Biomaterials 35 (2014) 7121e7132

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Biomaterials journal homepage: www.elsevier.com/locate/biomaterials

Tumor-specific delivery of siRNA using supramolecular assembly of hyaluronic acid nanoparticles and 2b RNA-binding protein/siRNA complexes Kyung-mi Choi a, Mihue Jang b, Jong Hwan Kim b, c, Hyung Jun Ahn b, * a b c

Institute of Global Environment and Department of Biology, Kyung Hee University, Dongdaemun-Gu, Seoul 130-701, South Korea Center for Theragnosis, Biomedical Research Institute, Korea Institute of Science and Technology, Seongbuk-Gu, Seoul 136-791, South Korea Department of Chemistry, Kyung Hee University, Dongdaemun-Gu, Seoul 130-701, South Korea

a r t i c l e i n f o

a b s t r a c t

Article history: Received 17 March 2014 Accepted 21 April 2014 Available online 20 May 2014

Anticancer therapeutics delivering exogenous siRNA have been explored to suppress the tumorassociated genes, but several limitations of siRNA delivery such as tumor-targeted delivery, controlled siRNA release at the sites of interest, or instabilities of siRNA in physiological fluids should be preferentially addressed for its clinical applications. As an attempt to meet these criteria, we designed a supramolecular assembly, which was composed of cholesterol-bearing hyaluronic acid (HA-Chol) conjugates and 2b RNA-binding protein (2b)/siRNA complexes. In contrast to the traditional siRNA polyplexes using electrostatic interactions, HA-Chol nanoparticles, as a results of self-assembly of HAChol conjugates, provide the hydrophobic core that acts as the container for 2b protein/siRNA complexes, where a high affinity of 2b protein for siRNA could neutralize the negative-charged siRNA. Here, we investigated the potential of HA-Chol/2b/siRNA complexes as the siRNA carriers that provide encapsulation, protection, and targeted delivery of siRNA. The HA-Chol nanoparticles could selectively deliver 2b protein/siRNA complexes to the tumor cells with up-regulated CD44 receptors and suppress the expression of target gene. The pH-associated binding properties of siRNA for 2b proteins allowed the controlled release of siRNA in the endocytic compartments, and ultimately the released siRNA could obtain the RNAi acitivities in the cells, whereas the encapsulated 2b proteins still stayed within the HAChol nanoparticles. Our delivery systems demonstrate the promising potential of the efficient siRNA carriers in the anticancer therapeutic applications. Ó 2014 Elsevier Ltd. All rights reserved.

Keywords: siRNA siRNA carrier Supramolecular assembly Hyaluronic acid-choleresterol conjugate

1. Introduction Recently, RNA interference (RNAi) has provided a promising tool to suppress the expression of target gene via degradation of mRNA [1,2]. RNAi mechanism is fundamentally triggered by small interfering RNA (siRNA), which initiates the cleavage and degradation of its complementary mRNA after assembled with RNA-induced silencing complex (RISC), and consequently, the synthesis of the corresponding protein is blocked. However, the inherent properties of naked siRNA such as its anionic property, immune response, large molecular weight, and enzymatic degradation in serum have

* Corresponding author. Korea Institute of Science and Technology, 39-1 Hawolgok-dong, Seongbuk-Gu, Seoul 136-791, South Korea. Tel.: þ82 2 958 5938; fax: þ82 2 958 5909. E-mail address: [email protected] (H.J. Ahn). http://dx.doi.org/10.1016/j.biomaterials.2014.04.096 0142-9612/Ó 2014 Elsevier Ltd. All rights reserved.

limited its applications in vitro or in vivo [3e5]. Also, the naked siRNA does not possess any functional moiety that selectively reaches the target tissues, an appropriate carrier system should address these criteria for therapeutic RNAi applications. A large range of delivery vehicles have been exploited for siRNA, including viruses, lipids, proteins, polymers, and gold nanoparticles [6e9]. In particular, the traditional polycationic polymers such as polyethylenimines (PEIs), chitosans, and reducible polyamidoamines (rPAAs) have been explored to achieve the effective siRNA delivery and gene knockdown [10e12]. The amino groups present in the polycationic polymers can complex with anionic siRNA via electrostatic interactions and thus provide a driving force to form the condensed siRNA-complexed nanoparticles (siRNA polyplexes), which protect the bound siRNA against RNases attack as well as neutralize the anionic siRNA for cellular internalization. Since the effective delivery systems should avoid potentially toxic side effects of the vehicles, there have been many efforts to

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reduce the inherent cytotoxicities derived from a high charge density or limited degradability of polycationic polymers, of which cytotoxicities lead to necrotic cell death or apoptosis in the uncontrolled manner [13]. For example, many researchers have modified polycationic polymers or synthesized new polycationic polymers, while keeping a balance between their complexing ability and cell viability [14]. Nevertheless, it is still challenging to condense siRNA into the less cytotoxic siRNA polyplexes. Interestingly, many globular proteins have been experimentally reported to complex with the self-assembled nanoparticles such as cholesterol-bearing pullulan through the hydrophobic interactions between these proteins and hydrophobic moieties within the nanoparticles [15,16]. In this respect, the self-assembled nanoparticles, composed of amphiphilic polymers, have extensively drawn much attention as a protein drug carrier because they permit the encapsulation of hydrophobic therapeutic proteins, thereby protecting them from harsh in vivo conditions [17]. In the current study, hyaluronic acids (HA) as a starting building block is a highly anionic mucopolysaccharide, of which the alternating disaccharide units are composed of D-glucuronic acid and Nacetyl-D-glucosamine with b (1e4) interglycosidic linkage [18]. HA is the biodegradable non-toxic biomaterial, and notably, has been used as the popular delivery agent for cancer treatment, because it specifically binds CD44 and RHAMM receptors abundantly presented in many types of tumor cells as well as regulates the angiogenesis during tumor growth [19,20]. For example, HAmodified macromolecular prodrugs and nanocarriers showed a specific binding to CD44-overexpressing tumor cells regardless of their negatively-charged surface charge [21,22]. Also, thiolfunctionalized HA nanogels encapsulating siRNA by an inverse emulsion method showed the cellular internalization and gene knockdown effects in the CD44-overexpressing HCT-116 cells [23]. For delivering siRNA to tumor cells, we prepared the selfassembled HA nanoparticles (HA-Chol NPs) in an aqueous environment after hydrophobically modifying HA backbone with cholesterols. Although the HA nanoparticles are the attractive tumorspecific carrier for siRNA delivery, their negatively charged property cannot encapsulate the anionic siRNA directly [24]. Instead of encapsulating the naked siRNA, we here proposed an alternative method to incorporate siRNA/2b protein complexes into the HA nanoparticles. The 2b proteins, derived from Tomato aspermy virus, are known to bind dsRNA to counter host defense during viral infection [25], and here showed the stable complexing ability with siRNA in the physiological pH condition. Based on the 3D structural analysis of siRNA/2b protein complex, 2b proteins neutralize the negative-charged phosphate backbone of siRNA when they wrap around the siRNA duplex [26]. Here, we demonstrate that the neutralized siRNA, as a result of complexing with 2b proteins, can be encapsulated into the HA-Chol NPs to form the HA-Chol/2b/ siRNA complexes in the absence of polycationic polymers (Fig. 1). We enzymatically degraded the HA-Chol/2b/siRNA complexes with hyaluronidase to show whether siRNA locates deeply inside the core of HA-Chol nanoparticles or is simply bound to the surface of nanoparticles, because the siRNA stabilities in serum can be explained by inaccessibility of RNases. Also, we investigated the CD44-mediated tumor-targeting abilities of the HA-Chol/2b/siRNA NPs and their cellular uptakes via endocytosis pathways. To better understand the siRNA dissociation in the acidity environments such as endosomes or endolysosomes, we assessed the variation of siRNA binding affinity for 2b proteins or HA-Chol NPs in the acidic condition. Using the FITC-labeled 2b proteins, we could examine the retention of 2b proteins within the nanoparticles after cellular internalization. Ultimately, we investigated whether the HA-Chol/ 2b/RFP-siRNA NPs effectively suppressed the expression of targeted RFP gene in tumor cells. Compared to the traditional delivery

Fig. 1. Schematic illustration of supramolecular assembly for tumor-specific siRNA delivery. The amphiphilic HA-Chol conjugates self-assemble to form HA-Chol nanoparticles, which provide the hydrophobic core that can act as a container for hydrophobic cargo, 2b protein/siRNA complexes. The molecular structures explain how 2b protein dimer recognizes siRNA duplex. 2b proteins and siRNA are colored magenta and yellow, respectively. The molecular structures were prepared by the Pymol program, and the structures of 2b protein, siRNA, and 2b/siRNA complex are from the Protein Data Bank (PDB IDs: 2ZI0). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

methods utilizing electrostatic interactions, these studies provide an alternative strategy to deliver siRNA to the tumor cells for cancer therapy and control siRNA release, as well as to efficiently encapsulate siRNA into HA nanoparticles. 2. Materials and methods 2.1. Materials Cholesteryl chloroformate, ethylenediamine, N-(3-dimethylaminopropyl)-N0 ethylcarbodiimide (EDC), N-hydroxysulfosuccinimide (sulfo-NHS), bovine testicular hyaluronidase, and pyrene were purchased from Sigma Aldrich (St. Louis, MO). Sodium hyaluronate (HA, Mw 234 kDa) was obtained from Lifecore Biomedical (Chaska, MN). NHS-Cy5.5 fluorescence dye was purchased from Amersham Bioscience (Piscataway, NJ). Primers and siRNA were available from Bioneer (Daejeon, Korea). 2.2. Synthesis of HA-Chol conjugates First, cholesteryl chloroformate was converted to 3b-Cholest-5-en-3-yl N-(2Aminoethyl)carbamate (Chol-Am) that could be reacted with the carboxylic acids of HA, as shown in the Fig. 2. In brief, cholesteryl chloroformate (2.25 g, 5 mmol) was dissolved in anhydrous toluene (50 mL), and to this solution, ethylenediamine (16.7 ml, 250 mmol) dissolved in anhydrous toluene (150 mL) was dropwisely added. After stirred for 16 h at room temperature, the mixture was washed out with

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solution and distilled water, respectively. We freeze-dried the dialyzed solution and could obtain HA-Cholesterol (HA-Chol) conjugates as a powder. The amount of cholesterol molecules conjugated in the backbone of HA was determined by 1H NMR in CDCl3. 2.3. Preparation and characterization of HA-Chol NPs To form HA-Chol nanoparticles via a self-assembling process, we dissolved the amphiphilic HA-Chol conjugates in PBS buffer with sonicating for 2 min (Digital Sonifier-250, Branson, MO). After filtering the nanoparticles (2 mg/mL) with a membrane filter (pore-size; 0.45 mm), we could measure their sizes and zeta potential charges using a Zetasizer Nano ZS (Malvern, UK), which was operated at 633 nm and 22  0.3  C. The autocorrelator collected the scattered light at an angle of 90 and each of diameter values reported in the current studies was an average of more than three measurements. Each of measurement composed of more than 11 data collections. The transmission electron microscopy images of HA-Chol nanoparticles could be obtained in a similar way as described previously [27]. Moreover, we measured the critical aggregation concentration (CAC) following a procedure described elsewhere [28], and ultimately, could estimate CAC value of HA-Chol conjugates by plotting the fluorescence intensity ratio (I338/I333) versus HA-Chol concentration. 2.4. Cy5.5-labeling to HA-Chol NPs and FITC-labeling to 2b proteins We added NHS-Cy5.5 (14 mmol) to an excess of ethylenediamine (700 mmol) dissolved in dichloromethane, and stirred for 6 h. We could remove the unreacted ethylenediamine with solvent extraction method using distilled water/dichloromethane (1/1, v/v), and then could prepare amine-bearing Cy5.5. Next, HA-Chol NPs (1 mg) dissolved in PBS buffer were reacted with the amine-bearing Cy5.5 (1 mmol, in 200 mL of THF) in the presence of EDC/sulfo-NHS (1 mmol/1 mmol) for 2 h, and then the reaction mixture was subjected to dialysis against distilled water. The content of Cy5.5 molecules in the Cy5.5-HA-Chol NPs was 4.5 wt.%, as determined using UV/VIS spectrophotometer (Agilent, Santa Clara, CA) at 686 nm. To label 2b proteins with FITC fluorescence dye, we chemically conjugated the amine functional groups of 2b proteins with NHS-Fluorescein, which was obtained from Thermo Scientific. Briefly, we dissolved 20 mg of NHS-Fluorescein in 1 mL of sodium carbonate (pH 9.3) and mixed the solution with 1 mg of 2b proteins, which was stored in PBS buffer. After incubating 3 h at room temperature, we dialyzed the reaction mixture against PBS buffer using membrane tubes (MWCO ¼ 3000) to remove the unreacted NHS-Fluorescein. When we calculated the molar concentrations of FITC and 2b proteins, the ratio of FITC/protein was between 2 and 5. 2.5. Biosynthesis and purification of 2b protein For cloning of expression vectors, we first amplified an insert gene corresponding to 2b proteins from Tomato aspermy virus by PCR reaction with forward and reverse primers. Based on the restriction enzyme sites NdeI and XhoI, we could ligate the insert gene into plasmid pET-22b(þ) using T4 DNA ligase. The insert gene sequences of plasmid pET-22b(þ)/2b were confirmed by DNA sequencing analysis, and then the plasmid was transformed into Escherichia coli strain BL21(DE3) on the ampicillin-containing agar plates. After 10 mL of seed culture was inoculated to 1 L of LB medium containing ampicillin antibiotics, cells were grown at 37  C. At OD600 of 0.5, we added 0.5 mM of IPTC into the cells to induce the protein expression. After overnight growth, we harvested the cells by using centrifugation. The collected cell pellet was resuspended with 30 mL of buffer A containing 50 mM TriseHCl (pH 8.0), and then fully sonicated. After centrifugation at 13,000 rmp for 1 h, we loaded the supernatant fractions into Ni-NTA resin (Qiagen affinity system) for protein purification. The resin was fully washed with 100 mL of buffer A, and then the purified proteins could be eluted by 10 mL of buffer B containing 50 mM TriseHCl (pH 8.0) and 300 mM imidazole. We confirmed the protein purity using SDS-PAGE analysis and calculated its concentration based on a Bradford method. To concentrate the protein solution, we used Amicon Ultra 3K MWCO centrifugal filters (Millipore). 2.6. Supramolecular assembly of HA-Chol NPs, 2b protein, and siRNA Fig. 2. Synthetic scheme and 1H NMR spectrum of HA-Cholesterol conjugate.

distilled water and subjected to freeze-drying. The resulting powder was then dissolved in dichloromethane/methanol solution (1/1, v/v, 100 mL) with stirring. We filtered off the suspension solution with a syringe filter (PTFE, MWCO: 1 mm, Whatman, Piscataway, NJ) to eliminate biscarbamate, and lyophilized the filtrate to obtain cholesteryl amine conjugate (Chol-Am) as a powder (750 mg, 1.60 mmol, 32%). We characterized its chemical structure using 1H NMR (Unity Plus 600, Varian, 400 MHz). Second, we chemically modified the backbone of HA with the hydrophobic CholAm in the presence of EDC and sulfo-NHS. Briefly, we dissolved sodium hyaluronate (100 mg, 244 mmol) in distilled water (10 mL) containing EDC (23.4e93.5 mg) and sulfo-NHS (26.4e106.0 mg), and to this solution, we slowly added Chol-Am conjugate (5.35 mge21.4 mg, 12.2 mmole48.8 mmol) dissolved in THF (10 mL). After stirred for 6 h, the reaction mixture was dialyzed against distilled water/THF (1/1, v/v)

First, we estimated the complexing ratio between 2b protein and siRNA using a gel retardation assay. FITC-siRNA (20 mg) dissolved in 20 mL of PBS buffer was incubated with varying amounts of 2b protein for 10 min. On 20% polyacrylamide gel, we examine the electrophoretic mobilities of each sample under TBE running buffer. We could detect the fluorescent bands corresponding to FITC fluorescence using a 12 bit CCD camera (KODAK Image Station 4000 MM, Japan). Second, we measured the loading content of siRNA in the self-assembled HAChol/2b/siRNA NPs. We dissolved each type of HA-Chol NPs (1.6 mg) in 20 mL of PBS buffer, and then mixed the nanoparticle solutions with 2b/FITC-siRNA complexes (1.5 mg, 2:1 M ratio). At the indicated incubation time, each sample was centrifuged at 13,000 rpm for 30 min and the supernatant was collected to separate the unbound 2b/FITC-siRNA from HA-Chol/2b/FITC-siRNA NPs, which were precipitated as a fluorescent pellet. When we measured the amounts of FITC-siRNA in the supernatant fraction, the amounts of FITC-siRNA assembled into HA-Chol/2b/FITC-siRNA NPs could be estimated. To calculate the loading content of siRNA in HA-Chol/2b/ FITC-siRNA NPs, we divided the amount of siRNA assembled into HA-Chol/2b/

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FITC-siRNA NPs by the weight of HA-Chol conjugates plus 2b proteins. In contrast of HA-Chol/2b/FITC-siRNA NPs, there was no precipitation seen when we centrifuged 2b/FITC-siRNA solution. The fluorescent intensities of FITC-siRNA were determined by a 12 bit CCD camera in a similar way as described above. 2.7. Hyaluronidase digestion With 50 U/mL of bovine testicular hyaluronidase dissolved in PBS buffer, we degraded the HA-Chol24/2b/FITC-siRNA NPs (10 mg/mL) for the given incubation time ranging from 0 to 48 h. After centrifuging the reaction solutions at 13,000 rpm for 30 min, we separated the supernatant from the pellet, and then measured the relative amounts of FITC-siRNA in the supernatant using a 12 bit CCD camera. The fluorescent pellet corresponding to HA-Chol24/2b/FITC-siRNA NPs was resuspended with PBS buffer and its fluorescent intensity was also quantitatively measured. 2.8. siRNA stability test and pH-associated dissociation of siRNA from HA-Chol/2b/ siRNA NPs We incubated HA-Chol24/2b/FITC-siRNA NPs (corresponding to 0.2 mg of siRNA) with PBS buffer containing 30% of fetal bovine serum (FBS) at 37  C. According to the given incubation times, we quenched the enzymatic degradation by adding RNase inhibitor (3 units), and then loaded each sample to 20% polyacrylamide gel. During the electrophoresis under TBE running buffer, the FITC fluorescence bands corresponding to HA-Chol24/2b/siRNA NPs were continuously seen only in the loading wells due to their large molecular weights, whereas the FITC fluorescence bands of free FITC-siRNA showed the fast electrophoretic mobility. Next, to investigate pH-associated dissociation behavior of siRNA from the assembled nanoparticles, we incubated HA-Chol24/2b/FITC-siRNA NPs for 30 min under the different buffer conditions ranging from pH 7.4 to pH 5.0. Subsequently, each sample was electrophoresised on 20% polyacrylamide gel and the resulting fluorescent bands were measured in a similar way as described above. For quantitative analysis of fluorescent bands, we used GelQuant software. Following the same procedures, we also examined the pH-associated dissociation behavior of siRNA from 2b/FITC-siRNA complexes and plotted the amounts of dissociated siRNA along the indicated pH conditions. 2.9. Cytotoxicity studies and TNF-a analysis For in vitro cytotoxicity studies, we carried out MTT assay following a procedure described elsewhere [27]. We examined the cytotoxicities of empty HA-Chol NPs and HA-Chol/2b/siRNA NPs in the B16F10/RFP murine melanoma cells, and plotted their cytotoxicities along the concentrations ranging from 5 mg/mL to 100 mg/mL. We also studied the cytotoxicities of branched polyethylenimine (PEI, 25 kDa), which is one of the representative polycationic polymers, and compared their cytotoxicities with those of HA-Chol nanoparticles. For the innate immunity studies, we performed TNF-a analysis following the same procedures as previously described [29]. Briefly, to the murine macrophage RAW264.7 cells (1  104 cell/mL) prepared on a 96-well plate, we treated empty HAChol24 NPs (10 mg/mL), 2b/siRNA complexes (50 nM), HA-Chol24/2b/siRNA NPs (corresponding to 50 nM siRNA), or Lipofectamine 2000 (LF)/siRNA complexes (corresponding to 50 nM siRNA), and then collected the cell culture supernatants at 4 h and 24 h post-treatment, respectively. Next, we measured the released TNF-a cytokines using TNF-a Platinum ELISA kit (eBioscience, San Diego, CA) according to the manufacturer’s instructions. As a positive control for TNF-a release, we treated 0.1 mg/mL of lipopolysaccharide and the resulting TNF-a secretion was also measured. 2.10. Cell internalization studies, blocking control experiments, endocytic inhibitor studies, and intracellular localization studies To estimate the amounts of CD44 on the cells, we incubated 200 mL of murine B16F10 cells melanoma cells with anti-CD44 antibody (PE tag, R&D systems) for 1 h and then washed three times with PBS buffer. In parallel, we treated human foreskin fibroblast (HFF) normal cells, for the negative control set, in the same procedure. After detaching both sets of cells from the plates, we could collect 1  104 cells/set. The relative amounts of CD44 on both sets of cells were measured by flow cytometer (FC-500 flow cytometer, Beckman Coulter, FL). For cellular uptake studies, we treated Cy5.5-HA-Chol24/2b/FITC-siRNA NPs (10 mg/mL) to B16F10 cells, which were cultured in RPMI-1640 medium supplemented with 10% FBS using a 5% CO2 incubator. At 2 h post-treatment, we washed the cells three times with PBS buffer. We could measure the Cy5.5 and FITC fluorescence images by using a DeltaVision Deconvolution microscope (Applied Precision, Issaquah, WA) equipped with 60 oil immersion lens, and quantitatively analyze their fluorescence intensities with CXP software. DAPI (10 mg/mL) was treated to cells for the nuclear staining. As a control, we treated the free FITC-siRNA to the cells and could obtain their fluorescence microscopic images. To perform the blocking studies, we pretreated free HA polymer (10 mg/mL) to B16F10 cells for 1 h prior to treatment with HA-Chol24/2b/FITC-siRNA NPs (10 mg/ mL) for 2 h. In parallel, we treated the cells only with HA-Chol24/2b/FITC-siRNA NPs (10 mg/mL) for 2 h. Next, we washed both cell samples with PBS buffer and obtained their fluorescence images by using the fluorescence microscope. The relative

fluorescence intensities of both cell samples were analyzed according to the same procedure as described above, quantified, and plotted. To examine each effect of four types of chemical endocytic inhibitors, we first searched the concentration ranges that would result in less than 10% of cytotoxicities in the B16F10 cells. Such nontoxic concentration ranges were as follows; chlorpromazine (10e20 mg/mL), filipin III (1e5 mg/mL), amiloride (50e80 mM), and cytochalasin D (10e30 mg/mL). We pretreated each drug in its non-toxic concentration range to the B16F10 cells, which then incubated with HA-Chol24/2b/FITC-siRNA NPs (10 mg/mL) for 2 h. After washing three times with PBS buffer, we visualized FITC fluorescence images by the fluorescence microscope and quantified the fluorescence intensities. We indicated the error bars as mean  standard deviation for three replicate measurements and the * represented p < 0.05 when determined using one-way ANOVA. For labeling of early endosomes, we transfected the B16F10 cells with CellLight Early Endosomes-RFP (Rab5a) markers 24 h prior. In parallel, we stained the B16F10 cells with Lysotracker Red DND-99 markers for labeling of late endosomes and endolysosomes. To both sets of stained cells, we treated HA-Chol/2b/FITC-siRNA NPs (10 mg/mL) for 1 h, and then acquired the cellular images by the fluorescence microscope, which was equipped with polychroic filter providing excitation and emission wavelength for FITC or RFP. CellLight Early Endosomes-RFP and Lysotracker Red DND-99 markers were purchased from Invitrogen and Life Technologies. 2.11. Nuclear localization of 2b protein To visualize the nuclear localization of 2b proteins, we prepared FITC-labeled 2b proteins (FITC-2b proteins) as described above and delivered them directly into B16F10 cells by using Xfect Protein Transfect Reagent (Clontech Laboratoreis, Inc., Mountain View, CA). First, we seeded the cells on a 6-well plate the day before doing transfection. To prepare the transfection reagent/protein mixture, we diluted 15 mL of Xfect Protein Transfection Reagent stock solution in 85 mL of distilled water and mixed them with 100 mL of FITC-2b protein/siRNA complexes (50 mg/L) following the manufacturer’s protocol. While incubating the mixture for 30 m, we washed the cells with PBS. After removing PBS, we added serum-free medium to each well. For transfection, we added the prepared transfection reagent/protein mixture to the cells and incubated for 1 h. In parallel, we prepared HA-Chol/FITC-2b protein/siRNA NPs and treated the cells with them in the absence of transfection reagents. We obtained the fluorescence images of both sets of cells by the fluorescence microscope. For clarity, we stained nucleus with DAPI and also measured its fluorescence images. The nuclear localization images of transfection reagents/FITC-2b proteins/ siRNA mixture were compared with those of HA-Chol/FITC-2b protein/siRNA NPs. 2.12. Targeted RFP gene knockdown in RFP-expressing B16F10 cells After RFP-expressing B16F10 (B16F10/RFP) cells were seeded on 96-well plates the day before siRNA treatments, the cells were grown in RPMI-1640 medium supplemented with 10% FBS using a 5% CO2 incubator. While preparing the mixture of medium and HA-Chol24/2b/siRNA NPs (corresponding to 50 nM of siRNA), we washed the cells with the medium. For RFP gene knockdown, the cells were treated with the mixture and incubated for 2 h. The cells were washed with the medium and then cultured further for the additional 22 h. To examine the RFP gene knockdown, we measured the RFP fluorescence images using the fluorescence microscope. In the same procedure, we treated the cells with HA-Chol24/2b/scrambled siRNA NPs (equivalent to 50 nM siRNA), or empty HA-Chol24 NPs (10 mg/mL) and obtained their fluorescence images. RFP targeting siRNA and scrambled (sc) siRNA could be prepared as previously described [27]. Similarly, the RFP gene silencing abilities of LF/ siRNA complexes, as the traditional gene transfection reagents, were examined. To quantify the RFP gene knockdown using RT-PCR, we completely removed DNA in the extracted total RNAs from the harvested cells following the procedures as previously described [27]. After transcribing RFP mRNA to its complementary DNA by using Reverse Transcriptase (Sigma Aldrich), we amplified the reaction mixture using PCR reaction (denaturing steps, 92  C for 1 m; annealing steps, 50  C for 1 m; elongation steps, 65  C for 1 m; 30 cycles). The amplified DNAs were analyzed on 2% agarose gel electrophoresis. Also, we estimated the relative amounts of RFP proteins in the RFP gene-suppressed cells following the Western blotting methods as previously described [27]. Finally, we performed a flow cytometry analysis (FC-500 flow cytometer) to quantify the RFP gene knockdown in the HA-Chol24/2b/siRNA-treated cells. For ten thousands of cells, we estimated the RFP fluorescence-positive fractions and the mean fluorescence intensities. The excitation and emission wavelength for RFP detection was 550 and 600 nm, respectively.

3. Results and discussion 3.1. Synthesis of cholesterol-bearing hyaluronic acids For the synthesis of amphiphilic HA-Chol conjugates, chloroformate group of cholesteryl chloroformate was first converted to amine group in the presence of excess ethylenediamine, leading to formation of 3b-Cholest-5-en-3-yl N-(2-Aminoethyl)carbamate

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(cholesteryl amine) (Fig. 2). Depending on a feed ratio of cholesteryl amine to HA polymers in the presence of EDC and sulfo-NHS, the number of cholesterol per 100 sugar residues on HA backbones (degree of substitution, DS) was tunable, and the cholesterolbearing HA conjugates (HA-Chol conjugates) could be obtained. When we examined the chemical structures of HA-Chol conjugates using 1H NMR (CDCl3), formation of cholesteryl amine could be confirmed by the peaks appearing from ethylene group (d ¼ 2.82 ppm [2H, eCH2NH2], d ¼ 3.22 ppm [2H, eNHCH2e]). When the amount of cholesteryl amine was calculated based on the integration ratio between the characteristic peak of N-acetyl group in HA (d ¼ 2.01 ppm, [3H, eCOCH3]) and that of methine group of cholesterol (d ¼ 5.37 ppm [1H, eCH]C]), the DS of cholesterol increased proportionally to the feed ratio of cholesteryl amine, as shown in the Table 1. 3.2. Characterization of HA-Chol nanoparticles The freshly prepared HA-Chol conjugates were sonicated in aqueous conditions to spontaneously form the nano-sized particles (HA-Chol NPs). When the particle sizes of nanoparticles from HAChol conjugates with different DS (HA-Chol3 e HA-Chol24) were evaluated by dynamic light scattering (DLS), the average sizes of HA-Chol NPs ranged from 190 to 410 nm. As the DS increased, the particle size of HA-Chol NPs decreased, and these results indicated the formation of dense nanoparticles was derived from the formation of more hydrophobic core. Table 1 shows that the x potentials of HA-Chol NPs are negative in the range of 64.3 to 45.1 mV, indicating that the negatively charged HA polymers surrounded the nanoparticle surface. Moreover, the moderately low polydispersity factors (0.22e0.298) showed that HA-Chol NPs had a narrow size distribution. According to the TEM images, the shape of HA-Chol NPs was uniformly spherical and the sizes of particles in a dehydrated state were moderately smaller than those determined from DLS analysis (Fig. 3A). When the sizes of particles were monitored for 6 days in the PBS buffer, there was no significant variation in size, and these stabilities might be due to the negatively charged surface of HA-Chol NPs (Fig. 3B). Moreover, the sizes of particles were rarely affected by their concentrations ranging from 0.5 to 5 mg/mL and not aggregated by interactions among nanoparticles (data not shown). The Fig. 3C shows the critical aggregation concentration (CAC) of HA-Chol NPs estimated by pyrene fluorescence studies. When the intensity ratio (I338/I333) of pyrene excitation spectra measured with varying the HA-Chol NPs concentrations, the CAC values of HA-Chol NPs ranged from 0.006 to 0.15 mg/mL depending on their DS. In particular, as the number of conjugated cholesterol molecule increased, the CAC values of HA-Chol NPs remarkably decreased, and thus these results show that the molecular associations required for self-assembled nanoparticle formation increases proportionally to the amounts of conjugated cholesterol molecules. Table 1 Characteristics of HA-Chol conjugates. The number behind HA-Chol indicates the degree of substitution of cholesterol. Samplea

DSb

M nc

Size (nm)d

x (mV)e

PDIf

HA HA-Chol3 HA-Chol13 HA-Chol24

e 2.6 12.7 24

234,000 241,000 270,000 301,000

e 410 268 190

e 64.3 54.7 45.1

e 0.282 0.22 0.298

a b c d e f

HA-Chol conjugates with different DS values. Degree of substitution (DS, the number of cholesterol per 100 sugar residue). Number-average molecular weight, estimated from 1H NMR spectrum. Average size of HA-Chol NPs, measured by dynamic light scattering in PBS. The zeta potential of HA-Chol NPs in distilled water (1 mg/mL). Polydispersity Index of HA-Chol NPs.

Fig. 3. (A) Size distribution of HA-Chol NPs, prepared from HA-Chol24 conjugates (2 mg/mL) in PBS buffer, is shown. The inset TEM image shows the particle shape of nanoparticles in distilled water. (B) Stability of HA-Chol nanoparticles, estimated by size change in PBS buffer as a function of time. (C) Determination of critical aggregation concentration (CAC). Fluorescence intensity ratios from pyrene excitation spectra were plotted against concentration of HA-Chol conjugates. CAC of HA-Chol3, HA-Chol13, and HA-Chol24 conjugates were 0.15, 0.025, and 0.006 mg/mL, respectively.

3.3. Supramolecular assembly of HA-Chol NPs, 2b protein, and siRNA Based on the structural simulation as shown in the Fig. 1, two 2b proteins recognize a single siRNA duplex. When a complexing ratio of 2b protein and FITC-siRNA was estimated on the gel shift assay, where the relative amounts of 2b proteins were varied for a fixed amount of FITC-siRNA, a single FITC-siRNA duplex was recognized

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by two 2b proteins (Fig. 4A), and these results coincided with those obtained from the structural simulation studies. Next, we loaded 2b/FITC-siRNA complexes (2:1 M ratio) into HAChol NPs to form a supramolecular assembly. When each type of HA-Chol NPs were incubated with 2b/FITC-siRNA complexes for the indicated time (1 he48 h), as shown in the Fig. 4B, a simple centrifugation step (13,000 rpm, 30 min) could separate the unbound 2b/FITC-siRNA complexes from a precipitated fluorescent pellet corresponding to the assembled HA-Chol/2b/FITC-siRNA NPs. By measuring the fluorescent intensities of FITC-siRNA in the supernatant, the loading content of siRNA in the nanoparticles could be estimated, as described in the experimental section. At 1 h incubation time, each of HA-Chol NPs (HA-Chol3, HA-Chol13, and HA-Chol24) showed more than 7% loading content of siRNA. As the incubation time increased, the loading content of siRNA continuously increased, and at 24 h, reached about 9.5%, 11%, and 11% on HA-Chol3 NPs, HA-Chol13 NPs, and HA-Chol24 NPs, respectively. As a control, we centrifuged a solution of 2b/FITC-siRNA complexes in the absence of HA-Chol NPs, but there was no precipitated pellet and 2b/FITC-siRNA complexes only existed as a soluble form (data not shown). Moreover, when FITC-siRNA was loaded to HA-Chol NPs in the absence of 2b protein, any detectable fluorescence intensity of FITC-siRNA was not seen within the HA-Chol NPs even up to 48 h incubation time. These studies showed that the anionic siRNA molecules could be efficiently encapsulated into HA-Chol NPs as a 2b/siRNA complex form, not as a free siRNA form. In these assembly studies, HA-Chol24 NPs exhibited the highest loading content for siRNA among the HA-Chol NPs with different DS, as well as the nano-sized particles favorable for systemic delivery [30], and therefore chosen as a candidate siRNA carrier. Notably, the particle sizes and x potentials of HA-Chol24/2b/siRNA NPs were determined as 192 nm and 44.2 mV, respectively and there was no substantial variation both in size and surface charge of nanoparticles after 2b/siRNA complexes were encapsulated into HA-Chol24 NPs. In the following experiments, we incubated HAChol24 NPs with 2b/siRNA complexes for at least 24 h and used the pellet obtained after centrifuging. 3.4. Enzymatic degradation of HA-Chol/2b/siRNA NPs The hyaluronidases are the enzymes that randomly hydrolyze the hyaluronic acids into polysaccharide fragments with a glucuronic acid [31]. To further examine whether siRNA was encapsulated into the core of nanoparticles or simply bound to the surface of nanoparticles, we treated the assembled HA-Chol/2b/FITC-siRNA NPs with hyaluronidases for the indicated time (Fig. 4C). After the resulting break-down products were centrifuged, each of fluorescence intensities from the supernatant and pellet fraction was measured by a 12 bit CCD camera. As the incubation time increased, the fluorescence intensities of supernatant fractions, which corresponded to the free FITC-siRNA or 2b/FITC-siRNA complexes released from the nanoparticles, gradually increased, and in 24 h incubation time, about 45% of siRNAs were found as a dissociated form. Also, the fluorescence intensities from the pellet fractions corresponding to the assembled HA-Chol/2b/siRNA NPs showed that about 60% siRNAs still remained encapsulated even in 24 h incubation time. These observations that siRNA dissociation increases proportionally to enzymatic degradation of hyaluronic acids indicate that siRNA may be located deeply inside the nanoparticles, rather than simply bound to the surface of nanoparticles. 3.5. siRNA stability test in serum condition Therapeutic RNAi strategies employing the exogenous siRNA should consider the stability of siRNA in the physiological fluid,

Fig. 4. (A) Complexing of siRNA with varying amounts of 2b protein for determination of a molar ratio of 2b/siRNA complexes. In the Figure AeC, FITC-labeled siRNAs were used to enhance the detection sensitivity instead of EtBr-staining. The FITC fluorescence intensities were measured by Kodak Image Station. (B) Determination of loading content of 2b/siRNA complexes in the HA-Chol carriers. At the indicated incubation time, each sample was centrifuged at 13,000 rpm for 30 min, and the supernatant was collected to separate the unbound 2b/FITC-siRNA from HA-Chol/2b/FITC-siRNA NPs, which were precipitated as a fluorescent pellet. In the absence of 2b protein, the loading content of FITC-siRNA in the HA-Chol carriers was also measured in a similar manner, but any detectable fluorescence intensity of FITC-siRNA was not seen within the HA-Chol NPs. Results are presented as mean  s.e. (n ¼ 5). (C) Release of siRNA from HA-Chol/2b/siRNA NPs during enzymatic degradation by hyaluronidase. After the resulting break-down products were centrifuged at 13,000 rmp, each of fluorescence intensities from the supernatant and pellet fraction was measured in a similar way to the Figure B. Results are presented as mean  s.e. (n ¼ 5).

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where the naked siRNA is rapidly degraded by enzymes and cleared [32]. In the RNase conditions containing 30% fetal bovine serum (FBS), the naked siRNA was completely degraded at 30 min incubation time, whereas the siRNA encapsulated into HA-Chol NPs showed the enhanced stability behaviors over the more extended period of time (Fig. 5A). Consequently, about 50% of siRNA within the HA-Chol/2b/siRNA NPs remained intact at 48 h incubation time. These siRNA stabilities might be due to the inaccessibility of RNases, as expected from the encapsulation of siRNA into HA-Chol NPs. 3.6. Dissociation of siRNA from HA-Chol/2b/siRNA NPs in the acidic conditions In the polymer-based gene delivery systems, polyamines such as PEI, PLL, or spermine are usually conjugated into the polymer backbone to promote cytosolic release of cargo genes in the acidic endosome or endolysosome [33]. We also examined the dissociation properties of siRNA from the HA-Chol/2b/FITC-siRNA NPs in the acidic conditions. Fig. 5B shows that dissociation of siRNA was not seen in the neutral condition (pH 7.4), but as the acidity shifted to pH 6.5, siRNA initiated to dissociate from the HA-Chol/2b/FITC-siRNA NPs. Since 2b proteins remained in the loading wells of 20% polyacrylamide gel and did not move due to their large molecular weights, the lower fluorescent bands represented the free siRNA, not 2b/siRNA complexes. As the solution conditions became more acidic, the degree of dissociation proportionally increased to the acidity, and ultimately in pH 5.0 condition, siRNA lost its association ability with the nanoparticles. In a similar way, the dissociation of siRNA from 2b proteins was also investigated in the acidic conditions. As the acidity increased from pH ¼ 7.4 to pH ¼ 5.0, dissociation of siRNA from 2b proteins significantly increased, and at pH 5.0, most siRNA was dissociated from the 2b proteins (Fig. 5C). This pH-associated assembly of siRNA and 2b proteins might be explained by 2b proteins’ histidine residues that are critically involved in the hydrogen bonds, electrostatic interactions, or van der Waals interactions with the bound siRNA. The His29, His38, and His51 residues, which are located on the interface between siRNA and 2b proteins and particularly have a pKa value of 6.5, are invariable or highly conserved. As the previous mutational studies performed on the His29 residue have reported that the binding affinity of mutated proteins was lowered by ten times [26], the increased protonation of those residues in the acidic conditions may consequently lower the affinity of 2b protein for siRNA. These studies showed that acidity could induce the release of siRNA from HA-Chol/2b/siRNA NPs, of which the negatively charged properties would accelerate release of the anionic siRNA due to the chargeecharge repulsion. Therefore, these results suggest that the acidity environments inside the endosome or endolysosome may facilitate the cytosolic release of encapsulated siRNA. 3.7. Cytotoxicity studies and TNF-a analysis Polyamines usually contribute positive charges to nanoparticle delivery systems for encapsulation of cargo siRNA and its cytosolic release, but their substantial cytotoxicities derived from high charge density and limited biodegradability imply that a critical balance of polyamine contents and cell viability should be considered when designing the safe nanoparticles for therapeutic siRNA delivery [34,35]. Fig. 6A shows that there was little or no cytotoxicity seen in the empty HA-Chol NPs- and HA-Chol/2b/siRNA NPs-treated cells when we monitored it up to 100 mg/mL using a MTT assay. In

Fig. 5. (A) Stability test of free siRNA or HA-Chol/2b/siRNA NPs in 30% FBS solution (pH 7.4). The free siRNA and HA-Chol/2b/siRNA NPs incubated for the indicated times were analyzed by a gel retardation assay. In the Figure AeC, FITC-labeled siRNAs were used to enhance the detection sensitivity instead of EtBr-staining, and their fluorescent images and intensities were obtained by a 12 bit CCD camera (Kodak Image Station 4000 MM). Results are presented as mean  s.e. (n ¼ 5). (B) pH effect on the supramolecular assembly (HA-Chol/2b/siRNA NPs). In the various pH conditions, dissociation of siRNA from the HA-Chol/2b/siRNA NPs was examined by a gel electrophoresis. The relative fluorescence intensities of upper and lower bands were plotted along the pH conditions. Results are presented as mean  s.e. (n ¼ 5). (C) pH effect on the 2b protein-siRNA interaction. The 2b protein also shows the pH-associated complexing/ dissociation behaviors with siRNA. Results are presented as mean  s.e. (n ¼ 5).

contrast, 25 kDa of polyethylenimine (a branched form), which is a representative polyamine used for gene delivery systems [13], resulted in a significant cytotoxicity at the relatively low concentrations as expected. These studies show that HA-Chol NPs assembled with siRNA/2b complexes do not cause the severe cytotoxicity in the cell culture systems and can be treated in

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Fig. 6. (A) In vitro cytotoxicity studies of B16F10 cells treated with empty HA-Chol NPs, HA-Chol/2b/siRNA NPs, or polyethylenimine (PEI, a branched form) were performed by a MTT assay. Results are presented as mean  s.e. (n ¼ 5). (B) TNF-a induction was analyzed at 4 or 24 h after mock (PBS) treatment, empty HA-Chol NPs, 2b/siRNA complexes, HAChol/2b/siRNA NPs, or Lipofectamine/siRNA complexes treatment. 0.1 mg/mL Lipopolysaccharide (LPS) represents a positive control of TNF-a induction. Results are presented as mean  s.e. (n ¼ 5). (C) Fluorescence microscopy images of B16F10 cancer cells or HFF normal cells after 1 h incubation with Cy5.5-HA-Chol/2b/FITC-siRNA NPs. Green and red signals inside the cells represent FITC-siRNA and Cy5.5-HA-Chol NPs, respectively. The nucleus in each cell was stained with DAPI (blue). The merged fluorescence images of HFF normal cells were presented because there was neither green nor red signal. Also, the relative amounts of CD44 on B16F10 cancer and HFF normal cells were estimated by FACS analysis. (D) The blocking studies of CD44 receptors by binding of free HA polymer ligands. To saturate the binding sites of CD44, the free soluble HA polymers were pre-treated to B16F10 cells prior to HA-Chol/2b/FITC-siRNA treatments, and then their fluorescence intensities were compared to those of the cells treated only with HA-Chol/2b/FITC-siRNA NPs. Results are presented as mean  s.e. (n ¼ 3). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

concentration ranges to facilitate the gene silencing without undesirable cytotoxicity. Since the exogenous siRNA is known to potentially activate the innate immune systems via TLR pathways, we investigated the

cytokine release in the HA-Chol/2b/siRNA NPs-treated RAW264.7 macrophage cells. Notably, siRNA structures or its carriers are closely associated with the innate immune responses, which are responsible for the immediate immune reactions and lead to the

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cytokine induction [36,37]. As shown in the Fig. 6B, TNF-a was remarkably produced in the lipopolysaccharide-treated cells after 4 h and 24 h, respectively, whereas there was little or no TNF-a production in the HA-Chol/2b/siRNA NPs-treated cells. Moreover, either of empty HA-Chol NPs or 2b/siRNA complexes did not induce any remarkable TNF-a response. Similarly, any significant activation of TNF-a response was not seen in the Lipofectamine 2000 (LF)/ siRNA complexes-treated cells, where LF/siRNA complexes represent the commercialized siRNA delivery carriers. Therefore, these studies suggest that the HA-Chol/2b/siRNA NPs may facilitate the efficient RNAi in vivo without activating the innate immune systems. 3.8. Cellular uptake of nanoparticles by target cells Notably, the receptor expression levels have drawn remarkable attention for delivering siRNA to the target sites of therapeutic interests, as well as efficient cellular internalization [38]. When we estimated the relative amounts of CD44 receptors on B16F10 cancer and HFF normal cells using FACS analysis, CD44 receptors on B16F10 cells were highly expressed (that is, 85%) in comparison with those on HFF cells (3%) (Fig. 6C). Next, using the HA-Chol NPs and siRNA labeled with the fluorescence dye Cy5.5 and FITC, we monitored the cellular internalization of Cy5.5-HA-Chol/2b/FITCsiRNA NPs in B16F10 or HFF cells. After 2 h post-treatment, each of FITC and Cy5.5 fluorescence was observed as green and red signals in the cytoplasm of B16F10 cells (Fig. 6C). However, any detectable cellular internalization was not seen in HFF cells. FITC-siRNAs alone or 2b/FITC-siRNA complexes were also treated to B16F10 cells, but either of their cellular uptakes was not seen (data now shown). These results indicate that HA-Chol NPs can selectively deliver siRNA to the target cells with overexpressed CD44, and also the assembled HA-Chol/2b/siRNA NPs might be internalized into the targeted cells via HA-CD44 interactions. To demonstrate that CD44 receptors on the cancer cells were involved in cellular internalization of HA-Chol NPs, we carried out the competition studies by using an excess of free HA polymers. Prior to treating HA-Chol/2b/FITC-siRNA NPs to cells, the free soluble HA polymers, which specifically bind CD44, were pre-treated to B16F10 cells to saturate the binding sites of CD44. The relative fluorescence intensities determined by the fluorescence microscopic images showed that the cellular uptakes of FITC-siRNA were highly reduced in the pre-treated cells with free HA polymers when compared with those of unblocked cells (Fig. 6D). These results indicated that HA-Chol NPs were internalized via CD44-associated pathways and could selectively target CD44-overexpressing cancer cells. HA-Chol/2b/siRNA NPs had a negative surface charge as mentioned above, nevertheless our results demonstrate that the cellular uptakes of HA-Chol NPs are both receptor-mediated and independent of the charge on their surface. Notably, it has already been reported that the PEI-modified HA nanoparticles complexed with siRNA exhibit a successful cellular uptake in the CD44overexpressing cells although HA nanoparticles reveal a completely negative surface charge when complexed with siRNA [35]. To better understand the CD44-associated endocytosis during the cellular uptake of HA-Chol NPs, we studied the effects of endocytic inhibitors including chlorpromazine, filipin III, cytochalasin D, and amiloride. The endocytosis pathways can be classified into macropinocytosis, clathrin-mediated, caveolin-mediated, and clathrin- or caveolin-independent type [39]. Each inhibitor, responsible for individual endocytic pathway, was pre-treated to B16F10 cells prior to HA-Chol/2b/FITC-siRNA NPs treatment, and subsequently we estimated its effect on the cellular uptake of nanoparticles by measuring fluorescent FITC intensities on flow

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cytometry. Fig. 7A shows that chlorpromazine, which is responsible for the clathrin-mediated endocytosis, strongly inhibited the cellular uptake by 42% when compared to those in the inhibitoruntreated cells. Filipin III, which is known to block formation of caveolae, resulted in a statistically significant reduction of cellular uptake (19%). Cytochalasin D drug that blocks the endocytosis by inhibiting cytoskeletal proteins showed 19% of reduction in the cellular uptakes. However, amiloride, responsible for sampling extracellular fluid and its contents, showed little or no reduction of cellular uptakes. These studies indicate that after CD44 receptors recognize the HA-Chol NPs, HA-Chol NPs are internalized into cells via these endocytosis pathways. Interestingly, 2b proteins from Cucumber mosaic cucumovirus (CMV) was reported to be localized at the nuclei via nuclear localization signal (NLS) [40]. When FITC-2b protein/siRNA complexes were internalized into B16F10 cells by transfection reagents, as described in the experimental section, the substantial amounts of 2b proteins were distributed to the nucleus as well as to the cytoplasm (Fig. 7B). These results indicate that 2b proteins from Tomato aspermy virus can be also accumulated at the nuclei by NLS sequence. Next, to investigate whether the encapsulated 2b proteins dissociate from the HA-Chol NPs after cellular internalized, we assembled the FITC-2b proteins together with HA-Chol systems and treated the resulting HA-Chol/FITC-2b/siRNA NPs to the cells. In contrast to the free FITC-2b proteins, HA-Chol/FITC-2b/siRNA NPs did not show any nuclear accumulation and the majority of NPs were distributed throughout the cytoplasm. Therefore, these results indicate that 2b proteins may not dissociate from the nanoparticles after cellular internalization. To investigate the intracellular localization of nanoparticles, we treated HA-Chol/2b/FITC-siRNA NPs to B16F10 cells either expressing Rab5a (an early endosome marker) or pre-stained with Lysotracker (a late endosome and endolysosome marker). Fig. 7C shows that the significant amounts of FITC fluorescence signals are colocalized with both early and late endosome markers. These studies indicate that the HA-Chol/2b/siRNA NPs may be sequestered in the endosomes immediately after cell uptakes, and ultimately reach the late endosomes and endolysosomes. These acidic compartments are expected to trigger the dissociation of siRNA from the nanoparticles due to the pHassociated binding properties of siRNA as mentioned above. The green signals distributed throughout the cytoplasm in both sets of cells represent the FITC-siRNA as a result of dissociation from the nanoparticles, whereas the encapsulated 2b proteins may not dissociate from the nanoparticles as shown in the Fig. 7B. Therefore, there would be no probability that the released siRNA bind the dissociated 2b proteins again in the cytoplasm. All the take together, these studies show that HA-Chol NPs successfully deliver siRNA to the cytoplasm of cancer cells in a CD44-associated manner. 3.9. Gene silencing of target RFP gene Using RFP-siRNA duplexes, we carried out the gene knockdown studies of HA-Chol/2b/siRNA NPs in the RFP-expressing B16F10 cancer cells (B16F10/RFP). When the amounts of RFP expression were measured by fluorescence microscopy, Fig. 8A shows that the B16F10/RFP cells treated only with PBS buffer highly express RFP proteins, but the cells treated with HA-Chol/2b/siRNA NPs significantly suppress RFP expression after 1 day post-treatment. Neither of HA-Chol/2b/scrambled (sc) siRNA NPs or empty HAChol NPs showed any RFP gene silencing. Based on the relative RFP fluorescence intensities, HA-Chol/2b/siRNA NPs showed the more efficient gene silencing ability in comparison with the Lipofectamine 2000/siRNA complexes. Moreover, after the degree of

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Fig. 7. (A) Effects of endocytic inhibitors on internalization of HA-Chol/2b/siRNA NPs. Prior to HA-Chol/2b/FITC-siRNA treatments, B16F10 cells were pre-incubated with each of inhibitor drugs for 1 h. In parallel, HA-Chol24/2b/FITC-siRNA NPs were treated to the control B16F10 cells in the absence of drug. The cellular internalization of HA-Chol/2b/FITCsiRNA NPs was analyzed by FACS, and then their reduced cell uptakes were plotted based on the FITC fluorescence-positive fractions of the cells. The FACS profiles for control cells and chlorpromazine-preincubated cells are representatively presented. Error bars indicate the standard deviation of the mean fluorescence (n ¼ 3). The * represents P  0.05, as determined using ANOVA. (B) Nuclear localization images of free 2b proteins/siRNA complexes and HA-Chol/2b/siRNA NPs. The free FITC-2b proteins/siRNA complexes were internalized into B16F10 cells via transfection reagents (TR) as mentioned in the experimental section. In parallel, another set of FITC-2b proteins/siRNA complexes were internalized into B16F10 cells via HA-Chol delivery systems. The nucleus in each cell was stained with DAPI (blue). In contrast to the free FITC-2b proteins/siRNA complexes, the FITC-2b proteins encapsulated in the nanoparticles did not show any nuclear accumulation. (C) Intracellular localization of HA-Chol/2b/FITC-siRNA NPs examined by early or late endosome marker. To label the early endosome, B16F10 cells were transfected with Rab5a 24 h prior (Early). To label the late endosome and endolysosomes, B16F10 cells were treated with 50 nM Lysotracker dye. To both sets of cells, HA-Chol/2b/FITC-siRNA NPs were treated and then their cellular fluorescence images were obtained. Images are pseudocolored for visualization of co-localization: blue ¼ DAPI; red ¼ Rab5a or Lysotracker; green ¼ HA-Chol/2b/FITC-siRNA NPs. Colocalization of HA-Chol/2b/FITC-siRNA NPs with the early or late endosome markers appears yellow. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

RFP gene silencing was maximized in 1 day, it continuously diminished, and finally we could observe approximately 30% of RFP genes silencing in 3 days. As an effort to semi-quantitatively measure the RFP gene knockdown, we performed RT-PCR and Western blot analysis. Fig. 8B shows that the exogenous siRNA delivered by HA-Chol NPs caused target RFP mRNA to be greatly reduced by 70% due to its degradation. The reduction of RFP mRNA lead to the robust decrease of RFP expression as shown in the Fig. 8C. Consistently, our HA-Chol/2b/siRNA NPs resulted in the more efficient RNAi activity than Lipofectamine 2000/siRNA complexes both in RT-PCR and Western blot studies. Moreover, FACS studies demonstrated that RFP fluorescence-positive fraction was significantly decreased from 90.2  5.2% to 19.2  2.6% when the untreated B16F10/RFP cells were compared with the HA-Chol/2b/RFP-siRNA NPs-treated cells (Fig. 8D). Also, the mean fluorescence intensity of the untreated

cells was decreased to 26.0  2.5%. The Lipofectamine 2000/siRNA complexes showed 36.2  4.1% of RFP-positive fraction and 45.8  4.4% of mean intensity, but their gene silencing efficacy was noticeably lower than that of HA-Chol/2b/RFP-siRNA NPs. Collectively, these results show that our siRNA delivery system can effectively suppress the expression of target protein by using the RNAi mechanisms. 4. Conclusions We here demonstrate the use of an alternative carrier to the traditional siRNA polyplexes. In the absence of polycationic polymers, the HA-Chol nanoparticles, as the nano-sized containers for hydrophobic cargos, 2b protein/siRNA complexes, could selectively deliver 2b protein/siRNA complexes to the tumor cells with upregulated CD44 receptors and suppress the expression of target

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Fig. 8. (A) RFP gene knockdown studies of HA-Chol/2b/RFP-siRNA NPs in RFP-expressing B16F10 cells. The relative amounts of RFP expression in the cells were visualized on fluorescence microscopy and their fluorescence intensities were plotted. Fluorescence images of the cells were obtained after 1 day post-treatment with the HA-Chol/2b/siRNA NPs (equivalent to 50 nM of siRNA) and compared with those of control cells, which were treated with PBS buffer. The fluorescence images and intensities of the HA-Chol/2b/scrambled siRNA NPs, Lipofectamine/siRNA complexes, and empty HA-Chol NPs were also compared. For the HA-Chol/2b/siRNA NPs-treated B16F10 cells, the quantitative analysis of RFP expression was also plotted versus the prolonged incubation time. Results are presented as mean  s.e. (n ¼ 300). (B) The amounts of reduced RFP mRNA were estimated by semiquantitative RT-PCR analysis. RFP cDNA transcribed from the extracted RFP mRNA was amplified by PCR reaction, and then the resulting PCR products were analyzed on 2% agarose gel electrophoresis. The relative RFP band intensities visualized by EtBr-staining were plotted below. Results are presented as mean  s.e. (n ¼ 5). (C) The amounts of RFP proteins in the cells were estimated by Western blotting analysis. Primary antibodies against RFP proteins were used and the relative amounts of RFP proteins were plotted below. (D) Semiquantitative determination of RFP gene silencing by FACS analysis. Representative FACS data including HA-Chol/2b/siRNA NPs- and LF/siRNA complexes-treated RFP/B16F10 cells were compared with those of control RFP/B16F10 cells. The percentage of RFP positive cells was determined by gating against RFP/B16F10 cells, and the fluorescence-positive proportions of the cells were also compared. Results are presented as mean  s.e. (n ¼ 5).

gene. Also, our HA-Chol nanoparticles encapsulated the 2b protein/ siRNA complexes deeply into their core, and thus could stabilize the siRNA against the enzymatic degradation. Our delivery systems form a non-toxic negatively charged nanoparticles and could allow the potential for multiple repeat doses. The pH-associated binding properties of siRNA for 2b proteins might allow the encapsulated

siRNA to be dissociated from the HA-Chol NPs in the endocytic compartments, and ultimately the released siRNA could obtain the RNAi activities in the cells, whereas the encapsulated 2b proteins still stayed within the HA-Chol nanoparticles. For the clinical cancer treatments, these results show the promising potential to accomplish targeted delivery of siRNA to the tumor cells, control the

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siRNA release at the sites of interest, and avoid the inherent cytotoxicities of the agents. Acknowledgments This research was supported by the Converging Research Center Program through the Korean Ministry of Education, Science and Technology (2013K000435), by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (2010-0029206), and by Intramural Research Program of KIST. References [1] Martinez J, Patkaniowska A, Urlaub H, Lührmann R, Tuschl T. Single-stranded antisense siRNAs guide target RNA cleavage in RNAi. Cell 2002;110:563e74. [2] Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 2001;411:494e8. [3] Lu PY, Xie F, Woodle MC. In vivo application of RNA interference: from functional genomics to therapeutics. Adv Genet 2005;54:117e42. [4] Oishi M, Nagasaki Y, Itaka K, Nishiyama N, Kataoka K. Lactosylated poly(ethylene glycol)-siRNA conjugate through acid-labile beta-thiopropionate linkage to construct pH-sensitive polyion complex micelles achieving enhanced gene silencing in hepatoma cells. J Am Chem Soc 2005;127:1624e5. [5] Shukla S, Sumaria CS, Pradeepkumar PI. Exploring chemical modifications for siRNA therapeutics: a structural and functional outlook. ChemMedChem 2010;5:328e49. [6] Herrero MA, Toma FM, Al-Jamal KT, Kostarelos K, Bianco A, Da Ros T, et al. Synthesis and characterization of a carbon nanotube-dendron series for efficient siRNA delivery. J Am Chem Soc 2009;131:9843e8. [7] Tseng YC, Mozumdar S, Huang L. Lipid-based systemic delivery of siRNA. Adv Drug Deliv Rev 2009;61:721e31. [8] Eguchi A, Meade BR, Chang YC, Fredrickson CT, Willert K, Puri N, et al. Efficient siRNA delivery into primary cells by a peptide transduction domain-dsRNA binding domain fusion protein. Nat Biotechnol 2009;27:567e71. [9] de Fougerolles A, Vornlocher HP, Maraganore J, Lieberman J. Interfering with disease: a progress report on siRNA-based therapeutics. Nat Rev Drug Discov 2007;6:443e53. [10] Lemkine GF, Demeneix BA. Polyethylenimines for in vivo gene delivery. Curr Opin Mol Ther 2001;3:178e82. [11] Saranya N, Moorthi A, Saravanan S, Devi MP, Selvamurugan N. Chitosan and its derivatives for gene delivery. Int J Biol Macromol 2011;48:234e8. [12] Lin C, Zhong Z, Lok MC, Jiang X, Hennink WE, Feijen J, et al. Linear poly(amido amine)s with secondary and tertiary amino groups and variable amounts of disulfide linkages: synthesis and in vitro gene transfer properties. J Control Release 2006;116:130e7. [13] Cho KC, Choi SH, Park TG. Low molecular weight PEI conjugated pluronic copolymer: useful additive for enhancing gene transfection efficiency. Macromol Res 2006;14:348e53. [14] Park TG, Jeong JH, Kim SW. Current status of polymeric gene delivery systems. Adv Drug Deliv Rev 2006;58:467e86. [15] Akiyoshi K, Nagai K, Nishikawa T, Sunamoto J. Self-aggregates of hydrophobized polysaccharide as a host for macromolecular guests. Chem Lett 1992;21:1727e30. [16] Nishikawa T, Akiyoshi K, Sunamoto J. Supramolecular assembly between nanoparticles of hydrophobized polysaccharide and soluble protein complexation between the self-aggregate of cholesterol-bearing pullulan and a-chymotrypsin. Macromolecules 1994;27:7654e9. [17] Glass JE. Polymers in aqueous media: performance through associationIn Advances in chemistry series 223. Washington, DC, USA: American Chemical Society; 1989.

[18] Ossipov DA. Nanostructured hyaluronic acid-based materials for active delivery to cancer. Expert Opin Drug Deliv 2010;7:681e703. [19] Akima K, Ito H, Iwata Y, Matsuo K, Watari N, Yanagi M, et al. Evaluation of antitumor activities of hyaluronate binding antitumor drugs: synthesis, characterization and antitumor activity. J Drug Target 1996;4:1e8. [20] Hua Q, Knduson CB, Knduson W. Internalization of hyaluronan by chondrocytes occurs via receptor-mediated endocytosis. J Cell Sci 1993;106:365e 75. [21] Lee H, Lee K, Park TG. Hyaluronic acid-paclitaxel conjugate micelles: synthesis, characterization, and antitumor activity. Bioconjug Chem 2008;19: 1319e25. [22] Eliaz RE, Szoka Jr FC. Liposome-encapsulated doxorubicin targeted to CD44: a strategy to kill CD44-overexpressing tumor cells. Cancer Res 2001;61:2592e 601. [23] Lee H, Mok H, Lee S, Oh YK, Park TG. Target-specific intracellular delivery of siRNA using degradable hyaluronic acid nanogels. J Control Release 2007;119: 245e52. [24] Ruponen M, Honkakoski P, Rönkkö S, Pelkonen J, Tammi M, Urtti A. Extracellular and intracellular barriers in non-viral gene delivery. J Control Release 2003;93:213e7. [25] Ding SW, Voinnet O. Antiviral immunity directed by small RNAs. Cell 2007;130:413e26. [26] Chen HY, Yang J, Lin C, Yuan YA. Structural basis for RNA-silencing suppression by tomato aspermy virus protein 2b. EMBO Rep 2008;9:754e60. [27] Choi KM, Choi SH, Jeon H, Kim IS, Ahn HJ. Chimeric capsid protein as a nanocarrier for siRNA delivery: stability and cellular uptake of encapsulated siRNA. ACS Nano 2011;5:8690e9. [28] Choi KM, Kwon IC, Ahn HJ. Self-assembled amphiphilic DNA-cholesterol/DNApeptide hybrid duplexes with liposome-like structure for doxorubicin delivery. Biomaterials; 2013:4183e90. [29] Choi KM, Kim K, Kwon IC, Kim IS, Ahn HJ. Systemic delivery of siRNA by chimeric capsid protein: tumor targeting and RNAi activity in vivo. Mol Pharm 2013;10:18e25. [30] Bartlett DW, Su H, Hildebrandt IJ, Weber WA, Davis ME. Impact of tumorspecific targeting on the biodistribution and efficacy of siRNA nanoparticles measured by multimodality in vivo imaging. PNAS 2007;104:15549e54. [31] Bulpitt P, Aeschlimann D. New strategy for chemical modification of hyaluronic acid: preparation of functionalized derivatives and their use in the formation of novel biocompatible hydrogels. J Biomed Mater Res 1999;47: 152e69. [32] Whitehead KA, Langer R, Anderson DG. Knocking down barriers: advances in siRNA delivery. Nat Rev Drug Discov 2009;8:129e38. [33] Putnam D, Gentry CA, Pack DW, Langer R. Polymer-based gene delivery with low cytotoxicity by a unique balance of side-chain termini. PNAS 2001;98: 1200e5. [34] Shim MS, Kwon YJ. Efficient and targeted delivery of siRNA in vivo. FEBS J 2010;277:4814e27. [35] Ganesh S, Iyer AK, Morrissey DV, Amiji MM. Hyaluronic acid based selfassembling nanosystems for CD44 target mediated siRNA delivery to solid tumors. Biomaterials 2013;34:3489e502. [36] Pang IK, Iwasaki A. Control of antiviral immunity by pattern recognition and the microbiome. Immunol Rev 2012;245:209e26. [37] Judge AD, Bola G, Lee AC, MacLachlan I. Design of noninflammatory synthetic siRNA mediating potent gene silencing in vivo. Mol Ther 2006;13: 494e505. [38] Kim TH, Nah JW, Cho MH, Park TG, Cho CS. Receptor-mediated gene delivery into antigen presenting cells using mannosylated chitosan/DNA nanoparticles. J Nanosci Nanotechnol 2006;6:2796e803. [39] Doherty G, McMahon H. Mechanisms of endocytosis. Annu Rev Biochem 2009;78:857e902. [40] Lucy AP, Guo HS, Li WX, Ding SW. Suppression of post-transcriptional gene silencing by a plant viral protein localized in the nucleus. EMBO J 2000;19: 1672e80.