Efficient siRNA Delivery into Tumor Cells by p19-YSA Fusion Protein

Dec 31, 2012 - ... a tumor-targeting siRNA delivery carrier. Eun Young Park , Mihue Jang , Jong Hwan Kim , Hyung Jun Ahn. Acta Biomaterialia 2014 10 (...
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Brief Article pubs.acs.org/molecularpharmaceutics

Efficient siRNA Delivery into Tumor Cells by p19-YSA Fusion Protein Kyung-mi Choi,† Ggon Lip Park,† Kwang Yeon Hwang,‡ Jeong-Won Lee,§ and Hyung Jun Ahn*,† †

Center for Theragnosis, Biomedical Research Institute, Korea Institute of Science and Technology, Seongbuk-Gu, Seoul 136-791, South Korea ‡ Division of Biotechnology, College of Life Sciences & Biotechnology, Korea University, Seoul 136-701, South Korea § Department of Obstetrics and Gynecology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul 135-710, South Korea ABSTRACT: For the efficient cytoplasmic delivery of siRNA in a receptor-specific fashion, we designed a p19-YSA fusion protein composed of p19 RNA binding protein and ephrin mimetic peptide (YSA peptide). The resulting recombinant protein had the high affinity for EphA2 receptor overexpressed on cancer cells as well as the complexing ability with siRNA, thus leading to tumor-targeted delivery of siRNA. The buried structure of siRNA within p19-YSA/siRNA complexes allowed the bound siRNAs to be protected from the external RNases, resulting in the enhanced stability of siRNA in serum conditions. The p19-YSA carriers could complex with siRNA in a size-dependent and sequence-independent manner and showed the pH-dependent complexing/dissocation behaviors with siRNA. In contrast to electrostatic interaction-mediated siRNA delivery systems such as cationic polymers/siRNA or cationic polypeptides/siRNA complexes, the bound siRNA within p19-YSA/siRNA complexes showed enhanced stability against large polyanions found outside cells, due to the nanomolar levels of affinity. Here, we demonstrated the superior efficiency of p19-YSA/siRNA complexes in RFP gene silencing, compared to untreated cells. These results provide an alternative approach to enhance the stability of siRNA as well as to achieve the targeted siRNA delivery. KEYWORDS: siRNA, cytoplasmic delivery, recombinant protein, tumor targeting, ephrin mimetic peptide

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protein24,25 have been demonstrated to reduce target gene expression in vitro and in vivo. Also, a peptide transduction domain−dsRNA binding domain (PTD-DRBD) fusion protein has been reported as an efficient siRNA carrier, especially in difficult-to-transfect primary cell types through PTD-mediated cellular uptake, although this approach could not target specific cell types such as tumor cells due to a lack of receptor specificity.26 To overcome these limitations in the siRNA delivery, we synthesized a p19-YSA carrier by genetically fusing a p19 RNA binding protein (p19 protein) with an ephrin mimetic peptide (called YSA peptide). Depending on the complexing property of p19 proteins with siRNA, the p19-YSA fusion protein binds siRNAs in a size-dependent and sequence-independent manner but does not bind ssRNA or dsDNA.27 In particular, p19-YSA/ siRNA complexes show the specific targeting ability of a variety of cancer cells because YSA peptide binds specifically to the ligand-binding domain of EphA2 receptor. The EphA2 receptors are highly expressed in many types of cancers and in tumor blood vessels, while they are not expressed in

INTRODUCTION RNA interference (RNAi) has been an important technology for manipulating cellular phenotypes, mapping genetic pathways, and discovering therapeutic targets, and it has attracted increased interest for potential therapeutics.1−4 However, the therapeutic applications of siRNA have been limited by its rapid enzymatic degradation and poor cellular uptake.5,6 siRNA is quickly degraded by ribonuclease (RNase) activity in serum, and due to their large size (∼14,000 Da) and high negative charge, siRNA does not readily enter cells. Chemically modified siRNA is known to have greatly prolonged stability in plasma,7,8 but it may reduce RNAi efficiency,9 and its degradation may generate metabolites that might be unsafe or trigger an unwanted effect.10 To achieve the efficient cytoplasmic delivery of siRNA, various nonviral siRNA delivery systems, including lipid-based agents,11,12 cationic polymers,13,14 and cationic polypeptides,15−17 have been efficiently employed as siRNA carriers, due to their relatively low immunogenicity, relatively low production cost or absence of oncogenicity.18,19 However, the dose-dependent toxicities found in the components of the carriers have been a hurdle to their in vivo applications. For example, polyethyleneimine and poly(L-lysine) have been shown to trigger necrosis and apoptosis in a variety of cell lines.20,21 Direct conjugations of siRNA to cholesterol22,23 or © 2012 American Chemical Society

Received: Revised: Accepted: Published: 763

June 25, 2012 October 25, 2012 December 31, 2012 December 31, 2012 dx.doi.org/10.1021/mp300344p | Mol. Pharmaceutics 2013, 10, 763−773

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quiescent vasculature and are at low levels in most adult tissues.28−31 Moreover, the shielding effect derived from the siRNA binding pocket within p19-YSA carriers does not allow access of RNases to the bound siRNA, thereby enhancing the stability of siRNA. The cellular uptake of p19-YSA/siRNA complexes was investigated by the competition experiment and endocytic inhibitor studies, and the efficiency of p19-YSA complexes for siRNA delivery was confirmed through in vitro red fluorescent protein (RFP) gene silencing.

For gene expression of the p19-YSA fusion protein, cells were grown until OD600 of 0.5 in LB medium containing 50 mg/mL kanamycin, and then protein expression was induced by 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG). After 18 h further growth, the cells were harvested and then the resulting cell pellet was resuspended in the lysis buffer (50 mM Tris-HCl pH 8.0, 100 mM sodium chloride, 1 mM phenylmethylsulfonyl fluoride). Using an ultrasonic processor, the cells were lysed and the soluble fractions were purified with two chromatographic steps; the first step was performed by metalchelate chromatography using Ni-NTA resin (Qiagen), and then the proteins were further purified by size exclusion chromatography (SEC) using a Superdex 200 10/300 GL column (GE Healthcare), previously equilibrated with a buffer containing 50 mM Tris-HCl (pH 8.0), 100 mM sodium chloride, and 1 mM mercaptoethanol. The 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). Labeling of p19-YSA Fusion Protein with Cy5.5 Dye. NIR fluorescence Cy5.5 dyes with NHS ester group were conjugated to the amine groups (−NH2) of lysine residues on the surface of p19-YSA fusion protein to track their cellular internalization in cell culture system. Based on the 3Dstructural analysis of the p19-YSA protein, there were seven lysine residues on its surface, and four lysine residues, which were not involved in complexing with RNA molecules, were expected to be the candidate sites for conjugation reaction of Cy5.5 dyes. The conjugation reaction mixture was stirred at room temperature for 2 h protected from light. To remove the unbound Cy5.5 dyes from the p19-YSA carriers, the reaction mixture was dialyzed by using PBS buffer. Quantitative Molar Ratio of p19-YSA/siRNA Complexes. The FITC-labeled siRNA (20 μg) in RNase-free distilled water (200 μL) was complexed with various amounts of p19-YSA carriers, at a molar ratio from 0.15 to 2 (fusion protein per siRNA). After incubation in the PBS buffer (pH 7.4) for 10 min at room temperature, the complex formation was confirmed by a gel retardation assay (20% polyacylamide gel) in the Tris/Borate/EDTA (TBE) buffer. The bands on the gel were visualized by a 12 bit CCD camera (KODAK Image Station 4000MM, Japan) equipped with a special C mount lens and FITC bandpass emission filter (Omega Optical; 488 nm to 530 nm). FITC-labeled siRNA/p19-YSA complexes ran down from the wells in 10% polyacrylamide gel under TBE buffer condition, but retained within the wells in 20% polyacrylamide gel. Therefore, 20% polyacrylamide gel was chosen for the present studies. siRNA Stability Test in Serum Condition and Heparin Polyanion Competition Assay. The free siRNA or p19YSA/siRNA complexes containing 0.2 μg of siRNA were incubated with RNases present in 30% fetal bovine serum (FBS) at 37 °C up to 24 h. The incubation experiments were performed in PBS conditions (pH 7.4). According to the incubation times, aliquots from each sample were electrophoresed on 20% polyacylamide gel in the TBE buffer. Under the electrophoresis condition, the siRNA complexed with the p19-YSA carriers remained in the loading wells, but the free siRNA entered the gel and ran as a clearly visible band. The FITC-labeled siRNA was used to enhance the detection limit,

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EXPERIMENTAL SECTION Materials. The YSA peptides with the sequence YSAYPDSVPMMS were synthesized by Peptron (Daejeon, Korea). The monoreactive hydroxysuccinimide 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). 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. Lipofectamine2000 was purchased from Invitrogen. Chlorpromazine hydrochloride, filipin III, and amiloride hydrochloride hydrate were purchased from Sigma. All other chemicals were purchased as reagent grade and used without further purification. All solution 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 siRNA/p19 RNA binding protein complex are from Protein Data Bank (www.pdb.org; PDB ID: 1RPU). Reagents for cell culture were purchased from Gibco BRL. Human foreskin fibroblast (HFF) cells were provided by M. J. Song, College of Life Sciences and Biotechnology, Korea University. The human EphA2 protein with an N-terminal GST-tag was obtained from Sigma. SKOV3/RFP cell line was purchased from Cell Biolabs Inc. (CA, USA). Biosynthesis and Purification of p19-YSA Fusion Protein. The full length of p19 RNA binding protein gene derived from Carnation Italian ringspot virus (CIRV) was used as a template to construct expression vectors of the p19-YSA fusion protein. The gene encoding the p19-YSA recombinant protein, which has the extended YSAYPDSVPMMS peptide at the C-terminal end of the p19 RNA binding protein, was amplified by PCR amplification using the proper forward and reverse primers and then inserted into pET-28a(+) vector (Novegen) via NdeI/XhoI restriction enzyme sites. This cloning strategy located the polyhisidine-tag and extra amino acid sequence (MGSSHHHHHHSSGLVPRGS) at the N-terminal end of p19-YSA recombinant protein. After the complete DNA sequencing of gel-purified plasmid expression vector, Escherichia coli strain BL21(DE3) was transformed with pET-28a(+)/ p19-YSA vector, and then kanamycin-resistant transformants were selected. The molecular weight and theoretical pI of p19YSA fusion protein, when calculated with its amino acid sequence, are 22691.0 Da and 6.45, respectively. 764

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and its fluorescent images were obtained by Kodak Image Station. For the heparin polyanion competition assay, the p19-YSA/ FITC-labeled siRNA complexes were incubated with a 10-, 20-, and 50-fold molar excess of heparin sodium in PBS for 1 h, and then siRNA released from the p19-YSA/siRNA complexes was analyzed by 20% polyacrylamide gel electrophoresis. The fluorescent images and intensities of FITC-labeled siRNA were examined by a 12 bit CCD camera (KODAK Image Station 4000MM, Japan). pH-Dependent Complexing of p19-YSA Carriers with siRNA. In the various pH conditions, dissociation of siRNA from the p19-YSA/siRNA complexes was examined by gel electrophoresis. The p19-YSA/siRNA complexes were incubated in the indicated pH conditions ranging from 7.4 to 5.0 for 10 min at room temperature, and then each sample were electrophoresed on 20% polyacylamide gel. The media conditions for each pH are as follows: pH = 7.4 (PBS buffer (7.4)), pH = 6.0 (20 mM sodium phosphate buffer (6.0), 100 mM NaCl), pH = 5.5 (20 mM sodium acetate buffer (5.5), 100 mM NaCl), and pH = 5.0 (20 mM sodium acetate buffer (5.0), 100 mM NaCl). Similarly to siRNA stability test, the FITClabeled siRNA was used to enhance the detection limit and its fluorescence intensities were visualized by a 12 bit CCD camera (KODAK Image Station 4000MM, Japan). Affinity Measurement Using ELISA: Free Capture Mode. The p19-YSA protein at various concentrations was first incubated in solution with the EphA2 at constant concentration until equilibrium was reached. The concentration of free EphA2 was then determined by an indirect ELISA. The p19-YSA at various concentrations (4 × 10−10 M to 2 × 10−7 M) was mixed with a constant amount of EphA2 (3 × 10−10 M) in 0.1 M potassium phosphate (pH = 7.8), 2 mM EDTA, supplemented with 10 mg/mL BSA. After overnight incubation at 4 °C, 150 μL of each mixture was transferred and incubated overnight at 4 °C in the wells of a nickel coated plate (Thermo Scientific Pierce, USA) previously coated with polyhistidinetagged p19-YSA (150 μL per well, at 1 μg/mL in 50 mM sodium carbonate (pH = 9.6), overnight at 4 °C), in which free EphA2 is captured by binding to p19-YSA on the well. After washing with PBS supplemented with 0.5% Tween 20, the bound EphA2 was recognized by primary anti-EphA2 antibody. After washing with PBS supplemented with 0.5% Tween 20, the bound immunoglobulins were detected by adding rabbit Ig with specificity against mouse IgG coupled with HRP and measuring the HRP activity retained in each well. Dissociation constant was calculated by the modified Scatchard equation.32 The affinities of EphA2 with polyhistidine-tagged p19-YSA/siRNA or polyhistidine-tagged YSA peptide were measured as described above. MTT Assay. In vitro cytotoxicity of the p19-YSA/siRNA complexes was studied in the human ovary cancer cell (SKOV3) culture system. Briefly, SKOV3 cells were seeded on a 96-well plate at a cell density of 1 × 104 cells/mL and allowed to grow for 24 h in RPMI1640 with 10% FBS. Cells were then transfected with 0.04−4.0 μM of p19-YSA/siRNA complexes per well. After 24 h incubation, 200 μL of MTT (0.5 mg/mL) dissolved in the PBS buffer was added to each well and the cells were incubated for 4 h to produce formazan crystals. The formazan crystals were subsequently dissolved in 200 μL of DMSO and 25 μL of Sorensen’s glycine buffer, and then their quantity was measured at 570 nm by a microplate reader (Spectra max340, Molecular Devices, Sunnyvale, CA).

The absorbance of the nontransfected cells was used as a control, and each absorbance was corrected relative to the control value. To examine the cytotoxicity of the p19-YSA/ siRNA complexes for the relatively small time range, the cells were incubated with the p19-YSA/siRNA complexes for 12 h, and then their cell viability was evaluated in a similar manner. In both cytotoxicity studies, there was no remarkable difference in the cell viabilities. The cytotoxicities of empty p19-YSA or polyethyleneimine (PEI, 25 kDa) were measured in a similar manner. Cellular Uptake Studies. With the FITC-labeled siRNAs and Cy5.5-labeled p19-YSA carriers, we tracked the cellular internalization of either siRNAs or p19-YSA carriers in the human ovary cancer (SKOV3). After p19-YSA/siRNA complex treatments (10 μg/mL), the cells were washed twice with PBS containing Mg2+ and Ca2+, and then fixed with formaldehyde/ glutaraldehyde-combined fixative for 15 min. The fluorescence images were obtained by a FV1000 confocal laser scanning microscope (Olympus) equipped with Argon (488 nm) and HeNe (543 nm) lasers. Cross-talk between FITC and Cy5.5 signal channels was removed by using Line Sequential Action software. In the competition experiment using YSA peptides, the SKOV3 cells were preincubated with a 100-fold molar excess of YSA peptides for 1 h, and then the cellular internalization of the Cy5.5-labeled p19-YSA carriers was examined by a confocal laser scanning microscope. The sequence of the YSA peptides was derived from the EphA2-binding YSA peptides indentified by phage display and has been found effective for cell binding and internalization.30 The effects of several membrane entry inhibitors on the p19YSA carrier’s uptake were examined by incubating the SKOV3 cell cultures with chlorpromazine (10 μg/mL) to inhibit the formation of clathrin vesicles, filipin III (1 μg/mL) to inhibit caveolae, or amiloride (50 μM) to inhibit macropinocytosis. Because the p19-YSA carriers were labeled with the fluorescent Cy5.5 dyes, their cellular internalization could be visualized under a confocal laser scanning microscope. The nucleus in each cell was stained with DAPI (blue) following the supplier’s protocol. The statistical analysis was carried out using ASW2.0c software. Statistical analysis for the quantification approach was carried out using one-way ANOVA, and the results were reported as mean ± standard deviation. In all cases, p < 0.05 was considered significant. FACS Analysis. Cellular uptake efficacy of Cy5.5-labeled p19-YSA carriers was assessed in SKOV3 cells by using fluorescence activated cell analysis (FACS) (FC-500 flow cytometer, Beckman Coulter, Miami, FL). Briefly, the Cy5.5labeled p19-YSA carriers were added to the cells at 37 °C for 1 h, and the cells were washed twice with PBS buffer. Subsequently, 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. RFP Gene Silencing in Cell Culture System, RT-PCR, and Western blot. In vitro gene silencing efficacy of p19YSA/siRNA complexes was evaluated with RFP geneexpressing SKOV3 cells. The cells were plated at a density of 1 × 104/well in slideglass bottomed 35 mm culture dishes. After 24 h incubation, cell culture media were replaced with serumfree transfection media, and p19-YSA/siRNA complexes 765

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variety of carcinoma cells compared with normal epithelium cells. To develop an efficient siRNA carrier, we genetically modified a p19 RNA binding protein by fusing with YSA peptide. In our recombinant fusion protein (i.e., p19-YSA carriers), p19 proteins are expected to form a homodimer with a concave surface made of eight β-strands in the middle of the dimer and recognize double stranded siRNA in a sequenceindependent manner,27,35 and thereby two p19 proteins can encompass a single siRNA helix (Figure 1A). In particular, each p19 protein dimer is known to bind the double-stranded 21 bp RNA the most efficiently. Denatured SDS−PAGE analysis showed that p19-YSA fusion protein, highly expressed in E. coli, was homogeneously purified with two liquid chromatographic steps (Ni-NTA affinity and size exclusion chromatography) (Figure 1B). The high affinity of p19 proteins for siRNA allows the p19YSA carriers to form p19-YSA/siRNA complexes, an important prerequisite for siRNA delivery. In the determination of the quantitative molar ratio between the bound siRNA and p19YSA carriers, relatively low concentration of p19-YSA carriers showed that the majority of siRNA molecules appeared in the unbound form at the lower band on a gel (Figure 1C). However, when the p19-YSA carriers’ concentration increased relative to the fixed amount of siRNA for each lane, the amount of the complexed siRNA proportionally increased and the shifted siRNA bands appeared. Over a certain concentration of the p19-YSA carriers, the uncomplexed free siRNA was not seen on a gel anymore. From these studies, we could determine the maximum molar ratio of the bound siRNA/p19-YSA carrier as 0.5; that is, approximately one siRNA duplex molecule can complex with one dimeric form of p19-YSA carriers. These results coincided with the theoretical molar ratio expected from the simulated structure of p19-YSA carriers. When considering the efficient RNAi in vivo by delivering exogenous siRNA, the naked siRNA is relatively unstable in its native form, and especially in blood because it is rapidly cleared via degradation by RNases.4 To investigate the stability of siRNA against RNases, free siRNA or p19-YSA/siRNA complexes were incubated with RNases present in 30% fetal bovine serum (FBS). The free siRNA was fully degraded within 30 min, but the p19-YSA/siRNA complexes showed a distinguishable protecting effect against the RNases during a more extended period (Figure 2A). This enhanced stability of siRNA was expected to be due to the shielding effect derived from the siRNA binding pocket within p19-YSA carriers. As shown in Figure 1A, the bound siRNA are buried within a concave surface of p19-YSA carriers, and thereby the resulting complex structure may not allow access of RNases to siRNA. When nonviral siRNA delivery systems including cationic polymers or cationic polypeptides are employed for RNAi, siRNA complexes might encounter unwanted exchange with large polyanions such as sulfated glycosaminoglycans, because the negative charge density of siRNA molecules is relatively lower than that of large polyanions found outside cells, and consequently such unwanted exchange may result in the reduction of siRNA delivery efficiency.36 In the polyanion competition studies using various concentrations of heparin relative to a fixed siRNA concentration, most siRNAs maintained the complexing ability with p19-YSA carriers up to a 20-fold molar excess of heparin condition, and even on a 50-fold molar excess of heparin, about 40% siRNAs were still observed to exist as a form of p19-YSA/siRNA complexes (Figure 2B). However, in the control studies with poly-

(equivalent to 200 nM siRNA), p19-YSA/scrambled siRNA complexes, or empty p19-YSA carriers were treated to the cells for 4 h. As a positive control for gene silencing test, Lipofectamine2000/siRNA (LF/siRNA) complexes were prepared according to the manufacturer’s protocol. After 4 h treatments, transfection media were removed and replaced with fresh RPMI media containing 10% FBS, and then the cells were further incubated for 20 h at 37 °C. After the cells were fixed, we observed RFP signals of each sample using a confocal laser scanning microscope. For RFP fluorescent imaging, autofluorescence originally emitted in SKOV3 cells was examined and could be removed by precisely controlling laser intensities. Offset action software equipped in a confocal microscope allowed the background signals to be removed. Also, we performed the reverse transcription-polymerase chain reaction (RT-PCR) to analyze the RFP gene silencing efficacy of p19-YSA/siRNA complexes in RFP/SKOV3 cell culture system. After 24 h postincubation, the cells were harvested and lysed, and total RNAs were extracted by using an RNeasy mini kit (QIAGEN, Valencia, CA), according to the manufacturer’s protocol. The residual DNA was removed by on-column DNase digestion using the RNase-Free DNase Set, and the DNase was efficiently removed in subsequent wash steps. The A260/A280 ratio of the extracted RNA was 1.91, and the RNA was expected to be of sufficient quality and suitable for RT-PCR application. 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′; and reverse, 5′-GCGTCCACGTAGTAGTAGCC-3′) were synthesized and purified by Bioneer (Daejeon, Korea). Twenty nanograms of cDNA per reaction was amplified during 20 cycles using the primers. The sizes of the PCR-amplified 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 the β-actin gene and quantified by TINA Image analysis software. When the extracted RNA was incubated without reverse transcriptase and subsequently amplified via PCR, PCR-amplified product was not detected on 2% agarose gel. The relative expression level of RFP gene was presented as mean ± SE (n = 3). RFP gene silencing efficacy of p19-YSA/siRNA complexes was also evaluated by Western blotting with an anti-RFP antibody. Primary antibodies against the RFP proteins were purchased from Cell Signaling Technology. RFP/SKOV3 cells treated with p19-YSA/siRNA complexes, p19-YSA/sc siRNA complexes, LF/siRNA complexes, or empty p19-YSA by the same method as described above were subsequently lysed with a lysis buffer (1% SDS, 10% glycerol, 10% 2-mercaptoethanol, 0.001% Bromophenol Blue, 50 mM Tris/HCl, pH 6.8). The samples of 10 μg of lysates and 1 μL of markers were electrophoretically separated with 18% SDS polyacrylamide gels and transferred onto a nitrocellulose membrane. Western blotting was carried out as described previously.33

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RESULTS AND DISCUSSION YSA peptide has been reported as an ephrin mimetic peptide that specifically binds to the receptor EphA2,30 and shown to deliver a wide variety of cargo into cancer cells and tissues,30,31,34 because EphA2 is more highly expressed on a 766

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under the environment of extracellular matrix compared to the cationic carrier/siRNA complexes. The binding affinity of p19-YSA carriers for siRNA was investigated in various pH conditions, because the gene delivery systems usually utilize the acidic environment of endosomal or lysosomal compartments for cytosolic release of the cargo gene.37 At neutral pH (7.4), most p19-YSA carriers showed complexing capability with siRNAs (Figure 2C). However, in the acidic condition at pH 6.0, the bound siRNAs began to dissociate from the p19-YSA carriers, and in a more acidic condition (pH 5.5), more than half of the bound siRNAs were released from the p19-YSA carriers. When the acidity of solution changed to pH 5.0, the majority of siRNAs were dissociated from the p19-YSA carriers. These results indicate that the pH drop inside the endosome or lysosome vesicles may lower the binding affinity of p19-YSA carriers for siRNA and facilitate the cytosolic release of siRNA. In the 3D structural simulation, the N-terminal polyhistidine-tag is far distant from the siRNA binding pocket and the siRNA binding is determined by the “lock and key” molecular interactions between p19 protein and siRNA molecule, and thus the polyhisitidine-tag is not expected to affect siRNA binding mode between p19-YSA and siRNA molecule. Synthetic gene delivery systems such as lipofection- or polyethylenimine-based siRNA carriers present a substantial toxic effect due to the high density of charge and limited biodegradability of cationic lipids or polymer,10 and thus have their limitations on the in vivo applications. However, as shown in Figure 2D, the p19-YSA/siRNA complexes did not cause any severe toxicity up to 2 μM, and even at 4 μM, the p19-YSA/ siRNA complexes showed reasonable in vitro cell viability. The empty p19-YSA showed a similar level of cell viability compared to p19-YSA/siRNA complexes, whereas 25 kDa of polyethyleneimine presented substantial toxicity even at 0.8 μM. Therefore, these results support that the p19-YSA fusion proteins are biocompatible in the cell culture system. Since siRNA is an anionic macromolecule that does not readily enter cells by passive diffusion mechanisms, an appropriate siRNA delivery system should enhance its cellular uptake efficiency. With the Cy5.5-labeled p19-YSA and FITClabeled siRNA, we tracked the cellular localization of the Cy5.5p19-YSA/FITC-siRNA complexes in SKOV3 cells. After 1 h incubation at 37 °C, we clearly noticed siRNAs (green spots) and p19-YSA carriers (red spots) inside the cells (Figure 3A). As the control experiments, the FITC-siRNA alone was employed for uptake studies, but its cellular uptake was not observed as we expected (data now shown). Therefore, these results show that the complexed siRNAs successfully enter the cells through the p19-YSA carrier system. Notably, even at the prolonged incubation time, either of siRNAs or p19-YSA carriers were not seen in the cell nucleus, and this observation indicates that the p19-YSA carriers do not enter the nucleus. Equilibrium binding data obtained by surface plasmon resonance reported that the YSA peptide bound to EphA2 with high affinity (KD = 186 nM ± 7).30 To examine whether the affinity of the YSA domain in the fusion protein p19-YSA was retained relative to the YSA peptide alone, we measured EphA2 binding affinity by using ELISA binding assay (Figure 3B). The dissociation constant, derived by the modified Scatchard equation,32 showed that the affinity of p19-YSA protein for EphA2 (KD = 211 nM ± 5) was similar to that of YSA peptide alone (KD = 240 nM ± 2). Also, there was no remarkable difference between the affinities of the p19-YSA/

Figure 1. (A) Three-dimensional structure of a p19-YSA carrier for siRNA delivery. As a result of structural simulation, p19-YSA/siRNA complex is drawn in molecular structure. A dimer form of p19-YSA fusion protein can encompass a siRNA duplex, and thus results in the partially buried structure of the siRNA within the binding pocket of a p19-YSA carrier. The molecular structure is rotated around a fixed axis to clearly show the bound siRNA within the p19-YSA carrier. A colorbar diagram explains how each component is connected to the other in a monomer. siRNA is colored yellow, and each color of the molecular structure corresponds to that of the color-bar diagram. The molecular structures were prepared by the Pymol program, and the structure of siRNA/p19 RNA binding protein complex is from Protein Data Bank (www.pdb.org; PDB ID: 1RPU). (B) Denatured SDS−PAGE demonstrates that p19-YSA fusion protein was highly purified through two purification steps (T, total cell lysate; S, soluble fraction of cell lysate; (−) uninduced, (+) induced by IPTG; M, molecular marker). (C) Complexing of the p19-YSA carriers with siRNA at various molar ratios. FITC-labeled siRNA (20 μg) in RNase-free distilled water was complexed with various amounts of p19-YSA carriers, at a molar ratio from 0.15 to 2 (p19-YSA carrier per siRNA), and then a gel retardation assay was performed: the amount of siRNA is kept constant for each lane while that of p19-YSA carriers is increasing from left to right. For quantitative analysis, the fluorescence intensities of the uncomplexed siRNA bands on a gel were measured by Kodak Image Station, and each was corrected relative to the value obtained from the control. This result shows the quantitative molar ratio between the bound siRNA and p19-YSA carriers.

ethyleneimine (PEI)/siRNA complexes, the relatively low concentration of heparin (equivalent to siRNA concentration) readily released the siRNA out of siRNA complexes (data not shown). The p19-YSA carriers have the nanomolar levels of affinity with siRNA molecules via “lock and key” molecular interactions, whereas the cationic siRNA carriers depend on the electrostatic interactions to complex with siRNA. Therefore, the p19-YSA/siRNA complexes are expected to be more stable 767

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Figure 2. (A) Stability test of free siRNA or p19-YSA/siRNA complexes in serum conditions. The free siRNA and p19-YSA/siRNA complexes were incubated in 30% FBS solution (pH 7.4) for the indicated times, and then analyzed by the gel retardation assay. FITC-labeled siRNAs were used to enhance a detection limit, and their fluorescent images and intensities were obtained by a 12 bit CCD camera (Kodak Image Station 4000MM) in panels A−C. Results are presented as mean ± SE (n = 5). (B) Heparin polyanion competition assay of p19-YSA/siRNA complexes in the presence of a 10-, 20-, and 50-fold molar excess of heparin sodium in PBS. The relative amounts of the complexed or released siRNAs were plotted with respect to the heparin concentrations by measuring the fluorescence intensities on the polyacrylamide gel. Results are presented as mean ± SE (n = 5). (C) pH effect on the p19-YSA carriers-siRNA interaction. The p19-YSA carriers show the pH-dependent complexing/dissociation behaviors with siRNA. In the various pH conditions, dissociation of siRNA from the p19-YSA/siRNA complexes was examined by gel electrophoresis. Results are presented as mean ± SE (n = 5). (D) In vitro cytotoxicities of RFP/SKOV3 cells treated with p19-YSA/siRNA complexes, empty p19-YSA, or polyethyleneimine (PEI) were measured by a MTT assay. The results represent the means ± SD (n = 5).

siRNA complexes and YSA peptide alone. These results demonstrate that the affinity of YSA peptide for EphA2 receptor is still retained even though YSA peptide is fused to p19 protein, and also indicate the polyhistidine-tag or any other sequence within the p19-YSA fusion protein does not affect the affinity of YSA domain. With the competition experiments using chemically synthesized YSA peptides, we examined whether the p19-YSA carriers could target the EphA2 receptors overexpressed on tumor cells. As we expected, preincubation of SKOV3 cells with a 100-fold molar excess of YSA peptides showed significantly reduced cellular uptake of p19-YSA carriers in both the confocal microscopic and flow cytometric analysis (Figure 3C,D). Moreover, p19 protein, which does not have the YSA domain, did not show any detectable cellular uptake compared to the control cells (data not shown). These results could be explained by the fact that a high excess of YSA peptides competed with p19-YSA carriers for binding with EphA2 receptors and thus the YSA peptides hindered the p19-YSA carriers from binding with EphA2 receptors on the cell surface. These results provide evidence that cellular uptake of p19-YSA carriers may be mediated by the YSA peptide−EphA2

interactions, and support that YSA peptides can be used to selectively deliver the p19-YSA carriers to EphA2 receptorexpressing tissues. To identify the uptake mechanisms involved in the cellular entry of p19-YSA/siRNA complexes, we investigated their cellular uptake in the presence of endocytic inhibitors that each block a component of the endocytosis pathway. The intracellular fate of the macromolecular carriers is strongly affected by the route of entry, and several endocytic pathways for macromolecules are identified to date. Chlorpromazine (CPZ) dissociates clathrin from the surface membrane to inhibit clathrin-mediated endocytosis.38 Filipin III (Filip) inhibits the caveolae-mediated endocytosis by the blocking of caveolae formation.39 Amiloride (Amil) inhibits the macropinocytosis by blocking the Na+/H+ exchange required for macropinocytosis.40 When SKOV cells were preincubated with CPZ and then treated with p19-YSA carriers, the uptake of p19-YSA was reduced by ca. 30% compared to that of the cells not treated with CPZ (Figure 4A). Thus, this result indicates that the clathrin-mediated endocytosis is involved in cellular uptake of p19-YSA carriers. Similarly, the Filip-treated cells and Amiltreated cells showed the relative uptake reduction by ca. 35% 768

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Figure 4. (A) Effects of endocytic inhibitors on internalization of p19YSA carriers. Prior to treatment of Cy5.5-p19-YSA carriers, SKOV3 cell cultures were preincubated with chlorpromazine (CPZ), filipin III (Filip), or amiloride (Amil) in serum-free media for 1 h. The cellular internalization was visualized by a confocal laser scanning microscope. Scale bar: 10 μm. The reduced cell uptake of Cy5.5-p19-YSA carriers was plotted by measuring the fluorescence intensities of the cells. Each was corrected relative to the value obtained from the image with p19YSA carriers alone. Results are presented as mean ± SE (n = 300). (B) Flow cytometry analysis data were quantified and plotted for several types of cancer or nonmalignant normal cells treated with Cy5.5-p19YSA carriers. The cellular uptakes of HeyA8, MDA-MB231, and SKOV3 cancer cells, which are the representative cancer cell lines known to have the highly expressed EphA2 receptors, were compared with those of the nonmalignant normal cells including HEK293 and HFF cells. The FACS profiles for HeyA8 and HEK293 cells are representatively shown. (C) Western blotting of EphA2 receptor was performed in several types of cancer or nonmalignant normal cells. Primary antibody against EphA2 protein was used for an immunoblotting analysis.

Figure 3. (A) Confocal microscopy images of SKOV3 cells after 1 h incubation with Cy5.5-p19-YSA/FITC-siRNA complexes. Red and green signals inside cells represent Cy5.5-labeled p19-YSA carriers and FITC-labeled siRNA, respectively. Scale bar: 10 μm. (B) Scatchard plot of the binding of p19-YSA to EphA2 measured by ELISA. ν is the fraction of the bound EphA2, and a is the concentration of free p19YSA at equilibrium. ν corresponds to (A0 − A)/A0, where A0 is the absorbance measured for the EphA2 in the absence of p19-YSA and A is absorbance measured in ELISA. The affinities of EphA2 with polyhistidine-tagged p19-YSA/siRNA or polyhistidine-tagged YSA peptide were measured by the same method. (C) Competition experiments with the YSA peptides were performed to inhibit the p19YSA carriers from binding the EphA2 receptors on cell surface. The SKOV3 cells were preincubated with a 100-fold molar excess of the YSA peptides prior to treatment with Cy5.5-p19-YSA carriers. As a control, the SKOV3 cells treated only with Cy5.5-p19-YSA carriers were compared. The fluorescence intensities of the Cy5.5-p19-YSA carriers were quantified and plotted. Each was corrected relative to the value obtained from the image with p19-YSA carriers alone. We tested three different samples per experiment, and 100 cells/experiment (total 300 cells) were randomly selected for imaging analysis. Results are presented as mean ± SE (n = 300). Scale bar: 10 μm. (D) The Cy5.5-p19-YSA-treated SKOV3 cells were investigated by flow cytometry analysis in the presence or absence of YSA peptides. The SKOV3 sample cells were prepared by the same method as described above. The gray dashed line represents the reduced cellular uptake of Cy5.5-p19-YSA carriers in the YSA-preincubated cells. As a control, FACS data for the SKOV3 cells treated only with PBS buffer was compared in the gray solid line.

Endocytosis of Eph receptor is critical for a number of biological processes. In particular, ephrin ligand stimulation of tumor cells induces Eph receptor internalization and degradation, a process that has been explored as a means to reduce tumor malignancy.41 The soluble ephrin-A5 ligand was rapidly internalized by the EphA8 receptor at the cell surface, and this process occurred via clathrin-mediated endocytosis.42 Moreover, both clathrin-mediated and caveolae-mediated endocytosis were reported to be linked to uptake of EphB receptor from the cell surface.43 Although the mechanism and regulation of ligand-induced Eph receptor internalization are not well understood, our endocytic inhibitor studies, consistent with the endocytosis results of these reports, might indicate that the p19-YSA carriers, after complexation with EphA2 receptor, are internalized via at least three distinct endocytic pathways. A therapeutic siRNA carrier for cancer treatment should possess two complementary characteristics: it should efficiently penetrate cell membranes, but it should also be cell typespecific by targeting only cancer cells while sparing normal cells.

and 30%, respectively. Taken together, these inhibition studies may indicate that at least three distinct endocytic pathways (clathrin-mediated endocytosis, caveolae-mediated endocytosis, and macropinocytosis) are involved in the p19-YSA uptake. 769

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Figure 5. (A) The p19-YSA/siRNA complex-mediated RFP gene silencing in RFP/SKOV3 cells. Confocal images of the cells were obtained after 1 day post-treatment with the p19-YSA/siRNA complexes (equivalent to 200 nM siRNA). The quantitative analysis of RFP expression is plotted by measuring the fluorescence intensities of the RFP expressed cells. Each measurement was corrected relative to a control. The control cells were treated with PBS buffer, and the results of the p19-YSA/scrambled siRNA complexes, empty p19-YSA carriers, and p19/siRNA complexes were also compared. Lipofectamine 2000/siRNA (LF/siRNA) complexes were prepared according to the manufacturer’s protocol. For the p19-YSA/siRNA complex-treated SKOV3 cells, the quantitative analysis of RFP expression was also plotted versus the prolonged incubation time by measuring the fluorescence intensities of the RFP expressed cells. Results are presented as mean ± SE (n = 300). Scale bar: 20 μm. (B) Semiquantitative RT-PCR analysis for RFP mRNA level. The RFP expression reduction was quantified by normalizing with β-actin expression. Results are presented as mean ± SE (n = 5). (C) Western blotting of RFP protein was performed in the SKOV3 cells treated with p19-YSA/siRNA complexes, p19-YSA/sc siRNA complexes, LF/siRNA complexes, empty p19-YSA, or p19/siRNA complexes. Primary antibody against RFP protein was used for an immunoblotting analysis, and the control cells were treated with PBS buffer. (D) Representative flow cytometry analysis data are shown for RFPexpressing SKOV3 cells either untreated or treated with the p19-YSA/siRNA complexes. The percentage of RFP positive cells was determined by gating against RFP/SKOV3 cells. The fluorescence-positive proportions of untreated or p19-YSA/siRNA complex-treated cells were also compared. Results are presented as mean ± SE (n = 5).

flow cytometry. The cellular uptakes of HeyA8, MDA-MB231, and SKOV3 cancer cells, which are known to have the highly expressed EphA2 receptors on the cell surface,44 were compared with those of the nonmalignant normal cells including HEK293 and HFF cells. When incubated with the p19-YSA carriers for 1 h at 37 °C, all cancer cell lines showed significantly increased cellular uptake efficacies as we expected, whereas the nonmalignant normal cells showed relatively much

The differential expression of EphA2 in normal cells compared to cancer cells signifies its importance as a therapeutic target.44 In particular, a high level of EphA2 is detected in malignant cancer-derived cell lines and advanced forms of cancer. To examine whether our siRNA carriers could provide a useful tool for targeted delivery of cargo molecules, we treated several types of cancer or nonmalignant normal cells with the p19-YSA carriers and compared their cellular uptake efficacies by using 770

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size (i.e., the size range of 20−23 nucleotides) and siRNA shape (i.e., RNA duplex).27,35 Thus, only double stranded siRNA with the size range of 20−23 nucleotides can complex with the p19-YSA carriers, whereas other nucleotides including DNA, single stranded siRNA, or rRNA cannot. In contrast to the high toxicity of cationic lipids or polymers utilized in siRNA delivery systems, our protein-based p19-YSA carriers did not show any severe cytotoxicity in the cell culture system. The pH drop inside the endosome or lysosome vesicles is expected to facilitate cytosolic release of the bound siRNAs due to endosomal escape, because an acidic condition lowered the binding affinity of p19-YSA carriers for siRNA. When complared to the charge−charge interactions utilized in the cationic polymers/siRNA or cationic polypeptides/siRNA complexes, the key-and-lock molecular interactions employed in the p19-YSA/siRNA complexes showed nanomolar levels of affinity and thus could explain the enhanced stability of the bound siRNA against the large polyanions found outside cells. Finally, we showed the efficiency of p19-YSA/siRNA complexes for both siRNA delivery and RNAi activity through in vitro RFP gene silencing. Recently, we have reported that a p19 protein-fused chimeric capsid protein, composed of a capsid shell, integrin targeting peptide (RGD), and p19 protein, assembled into a macromolecular container-like structure with capsid shell, and demonstrated the superior efficiency of siRNA/capsid nanocarrier complexes in RFP gene silencing.47 The RGD peptides on the exterior surface of capsid shell enabled the capsid nanocarriers to deliver siRNA into the cytosol of tumor cells and the capsid shell structure allowed the encapsulated siRNAs to be protected from the external nucleases, leading to the enhanced stability of siRNA in serum conditions. When compared to this protection effect expected in the previous chimeric capsid nanocarriers, the shielding effect derived from the siRNA binding pocket within p19-YSA carriers is also expected to enhance the stability of siRNA during body circulation in vivo. In addition, the YSA peptide-mediated tumor targeting ability of p19-YSA carriers may efficiently deliver the therapeutic siRNA to the tumor sites. Herein, our results provide an alternative approach to enhance the stability of siRNA as well as to achieve targeted siRNA delivery, and suggest that the p19-YSA carrier system has the potential of the efficient siRNA carrier in therapeutic applications.

lower levels of cellular uptake efficacies (Figure 4B). As similarly to the competition studies performed in the SKOV3 cells, all cancer cells also showed reduced cellular uptake efficacies in the presence of a 100-fold molar excess of YSA peptides (data not shown). Moreover, the immunoblot analysis using an anti-EphA2 antibody showed that the EphA2 receptor was highly expressed on malignant cancer cell lines, but not on nonmalignant normal cells (Figure 4C). Hence, these results indicate that the p19-YSA carriers can direct the bound siRNAs to the cancer cells with a high level of EphA2 receptors. In the electrostatic surface analysis of p19 protein/siRNA complexes, a concave surface that blankets siRNA molecule is noticeably positive-charged in the absence of siRNA, but the negativecharged siRNA molecule bound to p19 protein neutralizes such a positive-charged surface.45 These reports indicate that the cellular uptake of p19-YSA/siRNA complexes might not be mediated via electrostatic interactions with the anionic membrane, but via ligand−receptor interactions, although the electrostatic interactions between the anionic membrane and cationic carriers facilitate the cellular uptake.46 Next, we investigated the gene silencing efficacy of p19-YSA/ siRNA complexes in the RFP gene-expressing SKOV3 cells (RFP/SKOV3). The level of RFP expression was analyzed by visualizing RFP gene knockdown with a confocal laser scanning microscope. As shown in Figure 5A, the fluorescence images showed that the treatment of p19-YSA/siRNA complexes effectively suppressed the expression of RFP in the cells compared to the control cells, and their gene silencing efficacy was similar to that of Lipofectamine 2000 (LF)/siRNA complexes. However, the p19-YSA/scrambled siRNA complexes or empty p19-YSA carriers could not suppress the RFP gene expression in the RFP/SKOV3 cells. Moreover, the fluorescence images that did not suppress the RFP gene expression in the p19/siRNA complex-treated RFP/SKOV3 cells indicate the cellular uptake of siRNA is mediated through the interaction between YSA domain and EphA2 receptor. When the RFP/SKOV3 cells treated with p19-YSA/siRNA complexes were continuously monitored over more prolonged incubation time, the RFP gene silencing effect was seen until 3 days post-treatment, even though it gradually decreased after reaching the maximum at 24 h (Figure 5A). RT-PCR, which analyzes degradation of the RFP mRNA, also showed that the p19-YSA/siRNA complexes had a great gene silencing efficacy to RFP gene expression, wherein approximately 75% of RFP gene reduction was observed compared to untreated control cells (Figure 5B). In the Western blot analysis using anti-RFP antibodies, large amounts of RFP proteins were observed in the untreated control cells, p19-YSA/sc siRNA complex-treated cells, empty p19-YSAtreated cells, or p19/siRNA complex-treated cells, but not in the p19-YSA/siRNA complex-treated cells (Figure 5C). Flow cytometry cell analysis also showed that the RFP fluorescencepositive fraction of SKOV3 cells was 91.2 ± 2.4% in the untreated cells, but was significantly reduced to 29.6 ± 3.4% in the p19-YSA/siRNA complex-treated cells (Figure 5D). The mean fluorescence intensity was reduced to 32.0 ± 4.5% in the p19-YSA/siRNA complex-treated cells when compared to RFPexpressing SKOV3 cells. Taken together, these results indicate that the p19-YSA/siRNA complexes can break down the specific mRNA in the cell culture system. In the present studies, we have demonstrated that the p19YSA carriers had several functionalities favorable to the RNAi applications. The p19-YSA carriers have specificity to siRNA

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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]. Notes

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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