of nanoparticles with high density of R8 allows their escape from endocytic vesicles via membrane fusion. Based on this report, it is assumed that the escape of arginine peptide/ siRNA complexes may take place mainly through endosomal membrane fusion, which leads to efficient escape. In recent years, polycations have been widely studied as a siRNA delivery vehicle [14,22]. However, their RNAi efficiency has been described mostly in vitro. It is well known that in vitro siRNA delivery is typically far more efficient than the corresponding in vivo siRNA delivery, since the whole organism interposes the extracellular matrix, cytotoxicity, and myriad other supramolecular barriers. Here, the in vivo therapeutic efficiency of arginine/siRNA complex was investigated in nude mice bearing human ovarian cancer (SKOV3 cells) xenografts. Previously, inhibition of HER-2 expression by siRNA was reported as an effective and useful method for tumor inhibition therapy in vivo [20]. In this study, we showed that treatment with arginine/HER-2 siRNA complex inhibited tumor growth, as compared to other siRNA treatment groups, 7 days after initial treatment (Fig. 7). On 17 days later, the tumor growth rate after arginine/HER-2 siRNA treatment became comparable to the other treatment groups. In addition, a body weight-monitoring study showed no toxicity, suggesting that the arginine/siRNA complex itself was safe, which would be a prerequirement of any clinical application (Fig. 10). In conclusion, an arginine peptide-mediated siRNA delivery system has been developed. The arginine/siRNA complex at a charge ratio of 3:1 was shown to deliver siRNA efficiently both in vitro and in vivo. In addition, we showed that the perinuclear region of cells seems to play an important role in the RNAi mechanism. All these results suggest that arginine peptide in complex with siRNA opens a new avenue for the development of an efficient and safe delivery system for therapeutic applications. Acknowledgement This study was supported by a grant of the Korea Healthcare Technology R&D Project, Ministry for Health, Welfare & Family Affairs, Republic of Korea. (A085119). References
Fig. 10. Mouse body weight during the treatment with arginine/siRNA complexes. Data = mean, n = 6. S.D. of the data points is not shown for clarity.
[1] R.K. Leng, P.A. Whittaker, RNA interference: from gene silencing to gene-specific therapeutics, Pharmacol. Ther. 107 (2005) 222–239. [2] G.J. Hannon, J.J. Rossi, Unlocking the potential of the human genome with RNA interference, Nature 431 (2004) 371–378. [3] M. Amarzguioui, J.J. Rossi, D. Kim, Approaches for chemically synthesized siRNA and vector-mediated RNAi, FEBS Lett. 579 (2005) 5974–5981. [4] D.H. Kim, J.J. Rossi, Strategies for silencing human disease using RNA interference, Nat. Genet. 8 (2007) 173–184. [5] A. Fougerolles, H.P. Vornlocher, J. Maraganore, J. Liberman, Interfering with disease: a progress report on siRNA-based therapeutics, Nat. Drug Discov. 6 (2007) 443–453. [6] J. Martinez, A. Patkaniowska, H. Urlaub, R. Luhrmann, T. Tuschl, Single stranded anti siRNAs guide target RNA cleavage in RNAi, Cell 110 (2002) 563–574. [7] J. Liu, M.A. Carmell, F.V. Rivas, C.G. Marsden, J.M. Thomson, J.J. Song, Argonaute2 is the catalytic engine of mammalian RNAi, Science 305 (2004) 1437–1441. [8] T.A. Rand, S. Petersen, F. Du, X. Wang, Argonaute2 cleaves the anti-guide stranded of siRNA during RISC activation, Cell 123 (2005) 621–629. [9] C. Matranga, Y. Tomari, C. Shin, D.P. Bartel, P.D. Zamore, Passenger-strand cleavage facilitates assembly of siRNA into AGO2-containing RNAi enzyme complexes, Cell 123 (2005) 607–620.
[10] S.M. Elbashir, W. Lendeckel, T. Tuschl, RNA interferences is mediated by 21- and 22-nucleotide RNAs, Genes Dev. 15 (2001) 188–200. [11] S. Akhtar, I. Benter, Toxicogenomics of non-viral drug delivery systems for RNAi: potential impact on siRNA-mediated gene silencing activity and specificity, Adv. Drug Deliv. 59 (2007) 164–182. [12] M.A. Behlke, Progress towards in vivo use of siRNAs, Mol. Ther. 13 (2006) 644–670. [13] D. Bumcrot, M. Manoharan, V. Koteliansky, D.W. Sah, RNAi therapeutics: a potential new class of pharmaceutical drugs, Nat. Chem. Biol. 2 (2006) 711–719. [14] Y. Nakamura, K. Kogure, S. Futaki, H. Harashima, Octaarginine-modified multifunctional envelope-type nano device for siRNA, J. Control. Release 119 (2007) 360–367. [15] S. Futaki, T. Suzuki, W. Ohashi, T. Yagami, S. Tanaka, K. Ueda, Arginine rich peptides: an abundant source of membrane-permeable peptides having potential as carriers for intracellular protein delivery, J. Biol. Chem. 276 (2001) 5836–5840. [16] H.H. Kim, W.S. Lee, J.M. Yang, S. Shin, Basic peptide system for efficient delivery of foreign genes, Biochim. Biophys. Acta 1640 (2003) 129–136. [17] F. Valentin, M.C. Field, J.R. Tippins, The mechanism of oxidative stress stabilization of the thromboxane receptor in COS-7 cells, J. Biol. Chem. 279 (2004) 8316–8324. [18] S.H. Kim, J.H. Jeong, K.C. Cho, S.W. Kim, T.G. Park, Target-specific gene silencing by siRNA plasmid DNA complexed with folate-modified poly (ethylenimine), J. Control. Release 104 (2005) 223–232. [19] H.S. Choi, H.H. Kim, J.M. Yang, S. Shin, An insight into the gene delivery mechanism of the arginine peptide system: role of the peptide/DNA complexes size, Biochim. Biophys. Acta 1760 (2006) 1604–1612. [20] B. Urban-Klein, S. Werth, S. Abuharbeid, F. Czubayko, A. Aigner, RNAi-mediated gene-targeting through systemic application of polyethylenimine-complexed siRNA in vivo, Gene Ther. 12 (2005) 461–466.
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Journal of Controlled Release j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j c o n r e l
RNA interference in vitro and in vivo using an arginine peptide/siRNA complex system Sung Wook Kim 1, Na Young Kim 1, Yu Bin Choi, Seo Hyun Park, Jai Myung Yang, Sungho Shin ⁎ Department of Life Science, Sogang University, Shinsu-Dong, Mapo, Seoul, 121-742, South Korea
a r t i c l e
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Article history: Received 14 August 2009 Accepted 5 January 2010 Available online 14 January 2010 Keywords: Arginine peptide siRNA cellular location siRNA delivery system Xenograft tumor model
a b s t r a c t Efficient delivery systems are required to exploit the enormous potential of RNA interference. We introduced an arginine peptide-based small-interference RNA (siRNA) delivery system for in vitro and in vivo RNA interference. Arginine peptides formed stable complexes with siRNA and transduced siRNA into COS-7 cells in vitro, resulting in efficient gene silencing. The intracellular path of the peptide/siRNA complex was investigated in live cells using fluorescent labeling and confocal microscopy. At 24 h after transfection, most of the siRNA signals were observed in the perinuclear region, indicating that siRNA was targeted to the perinuclear region for interactions with RNA-induced silencing complex (RISC). Effective in vivo RNA interference was achieved in a mouse model bearing a subcutaneous tumor. Intratumoral administration of HER-2-specific siRNA/peptide complexes resulted in a marked reduction of tumor growth. Body weight monitoring during treatment showed that our delivery system was nontoxic. Our approach offers the potential for siRNA delivery in various in vitro and in vivo applications. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Post-transcriptional gene silencing by RNA interference (RNAi) is an attractive approach for the targeted inhibition of gene expression both in vitro and in vivo [1–3]. One of main advantages of using RNAi is its ability to specifically and potently knock down the expression of target genes. Gene silencing can be induced by small-interference RNA (siRNA) through sequence-specific cleavage of perfectly complementary mRNAs [4,5]. Introduction of siRNA into the mammalian cell cytoplasm triggers mRNA cleavage by RNA-induced silencing complexes (RISC) [6]. RISC activation is thought to initially involve endonuclease Argonaute 2 (AGO2)-mediated cleavage of the passenger or sense strand of the double-stranded siRNA, whereby the single-stranded antisense strand produced serves to guide the RISC to complementary sequences in target mRNAs [7,8]. The activated AGO2-RISC complex then binds to an mRNA strand and cleaves the mRNA strand between the bases that are complementary to bases 10 and 11 relative to the 5′ end of the siRNA guide strand, thereby leading to subsequent degradation of the cleaved mRNA transcript by cellular exonucleases [9,10]. Although their endogenous mechanism is not yet fully understood, the potential of siRNA molecules for treating a wide range of diseases has become the focus of the pharmaceutical industry in recent years [5,11]. Since the biological functions of the siRNA molecules take place in the cytoplasm, intracellular delivery of siRNA is a key factor in RNAi ⁎ Corresponding author. Tel.: + 82 2 701 8550; fax: + 82 2 704 3601. E-mail address: [email protected] (S. Shin). 1 These two authors contributed equally to this work. 0168-3659/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2010.01.009
technology. Because of the high molecular weight and net-negative charge, naked siRNA cannot freely access the intracellular environment [12]. Thus, delivery systems are required to facilitate cellular accumulation of siRNA molecules. Currently, various siRNA-delivery systems have been developed including liposomes, polymers, and peptides [13]. In recent times, membrane penetrating peptides (MPPs) have been widely used as cellular delivery vectors. These peptide-mediated delivery systems are advantageous because of the low costs incurred for reagents, nontoxic effects, and the ease of preparation. A common characteristic of MPPs is the presence of highly basic amino acids, such as arginine and lysine, thus suggesting that basic amino acids are critical motifs for the efficient delivery of exogenous biomolecules into cells [14]. Further investigation demonstrated that peptides composed only of arginine residues were able to translocate efficiently through the cell membrane [15]. Thus, the transduction properties of arginine peptides are not limited only to protein delivery, because they can deliver DNA as well. In a previous study, we demonstrated that short arginine peptides capable of forming complexes with DNA, promote efficient transfection in various mammalian cells types [16]. Despite the high proficiency of arginine peptide as a carrier for protein and DNA transfer, the molecular mechanism for the siRNA delivery process is poorly understood. In the present work, we report that functional siRNA can be delivered into cells by using a short arginine peptide (R15), and the siRNA can be localized to specific cytoplasmic compartments in the perinuclear region. In addition, arginine peptide-mediated delivery of HER-2 siRNA was found to downregulate HER-2 mRNA efficiently; inducing inhibition of tumor growth in vivo. Taken together, these
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results support the potential for an arginine peptide-mediated siRNA delivery system for therapeutic application. 2. Materials and methods 2.1. Materials GenePorter2 was obtained from Invitrogen (Carlsbad, CA, USA). Heparin was provided by Amersham/USB (Piscataway, NJ, USA). A luciferase assay kit was purchased from Promega (Madison, WI, USA). All chemicals used in the study were provided by Sigma-Aldrich (St. Louis, MO, USA), unless otherwise mentioned.
2.7. Arginine peptide-mediated siRNA delivery COS-7 cells stably expressing luciferase were seeded into the 24well plates at a density of 5 × 104 cells in 1 ml of culture medium. After overnight incubation, the cells were treated with the peptide/siRNA complexes. The solution containing these peptide/siRNA complexes (300 μl) was gently added to the cells, followed by incubation at 37 °C for 2 h in a 5% CO2 atmosphere. The cultures were then washed three times with serum-free medium and transferred to the complete medium for growth. Twenty-four hours after treatment with peptide/ siRNA complexes, cells were lysed and analyzed for luciferase activity. 2.8. Luciferase activity measurement
2.2. Peptide and siRNA synthesis Arginine peptide (R15) was prepared by solid-phase peptide synthesis using standard Fmoc (9-fluorenylmethyloxycarbonyl) chemistry. High performance liquid chromatography (HPLC) analysis indicated that the synthetic peptide was at least 95% pure. The peptide was dissolved in phosphate-buffered saline (PBS, pH 7.4) to appropriate concentrations. The siRNA used for luciferase gene silencing (sense-5′ CUU ACG CUG AGU ACU UCG ATT 3′, antisense5′ UCG AAG UAC UCA GCG UAA GTT 3′) and 5′ Cy3-labeled siRNAs were ordered from Samchully Pharm (Seoul, South Korea).
Cells were lysed by the addition of 150 μl of passive lysis buffer per well. The cell lysate (100 μl) was then recovered for the luciferase activity assay. Light emission was measured by integration over 10 s at 25 °C using a luminometer (MicroLumat Plus LB96V, Berthold, Germany). The total protein concentration in the cell lysate was determined using the BCA assay (Pierce Chemical, Rockford, IL, USA). Luciferase activity in each sample was normalized to relative light units (RLU) per mg of cell lysate proteins. 2.9. Observation of labeled peptide/siRNA complexes by confocal microscopy
2.3. Cell culture The COS-7 cells were cultured in Dulbecco's Modified Eagle Media (DMEM) (Invitrogen) with 10% heat-inactivated fetal bovine serum and 1% antibiotics (streptomycin + penicillin). For RNAi assay, cells were grown in 24-well plates and incubated at 37 °C in an incubator containing 5% CO2 atmosphere. For confocal laser scanning microscopy, cells were grown in glass bottom cell culture dishes. 2.4. Establishment of stable luciferase-expressing cells To obtain a stable luciferase-expressing cell line, COS-7 cells were transfected with luciferase gene by pcDNA 3.1/zeo-expression vector and maintained in DMEM in the presence of zeocin. Stable COS-7 cell clones were selected with zeocin (200 μg/ml) as described previously [17]. 2.5. Preparation of peptide/siRNA complexes The siRNA was diluted with 50 µl of PBS to a concentration of 10 µg/ml, and arginine peptides were dissolved in 250 µl of serumfree medium. To form the peptide/siRNA complex, the siRNA solution was pipetted into the peptide solution and mixed vigorously by vortexing. The complexes were incubated at room temperature (25 °C) for l h. The particle size of complexes was measured by dynamic light scattering (DLS) method and zeta-potential was determined by laser Doppler electrophoresis (LDE) method with Zetasizer Nano ZS (Malvern, UK). 2.6. Gel shift assay To determine the optimal relative concentration at which peptide/ siRNA complex formation would be facilitated, 0.5 µg of siRNA was incubated with various amounts of arginine peptide for 1 h at 25 °C in serum-free media, with a peptide/siRNA charge ratio ranging from 0 − to 20. The ratio between peptide (NH+ 3 ) and siRNA (PO4 ) was defined as the nitrogen/phosphate (N/P) ratio [18]. The peptide/siRNA complexes were then analyzed by electrophoresis on a 20% polyacrylamide gel in TAE (40 mM Tris acetate/2 mM EDTA) buffer at 100 V for 70 min, which was followed by staining with ethidium bromide (EtBr) for 20 min.
To investigate the distribution of the peptide/siRNA complexes in live cells, Cy3-labeled siRNA and fluorescein isothiocyanate (FITC)tagged arginine peptides were used. Double-labeled complexes were gently added to cells and incubated for various time periods. Following incubation, the cells were washed three times with PBS; they were again extensively washed with heparin-containing PBS (1 mg/ml) to completely remove the cell surface-bound complexes as described previously [19]. Distribution of the fluorescently labeled peptide/DNA complexes was analyzed using a confocal laser scanning microscope (LSM 510; Carl Zeiss, Germany) without fixing the cells. 2.10. Cytotoxicity assay The MTT assay was conducted essentially according to the manufacturer's protocol (Boehringer Mannheim, Mannheim, Germany). Briefly, cells were plated on to 96-well plates (4× 104/well) in DMEM medium with 10% heat-inactivated fetal bovine serum in the presence of peptide/siRNA complexes (3:1). After various incubation periods, MTT (200 μg/ml) was added to each well. Cells were further incubated with MTT for 4 h. The insoluble formazan was dissolved in DMSO solution. Cell viability was assessed by measuring the absorbance at 570 nm and expressed as the ratio of the A570 of cells treated with arginine peptide/ siRNA over the control samples. LDH assay was performed according to the manufacturer's protocol (BioVision, USA). 2.11. In vivo experiments In order to generate tumors, Balb/c mice (5 weeks of age; Samtako, Osan, South Korea) were injected subcutaneously in the left flank with 100 μl of cell suspension containing 5 × 106 SKOV-3 cells. Tumor size was measured using a Vernier caliper and the volume of tumor was calculated using the formula V = 0.523 × longest diameter× shortest diameter2. Treatment of the tumors was started after 10–15 days, based on when the subcutaneous tumors reached a size of 100 mm3. Argininecomplexed HER-2-specific siRNA (N/P ratio of 3.0), arginine-complexed mismatched siRNA, or naked siRNA were prepared in 5% glucose solution, and the complexes were injected directly into the tumors every 3 days at a dose of 4-μg siRNA per mouse. For HER-2 targeting, three designed siRNAs (CCUGGAACUCACCUACCUGdTdT/CAGGUAGG UGAGUUCCAGGdTdT, CUACCUUUCUACGGACGUGdTdT/CACGUCCGUA
GAAAGGUAGdTdT, and GAU CCGGAAGUACACGAUGdTdT/CAUCGUGUACUUCCG GAUCdTdT) were synthesized, and the three duplexes were mixed at equimolar ratios as described previously [20]. Mouse body weight was also monitored. 2.12. Western blot The protein (30 μg) from nude mouse xenograft for each sample was loaded on 8% SDS-PAGE gel and electrophoresed. Protein bands in the gel were then transferred to a nitrocellulose membrane. Membranes were blocked for 1 h in 5% nonfat dry milk in TBST (10 mM Tris–HCl, pH 7.5; 150 mM NaCl, and 0.1% Tween 20) and probed for 1.5 h with primary HER-2 antibodies diluted in TBST/5% nonfat dry milk (1:8000) as per the supplier's recommendations (Thermo SCIENTIFIC, Fremont, CA, USA). Membranes were then washed with TBST for 20 min × 3 and incubated for 1 h with horseradish peroxidase-labeled secondary antibodies in TBST. After an additional four washes with TBST, bound antibody was visualized by X-ray film. Membranes were tested for GAPDH (0.1 µg/ml antiGAPDH primary antibody) to confirm equal loading.
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Table 1 Characteristics of siRNA complex formation with arginine peptide at various N/P ratios. N/P ratio
Complex size (nm)
Zeta-potential (mV)
0.5 1.0 3.0 10.0
113.65 ± 0.78 182 ± 0.1 224.85 ± 4.17 270.45 ± 31.04
−10.24 ± 2.91 −1.46 ± 1.00 7.06 ± 0.10 15.63 ± 0.06
Experiments were performed at least in triplicate each time; data shown are mean value and S.D. for three different measurements.
Table 1, at N/P ratio 3.0, arginine peptide was able to condense the siRNA into small positively charged particles suitable for intracellular trafficking and biodistribution. In order to use arginine peptide as a siRNA delivery agent, the degree of cytotoxicity of peptides/siRNA complex is critical. We measured the cytotoxicity of peptide/siRNA complexes by MTT assay. To check the cell membrane integrity, LDH assay was also carried out (Fig. 2). The cell viability of peptide/siRNA complex-treated cells was almost same as that in nontreated cells. We observed no significant impairment of cell survival at a 3:1 arginine peptide/siRNA ratio. After 48 h, COS-7 cells displayed greater than a 98% survival rate.
2.13. Statistical analysis The quantitative data collected were expressed as means ± S.D. Statistical significance was analyzed by a paired, two-sample Student's t test. Statistical significance was inferred at a value of P b 0.05. 3. Results 3.1. Complex formation of arginine peptide with siRNA To assess formation of peptide/RNA complexes, a gel shift assay was employed to measure the electrostatic interaction in the complexes as a function of the positive peptide (NH+ 3 )/negative RNA (PO− 4 ) charge ratios. Luciferase siRNA (0.5 μg) was mixed with arginine peptides at various N/P ratios (0.5–20). The results showed that the electrophoretic migration of RNA was retarded with an increasing ratio of peptides/RNA (Fig. 1). The intensity of the siRNA band decreased as the N/P ratio increased. No migration RNA band was observed at N/P ratio 3.0 or higher. This absence of migration was probably due to the neutralization of nucleic acid by the arginine peptide and/or formation of a large complex between the arginine and the RNA [16]. These results suggest that an arginine peptide is sufficient to form a complex with siRNA. We next evaluated physical properties such as size and zetapotential of peptide/siRNA complexes at various N/P ratios. As seen in
Fig. 1. Gel shift assay of arginine/siRNA complex formation. Luciferase siRNA was incubated for 1 h at 25 °C with different concentrations of arginine peptides, corresponding to peptide (N)/siRNA (P) charge ratios in a range between 0 and 20.0, as indicated above each lane. The complexes were analyzed by electrophoresis on 20% polyacrylamide gel stained with ethidium bromide.
3.2. Silencing effect of arginine/siRNA complexes Since arginine peptides are known to bind siRNA, and the complexes show no cytotoxicity, we tested the biological effectiveness of this system. We investigated the inhibitory activity of siRNA against the expression of luciferase. COS-7 cells that stably express luciferase were used as a gene knockdown assay system [17]. A wide range of N/P ratios were used for transfection to explore the effect of charge parameters on delivery efficiency. As indicated in Fig. 3A, reduction of luciferase expression was observed in COS-7 cells treated with arginine/siRNA complexes when the siRNA concentration was kept constant at 0.5 μg /ml and N/P ratio was varied from 0.5 to 10.0. The expression of luciferase was decreased with increasing N/P ratio up to 3.0. At an N/P ratio of 3.0, the complex suppressed luciferase expression by 50% compared with the untreated control. The delivery of siRNA using a commercially available nonviral transfection reagent, liposome (GenePORTER2), resulted in a smaller decrease in luciferase expression compared with arginine peptide. A mismatched siRNA complexed with peptide did not result in a change in luciferase expression levels, demonstrating the selective inhibition by siRNA. Once the N/P ratio parameter was set, the time-course of inhibition produced by arginine/siRNA complexes was studied. Inhibition of
Fig. 2. Cytotoxicity of peptide/siRNA complexes at N/P ratio 3:1 for COS-7 cells was evaluated by MTT and LDH assay. Experiments were performed at least in triplicate each time; data shown are mean value and S.D. for three different measurements.
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3.3. Cellular localization of arginine/siRNA complexes Despite the current widespread use of MPPs as siRNA delivery vectors, the cellular localization of peptide/siRNA complexes has remained controversial. Previous reports have suggested that siRNA was localized in the cytoplasm around the periphery of the nucleus [21,22]. In contrast, other reports have indicated that siRNA was detected in the nucleus [14,23]. Therefore, we investigated the cellular location of siRNA in live cells. We double-labeled the complexes with Cy3 and FITC and evaluated the cellular locations of the complexes using confocal microscopy. Fig. 4A shows a representative confocal image of live COS-7 cells treated with FITC-tagged arginine peptide and Cy3-labeled siRNA complexes. Since RNAi efficiency was maximized at 24 h after transfection, microscopic observations were performed 24 h after initial treatment with peptide/siRNA complexes. Red (siRNA) and yellow fluorescence (peptide/siRNA complexes) was found in the cytoplasm in the form of a clustered-dot pattern. Notably, the complexes appeared to be heterogeneously distributed in the perinuclear region of the cells, and no signals were observed inside of the nucleus in a Z-stack image (Fig. 4B), indicating that siRNA was localized in the cytoplasm. Our results are consistent with previous findings that suggested that TAT peptide-mediated delivery of siRNA resulted in cytoplasm location of siRNA [20]. 3.4. Intracellular trafficking of arginine/siRNA complexes
Fig. 3. Luciferase activity after transfection of arginine/siRNA complexes in COS-7 cells stably expressing luciferase. Cells were incubated in the presence of preformed arginine/ luciferase siRNA complexes for 2 h, after which they were washed with serum-free medium and replaced in complete medium. Twenty-four hours later, cell extracts were prepared, and their luciferase activities were determined as described in Materials and methods. (A) Charge (N/P)-dependent silencing effect of arginine/siRNA complexes. (B) Time-dependent silencing effect of arginine/siRNA complexes. (C) Dosedependent silencing effect of arginine/siRNA complexes. Experiments were performed at least in triplicate each time; data shown are mean value and S.D. for each measurement. *, P b 0.05 compared with control.
luciferase expression remained essentially constant between 24 h and 36 h (Fig. 3B). Arginine/siRNA complexes showed maximum inhibition effect at 24 h after treatment. However, 48 h later, the luciferase activity returned to normal levels. To further identify the optimal experimental conditions, we then assessed the optimal concentration of siRNA necessary to maximize luciferase gene silencing. As shown in Fig. 3C, the silencing effect was dose-dependent. Inhibition of luciferase expression gradually increased from 20% in the presence of 15 nM siRNA to over 50% inhibition with 150 nM siRNA.
Next, to improve understanding of the cellular location of siRNA, we investigated the intracellular pathway taken by the arginine/siRNA complexes from the margin of the cell surface until their entry into the perinuclear region of live cells. When cells were incubated with the complexes for 1 h, various sized fluorescent spots were detected predominantly on the extracellular surface of the plasma membrane (Fig. 5A). After 3 h of incubation, punctuated fluorescence was observed in the cytoplasm, indicating the entry of the complexes into the cells (Fig. 5B). However, some portions of the complexes were still on the cell surface. In the cytoplasm, labeled complexes appeared as vesicles rather than as a diffuse distribution, suggesting an endocytotic uptake mechanism. With increasing incubation time, fluorescence appeared to aggregate to more discrete areas in the cytoplasm. Following 6–24 h of incubation, we noticed the appearance of fluorescence in a perinuclear location (Fig. 5C–E). The fluorescence revealed round structures restricted primarily to the external surface of the nuclear membrane. Finally, 48 h after incubation, most of the FITC signals of the arginine/ siRNA complexes disappeared, and only faint Cy3 signals were observed (Fig. 5F). All these observations suggest that the arginine/siRNA complexes make their way into the cytoplasm of cells. In addition, we assessed whether the cellular location of siRNA in the perinuclear region of the cytoplasm was affected by the presence or absence of the mRNA target. As shown in our results, siRNA localization in cells was similar without (Fig. 6) or with luciferase reporter plasmid (Fig. 4A), indicating that siRNA cellular localization was independent of the endogenous mRNA target. 3.5. Inhibition of tumor growth by arginine/siRNA complexes Although a number of MPP-mediated siRNA delivery systems have been shown to be powerful methods for inhibiting gene expression in vitro, little is known about in vivo application of these systems for therapeutic purposes. To examine the in vivo therapeutic efficiency of the arginine peptide-mediated siRNA delivery system, we investigated the effect of arginine/siRNA complexes on growth inhibition in tumors. A previous report demonstrated that the stable reduction of HER-2 expression in SKOV-3 cells inhibited subcutaneous tumor growth in a mouse model, indicating that expression of HER-2 is rate-limiting for tumor growth in vivo [20]. Therefore, we used this model to test
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Fig. 4. Cellular localization of double-labeled arginine/siRNA complexes. COS-7 cells stably expressing luciferase (5 × 104) were incubated in the presence of arginine/siRNA complexes for 24 h. After incubation, samples were prepared as described in Materials and methods and examined by confocal laser scanning microscopy. Nuclei (blue) were stained with Hoechst 33258 dye. (A) a, FITC-labeled arginine (green); b, Cy3-labeled siRNA (red); c, differential interference contrast (DIC); and d, merged image (yellow fluorescence indicates colocalization of arginine and siRNA). (B) Z-stack image of the cells. The green and red lines indicate the position within the image of the projections given in the upper and right, respectively.
whether application of arginine/HER-2-specific siRNA complexes resulted in the growth inhibition of subcutaneous tumors. SKOV-3 cells (5× 106) were subcutaneously injected into the flanks of mice. When the tumors reached a size of approximately 100 mm3, we started intratumoral treatment with arginine/HER-2-specific siRNA, arginine/ HER-2-mismatched RNA complexes, or naked siRNA and repeated treatment every 3 days for a total of five treatments. As shown in Figs. 7 and 8, arginine/HER-2-specific siRNA complex significantly reduced the tumor growth compared with arginine/HER-2-mismatched siRNA
complex, naked siRNA, or 5% glucose solution used as control. Differences were obvious as soon as 1 week after onset of treatment (Pb 0.05 at day 7). These findings were confirmed by western blot analysis of tumor sections. Measurement of HER-2 by western blot of tumor lysate showed that tumor collected 17 days following administration of arginine/siRNA complexes showed significantly decreased HER-2 expression compared with treatment with mismatched siRNA, naked siRNA, or control (Fig. 9).
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Fig. 5. Intracellular tracking of double-labeled arginine/siRNA complexes in COS-7 cells. Cells were incubated with FITC-labeled peptide and Cy3-labeled siRNA complexes at 37 °C for the indicated time periods. After incubation, the cells were washed as described in Materials and methods to remove surface-bound materials. The live cells were examined by confocal laser scanning microscopy. (A) 1 h, (B) 3 h, (C) 6 h, (D) 12 h, (E) 24 h, and (F) 48 h.
Finally, the in vivo toxicity of arginine/siRNA complexes was studied. As shown in Fig. 10, mouse body weight did not change significantly during the arginine/siRNA complex treatment. From these results, we inferred that the arginine peptide-mediated siRNA delivery system was safe. 4. Discussion Interest in siRNA as a tool for genome-wide analysis of mammalian cells is growing. As a consequence, siRNA holds great potential as a therapeutic agent against genes causing a wide range of diseases. In the current study, we introduced the arginine peptide-mediated delivery system for RNA interference in vitro and in vivo. Furthermore, to obtain the best delivery efficiency, we optimized the various technical factors such as charge ratio of arginine peptide/siRNA complex, cell/complex incubation period, and concentration of siRNA.
Using gel shift assay, we showed the ability of arginine peptides to form complexes with siRNA, as reported previously for condensing DNA [16]. Thus, siRNA may be protected from cellular enzymatic degradation and is suitable for intracellular transport. To investigate whether this complex formation ability was sufficient to induce RNAi, a COS-7 cell line stably expressing luciferase was used as a gene knockdown assay system. As much as a 50% reduction of luciferasespecific expression was obtained in cells transfected for 24 h with arginine/siRNA complex at a charge ratio of 3:1. However, further increase of the peptide/siRNA charge ratio showed decreased RNAi efficiency. One possible explanation for this result is that an overly strong peptide-siRNA interaction may prevent spatially appropriate release of siRNA to the cytoplasm, as in peptide-DNA interaction [24]. The silencing effect was also dose-dependent (Fig. 3C). A significant reduction of luciferase expression was observed when peptide was complexed with luciferase siRNA at a concentration of
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Fig. 6. Cellular localization of arginine/siRNA complexes in wild COS-7 cells. Cells (5× 104) were incubated in the presence of arginine/siRNA complexes for 24 h. After incubation, samples were prepared as described in Materials and methods and examined by confocal laser scanning microscopy. Nuclei (blue) were stained with Hoechst 33258 dye. A, FITC-labeled arginine (green); B, Cy3-labeled siRNA (red); C, differential interference contrast (DIC); and D, merged image.
150 nM. A similar effective siRNA concentration range was observed in a previous MPG peptide-mediated siRNA delivery study [25]. Prior to this study, Kim et al. investigated the potency of cholesteryl-R9 conjugates as a siRNA delivery vehicle in vitro and in vivo model [26]. Adding to hydrophobic moieties to oligoarginine was shown by others to improve the plasmid gene transfection efficiency [27], presumably due to the hydrophobic character to the arginine oligopeptides would enhance their ability to cross the plasma membrane, and hence, facilitate intracellular delivery of the plasmid DNA cargo. Comparing the R9 with cholesteryl, the maximal
Fig. 7. Arginine/siRNA complex inhibited tumor growth in nude mice. SKOV-3 cancer cells (5 × 106 cells/mouse) were implanted in the mice. Once the tumors reached approximately 100 mm3, the mice were treated intratumorally with arginine/HER-2specific siRNA, arginine/HER-2-mismatched siRNA, or naked siRNA. As a control, 5% glucose solution was injected. Data = mean, n = 6. S.D. of the data points is not shown for clarity.
achievable levels of siRNA-mediated target protein down-regulation are roughly same as arginine peptide (R15) only in our study. However, the observed RNAi effects between our study and theirs must reflect the experimental conditions, such as number of arginine residues, target genes and cells utilized for siRNA delivery. The present study was not intended to investigate this issue, and future studies will be needed to elucidate RNAi effect of hydrophobic molecule-conjugated R15 peptides.
Fig. 8. SKOV-3 xenograft tumor growth inhibition by arginine/siRNA complexes. Representative example of tumors in the four treatment groups. On day 17 tumors were taken. A, control; B, naked siRNA; C, arginine/HER-2-mismatched siRNA; and D, arginine/ HER-2-specific siRNA.
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Fig. 9. Western blot analysis of HER-2 in the SKOV-3 xenograft tumor after treatment with various formulations. HER-2 levels in tumors at 17 days after treatment initiation. A, control; B, naked siRNA; C, arginine/HER-2-mismatched siRNA; and D, arginine/HER2-specific siRNA.
Identifying the cellular location of siRNA is crucial to developing efficient peptide-mediated siRNA delivery systems. Although a considerable body of data has accumulated, the cellular location of siRNA is still controversial. Previous studies have demonstrated that siRNA delivery mediated by TAT peptide resulted in a cytoplasmic location of siRNA [21]. Further investigations have reported that part of siRNA was identified in the nucleus with the R-8 MEND carrier [14]. In addition, Veldhoen et al. suggested that the cellular location of siRNA was dependent on the type of carrier peptide used [25]. As shown in a Z-stack scanning confocal microscopy image (Fig. 4B), in our study, siRNAs were localized in the cytoplasm, especially in the perinuclear region. Furthermore, previous experiments have shown that Dicer and RNAi activity was located in cytoplasm [28,29]. Combining all these results, the present report supports the view that subcellular compartmentalization may play an important role in the function of the RNAi machinery. Since siRNA was localized in the cytoplasm in our system, we next investigated whether this cellular location of siRNA was target mRNAspecific. The luciferase siRNA was localized in the peripheral region of the nucleus in wild COS-7 cells (Fig. 6). These results suggest that the subcellular location of siRNA is not specific to the targeted mRNA. Similar results have shown that siRNA localization of both functional and nonfunctional siRNA sequences was in the perinuclear region of cytoplasm [21]. To examine further the cellular location siRNA, we investigated the intracellular path of double-labeled arginine/siRNA complexes. To avoid any artifacts due to cell fixation, our experiments were performed with living cells. The course of the intracellular pathway of complexes in our experiments is summarized as follows. At 1 h after incubation, arginine/siRNA complexes began to attach to the cell surface. At 3 h after incubation, arginine/siRNA complexes were observed in the cytoplasm. However, some of the complexes were still on the cell surface. With time, complexes gradually migrated closer to the nucleus and, 6 h after incubation, a significant fraction of the
arginine/siRNA complexes reached the cytoplasm and appeared as vesicles. The localization of complexes peripheral to the nucleus was relatively prevalent after 12–24 h following treatment of the cells. Finally, 48 h after incubation, most of the FITC signals from the arginine/siRNA complexes disappeared and only red signals (siRNA) were observed in the perinuclear region, indicating that the siRNA was released from the arginine peptides and was in a stable condition. Therefore, these results suggest that after uptake via endocytosis, peptide/siRNA complexes can escape from endosome and finally move to the perinuclear region of cytoplasm. Recently, El-Sayed et al. [30] reported that modification of nanoparticles with high density of R8 allows their escape from endocytic vesicles via membrane fusion. Based on this report, it is assumed that the escape of arginine peptide/ siRNA complexes may take place mainly through endosomal membrane fusion, which leads to efficient escape. In recent years, polycations have been widely studied as a siRNA delivery vehicle [14,22]. However, their RNAi efficiency has been described mostly in vitro. It is well known that in vitro siRNA delivery is typically far more efficient than the corresponding in vivo siRNA delivery, since the whole organism interposes the extracellular matrix, cytotoxicity, and myriad other supramolecular barriers. Here, the in vivo therapeutic efficiency of arginine/siRNA complex was investigated in nude mice bearing human ovarian cancer (SKOV3 cells) xenografts. Previously, inhibition of HER-2 expression by siRNA was reported as an effective and useful method for tumor inhibition therapy in vivo [20]. In this study, we showed that treatment with arginine/HER-2 siRNA complex inhibited tumor growth, as compared to other siRNA treatment groups, 7 days after initial treatment (Fig. 7). On 17 days later, the tumor growth rate after arginine/HER-2 siRNA treatment became comparable to the other treatment groups. In addition, a body weight-monitoring study showed no toxicity, suggesting that the arginine/siRNA complex itself was safe, which would be a prerequirement of any clinical application (Fig. 10). In conclusion, an arginine peptide-mediated siRNA delivery system has been developed. The arginine/siRNA complex at a charge ratio of 3:1 was shown to deliver siRNA efficiently both in vitro and in vivo. In addition, we showed that the perinuclear region of cells seems to play an important role in the RNAi mechanism. All these results suggest that arginine peptide in complex with siRNA opens a new avenue for the development of an efficient and safe delivery system for therapeutic applications. Acknowledgement This study was supported by a grant of the Korea Healthcare Technology R&D Project, Ministry for Health, Welfare & Family Affairs, Republic of Korea. (A085119). References
Fig. 10. Mouse body weight during the treatment with arginine/siRNA complexes. Data = mean, n = 6. S.D. of the data points is not shown for clarity.
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