Bioreducible hyaluronic acid conjugates as siRNA carrier for tumor targeting

GENE DELIVERY

COREL-06882; No of Pages 10 Journal of Controlled Release xxx (2013) xxx–xxx

Contents lists available at ScienceDirect

Journal of Controlled Release journal homepage: www.elsevier.com/locate/jconrel

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Hong Yeol Yoon a,b,1, Hyun Ryoung Kim c,1, Gurusamy Saravanakumar a, Roun Heo d, Su Young Chae c, Wooram Um a, Kwangmeyung Kim b, Ick Chan Kwon b, Jun Young Lee a, Doo Sung Lee a, Jae Chan Park c,⁎, Jae Hyung Park a,d,⁎⁎

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Bioreducible hyaluronic acid conjugates as siRNA carrier for tumor targeting

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Departments of Polymer Science and Chemical Engineering, Sungkyunkwan University, Suwon 440-746, Republic of Korea Center for Theragnosis, Biomedical Research Institute, Korea Institute of Science and Technology, Seoul 136-791, Republic of Korea c Bio Research Center, Samsung Advanced Institute of Technology, Yongin 446-712, Republic of Korea d Department of Health Sciences and Technology, Sungkyunkwan University, Suwon 440-746, Republic of Korea b

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Article history: Received 24 March 2013 Accepted 4 September 2013 Available online xxxx

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The successful clinical translation of siRNA-based therapeutics requires efficient carrier systems that can specifically deliver siRNA within the cytosol of the target cells. Although numerous polymeric nanocarriers forming ionic complexes with siRNA have been investigated for cancer therapy, their poor stability and lack of tumor targetability have impeded their in vivo applications. To surmount these limitations, we synthesized a novel type of biodegradable hyaluronic acid-graft-poly(dimethylaminoethyl methacrylate) (HPD) conjugate that can form complexes with siRNA and be chemically crosslinked via the formation of the disulfide bonds under facile conditions. The crosslinked siRNA-HPD (C-siRNA-HPD) complexes exhibited high stability in a 50% serum solution, as compared to the uncrosslinked siRNA-HPD (U-siRNA-HPD) complexes and free siRNA. Both the C-siRNA-HPD and U-siRNA-HPD complexes were efficiently taken up by the CD44-overexpressing melanoma cells (B16F10), but not by the normal fibroblast cells (NIH3T3). When the RFP-expressing B16F10 cells were treated with the complexes or free siRNA, the C-siRNA-HPD complexes showed the highest decrease in RFP expression. In vivo studies demonstrated the selective accumulation of C-siRNA-HPD complexes at the tumor site after their systemic administration into tumor-bearing mice, resulting in an efficient gene silencing effect. Overall, these results suggest that the HPD conjugate could be used as an efficient carrier for the tumor-targeted delivery of siRNA. © 2013 Published by Elsevier B.V.

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Keywords: Hyaluronic acid Bioreducible conjugate Stability Tumor targetability siRNA

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1. Introduction

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In recent years, owing to their potent and specific gene silencing effects, siRNAs have received increasing attention as therapeutics for the treatment of intractable diseases, including cancer [1,2]. Nevertheless, their translation to clinical settings has been limited because naked siRNAs are associated with several inherent problems such as instability in physiological fluids, lack of targetability, and poor cell membrane permeability [3]. To surmount these problems, numerous carrier systems have been developed based on viral and non-viral vectors. In particular, since viral vectors have the potential to elicit immune responses, a great deal of attention has been focused on the development of nonviral carriers (e.g., cationic liposomes, polymers, and dendrimers) which can form complexes with siRNAs [4–7]. However, after systemic

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⁎ Corresponding author. Tel.: +82 31 280 6990; fax: +82 31 280 6816. ⁎⁎ Correspondence to: J.H. Park, Departments of Polymer Science and Chemical Engineering, Sungkyunkwan University, Suwon 440-746, Republic of Korea. Tel.: +82 31 290 7288; fax: +82 31 292 8790. E-mail addresses: [email protected] (J.C. Park), [email protected] (J.H. Park). 1 These authors contributed equally to this paper.

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administration, the positively charged complexes tend to aggregate in the blood due to their interactions with the negatively charged serum proteins [8]. In addition, the complexes are often unstable and readily dissociated in physiological fluid, because the carriers do not form robust particles with siRNA. Recently, a few strategies have been proposed to improve the stability of the complexes, such as the use of polymerized siRNA with a high charge density and chemical crosslinking of the complexes [9–11]. To realize the therapeutic potential of siRNA, it is also essential to impart the complexes with suitable properties, including high targetability to the disease site, efficient uptake behavior by specific cells, and the ability to release siRNA selectively within the cytosol. Hyaluronic acid (HA), a naturally occurring anionic polysaccharide, is a major constituent of the extracellular matrix in the body. It plays a vital role in the regulation of various cellular functions, including cell proliferation, migration, and differentiation [12,13]. Also, HA has binding ability to receptors such as CD44 and RHAMM which are over-expressed on various tumor cells. These unique characteristics, along with its other desirable properties such as biocompatibility and biodegradability, have encouraged researchers to investigate HA as a carrier for tumor-targeted drug delivery [13–18]. Poly(2dimethylaminoethyl methacrylate) (pDMAEMA) has been extensively

0168-3659/$ – see front matter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.jconrel.2013.09.008

Please cite this article as: H.Y. Yoon, et al., Bioreducible hyaluronic acid conjugates as siRNA carrier for tumor targeting, J. Control. Release (2013), http://dx.doi.org/10.1016/j.jconrel.2013.09.008

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system. The concentration of glutathione (GSH), a reducing agent for disulfide bonds, is found to be much lower in the extracellular milieu (~2 μM) than in the intracellular environment (1–10 mM). Therefore, the intramolecular disulfide bond in the complex would inhibit its dissociation during circulation and prevent the premature release of siRNA. Second, after its accumulation in the tumor, the HA surface would allow the complexes to effectively internalize into the cells through CD44-mediated endocytosis. Third, the dissociation of the complexes, through the reduction of the disulfide bond by the elevated level of GSH and hyaluronidase (HAdase)-mediated degradation of the HA backbone in the intracellular compartments, would facilitate the release of siRNA to activate the RNA interference mechanism.

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2. Materials and methods

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used as a potential carrier for siRNA, because its tertiary amine group allows strong complexation with siRNAs, leading to high gene silencing effects in vitro [19–22]. However, its extended applications have been limited because its complexes do not show high tumor targetability in vivo [23]. Herein, in an attempt to improve the stability and targetability of siRNAs, we prepared a novel type of bioreducible HA-graft-pDMAEMA (HPD) conjugate (Fig. 1a) by chemically attaching 2-(2-pyridyldithio) ethylamine hydrochloride (PDA⋅HCl) and amine-functionalized pDMAEMA to the carboxyl group of HA. By employing this HPD conjugate, crosslinked siRNA-HPD (C-siRNA-HPD) complexes (Fig. 1b) were prepared via simple ionic complexation with siRNA, followed by chemical crosslinking of the HA backbone through disulfide bonds using a minimal amount of dithiotheritol (DTT). We hypothesized that the CsiRNA-HPD complexes could efficiently deliver siRNA to the tumor cells through a three-step mechanism, as shown in Fig. 1c. First, the hydrophilic HA surface of the complex may shield siRNA and prolong its circulation time in the blood, resulting in high accumulation in tumor sites with the porous tumor vasculature and poor lymphatic drainage

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Sodium hyaluronate (MW = 6.7 × 104 Da) was purchased from 111 Lifecore Biomedical LLC (Chaska, USA). DMAEMA (98%), aminoethanethiol 112

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Fig. 1. (a) Chemical structure of HPD conjugate. (b) Formation of crosslinked C-siRNA-HPD complex. (c) Schematic illustration of three-step tumor-targeting mechanism of C-siRNA-HPD complex for in vivo gene silencing.

Please cite this article as: H.Y. Yoon, et al., Bioreducible hyaluronic acid conjugates as siRNA carrier for tumor targeting, J. Control. Release (2013), http://dx.doi.org/10.1016/j.jconrel.2013.09.008

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2.2. Synthesis of amine-functionalized pDMAEMA

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pDMAEMA was synthesized by the free radical chain transfer polymerization of DMAEMA (Supplementary Information Fig. S1a). In brief, DMAEMA (3.22 ml), AESH⋅HCl (72.28 mg), and AIBN (31.4 mg) were dissolved in 10 ml of methanol. The solution in the flask was deoxygenated by three freeze-thaw cycles and sealed under vacuum. The flask was transferred to a preheated oil bath at 70 °C and stirred for 6 h. The resulting solution was dialyzed against water/methanol (1v/1v) for 3 days using a dialysis membrane (MW cutoff = 1000 Da, Spectrum®, Rancho Dominquez, CA) and lyophilized to obtain a white powder (pDMAEMA). The chemical structure of pDMAEMA was confirmed using 1H NMR (Unity Inova 500, Varian, USA).

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2.3. Synthesis of HPD conjugates

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The HPD conjugate was synthesized by a two-step reaction. First, the HA-PDA conjugate was prepared through carbodiimide-mediated amide formation (Supplementary Information Fig. S1b). In brief, HA (100 mg) and EDC (60.58 mg) were dissolved in 25 ml of distilled water. After the addition of PDA⋅HCl (17.54 mg) and HOBt (42.7 mg) in 25 ml of methanol, the reaction mixture was stirred at room temperature for 24 h. The solution was dialyzed using a membrane tube (MW cutoff = 12–14 kDa) against water/ethanol (1v/1v) for 3 days, followed by lyophilization. The degree of substitution (DS) of PDA in the conjugate, defined as the number of pyridyldisulfide groups per 100 sugar residues, was determined by measuring the absorbance of pyridine-2-thione at 343 nm after the addition of DTT using the UV–Visible spectrophotometer (Optizen 3320 UV, Korea). Second, pDMAEMA was chemically attached to the HA-PDA conjugate in the presence of EDC and HOBt. In brief, HA-PDA (100 mg) and EDC (20.16 mg) were dissolved in 25 ml of distilled water. After the addition of pDMAEMA (213.54 mg) and HOBt (14.25 mg) in 25 ml of methanol, the mixture was stirred for 24 h at room temperature. The resulting solution was dialyzed against water/ethanol (1v/1v) for 3 days using a dialysis membrane tube (MW cutoff = 12–14 kDa) and lyophilized to obtain the HPD conjugate as a white powder. The chemical structure of the conjugate was analyzed using 1H NMR. The DS of pDMAEMA in the HPD conjugate and its molecular weight were determined by the static light scattering (SLS) method [25].

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To prepare the siRNA-HPD complexes, the HPD conjugate (5 mg/ml in PBS, pH 5.0, 150 mM NaCl) was mixed with siRNA (1 mg/ml in RNase-free distilled water) at different weight ratios of HPD to siRNA, followed by incubation for 1 h at 37 °C. For chemical crosslinking, 1 μl of the DTT solution (15 μM) was slowly added to 5 μl of the complex solution. After 10 min of incubation at 37 °C, the complexes were purified by exchanging the solution buffer three times with PBS (pH 8.0, 150 mM NaCl) using centrifugal filters (Ultracel®-3 K, Millipore Ireland Ltd., Ireland). The formation of C-siRNA-HPD was evaluated by a gel retardation assay using 8% polyacrylamide gel electrophoresis. siRNA was stained with ethidium bromide and visualized using a GelDoc-it 310 imaging system (UVP LLC, CA, USA). The size and zeta-potential of the complexes were measured using dynamic light scattering (DLS, Zetasizer Nano ZS, Malvern Instrument Ltd., Worcestershire, UK) at 632 nm and 25 °C. The morphology of the complexes was observed using transmission electron microscopy (TEM, CM30 Philips).

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2.5. Serum stability of the complexes

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The stability of the complexes was evaluated by exposing them to 50% (v/v) serum solution [26]. In brief, the free siRNA, uncrosslinked siRNA-HPD (U-siRNA-HPD), and C-siRNA-HPD were incubated with rat serum solution, in which the weight ratios of siRNA to the conjugate in the complexes were fixed at 1:20. Samples were taken at predetermined time points. The siRNA-HPD complexes were treated with 1 μl of heparin solution (12 kDa, 40 mg/ml) to de-complex the siRNA and polymer. For the C-siRNA-HPD complexes, 1 ml of DTT solution (1 M) was additionally used to cleave the disulfide bonds. The released siRNAs were visualized by electrophoresis, as described above. The relative bands of siRNA at various time points were quantitatively determined by measuring the intensity at the region of interest (ROI) using ImageJ software (NIH, USA).

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2.6. In vitro cytotoxicity of U-siRNA-HPD and C-siRNA-HPD complexes

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The cytotoxicity of the complexes was assessed using an MTT assay against tumor cells (B16F10), normal fibroblast cells (NIH3T3), and green monkey kidney cells (CV-1). The cells were cultured in RPMI1640 or DMEM medium at 37 °C in a CO2 incubator, respectively. The medium contained 10% fatal bovine serum and 1% penicillin–streptomycin. The cells were seeded in 96-well plates at 5 × 103 cells/well and incubated for 24 h at 37 °C. Thereafter, the medium was replaced with serum-free medium containing the U-siRNA-HPD or C-siRNA-HPD complexes, and incubated for 12 h at 37 °C. Finally, the cell viabilities were evaluated by the MTT colorimetric procedure.

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2.7. In vitro cellular uptake of complexes

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The cells were seeded into an 8 well-chamber slide at 1 × 104 cells/ well and incubated for 24 h at 37 °C. To obtain intracellular fluorescence images, siRNA was labeled with YOYO-1 dye (λex = 491, λem = 509). The cells were treated with serum-free OPTI-MEM medium containing free siRNA, U-siRNA-HPD or C-siRNA-HPD (at a concentration of 50 nM siRNA) for 30 min at 37 °C in a CO2 incubator. Untreated cells were used as the control. Then, the cells were washed twice using Dulbecco's PBS (DPBS) and fixed with 4% paraformaldehyde solution. After fixation, the cells were stained using 4,6-diamidino-2phenylindole (DAPI) solution for 10 min and washed twice using DPBS. Finally, the cells were observed using a focus drift compensating microscope (IX81-ZDC, Olympus, Tokyo, Japan).

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hydrochloride (AESH⋅HCl, 98%), 1-ethyl-3(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), 1-hydroxybenzotriazole (HOBt), thiazolyl blue tetrazolium bromide (MTT), 2,2′-azobisisobutyronitrile (AIBN), DTT, heparin sodium salt (MW = 12000 Da), ethidium bromide (EtBr), ammonium peroxodisulfate (APS), and 4,6-diamidino-2phenylindole (DAPI) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). RFP siRNA (sense strand: 5′-UGU AGA UGG ACU UGA ACU CdTdT-3′ and anti-sense strand: 5′-GAG UUC AAG UCC AUC UAC AdTdT-3′) and 40% bis-acrylamide solution were purchased from Bioneer Corporation (Daejeon, Korea). YOYO-1 fluorescence dye was purchased from Life Technologies Korea LLC (Seoul, Korea). The near infrared fluorescence (NIRF) dye, FPR675 (λex = 675, λem = 720), was purchased from Bioacts (Incheon, Korea). The murine melanoma cell line (B16F10) and mouse embryo fibroblast cell line (NIH3T3) were purchased from the American Type Culture Collection (ATCC, Rochkville, MD, USA). RFP-expressing melanoma cells (RFP-B16F10) were kindly donated by Kyungpook National University (Daegu, Korea). RPMI1640 and Opti-MEM media were obtained from Welgene Inc. (Daegu, Korea). All other chemicals were purchased as reagent grade and used without further purification. All solutions were made up in RNase-free distilled water. PDA⋅HCl was synthesized according to the procedure reported elsewhere [24].

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H.Y. Yoon et al. / Journal of Controlled Release xxx (2013) xxx–xxx

Please cite this article as: H.Y. Yoon, et al., Bioreducible hyaluronic acid conjugates as siRNA carrier for tumor targeting, J. Control. Release (2013), http://dx.doi.org/10.1016/j.jconrel.2013.09.008

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2.8. In vitro RFP gene silencing

2.11. Statistical analysis

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To assess the gene silencing efficiency of the complexes, the RFPB16F10 cells (5 × 103 cells/well) were treated with the free-siRNA, siRNA-Lipofectamine, U-siRNA-HPD, or C-siRNA-HPD complexes for 30 min (at a concentration of 50 nM siRNA) in serum-free OPTI-MEM medium. Untreated cells were used as the control. Thereafter, the cells were washed twice using DPBS and incubated for 48 h. After fixation using 4% parafarmaldehyde solution, the cells were stained using DAPI solution for 10 min and washed twice using DPBS. The cells were observed using a focus drift compensating microscope.

The statistical significance of the differences between each group tested was evaluated using one-way ANOVA. A p-value of less than 0.05 was considered significant and is highlighted in the Figures with an asterisk.

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3. Results and discussion

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In order to achieve high gene transfection efficiency, it is essential to develop a safe and efficient carrier system that is capable of overcoming the various extracellular and intracellular barriers. In principle, for the effective delivery of siRNA, the carrier should at least hold the ability to condense siRNA into compact nano-sized complexes, protect siRNA from degradation by RNase, prolong circulation of the complexes in vivo, prevent dissociation of complexes in blood during circulation, accumulate at the target tissue, internalize into the cells effectively, and release the siRNA at the cytosol [27,28]. However, in reality, infusing all of these attributes into a single carrier system is quite a challenge. The most widely explored carriers for gene delivery are cationic polymers, primarily due to their ability to form complexes with negatively charged siRNAs. For in vivo therapeutic applications, however, these positively charged complexes are confronted with many obstacles. Particularly, upon their systemic administration into the body, they may easily form aggregates through their interactions with the blood components, resulting in rapid clearance by the reticuloendothelial system and poor gene silencing efficiency [29,30]. This problem can be somewhat alleviated by neutralizing the complexes with anionic polymers or modifying them with poly(ethylene glycol) (PEG) to shield the positive surface during their circulation [31–33]. Although PEGylated complexes exhibit prolonged in vivo circulation and improved accumulation at the tumor tissue through the enhanced permeation and retention (EPR) effect, many studies have also demonstrated that PEGylation may hinder the internalization of the complexes into the target cells [34–36]. In addition, the PEGylated surfaces have the potential to induce a hypersensitivity reaction, complement activation, and anti-PEG antibody formation [37,38]. To improve their cellular uptake, the complexes should be modified with targeting moieties or ligands, which facilitate their internalization into the cells through receptor-mediated endocytosis. In an effort to improve the stability and tumor targetability of siRNA for efficient gene silencing, in this study, we designed and synthesized a novel bioreducible HPD conjugate (Fig. 1).

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3.1. Synthesis of bioreducible HPD conjugates

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The synthetic strategy for the HPD conjugate is shown in the Supplementary Information S1. DMAEMA is highly useful in preparing cationic gene carriers with different molecular weights and architectures through various controlled radical polymerization techniques [23]. Herein, we synthesized a linear, amine-functionalized pDMAEMA by the free radical chain transfer polymerization of DMAEMA using AET⋅HCl as the chain transfer agent and AIBN as the initiator. The chemical structure of the polymer was confirmed using 1H NMR (Supplementary Information S2a.). The number average molecular weight of the amine-functionalized pDMAEMA, calculated using 1H NMR, was 8 kDa. The HPD conjugate was then synthesized by a two-step reaction. First, PDA · HCl was conjugated to the carboxyl group of HA in the presence of EDC and HOBt. The DS of PDA in the conjugate, which was estimated by measuring the quantity of pyridine-2-thione released from it upon the addition of DTT, was 6.7. Since PDA is useful to generate the reactive thiol group by means of reducing agents such as DTT and GSH, it is expected that the PDA-bearing HA conjugate can be readily crosslinked after complexation with siRNA. Second, the aminefunctionalized pDMAEMA was chemically attached to the HA-PDA conjugate via the carbodiimide-mediated coupling reaction. The

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All animal experiments with live animals were performed in compliance with the relevant laws and institutional guidelines of Sungkyunkwan University and the institutional committees approved the experiments. Tumor-bearing mice were prepared by the subcutaneous injection of 1 × 106 SCC7 cells into the left flank of 5 weeks old Balb/c nude mouse. When the tumor grew to approximately 80–150 mm3 in volume, 200 μl of the FPR675-labled complexes were injected into the tail vein of the mice (n = 3 for each group). The in vivo biodistribution of the complexes was investigated by monitoring the fluorescence signal for 48 h using a non-invasive whole-body animal imaging system (eXplore Optix, ART Advanced Research Technologies, Inc., Montreal, Canada), where the laser power and integration time settings were optimized at 6 mW and 0.3 s per count point, respectively. The excitation and emission points were raster-scanned in 1 mm steps over the selected polygon ROI to obtain whole body NIRF images. The tumor accumulation profiles of the complexes were evaluated by measuring the NIRF intensity at the tumor site. All of the data were calculated using the ROI function of the Analysis Workstation software (n = 3, ART Advanced Research Technologies Inc., Montreal, Canada).

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2.10. In vivo RFP gene silencing

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The in vivo gene silencing efficacy of the complexes was evaluated using the RFP-B16F10 tumor-bearing mouse model. In brief, a suspension of 1 × 106 cells was subcutaneously injected into the left flank of 5 weeks old Balb/c nude mice. When the tumor grew to approximately 60–80 mm3 in volume, 100 μl of the complexes (10 μg of siRNA/mouse) were injected daily into the tail vein of the mice for 4 days. The control mice were treated with the saline solution (pH 7.4). The red fluorescence signal, expressed at the tumor tissue, was estimated using an IVIS spectrum system (Xenogen Corporation, CA, USA) for 5 days. Fluorescence images were acquired with excitation at 570 nm and emission at 620 nm using 3 s acquisition, a binning factor of 8 and field of view of 6.6 cm. To distinguish the auto-fluorescence and tumor tissue fluorescence, spectral unmixing was performed using the Living Image® 4.3.1 software (Caliper Life Sciences, MA, USA). Normalized data for the fluorescence was quantified as the radiant efficiency. At 5 days after observation, the tumor tissues were removed and the fluorescence signals were obtained using a 12-bit CCD camera equipped with a special Cmount lens (Kodak Image Station 4000MM, New Haven, CT, USA) and TRITC bandpass emission filter. The fluorescence intensity of the tumors was quantified by measuring the TRITC intensity at the ROI (n =3). In vivo tumor growth was also observed using the B16F10 tumor-bearing mice (n = 3). When the tumor grew to approximately 50–70 mm3 in size, 100 μl of the complexes (10 μg of siRNA/mouse) were injected every 3 days into the tail vein of the mice for 6 days. Tumor volumes were calculated as a × b2/2, where a and b were the largest and smallest diameters, respectively.

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Please cite this article as: H.Y. Yoon, et al., Bioreducible hyaluronic acid conjugates as siRNA carrier for tumor targeting, J. Control. Release (2013), http://dx.doi.org/10.1016/j.jconrel.2013.09.008

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Fig. 2. (a) Gel retardation assay of complexes U-siRNA-HPD and C-siRNA-HPD at different weight ratios (1:1, 5:1, 10:1, 20:1). Stability of U-siRNA-HPD, C-siRNA-HPD and free siRNA, under serum conditions, (b) gel images and (c) relative intensity of bands, where the asterisks (*) and (**) denote statistically significant differences (p b 0.05) calculated by one-way ANOVA.

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C-siRNA-HPD complexes were prepared by physically mixing siRNA and the HPD conjugate at different weight ratios of HPD to siRNA (ranging from 1:1 to 20:1), followed by chemical crosslinking through the formation of the disulfide bonds. To observe the

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complex formation behavior, polyacrylamide gel electrophoresis experiments were carried out for both U-siRNA-HPD and C-siRNA-HPD. As shown in Fig. 2a, the crosslinked HPD conjugate was capable of effectively condensing siRNA to form stable complexes above a weight ratio of 5:1. On the other hand, the uncrosslinked HPD conjugate produced loose complexes even at a weight ratio of 20:1. These results clearly confirm that the chemical crosslinking plays a vital role in the formation of stable complexes. Kataoka et al. also demonstrated that the chemical crosslinking of PEG-b-poly(L-lysine) micelles with the disulfide bonds at the core improves the stability of the micelles, thereby enhancing the delivery of siRNA [11]. Since a redox potential gradient exists between the extracellular (~ 2 μM of GSH) and intracellular (1–10 mM of GSH) environments, the structure of the disulfide-crosslinked carriers would remain intact in the bloodstream and release siRNA into the cytosol [39]. As the target

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pDMAEMA in the conjugate allows for strong complexation with siRNA via an ionic interaction. The successful formation of the HPD conjugate was confirmed by the 1H NMR spectrum (Supplementary Information S2b), which showed the presence of the characteristic peaks at 7.22 ppm (aromatic proton from the PDA moiety), 2.0 ppm (methyl proton of N-acetyl glucosamine, -NH-CO-CH3), and 4.02 ppm (methylene proton of pDMAEMA,-CO-CH2-). The DS of pDMAEMA in the HPD conjugate determined by the SLS method was 4.5.

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Fig. 3. In vitro cytotoxicity of U-siRNA-HPD and C-siRNA-HPD against (a) B16F10, (b) CV-1, and (b) NIH3T3 cells. The error bars in the graph represent standard deviations (n = 3).

Please cite this article as: H.Y. Yoon, et al., Bioreducible hyaluronic acid conjugates as siRNA carrier for tumor targeting, J. Control. Release (2013), http://dx.doi.org/10.1016/j.jconrel.2013.09.008

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disulfide bonds and degradation of HA in the intracellular environment, the C-siRNA-HPD complexes might be useful for the intracellular delivery of siRNA. The physicochemical characteristics of the complexes such as their size and surface charge significantly influence their fate in vivo, including their biodistribution and cellular uptake [29]. Therefore, we carefully studied the properties of the complexes before and after crosslinking. The hydrodynamic sizes of U-siRNA-HPD and C-siRNA-HPD were 416.0 ± 71.42 nm and 314.4 ± 15.14 nm, respectively. This implies that the chemical crosslinking produced compact complexes. The zeta potential values of the complexes (−6.02 ± 1.4 mV) were not significantly affected by the chemical crosslinking, which might be due to the absence of any significant changes in the amount of carboxyl groups on their surface. These results indicate that the pDMAEMA domain of the conjugate condensed the siRNA through the electrostatic interaction and that the hydrophilic HA shielded the surface of the complexes. The

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site of siRNA to mediate the RNAi mechanism is the cytosol, the disulfide-crosslinked carriers may ensure effective gene silencing. For the subsequent experiments, the complexes with the highest weight ratio (20:1) were used as representative samples. HAdase, the enzyme that degrades HA, is found to be elevated in various malignant tumors, and the catabolism of HA by the enzyme (Hyal-1) primarily occurs at the intracellular level [13]. Therefore, the HAdase-mediated degradation of the conjugate may further enhance the intracellular delivery of siRNA. In order to gain insight into the C-siRNA-HPD complexes under intracellular conditions, the gel retardation assay was additionally carried out in the presence of HAdase for the analysis of the complexes. Interestingly, after treating them with HAdase, free siRNA bands were clearly observed for both the U-siRNAHPD and C-siRNA-HPD complexes. This result indicates that the degradation of HA facilitates the disassociation of the complexes, thus releasing the siRNA from them. Considering the reduction of the

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Fig. 4. In vitro cellular uptake of U-siRNA-HPD and C-siRNA-HPD complexes (siRNA concentration = 50 mM). The scale bar represents 25 μm.

Please cite this article as: H.Y. Yoon, et al., Bioreducible hyaluronic acid conjugates as siRNA carrier for tumor targeting, J. Control. Release (2013), http://dx.doi.org/10.1016/j.jconrel.2013.09.008

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Despite its high transfection efficiency in vitro, the potential of pDMAEMA in gene delivery has been limited by its high cytotoxicity

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and poor tumor targetability in vivo. Some attempts were made to improve its cytotoxicity, such as by decreasing its molecular weight and copolymerizing it with biocompatible PEG [31,40,41]. Although these strategies reduced the cytotoxicity of pDMAEMA, the transfection efficiency was significantly decreased by the chemical modification, which might be due to the lowering of the cellular uptake. The cytotoxicity of the complexes against cancer cell (B16F10) and normal cells (CV-1 and NIH3T3) was evaluated using the MTT assay. For B16F10, chosen as the representative cancer cell which overexpresses CD44, both U-siRNA-HPD and C-siRNA-HPD showed considerable cytotoxicity at higher concentrations (N10 μg/ml) (Fig. 3a). This might be due to cytotoxicity of the pDMAEMA block in the conjugate, following receptor-mediated cellular uptake of the complexes. You et al. also reported that the bare pDMAEMA and bioreducible pDMAEMA polymers exhibited significant cytotoxicity against human pancreatic adenocarcinoma cells and endothelial cells [42]. For CV-1, chosen as the normal cell expressing the low level of CD44 [43], both complexes also showed cytotoxicity at higher concentrations (N 10 μg/ml) (Fig. 3b). However, B16F10 showed much lower cell viability than CV-1 at high concentrations of the complexes, implying high toxicity of the complexes to the cancer cells overexpressing CD44. Interestingly, neither of the complexes in this study exhibited significant cytotoxicity against CD44-deficient NIH3T3 cells even at high concentrations of up to 100 μg/ml (Fig. 3c). These results imply that the cytotoxicity of the complexes is dependent on the expression level of CD44 on the cells, resulting in high toxicity against the CD44-overexpressing cancer cells.

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TEM image supported the formation of stable and compact C-siRNAHPD complexes with a spherical morphology (Supplementary Information S3). In order to investigate the stability of the complexes under physiological conditions, the free siRNA, U-siRNA-HPD, and C-siRNA-HPD complexes were incubated in the presence of 50% rat serum. The siRNA bands were observed at various time intervals by the gel retardation assay. As shown in Fig. 2b, the C-siRNA-HPD complexes maintained the structural integrity of siRNA for up to 24 h, whereas considerable degradation was observed for the U-siRNA-HPD complexes and free siRNA after 3 h. In particular, the free siRNA was completely degraded after 24 h of incubation. For the quantitative analysis, the intensity of each band was measured (Fig. 2c). As expected, the free siRNA exhibited the highest rate of degradation. For the U-siRNA-HPD complexes, the intensity gradually decreased and 41% of the siRNA was degraded after 24 h. Interestingly, the C-siRNA-HPD complexes showed no significant decrease in intensity during the whole period of time tested and 92% of the siRNA was preserved after 24 h. Overall, these results indicate that the crosslinking of the complexes using the disulfide bond improved their stability in the serum condition, thereby maintaining the structural integrity of siRNA for a prolonged period of time.

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Fig. 5. In vitro gene silencing efficacy of U-siRNA-HPD and C-siRNA-HPD complexes (siRNA concentration = 50 mM). The scale bar represents 25 μm.

Please cite this article as: H.Y. Yoon, et al., Bioreducible hyaluronic acid conjugates as siRNA carrier for tumor targeting, J. Control. Release (2013), http://dx.doi.org/10.1016/j.jconrel.2013.09.008

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siRNA complexes, exhibiting promising in vitro characteristics, often fail to show the same level of performance under in vivo conditions. Therefore, it is of high importance to examine the in vivo fate of the complexes. To investigate the in vivo biodistribution of the complexes, their time-dependent in vivo NIRF images were obtained after the systemic administration of fluorescently labeled complexes into SCC7 tumorbearing mice. As shown in Fig. 6a, for an initial 3 h, both the U-siRNAHPD and C-siRNA-HPD complexes showed considerable fluorescent signals in their whole bodies, implying the circulation of complexes in the bloodstream. In addition, the strong signals for both complexes were found at the tumor site. This might be due to the characteristics of HA, allowing the selective accumulation into the tumor site by a combination of active and passive targeting mechanisms [15,16]. Interestingly, the fluorescent signals for the C-siRNA-HPD complexes gradually increased at the tumor site during the first 9 h. In addition, the tumors for the C-siRNA-HPD complexes always exhibited stronger signals than those for the U-siRNA-HPD complexes, indicating that crosslinked complexes effectively reached the tumor site. In order to understand the results more clearly, we quantitatively measured the time-

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In order to observe the cellular uptake behavior of the complexes, the YOYO-1-labeled siRNA complexes were incubated with B16F10 and NIH3T3 cells and their uptake was monitored using a fluorescent microscope. Strong green fluorescent signals were observed on the CD44positive B16F10 cells treated with U-siRNA-HPD and C-siRNA-HPD, whereas no detectable signals were found on the NIH3T3 cells (Fig. 4), indicating the receptor-mediated cellular uptake of the complexes. In our previous studies, we also demonstrated the efficient and fast cellular uptake of HA-based nanoparticles by CD44-overexpressing cancer cells [15,44]. These results clearly confirmed the assumption that the high cytotoxicity of U-siRNA-HPD and C-siRNA-HPD on the B16F10 cells is due to the enhanced internalization of the complexes through receptor-mediated endocytosis. It is also interesting to note that the fluorescence signals were evenly distributed all over the cytosol, implying that the complexes were able to release siRNA into the cytosol without aggregation. The in vitro gene silencing effect of the complexes was also investigated using RFP-siRNA against RFP-B16F10 cells (Fig. 5). The red fluorescence signals of the RFP-B16F10 cells, treated with the U-siRNAHPD and C-siRNA-HPD complexes, were remarkably reduced compared to those of the control, free siRNA, and Lipofectamine-siRNA complexes. This high transfection efficiency of the HPD-based complexes is attributed to the enhancement of the cellular uptake through receptor-

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Fig. 6. In vivo biodistribution of U-siRNA-HPA and C-siRNA-HPD in SCC7 tumor-bearing mice. (a) Whole body images of the mice. (b) Fluorescence intensity at the tumor site for up to 2 days. The asterisk (*) denotes statistically significant differences (p b 0.05) calculated by one-way ANOVA. The error bars in the graph represent standard deviations (n = 3).

Please cite this article as: H.Y. Yoon, et al., Bioreducible hyaluronic acid conjugates as siRNA carrier for tumor targeting, J. Control. Release (2013), http://dx.doi.org/10.1016/j.jconrel.2013.09.008

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siRNA in the tumor cells after receptor-mediated endocytosis, as shown in Figs. 4 and 5. Overall, these results indicate that the CsiRNA-HPD complexes possess excellent gene silencing ability in vivo, primarily owing to their high tumor targetability. The tumor volume was also measured after systemic administration of the complexes to observe their toxicity to tumor tissue. As shown in Fig. 7d, the mice treated with C-siRNA-HPD showed slower tumor growth than did animals with saline and U-siRNA-HPD. This indicates that high tumor targetability of C-siRNA-HPD resulted in suppression of tumor growth, owing to its cytotoxicity against cancer cells (Fig. 3). For clinical applications of the C-siRNA-HPD complex, it should be emphasized that its potential limitations have to be investigated as the further study. For instance, although CD44 is over-expressed on the cancer cells, it also exists on some of normal cells in the body, implying the potential toxicity of the complex to normal organs. There is also another HA receptor, called HARE, which is overexpressed on the liver sinusoidal endothelial cells [45]. In fact, studies have demonstrated that HA-based conjugates or nanoparticles can be accumulated into the liver [14,43,45]. Some reports suggested that the oligosaccharides, produced by degradation of HA, stimulate production of matrix metalloproteinases and proliferation of endothelial cells, resulting in significant angiogenesis [12,46]. Therefore, for the clinical use of the current system based on the HPD conjugate, the detailed pharmacokinetic and pharmacodynamic studies have to be carried out using the therapeutic siRNAs.

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dependent fluorescence intensity at the tumor site (Fig. 6b). For CsiRNA-HPD complexes, the signal intensity increased for 9 h, owing to their accumulation at the tumor. On the other hand, U-siRNA-HPD showed no remarkable increase in the fluorescence intensity at the tumor site. This result clearly suggests that increasing the stability of the complexes greatly improves the tumor targetability in vivo. Fig. 7 shows the in vivo gene silencing efficacy of U-siRNA-HPD and C-siRNA-HPD in RFP-B16F10 tumor-bearing mice, which is evaluated using a real-time NIRF technique. For this experiment, the mice were injected with the complexes (10 μg of siRNA/mouse) or the saline solution as the control on a daily basis for 4 days. The gene silencing effects of the complexes were estimated by comparing the fluorescence images (Fig. 7a). The RFP signal intensity at the tumor site after 5 days was significantly reduced for the mice treated with C-siRNA-HPD, whereas those treated with U-siRNA-HPD and the control showed enhanced fluorescence signals at the tumor. The fluorescence images of the excised tumors demonstrated that both of the complexes showed significantly lower fluorescent signals, compared to the control (Fig. 7b). As expected, the lowest fluorescence signal was found for the tumor treated with the C-siRNA-HPD complex. The quantitative analysis of the excised tumor exhibited a 6.6-fold lower signal intensity for the mice treated with the C-siRNA-HPD complex, compared to the control (Fig. 7c). Since Hyal-1 is abundant at the intracellular level [13,16], it was expected that the cross-linked complexes released effectively

Fluorescence intensity in tumor ROI (A.U.)

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Time (day) Fig. 7. In vivo gene silencing effect of U-siRNA-HPD and C-siRNA-HPD in RFP expressing-B16F10 tumor-bearing mice. (a) Whole body images of the mice. (b) Ex vivo fluorescence images. (c) Fluorescence intensity of the excised tumors 5 days post-injection. (d) Tumor volume as a function of time after systemic administration of complexes. The red arrow indicates the time point of injection. The asterisks (*) and (**) denote statistically significant differences (p b 0.05) calculated by one-way ANOVA. The error bars in the graph represent standard deviations (n = 3). (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)

Please cite this article as: H.Y. Yoon, et al., Bioreducible hyaluronic acid conjugates as siRNA carrier for tumor targeting, J. Control. Release (2013), http://dx.doi.org/10.1016/j.jconrel.2013.09.008

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This research was supported by the Basic Science Research Programs of MEST (20100027955 & 2012012827) and Samsung Advanced Institute of Technology.

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Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.jconrel.2013.09.008.

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