Systemic Delivery of siRNA by Chimeric Capsid Protein: Tumor

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Article pubs.acs.org/molecularpharmaceutics

Systemic Delivery of siRNA by Chimeric Capsid Protein: Tumor Targeting and RNAi Activity in Vivo Kyung-mi Choi,† Kwangmeyung Kim,† Ick Chan Kwon,† In-San Kim,‡ and Hyung Jun Ahn*,† †

Center for Theragnosis, Biomedical Research Institute, Korea Institute of Science and Technology, Seongbuk-Gu, Seoul 130-650, South Korea ‡ Department of Biochemistry and Cell Biology, Cell and Matrix Research Institute, School of Medicine, Kyungpook National University, Daegu 700-422, South Korea ABSTRACT: Recently, we reported that a chimeric capsid protein assembled into a macromolecular container-like structure with capsid shell and the resulting siRNA/capsid nanocarrier complexes efficiently suppressed RFP gene expression in the cell culture system. To extend RNAi to the in vivo applications, we here demonstrated that the siRNA/ capsid nanocarrier complexes could have tumor-specific targeting ability in vivo as well as the increased stability of siRNA during body circulation. When systemically administered, our siRNA/capsid nanocarrier complexes delivered siRNA to tumor tissues and efficiently suppressed RFP gene expression in tumor-bearing mice. The enhanced longevity of siRNA in vivo could be explained by shielding effect derived from the capsid shell, where the encapsulated siRNAs are protected from nucleases in plasma. The multivalent RGD peptides on shell surface, as a result of self-assembling of capsid protein subunits, showed efficient delivery of siRNA to the tumor tissues in vivo, due to the RGD-mediated binding to integrin receptors overexpressed on tumor cells. Moreover, the prolonged in vivo circulation time of our siRNA/capsid nanocarrier complexes increased the potential to serve as siRNA carriers for optimal in vivo RNAi. These results provide an alternative approach to systemically deliver siRNA to the tumor sites as well as to enhance the stability of siRNA in vivo. Therefore, our results revealed the promising potential of our capsid nanocarrier system as a therapeutic siRNA carrier for cancer treatment. KEYWORDS: siRNA, systemic delivery, capsid protein, tumor targeting, recombinant protein

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INTRODUCTION RNA interference (RNAi) has been of great interest not only as a powerful research tool to downregulate the expression of a target gene but also as a potent therapeutic strategy to silence disease genes.1−4 RNAi is fundamentally induced by 20−30 nucleotide double stranded small interfering RNA (siRNA), which becomes incorporated with the RNA-induced silencing complex (RISC) and guides endonucleolytic cleavage of the complementary target mRNA. The advantages of siRNA as a potential therapeutic agent are due to its naturally occurring conserved phenomenon with high specificity, and there is no limitation for its target gene/mRNA. mRNA silencing has been studied in the various target cells and tissues, including tumor and viral infection.5 Moreover, several clinical studies, focused on treatment for age-related macular degeneration (AMD) or respiratory syncytial virus (RSV), showed the successful in vivo applications of siRNA.5−9 However, siRNA has still several limitations in therapeutic applications.10,11 For example, direct administration of naked siRNA has proven efficacious through local injection, but the naked siRNA is susceptible to nuclease degradation within physiological fluids and is not able to easily penetrate the cell membrane due to its negative charge. © 2012 American Chemical Society

To achieve efficient in vivo siRNA delivery, various siRNA delivery approaches have been devised. Lentiviral vectors showed the highest efficacy of gene silencing for an extended period in mice, but safety concerns such as the risk of mutagenesis and carcinogenesis, which may take place when integrating their DNA into the host’s genomic DNA, limit their use for clinical applications.12,13 Also, pseudovirions including phagemids, herpes simplex virus (HSV) amplicons, simian virus 40 (SV40) in vitro-packaged vectors, influenza virosomes, and hemagglutinating virus of Japan (HVJ)-envelope vectors, have been developed as useful delivery vehicles in various cell types in vitro, and in vivo using mouse models.14 The pseudoviral systems are directly derived from viruses but whose viral coding sequences are eliminated, and thereby combine the most appealing features of viral and nonviral systems. However, there are some fundamental problems that still remained to be Special Issue: Viral Nanoparticles in Drug Delivery and Imaging Received: Revised: Accepted: Published: 18

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mide ester of Cy5.5 (Cy5.5-ester) was from Amersham Biosciences (Piscataway, NJ). Dimethyl sulfoxide (DMSO) and methanol were purchased from Merck (Darmstadt, Germany). Ethidium bromide (EtBr) was obtained from Sigma (St. Louis, MO). All other chemicals were purchased as reagent grade and used without further purification. All solutions were made up in RNase-free distilled water and autoclaved prior to use. The Pymol program (version 1.4.1) for 3D structural analysis of capsid architecture was obtained from DeLano Scientific LLC, and the structures of HBV capsid protein and siRNA/p19 RNA binding protein complex are from the Protein Data Bank (www.pdb.org; PDB ID: 1QGT, 1RPU). Biosynthesis and Purification of Capsid Nanocarriers. All the experimental procedures regarding molecular cloning, protein expression in Escherichia coli culture system, and liquid chromatographic purification were described in our previous report.31 Homogeneity of the purified protein was assessed by SDS−PAGE, and the protein concentration was calculated by a Bradford assay with bovine serum albumin as a standard. The protein solution was concentrated using an YM10 ultrafiltration membrane (Amicon). To analyze the assembled structure of the capsid protein, a standard protein curve on size exclusion chromatography (Superdex 200 10/300 GL column, GE Heathcare) was prepared: 24-mer of wild-type ferritin (Mr 440 kDa), BSA (Mr 67 kDa), and cytochrome C (Mr 14 kDa) were eluted at Ve = 9−10 mL, 15−16 mL, and 20−21 mL, respectively. Encapsulation of siRNA and Characterization of Capsid Nanocarriers. An excess of siRNA in RNase-free distilled water was incubated with the capsid nanocarriers in PBS buffer (pH 7.4) for 1 h at room temperature, and then the resulting mixture was loaded into a Ni-NTA affinity column (GE Healthcare). By using polyhistidine tags at the N-terminus of capsid nanocarriers, uncomplexed siRNAs were removed by wash buffer (50 mM Tris-HCl (8.0)), and then the siRNA/CN complexes bound to Ni-NTA resin were eluted with elution buffer containing 250 mM imidazole. The remaining imidazole salts were removed by a dialysis process. The protein concentration of capsid nanocarriers was determined by a Bradford assay, and the complexed siRNA to capsid nanocarriers was quantified by measuring UV absorbance at 260 nm, where siRNA concentration corresponding to A260 = 1 was 40 μg/mL. Consequently, the encapsulation efficiency of siRNA in the capsid nanocarriers was determined as siRNA/capsid nanocarrier = 120, that is, approximately 120 siRNA molecules can complex with one capsid nanocarrier. These results coincided with those of the gel retardation assay in our previous report31 and the theoretical molar ratio expected from the simulated structure of the capsid nanocarrier. For the particle size and morphology measurement, dynamic light scattering (DLS) measurement was conducted at 633 nm and 25 °C using a model 127-35 laser (Spectra Physics). We used a CM30 electron microscope (Philips) for transmission electron microscopy (TEM) images, operating at an acceleration voltage of 80 kV. In the TEM measurement, a drop of sample solution (1 mg/mL) was placed onto a 300-mesh copper grid coated with carbon. Approximately 2 min after deposition, the grid was tapped with filter paper to remove surface water and then air-dried. Negative staining was performed using a droplet of a 2 wt % aqueous uranyl acetate solution.

solved, such as immunogenicity or cell-specific targeting. The synthetic nonviral delivery systems including lipid-based agents,15 cationic polymers,16,17 and cationic polypeptides18,19 have been efficiently employed as an alternative method due to their low immunogenicity and relatively low production cost. However, the dose-dependent toxicities found in the components of the carriers should be preferentially addressed for their in vivo applications.20,21 Recently, direct conjugations of siRNA to cholesterol22,23 or protein24,25 have been demonstrated to reduce target gene expression in vitro and in vivo. High-pressure, hydrodynamic tail vein injection of nucleic acids has also been used for siRNA delivery,26 and chemically modified siRNA was shown to have a greatly prolonged half-life in plasma,27,28 although it may reduce RNAi efficiency and its degradation may trigger unwanted effects.29,30 When therapeutic siRNAs are applied to treat human diseases such as tumors or genetic disorders, their delivery carriers require several specific functionalities; the delivery carriers should have disease-specific targeting ability while stabilizing siRNA during body circulation, and also should be systemically delivered in a stable manner. Recently, we have reported that the chimeric capsid protein, which was composed of a hepatitis B virus (HBV) capsid shell, p19 RNA binding protein, and integrin-binding peptide (RGD peptide), assembled into a macromolecular container-like structure with capsid shell, and had the potential as an efficient siRNA nanocarrier in the cell culture system.31 A C-terminally truncated HBV capsid protein after residue 149, when fused with p19 RNA binding protein, assembled into capsid shell particles that contained 240 subunits and had an overall diameter of 36 nm. In particular, the shielding effect, derived from the capsid shell, could enhance stability of siRNAs by protecting them from nuclease attack, and consequently increased efficiency and longevity of RNAi. Therefore, our strategy was expected to improve the longevity of siRNA during body circulation in vivo, and we have proposed the potential of our chimeric capsid protein as in vivo siRNA delivery carriers. To extend RNAi to the in vivo applications, we investigated the siRNA delivery efficacy of our siRNA/capsid nanocarrier complexes using tumor-bearing mice. When administered intravenously through the lateral tail vein, our capsid nanocarriers showed efficient tumor targeting ability, via interaction of RGD peptides with αvβ3 integrins overexpressed in tumor tissues. The prolonged in vivo circulation time of our chimeric capsid proteins increased the potential as in vivo siRNA carriers, in particular, for an extended period. To investigate the gene silencing efficacy of our siRNA/CN complexes, red fluorescent protein (RFP) expressing B16F10 cells (RFP/B16F10) were used for in vivo animal testing, and a noninvasive optical imaging system could reveal the distinguished RFP gene silencing of siRNA/CN complexes in the tumor tissues.

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EXPERIMENTAL SECTION Materials. RFP siRNA and a mismatched scrambled (sc) RFP siRNA for target RFP gene silencing were synthesized and annealed from Bioneer (Daejeon, Korea) with the following sequences; RFP sense strand, 5′-UGUAGAUGGACUUGAACUCdTdT-3′; RFP antisense strand, 5′-GACUUCAAGUGCAACUUCAdTdT-3′; sc sense strand, 5′-UGAAGUUGCACUUGAAGUCdTdT-3′; and sc antisense strand, 5′-GACUUCAAGUGCAACUUCAdTdT-3′. FITC labeled-siRNA (the 5′end of RFP sense strand conjugated with FITC dye) was also purchased from Bioneer. The monoreactive hydroxysuccini19

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Labeling of Capsid Nanocarriers with Cy5.5 Dyes. NIR fluorescence Cy5.5 dyes with NHS ester group were conjugated to the amine groups (−NH2) of lysine residues on the exterior surface of capsid nanocarriers to monitor their cellular uptake in tumor cells or in vivo biodistribution in tumor-bearing mice. Based on the 3D-structural simulation, there were more than 480 lysine residues located on the exterior surface of a single capsid nanocarrier. p19 proteins were buried within the interior of capsid shell, and thereby the conjugation reaction of Cy5.5 dyes was expected to mainly occur with the reactive lysine residues on the capsid’s exterior. The gel retardation assay also showed that the Cy5.5 labeling did not change the complexing ability of capsid nanocarriers with siRNA (data not shown). The conjugation reaction was stirred at room temperature for 2 h protected from light. To remove unbound Cy5.5 dyes from the capsid nanocarriers, the reaction mixture was injected into a size exclusion column, which simultaneously could monitor UV absorbance by the protein sample at 280 nm and NIRF from Cy5.5 molecules at 674 nm, respectively. When compared to the standard protein curve, the Cy5.5-capsid nanocarriers were eluted as a single peak at an elution volume corresponding to that of the self-assembled native capsid particles. These results indicated that self-assembling of the capsid structure was not affected by the Cy5.5 conjugation process. Moreover, the fluorescence signal from Cy5.5 dyes coincided with an elution peak of the capsid nanocarriers, and these results revealed that Cy5.5 dyes were covalently bonded to the capsid nanocarriers. FACS Analysis. Cellular uptake efficacy of Cy5.5-labeled capsid nanocarriers was assessed in B16F10 cells by using fluorescence activated cell analysis (FACS) (FC-500 flow cytometer, Beckman Coulter, Miami, FL). B16F10 cells were preincubated with a 150-fold molar excess of cyclic RGD peptides at 37 °C for 1 h, and subsequently, Cy5.5-labeled capsid nanocarriers were added to the cells at 37 °C for 1 h. After washing with PBS buffer, the cells were detached from plates with trypsin−ethylenediamine tetraacetic acid and taken up in PBS buffer. Ten thousand cells were investigated on FACS, and their cellular uptake was assayed by excitation of Cy5.5 at 635 nm and detection of emission at 665 nm. Data were evaluated by using CXP software. TNF-α and INF-α Analyses. Mouse macrophage RWA264.7 cells were cultured 37 °C in 5% CO2 in RPMI 1640 medium supplemented with 10% of heat inactivated fetal bovine serum (FBS), 1% of L-glutamine, and 1% of penicillin− streptomycin (all from Gibco BRL, Paisley, U.K.). Macrophages were dispensed to 96-well plates and allowed to adhere for 24 h. Fresh complete RPMI medium was added, and cells were exposed to 200 nM siRNA plus either capsid nanocarriers or lipofection and seeded into a 96-well plate (5× 105 cells/well). As a positive control, cells were treated with 0.1 μg/mL lipopolysaccharide (LPS) for TNF-α induction. Culture supernatants were collected at 4 and 24 h postaddition, and assayed to analyze TNF-α induction by using TNF-α Platinum ELISA kit (eBioscience), according to the manufacturer’s protocol. Also, INF-α induction was analyzed with the cell culture medium derived from human Burkitt’s lymphoma Ramos cells. INF-α was quantified by VeriKine human INF-α ELISA kit (Pestka Biomedical Laboratories), and as a positive control, 10 μg/mL imiquimod was treated for INF-α induction. After measuring on an ELISA microplate reader, samples were analyzed by comparing their absorbances to the standard curve. In Vivo Biodistribution of Capsid Nanocarrier in Tumor-Bearing Mice. To investigate real-time in vivo

biodistribution of capsid nanocarriers, we intravenously administered Cy5.5-labeled RGD(+)- and Cy5.5-labeled RGD(−)-capsid nanocarriers (100 μg/mouse) to B16F10 tumor-bearing mice with a diameter about 8−10 mm. B16F10 cells were originally obtained from the American Type Culture Collection (Rockville, MD) and were cultivated in RPMI 1640 medium supplemented with 10% FBS, 100 U/ mL penicillin, 100 μg/mL streptomycin, and 5% CO 2 atmosphere at 37 °C. Six week old athymic male Balb/c nude (nu/nu) mice (Japan SLC, Inc.) were housed according to the guidelines by the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The CN-treated mice were monitored in a noninvasive manner by using the eXplore Optix system (Advanced Research Technologies Inc., Montreal, Canada) for up to 4 days. Laser power and count time settings were optimized at 25 μW and 0.3 s per point. Excitation and emission spots were raster-scanned in 1 mm steps over the selected region of interest to generate emission wavelength scans. A 670 nm pulsed laser diode was used to excite Cy5.5 molecules. Near infrared (NIR) fluorescence emission at 700 nm was collected and detected with a fast photomultiplier tube (Hamamatsu, Japan) and a time-correlated single photon counting system (Becker and Hickl GmbH, Berlin, Germany). Ex vivo fluorescence imaging of the excised tumors was performed by using a 12-bit CCD camera (Kodak Image Station 4000 MM, Kodak, New Haven, CT) equipped with a special C-mount lens and TRITC bandpath excitation/ emission filter set (Ex = 561 nm and Em = 580 nm; Omega Optical). The quantitative fluorescence intensities were presented as mean ± SE (n = 3). In Vivo Gene Silencing Efficacy of siRNA/CN Complexes in Tumor-Bearing Mice. To monitor in vivo gene silencing efficacy of siRNA/CN complexes, we used RFP expressing B16F10 tumor-bearing mouse model and observed its noninvasive fluorescence imaging, according to our previous studies.32 To evaluate the RFP expressing level of different number of tumor cells models, different numbers of RFP expressing tumor cells (B16F10/RFP cells; 1 × 106, 3 × 106, and 1 × 107) were inoculated into the dorsal sides of live Balb/ c nude mice. A week after tumor cell inoculation, strong red tumor fluorescent signals could be noninvasively monitored using a 12-bit CCD camera. All animal imaging experiments were performed under the identical illumination setting conditions (lamp voltage, filter, and exposure time), and all the RFP emission data were normalized to photons per second per centimeter squared per steradian (p/s/cm2/sr). The exposure time was ten seconds for all fluorescence images. The fluorescence intensity were presented as mean ± SE (n = 3). The strong red fluorescent signals showed that the RFP intensities within the tumors increased proportionally with respect to the number of inoculated tumor cells, up to 19 days. This imaging system represented RFP intensity with a sharply different value of region of interests (ROI), which was due to the different numbers of subcutaneously injected RFP expressing tumor cells. These results indicated that RFP expressing levels of various tumor tissues could be quantified and correlated with the tumor growth rate. Next, to evaluate the in vivo gene silencing efficacy of siRNA/ CN complexes, each mouse was subcutaneously injected in its flank with B16F10/RFP (1 × 106 cells/mouse) cells. Since the day of the first RFP fluorescence detection from tumor, mice received siRNA/CN complexes (33 μg of siRNA/mouse) via tail vein injection every 2 days (days 0, 2, and 4). During the 20

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experimental period, RFP expression within the tumor tissues was monitored by using a 12-bit CCD camera. The day after the final injection, mice were sacrificed and the tumor tissue was extracted for immunohistochemical staining studies. Paraffin-embedded tumor section samples with 6 μm thickness were immunostained with RFP primary antibodies and Histostain-Plus kit (Invitrogen). For semiquantitation of RFP gene silencing efficacy, we performed reverse transcription-polymerase chain reaction (RT-PCR) with the excised tumor tissues. The total RNA was isolated from the excised tumor tissues using RNeasy plus mini kit (Qiagen, Valencia, CA), following the manufacturer’s protocol. Reverse transcription was performed by MultiScribe Reverse Transcriptase contained in the High-capacity cDNA Reverse Transcription Kits (Applied Biosystems, Foster city, CA). The PCR primers (for β-actin, forward 5′-AGAGGGAAATCGTGCGTGAC-3′, reverse 5′-CAATAGTGATGACCTGGCCGT-3′; for RFP, forward 5′GGCTGCTTCATCTACAAGGT-3′, reverse 5′GCGTCCACGTAGTAGTAGCC-3′) were synthesized and purified by Bioneer (Daejeon, Korea). The sizes of the PCRamplified products were 138 bp and 245 bp, respectively, and they were separated in 2% agarose gel electrophoresis. The relative expression levels of RFP gene were normalized against expression of β-actin gene, and quantified by TINA Image analysis software. The relative expression level of RFP gene was presented as mean ± SE (n = 3).

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RESULTS AND DISCUSSION In previous reports, we have showed that p19 RNA binding protein, which was genetically fused to C-terminus of the truncated HBV capsid subunit protein, could incorporate siRNAs of interest into the capsid shell structure.31 p19 homodimer is an important prerequisite for specifically binding double stranded siRNA with nanomolar affinity, especially double-stranded 21 bp RNA with a 2-base overhang and a 5′ phosphate the most efficiently. To explain the encapsulation of siRNA molecules, we have proposed that the self-assembling process of capsid shell did not hinder each p19 protein from forming a homodimer, as shown in the Figure 1A. However, the exact molecular interactions between capsid shell and p19 homodimers were not clear. First, we investigated the siRNA binding ability of chimeric capsid proteins in their disassembled or assembled structure by varying urea concentration, because urea was reported to cause reversible dissociation of HBV capsids without denaturing protein dimer, which is a minimal building block of capsid shell.33 At 3.0 M urea, the FITC-labeled siRNA/CN complexes behaved as an apparent dimer on size exclusion chromatography (SEC) analysis, compared to the assembled shell structure in the absence of urea (Figure 1B). Also, the representative emission spectra (λex = 488) of the FITC fluorophore were observed on the dimer peak, rather than on the assembled shell peak, and the quantitative molar ratio between the bound siRNA and protein dimer was determined as 1, that is, one double stranded siRNA complexes with one protein dimer. These results show that a dimer clustering of capsid proteins allows each p19 protein to form a homodimer without losing the siRNA binding ability. When this disassembled form of siRNA/CN complexes was dialyzed under urea-free condition, each of the protein and FITC-labeled siRNAs coeluted at the assembled shell peak (about 9 mL) on SEC analysis (Figure 1B), and the

Figure 1. (A) A schematic diagram shows the self-assembling process of a chimeric capsid nanocarrier for siRNA delivery. A dimer clustering of HBV subunits drives each p19 protein to form a dimer, which is a prerequisite for complexing with siRNA, and then leads to a macromolecular container-like structure without disrupting a shell. The molecular structure of siRNA/CN complexes is drawn with partially uncovered surface to depict the encapsulated siRNAs. A colorbar diagram explains how each component is connected to the other in a monomer. siRNA is colored magenta, and each color of the molecular structure corresponds to that of the color-bar diagram. Inset image presents TEM image of the reassembled siRNA/CN complexes. (B) Size exclusion chromatography elution profile of FITC-siRNA/ CN complexes was monitored under 3.0 M urea (red line) or in the absence of urea (blue line), using UV absorbance at 280 nm. The emission spectra of the highlighted fractions were scanned at a fixed excitation wavelength (λex = 488) by fluorescence spectrophotometer (blue line at the bottom). As a positive control, the emission spectrum of uncomplexed FITC labeled-siRNA was obtained (red line). For comparison, the emission spectrum of the uncomplexed capsid nanocarriers with the equal protein concentration was scanned at λex = 488 (brown line). The emission spectra observed in the disassembled or reassembled structures were similar to each other in the peak shape and showed that FITC labeled-siRNA could complex with both the disassembled and reassembled capsid nanocarriers. 21

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quantitative molar ratio of siRNA/capsid nanocarrier in the highlighted fractions was 120, as expected from the simulated structure of siRNA/CN complexes. These reassembled siRNA/ CN complexes had average diameter of 36 ± 0.2 nm [mean ± SD] on dynamic light scattering analysis, and TEM image analysis also showed that these reassembled siRNA/CN complexes formed a capsid shell structure (Figure 1A). Notably, either of particle shape or particle size did not change compared to the native HBV capsid particles or the previously assembled siRNA/CN complexes.31 Taken together, these results indicate that each p19 protein responsible for complexing with siRNAs is located within a macromolecular containerlike structure of capsid shell, and consequently siRNAs can be encapsulated into the vacant cavity of capsid shell. Moreover, the bacterially originated RNA or DNA in the cultured E. coli did not seem to occupy the interior of the capsid, because our capsid nanocarriers without any pretreatment of RNase or DNase could complex with 120 FITC-labeled siRNA molecules on a gel retardation assay (data not shown). These results also support that our capsid nanocarriers have specificity to siRNA size and siRNA shape. Generally, the anionic siRNA molecules do not readily enter cells through a passive diffusion mechanism, and particularly, for tumor-specific targeting in vitro and in vivo, efficient siRNA delivery carriers should be devised. For tumor targeting ability, we replaced A80 and S81 in the loop segment of the surfaceexposed spike tip with the RGD peptides (CDCRGDCFC) to multivalently expose them on the shell surface (Figure 1A). Confocal microscopy images could demonstrate that the RGD peptides of our capsid nanocarriers could target cancer cells expressing αvβ3 integrin in vitro, and pH-dependent siRNA binding property of the capsid nanocarriers allowed the encapsulated siRNA to be efficiently delivered to the cytosol of cancer cells.31 In the quantitative analysis judged by flow cytometry analysis, the cellular uptake of Cy5.5-labeled capsid nanocarriers into melanoma B16F10 cells was determined as 92% at 1 h post-treatment (Figure 2A). Moreover, “cyclic” RGD peptide competition studies quantitatively showed that preincubation of the cells with a 150-fold molar excess of cyclic RGD peptides considerably reduced the cellular uptake of capsid nanocarriers up to the background level, due to the competitive binding of cyclic RGD. Notably, the confocal microscopy images in the presence of competitive RGD peptides clearly showed that our capsid nanocarriers were internalized into the cells, rather than bound on the cell surface.31 Taken together, these results indicate that our capsid nanocarriers are successfully internalized into the cancer cells through RGD-mediated integrin binding. Next, we assessed the innate immune responses of siRNA/ CN complexes by using the enzyme-linked immunosorbent assay. Innate immunity is responsible for the immediate immune response to pathogens,34 and particularly, siRNAs were reported to stimulate activation of toll-like receptors (TLR) 3, 7, 8 and consequently induce innate immune responses.35 When lipopolysaccharide or imiquimod was treated as a positive control for TNF-α or INF-α, significantly increased levels of TNF-α or INF-α induction were measured after 4 and 24 h post-treatment (Figure 2B). However, siRNA/ CN complex mediated delivery of immunostimulatory siRNAs failed to activate TNF-α or INF-α response above background levels and did not show any severe innate immune responses. Also, when compared to the innate immune response of siRNA/lipofection, which represents the commercialized

Figure 2. (A) Representative flow cytometry analysis data are shown for B16F10 cells either untreated or treated with Cy5.5-labeled capsid nanocarriers. For RGD peptide competition study, cells were preincubated with a 150-fold molar excess of cyclic RGD peptides before treatment with Cy5.5-labeled capsid nanocarriers. (B) TNF-α and INF-α induction were analyzed at 4 or 24 h after mock (PBS) treatment, RFP siRNA/CN complexes or RFP siRNA/lipofection, as indicated. 0.1 μg/mL Lipopolysaccharide (LPS) or 10 μg/mL imiquimod was used as a positive control for TNF- α and INF-α, respectively. Results are presented as mean ± SE (n = 5).

siRNA delivery method with no immunogenicity, any significant difference was not observed. Therefore, these results suggest that our capsid nanocarriers may have the potential as a siRNA carrier to achieve efficient RNAi in vivo. Targeting tumor cells or tumor vasculature by peptides is a promising strategy for delivering therapeutic drugs for cancer therapy.36 RGD peptide-inserted caged proteins found in the engineered ferritin and heat shock protein (Hsp) proteins showed their specific binding ability to cancer cells, which expressed high levels of αvβ3 integrins.37,38 In particular, the RGD-inserted Hsp proteins could be specifically accumulated to tumor tissues in the tumor-bearing mice when administered intravenously through the lateral tail vein.39 To obtain substantive evidence for tumor-targeting in vivo, we examined the in vivo biodistribution of Cy5.5-labeled RGD(+)- or RGD(−)-capsid nanocarriers on B16F10 tumor-bearing mice. The NIR photons emitted from Cy5.5 are minimally absorbed by water or hemoglobin and thus result in noninvasive live animal imaging. The RGD(+)-capsid nanocarriers, which have multivalently displayed RGD peptides on the shell surface, showed continuously increased tumor accumulation immediately after intravenous injection, and their NIRF intensities reached a maximum at 72 h postinjection (Figure 3A). Moreover, the NIRF intensities of tumor tissue could still be seen until 96 h postinjection by the eXplore Optix system. 22

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Figure 3. (A) In vivo real-time NIR fluorescence imaging of intravenously injected Cy5.5-labeled capsid nanocarriers to B16F10 tumor-bearing mice. Time-dependent tumor-targeting specificities of RGD(+)- and RGD(−)-capsid nanocarriers were monitored by using the eXplore Optix system. The circled region and arrow represent the solid tumors grown after subcutaneous injection. (B) Ex vivo imaging of the excised B16F10 tumors treated with Cy5.5-RGD(+)- or Cy5.5-RGD(−)-capsid nanocarriers after 72 h postinjection. Fluorescent images were obtained by Kodak Image Station, and their quantitative results are presented as mean ± SE (n = 3).

siRNA/CN-treated mice showed the increase of RFP intensities proportionally to the tumor growth, because the sc siRNA did not result in any substantial gene silencing efficacy. After 6 day postinjection, the fluorescence images of ex vivo excised tumor specimens from the siRNA/CN-treated mice showed remarkable RFP gene reduction compared to those of the sc siRNA/ CN-treated mice, and in particular, the RFP total photon counts per gram of tumor in the siRNA/CN-treated mice were 6.7-fold lower than those from the sc siRNA/CN-treated tumors (Figure 4B,C). However, either of siRNA/RGD(−)CN complexes or free siRNA did not show any detectable gene silencing effect (data not shown). In the immunohistochemical staining assay using an anti-RFP antibody, large amounts of RFP proteins were observed in the sc siRNA/CN-treated tumors, but not in the siRNA/CN complex treated tumors (Figure 4D). RT-PCR, which analyzes degradation of RFP mRNA in the ex vivo excised tumor tissues, also showed about 75% of RFP gene reduction in the siRNA/CN-treated tumors compared to the sc siRNA/CN-treated tumors (Figure 4E). Taken together, these results indicate that our siRNA/CN complexes can efficiently suppress the gene expression of target protein in tumor tissues. According to our previous studies,31 the capsid nanocarriers had several functionalities favorable to in vivo RNAi applications. First, the shielding effect, which is derived from the capsid shell, enhanced stability of siRNA by protecting from nuclease attack, and consequently may increase efficiency and longevity of in vivo RNAi. Second, pH drop inside the endosome or lysosome vesicles is expected to facilitate cytosolic release of the bound siRNAs because an acidic condition lowered the binding affinity of capsid nanocarriers for siRNA. Third, the recombinant protein-based capsid nanocarriers showed increased cell viability in the cell culture system, in contrast to the high toxicity of cationic lipids or polymers utilized in siRNA delivery systems, and, due to the lack of viral nucleic acids, do not have the risk of mutagenesis and

However, the control sample, free Cy5.5, was rapidly excreted from the body within 1 day and tumor accumulation was not observed (data not shown). A considerable liver uptake of our RGD(+)-capsid nanocarriers, found together with the specific tumor accumulation, may be partially integrin-mediated, as consistently shown in RGD-mediated imaging studies.40,41 However, a more detailed mechanism that explains the undesirable liver uptake still remains unclear, although RGD peptides have been widely utilized as a targeted-therapy ligand in the field of cancer imaging and drug delivery. As an effort to avoid liver uptake, modification of RGD peptides with sugar molecules has been reported to increase the tumor-to-liver ratio in mouse model.42 In contrast to the RGD(+)-capsid nanocarriers, the RGD(−)-capsid nanocarriers, that is, the truncated HBV capsid particles, did not show any tumor accumulation ability, and detectable NIRF signals could be seen only in the spleen and kidney. Also, ex vivo images of the excised tumors after 72 h postinjection showed significantly higher fluorescence intensities in the RGD(+)-CN-treated tumors rather than in the RGD(−)-CN-treated (Figure 3B). Finally, we evaluated in vivo gene silencing efficiency of siRNA/CN complexes using an animal imaging system (KODAK Image Station 400MM) on mice bearing lateral B16F10/RFP tumors. In the in vitro gene silencing studies using siRNA/CN complex treated B16F10/RFP cells, RFP gene silencing by the siRNA/CN complexes persisted until 2 days, but further gene silencing effect was not seen 3 days post treatment.31 Therefore, each mouse received the scrambled (sc) siRNA/CN or siRNA/CN complexes via tail vein injection every 2 days (days 0, 2, and 4). The in vivo optical imaging in the RFP channels on mice clearly showed substantial RFP signal reduction after the first siRNA/CN complex injection, although B16F10 tumors were continuously growing (Figure 4A). Notably, the siRNA/CN complex injected mice did not show any increase in RFP signal at the tumors up to 6 days, compared to the sc siRNA/CN-injected mice. However, the sc 23

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Figure 4. In vivo gene silencing images by systemic administration of RFP siRNA/CN complexes to tumor-bearing mice. (A) Fluorescence images of RFP gene silencing from scrambled siRNA/CN or siRNA/CN complexes, all with 33 μg of siRNA/mouse. (B) Ex vivo fluorescence imaging of the excised B16F10 tumors treated with sc siRNA/CN or siRNA/CN complexes after 6 day postinjection. (C) RFP intensities at different tumors were recorded as total photons per centimeter squared per steradian (p/s/cm2) per milligram (n = 3 mice per group). (D) Immunohistochemical staining of the excised tumors with RFP antibody. Brown spots represent RFP antibodies. (E) Semiquantitative RT-PCR analysis for RFP mRNA level from the excised tumor tissues. The RFP expression reduction was quantified by normalizing with β-actin expression. Results are presented as mean ± SE (n = 3).

Notes

carcinogenesis expected in the viral vectors such as retroviruses and lentiviruses.12 In the present studies, we also demonstrated that the capsid nanocarriers could systemically deliver siRNAs to the tumor tissues on mice and efficiently suppress target gene expression. Based on noninvasive NIR fluorescence imaging, this tumorspecific targeting ability of our capsid nanocarriers can be explained by prolonged in vivo circulation as well as RGDmediated binding to cancer cells. Moreover, the multivalency of the RGD peptides on the shell surface may increase the in vivo targeting abilities of our capsid nanocarriers, because multivalent versions of RGD peptide, as a homing peptide, exhibited much greater levels of tumor uptake than monomeric RGD peptide.41 Therefore, our siRNA delivery system provides an alternative approach to systemically deliver siRNA to the tumor sites as well as to enhance the stability of siRNA in vivo, and also suggests the potential as a therapeutic siRNA carrier for cancer treatment.

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by the Converging Research Center Program through the Ministry of Education, Science and Technology (2010K001205) and by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (2010-0029206).

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AUTHOR INFORMATION

Corresponding Author

*Korea Institute of Science and Technology, 39-1 Hawolgokdong, Seongbuk-gu, Seoul 136-791, Korea. Tel: +82-2-9585938. Fax: +82-2-958-5909. E-mail: [email protected]. 24

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Molecular Pharmaceutics

Article

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