COREL-07498; No of Pages 9 Journal of Controlled Release xxx (2014) xxx–xxx

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Article history: Received 5 September 2014 Received in revised form 18 November 2014 Accepted 24 December 2014 Available online xxxx

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Keywords: Hyaluronic acid Stability Crosslinked nanoparticle Doxorubicin Drug delivery

School of Chemical Engineering, Sungkyunkwan University, Suwon 440-746, Republic of Korea Biomedical Research Institute, Korea Institute of Science and Technology, Seoul 136-791, Republic of Korea Department of Health Sciences Technology, SAIHST, Sungkyunkwan University, Suwon 440-746, Republic of Korea d NanoBio Fusion Research Center, Korea Research Institute of Chemical Technology, Daejeon 305-600, Republic of Korea b c

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a b s t r a c t

For drug delivery nanocarriers to be a safe and effective therapeutic option, blood stability, tumor-targetability, and intracellular drug release features should be considered. In this study, to develop a potent drug delivery carrier that can meet the multiple requirements, we engineered a bioreducible core-crosslinked polymeric micelle based on hyaluronic acid (CC-HAM) by a facile method using D,L-dithiothreitol in aqueous conditions. The CCHAM exhibited enhanced structural stability under diluted conditions with PBS containing FBS or sodium dodecyl sulfates. We also successfully encapsulated doxorubicin (DOX), chosen as a hydrophobic anti-cancer drug, in CCHAMs with high loading efficiency (N80%). The drug release rate of CC-HAMs was rapidly accelerated in the presence of glutathione, whereas the drug release was significantly retarded in physiological buffer (pH 7.4). An in vivo biodistribution study demonstrated the superior tumor targetability of CC-HAMs to that of noncrosslinked HAMs, primarily ascribed to robust stability of CC-HAMs in the bloodstream. Notably, these results correspond with the improved pharmacokinetics and tumor accumulation of DOX-loaded CC-HAMs as well as their excellent therapeutic efficacy. Overall, these results suggest that the robust, bioreducible CC-HAM can be applied as a potent doxorubicin delivery carrier for targeted cancer therapy. © 2014 Published by Elsevier B.V.

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1. Introduction

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Self-assembled polymeric micelles (PMs) have been extensively investigated as a targeted drug carrier given its ability to solubilize water-insoluble anticancer drugs as well as their excellent tumortargeting activity [1–3]. Due to their amphiphilic properties, PMs can encapsulate hydrophobic small molecule drugs in their hydrophobic core [4–6]. Their nano-sized structure enables PMs to preferentially accumulate in angiogenic tumor tissues through the fenestrate vasculature following systemic administration [7–9]. Moreover, surface modification of the PMs with tumor-specific targeting ligands such as hyaluronic acid, folate or RGD molecules has been known to improve their tumor targetability and permeability into the tumor cells [10–14]. Tumor-specific, intracellular drug release features of smart PMs in response to intracellular stimuli such as a low endosomal/lysosomal pH, a higher concentration of glutathione (GSH) or intracellular enzymes in target cells have shown to further enhance the therapeutic efficacy [15–21].

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Hwa Seung Han a, Ki Young Choi b, Hyewon Ko c, Jueun Jeon a, G. Saravanakumar a, Yung Doug Suh a,d, Doo Sung Lee a, Jae Hyung Park a,c,d,⁎

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Bioreducible core-crosslinked hyaluronic acid micelle for targeted cancer therapy

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⁎ Corresponding author at: School of Chemical Engineering, Sungkyunkwan University, Suwon 440-746, Republic of Korea. E-mail address: [email protected] (J.H. Park).

However, a series of recent studies unveiled that self-assembled PMs are still in a dynamic state and exhibit low structural stability in the bloodstream upon intravenous injection, which results in premature drug release at unwanted sites [22,23]. The early drug release does not only lower the local therapeutic efficacy, but also causes undesirable toxicity to normal organs. For the PMs to be a safe and effective drug delivery system, therefore, PMs' blood stability should be guaranteed above all. Only when the blood stability is assured, tumor targeting features of PMs can lead to an increase in local drug concentration at the tumor. With the blood stability and the tumor targetability warranted, moreover, smart functions of PMs including stimuli-responsive, disease-specific drug release utility of PMs can generate synergistic effects on tumor therapy. In this study, to develop a drug carrier system that can meet the multiple requirements for an effective and tumor-targeted drug delivery nanocarrier, we engineered bioreducible, core-crosslinked polymeric micelles based on hyaluronic acid (CC-HAM), which can enable (i) secure encapsulation of drug cargos in the bloodstream by inner core-crosslinking with a bioreducible disulfide linkage, (ii) preferential accumulation in tumor cells by permeating leaky blood vessels in tumor tissues and by targeting CD44, a HA receptor that overexpresses on various tumor cells [24], and also enable (iii) intracellular drug

http://dx.doi.org/10.1016/j.jconrel.2014.12.032 0168-3659/© 2014 Published by Elsevier B.V.

Please cite this article as: H.S. Han, et al., Bioreducible core-crosslinked hyaluronic acid micelle for targeted cancer therapy, J. Control. Release (2014), http://dx.doi.org/10.1016/j.jconrel.2014.12.032

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2.1. Materials

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Sodium hyaluronate (MW = 7.4 × 103 Da) was purchased from Lifecore Biomedical (Chaska, MN, USA). Aldrithiol-2 was received from TCI (Tokyo, Japan). Propargylamine (98%), sodium cyanoborohydride (reagent grade, 95%), triethylamine (≥ 99%), copper (I) bromide (Cu (I)Br), sodium azide, doxorubicin hydrochloride (DOX.HCl), and DLdithiothreitol (DTT) were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). The NIR dye, Cy5.5, was purchased from Bioacts (Incheon, Korea). Water, used for synthesis and characterization, was purified using an AquaMax-Ultra water purification system (Younglin Co., Anyang, Korea). Squamous cell carcinoma (SCC7) cells were purchased from the American Type Culture Collection (Rockville, MD, USA). All other chemicals were of analytical grade and used as received.

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2.2. Preparation and characterization of HAMs

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HAMs were prepared by click chemistry using α-alkyne HA and azide-functionalized pyridyl disulfide methacrylate (PDSMA-N3). The detailed synthetic scheme is shown in Fig. 1a. To prepare α-alkyne HA, HA (100 mg) and propargylamine (117.32 mg; 2.13 mmol, ~ 100 equiv) were dissolved in acetate buffer (pH 5.6) at 2% W/V. Afterwards, sodium cyanoborohydride (133.84 mg; 2.13 mmol, ~ 100 equiv) was added and the reaction mixture was stirred at 50 °C for 5 days. The resulting solution was dialyzed against distilled water for 3 days using a dialysis tube (MWCO = 3.5 kDa, Spectrum Laboratories, Inc., CA, USA), followed by lyophilization. P(PDSMA)-N3 was synthesized as follows. First, aldrithiol-2 (15 g, 0.068 mmol) was dissolved in 75 ml of methanol with 1 ml glacial acetic acid. Mercaptoethanol (2.65 g, 33.97 mmol) in 15 ml methanol was added drop-wise at room temperature with stirring for 3 h. After the stirring was over, the solvent was evaporated to get the crude 2(pyridine-2-yldisulfanyl)ethanol as yellow oil, which was purified by silica gel column chromatography using a mixture of ethyl acetate/ hexane (v/v) as eluent. To get the desired products as colorless oil, the polarity of the eluent gradually increased with the portion of ethyl acetate increased from 0% (i.e. 100% hexane) to 40% (i.e. ethyl acetate/ hexane (40:60 v:v)). Then, purified monomer (4.62 g, 24.7 mmol) in 20 ml of dry dichloromethane was mixed with 3 g (29.7 mmol) of triethylamine and the solution was cooled in an ice-bath. When the

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2.3. Preparation of DOX-loaded CC-HAMs

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DOX-HA-SS-P(PDSMA) was fabricated by encapsulating DOX in the HA-b-P(PDSMA) through the dialysis method in the dark. Briefly, DOX (2.3 mg) was dissolved in 1.0 ml of distilled water/DMF solution (1:1 v/v) and mixed with 4 ml of the same co-solvent containing HAb-P(PDSMA) (5 mg/ml). Then, 16 ml of distilled water was slowly added under sonication using a probe-type sonicator (VCX-750, Sonics & Materials, CT, USA) and dialyzed for 9 h against distilled water using the dialysis membrane (MW cutoff = 3.5 kDa). Then, the resulting solution was filtered with a 0.8-μm syringe filter to remove unloaded DOX, and lyophilized to obtain a red powder (DOX-HA-b-P(PDSMA)). For crosslinking, an excess amount of DTT was added to the solution containing DOX-HA-b-P(PDSMA) before dialysis purification. After that, the red powder product was obtained by the lyophilization of this solution for 3 days.

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2.4. Characterization of CC-HAMs

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The amount of conjugated P(PDSMA) molecules was analyzed using H-NMR. All samples were prepared by dissolving the polymer 1:1 (v/v) CD3OD/D2O. The average diameters of HAMs, CC-HAMs, DOX-HAMs, and DOX-CC-HAMs were measured with a dynamic light scattering analyzer (DLS, FPAR-1000, Otsuka Electronics, Osaka, Japan). For measuring particle size, the sample was dispersed in PBS (pH 7.4, 1 mg/ml) and sonicated for 3.0 min using a probe-type sonicator (VCX-750, Sonics & Materials, CT, USA) at 180 W. The morphology of the nanoparticles was observed using a transmission electron microscope (TEM) (JEOL-2100F, Tokyo, Japan), which was operated at an accelerating voltage of 200 kV. For TEM images, all samples were dispersed in distilled water and stained by 1% uranyl acetate. To determine the drug loading content and loading efficiency, DOX-HAMs and DOX-CC-HAMs were dissolved in DMF, and the DOX concentration was estimated by measuring the absorbance at 480 nm using a UV/visible spectrophotometer (Optizen 3320, Mecasys Inc., Korea).

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mixture became cold, a solution of methacryloyl chloride (2.58 g, 24.7 mmol) in 10 ml dichloromethane was added drop-wise with stirring at room temperature for 6 h. Additionally, the reaction mixture was washed with 3 × 30 ml distilled water and then with 30 ml of brine. The organic layer was collected and concentrated to get the crude 2-(pyridine-2-yldisulfanyl) ethyl methacrylate as yellow oil. The purified monomer was obtained through silica gel column chromatography in the same way as in the above procedures. The polarity of the eluent gradually increased from 0 to 25% to get the pure product. To polymerize the monomer with an azide group using initiator, 0.5 g of the monomer (1.958 mmol) in 0.5 ml anisole with 7.03 mg (0.0490 mmol) of Cu(I)Br and 0.2048 mL (0.098 mmol) of N,N,N′,N ′,N″-pentamethyl diethylenetriamine (PMDETA) was stirred for 15 min. Then, 11.56 mg (0.0490 mmol) of 2-azidoethyl-2-bromo-2methylpropanoate was added to this homogeneous mixture and the mixture was stirred at 65 °C for 6 h under nitrogen. After that, the reaction was dialyzed against methanol/distilled water and distilled water only. HA-b-P(PDSMA) copolymer was synthesized by Huisgen's 1,3-dipolar cycloaddition (click chemistry) [26]. P(PDSMA)-N3 (28 mg, 4.05 μmol), α-alkyne HA (100 mg, 6.08 μmol), and pentamethyldiethylenetriamine (PMDETA, 3.38 μL, 8.10 μmol) were dissolved in 10 ml of dimethylformamide. The mixture was mixed with Cu(I)Br (2.32 mg, 8.10 μmol) under nitrogen atmosphere and stirred at 45 °C for 48 h. The reaction medium was dialyzed against water (MW cutoff = 5 kDa) for 3 days to remove excess α-alkyne HA, followed by lyophilization. For in vitro and in vivo experiments, HA-b-P(PDSMA) copolymer was labeled with Cy5.5 (λex = 675 nm, λem = 694 nm) as described previously [27].

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release triggered by glutathione (GSH), a thiol-containing tripeptide that can reduce disulfide bonds in the PM core by the thiol-disulfide exchange reaction. The GSH concentration (1–10 mM) is known to be considerably higher in the intracellular environment than that in the cellular exterior (~ 2 μM) [25]. This difference in concentration can contribute to the intracellular release of anticancer drugs from the CCHAMs. The bioreducible CC-HAM was synthesized by a click chemistry reaction between alkyne-functionalized hyaluronic acid (HA) and azidefunctionalized poly(pyridyl disulfide methacrylate) (P(PDSMA)). After encapsulation with doxorubicin (DOX) as a model anticancer drug, the HA conjugates were further crosslinked via a facile method using DTT, which is a small-molecule redox reagent. The conjugates were characterized using 1H-NMR, dynamic light scattering (DLS), transmission electron microscopy (TEM) and UV/VIS spectroscopy. We also investigated the in vitro stability of DOX-loaded CC-HAMs (DOX-CC-HAMs) under harsh conditions such as 50% serum (FBS) or concentrated surfactant (SDS) solution. The in vitro release behaviors of DOX were measured in the presence and absence of GSH. In addition, we confirmed the in vivo biodistribution and tumor targeting activities of Cy5.5-labeled CC-HAMs and also assessed pharmacokinetic (PK) profiles of DOX-CC-HAMs compared to those of DOX-loaded HAMs (DOX-HAMs) or free DOX. Finally, anti-tumor efficacies of DOX-loaded PMs were evaluated in vivo.

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Please cite this article as: H.S. Han, et al., Bioreducible core-crosslinked hyaluronic acid micelle for targeted cancer therapy, J. Control. Release (2014), http://dx.doi.org/10.1016/j.jconrel.2014.12.032

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Fig. 1. (a) Synthetic scheme of hyaluronic acid (HA)-poly(pyridyl disulfide methacrylate) (P(PDSMA)) conjugate. (b) Schematic illustration of core-crosslinked hyaluronic acid polymeric micelles loaded with doxorubicin (DOX-CC-HAMs).

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2.5. Stability of DOX-CC-HAMs

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The kinetic stability of the DOX-CC-HAMs was investigated in PBS containing 50% fetal bovine serum (FBS) using a DLS. In brief, the nanoparticles (10 mg) were dispersed in 10 ml of PBS containing 50% FBS at 37 °C. Thereafter, the changes in scattering intensities of the solutions were monitored as a function of time using a DLS. For SDS treatment, a SDS solution (1 ml, 7.5 g/l) was added to the DOX-CC-HAMs solution (2 ml, 0.75 g/l), and the solution was stirred using an orbital shaker

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(KE011-200, Komabiotech, Korea). At predetermined time points, the 208 ratio of scattered light intensity was monitored. For comparison, the 209 stability of DOX-HAMs was estimated under the same conditions. 210 2.6. Glutathione-mediated controlled release of DOX

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To examine the cross-linking effect, the DOX release profiles from 212 the DOX-CC-HAMs and the DOX-HAMs were compared in the PBS solu- 213 tion. For the effect of GSH on the release of DOX, DOX-CC-HAMs in the 214

Please cite this article as: H.S. Han, et al., Bioreducible core-crosslinked hyaluronic acid micelle for targeted cancer therapy, J. Control. Release (2014), http://dx.doi.org/10.1016/j.jconrel.2014.12.032

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2.9. In vivo antitumor efficacy

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To evaluate the antitumor efficacy of DOX-CC-HAMs, SCC7 tumorbearing mice were prepared as follows. A suspension of 1 × 106 SCC7 cancer cells in physiological saline (100 μl) was intravenously injected into the dorsa of athymic nude mice. The mice were divided into four groups: (i) normal saline (the control group), (ii) free DOX (5 mg/kg), (iii) DOX-HAM (DOX 5 mg/kg), and (iv) DOX-CC-HAM (DOX 5 mg/kg). When the tumor volume reached 200 mm3, each treatment was injected every 3 days via the tail vein (4 injections per mouse, n = 5 mice per each group). The volumes were observed for 12 days, once per day. Tumor volumes were calculated as a × b2 / 2, where a was the largest and b the smallest diameter. The values were presented as mean with standard deviations for groups of at least five animals.

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2.10. Statistical analysis

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To observe the in vivo biodistribution of HAMs and CC-HAMs, SCC7 tumor-bearing mice were prepared by injecting a suspension of 1 × 106 SCC7 cells in saline (60 μl) into the intravenous dorsa of athymic nude mice. When tumors grew to approximately 300–500 mm3 in volume, 200 μl (pH 7.4) of solution containing nanoparticles (1.0 mg/ml) was injected into the mice via the tail vein (n = 3 per each group). The time-dependent biodistribution and accumulation profiles of the nanoparticles were observed with the eXplore Optix system (ART advanced Research Technologies, Inc., Montreal, Canada). All the data were calculated using the region of interest (ROI) function of the Analysis Workstation software (ART Advanced Research Technologies, Inc., Montreal, Canada), and values are presented as the means with standard deviations for groups of at least three animals. To observe the organ distribution of the sample, each group of mice was sacrificed 24 h post-injection. Then, major organs and tumors were excised and observed using a Kodak image station (Kodak Image Station 4000MM, New Haven, CT, USA).

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To determine DOX pharmacokinetics, free DOX (5 mg/kg), DOXHAM (DOX 5 mg/kg), or DOX-CC-HAM (DOX 5 mg/kg) was intravenously injected into male Sprague–Dawley rats (average body weight, 200 g) through the tail vein (n = 5 SD rats per each group). Animal were randomly divided into three groups. A blood sample(500 μl) was collected and mixed with 3.8% sodium citrate solution at different time points (30 min and 1, 2, 4, 6, 9, 12, 48 and 72 h) after intravenous injection. The plasma was obtained by centrifugation at 6000 g for 3 min at 4 °C and stored at −70 °C. To extract DOX, acetone was added to the plasma, vortexed, and then the solution was centrifuged at 6000 g for 10 min. Fluorescence of DOX was measured using a microplate reader (Synergy HT Multi-Mode microplate reader, Biotek, USA) with excitation at 470 nm and emission at 590 nm. A linear standard curve of DOX was created and used for measuring the concentration of DOX in blood.

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In this study, the difference between experimental and control 302 groups was analyzed using one-way ANOVA and considered statistically 303 significant (marked with an asterisk (*) in figures) if p b 0.05. 304

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To evaluate the tumor tissue distribution of DOX molecules, SCC-7 tumor bearing mice were prepared as we described hereinbefore (Section 2.8). Free DOX (5 mg/kg), DOX-HAM (DOX 5 mg/kg), or DOX-CC-HAM (DOX 5 mg/kg) was intravenously injected into SCC-7 tumor bearing nude mice. Tumors were collected 12 h after injection, and the tumor tissues were homogenized for 1 min using a hand-held tissue homogenizer and suspended in cold buffer (50 mM Tris–HCL, pH 7.5, 0.2 M NaCl, 5 mM CaCl2, 1% Triton X-100). The tissue extracts were clarified by centrifugation at 15,000 rpm for 15 min. To estimate DOX concentration in tumor tissue, the amount of extracted tumor tissue was quantified by BCA protein assay and fluorescence intensities of DOX also were monitored using the microplate reader.

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PBS solution (1 ml, 1 g/l, pH 7.4) were placed in a dialysis membrane bag (MW cutoff = 1000). The release experiment was initiated by placing the dialysis bag in release media of 10 mM GSH. The release medium was shaken at a speed of 100 rpm at 37 °C. At predetermined time intervals, the release medium was refreshed by transferring the tube to the fresh release medium. The amount of DOX released was determined by measuring the absorbance at 480 nm using a UV/Vis spectrophotometer. DOX release profiles were also monitored by a fluorescence imaging technique. As we previously described [16], fluorescence signals of DOX can be quenched when DOX molecules are closely aggregated in the nanocarriers; however, the signals are recovered when DOX molecules are released from the PMs. To evaluate DOX release profiles from DOXHAMs and DOX-CC-HAMs in a reductive environment, they were dispersed in PBS buffer containing GSH at different concentrations (0, 5, 10 and 20 mM) or in SCC7 tumor tissue lysate. To obtain the tumor tissue lysates, a suspension of SCC-7 cells was subcutaneously injected into the dorsa of athymic nude mice (5.5 weeks old, 20–25 g). When the tumor reached 10 mm in diameter, the tumor tissues were homogenized for 1 min using a hand-held tissue homogenizer and suspended in cold buffer (50 mM Tris–HCl, pH 7.5, 0.2 M NaCl, 5 mM CaCl2, 1% Triton X-100). The tissue extracts were clarified by centrifugation at 15,000 rpm for 15 min. Then the DOX-HAM and DOX-CC-HAM solutions dispersed in a reductive environment were transferred into black 96-well flat-bottomed plate and incubated for 30 min at 37 °C. The fluorescence images of DOX were obtained using an IVIS spectrum system (Xenogen Corporation, CA, USA).

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3. Results and discussion

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3.1. Synthesis and characterization of DOX-CC-HAMs

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Amphiphilic HAMs were synthesized by click chemistry, which is a facile method to prepare conjugates between α-alkyne HA and azidefunctionalized poly pyridyl disulfide methacrylate (P(PDSMA-N3) (Fig. 1a)). First, an alkyne group of α-alkyne HA was introduced by reductive amination with propargylamine in the presence of sodium cyanoborohydride as a reducing agent as we previously reported [26]. Second, P(PDSMA)-N3 was prepared in three steps: the monomer, called 2-(pyridine-2-yldisulfanyl)ethyl methacrylate (PDSMA), was prepared from aldrithiol-2 in two simple steps. When the polymerization of PDSMA was carried out under ATRP conditions using 2-azidoethylbromo-2-methylpropanoate as the azide initiator in the presence of copper(I)bromide and N,N,N′,N′,N″-pentamethyldiethylenetriamine (PMDETA) in anisole at 65 °C. The number-average molecular weight of P(PDSMA)-N3 was 2.12 kDa from the 1H-NMR spectrum. The P(PDSMA)-N3 was confirmed based on the characteristic aromatic proton ortho-N peak (at δ 8.52 ppm) and methylene proton peak at δ 4.1 ppm (Fig. S1). The successful chemical conjugation of HAMs was also confirmed by FT-IR spectra (Fig. S2). The azide peak of P(PDSMA)-N3 was observed at 2100 cm−1, while the HAMs exhibited the characteristic peaks of both P(PDSMA) and HA except for the azide peak. Owing to its amphiphilicity, the HAMs can readily form self-assembled nanoparticles under aqueous conditions. The CC-HAMs prepared from the HAMs were easily crosslinked by the addition of reducing agents, in which core-crosslinking in HAMs was initiated by DTT to allow for the thiol exchange reaction (Fig. 1b). Both CC-HAMs and HAMs were spherical in shape and had unimodal size distributions with average diameters of 215 nm and 187 nm, respectively (Fig. 2a,b). The size distribution of the nanoparticles indicated that

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inter-particular chemical crosslinking did not occur substantially during the addition of DTT because the crosslinking process occurs in the interior of the nanoparticles. DOX was physically loaded onto the HAMs by the dialysis method and then it was further crosslinked via addition of DTT in this study. As shown in Table 1, the loading efficiencies of DOX for HAMs and CCHAMs were 80% and 87%, respectively, showing that DOX was effectively encapsulated in the nanoparticles by the dialysis method. This suggests that the DTT added for the crosslinking procedure may have considerable effects on the high encapsulation efficiency of DOX compared with non-crosslinked HAM (Table 1).

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Fig. 2. Size distribution and transmission electron microscopy (TEM) images of HAM (a) and CC-HAM (b). (c) Time-dependent change in the ratio of scattered light intensities of DOX-CCHAMs and DOX-HAMs in the FBS-containing PBS solution (50% FBS). (d) Kinetic changes in relative scattered light intensity for DOX-CC-HAMs and DOX-HAMs in the presence of SDS (2.5 g/l). The error bars in the graph represent standard deviations (n = 5). Asterisks (*) denote statistically significant differences (*p b 0.05) calculated by one-way ANOVA test.

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Table 1 Characteristics of HAMs, CC-HAMs, DOX-HAMs and DOX-CC-HAMs.

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215 ± 6 187 ± 2 201 ± 3 148 ± 5

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– – 8.0 ± 0.3 8.7 ± 0.1

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Weight percentage of the initially feeding amount of DOX relative to the amount of HAM. b Weight percentage of the amount of DOX loaded in HAM relative to the amount of HAM.

3.2. Effects of core-crosslinking on the stability of the DOX-CC-HAMs

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The structural stability of drug-loaded nanoparticles under in vivo conditions is an issue of great importance because it is a major factor for the successful delivery to target tissues. In order to investigate the structural stability of DOX-CC-HAMs under serum-mimicking conditions, we monitored the changes in scattering intensities of the DOXCC-HAMs and DOX-HAMs in the presence of 50% FBS as a function of time using DLS. As shown in Fig. 2c, the DOX-CC-HAMs maintained long-term stability in the 50% FBS-containing PBS solution, which was determined by measuring the scattering light intensity. The loss of scattered light intensity of the DOX-CC-HAMs was not extensive, and 82% of the initial intensity was maintained after 3 h. However, the scattering intensities of the DOX-HAMs without cross-linking dramatically decreased to 58% of the initial intensity. These results suggest the significant dissociation of a large portion of DOX-HAMs in the serum solution. Observing the nanoparticle stability in the presence of a surfactant, sodium dodecyl sulfate (SDS) has been recognized as a common method to monitor the nanoparticles' stability [28,29]. Hence, we also evaluated the kinetic stability of DOX-CC-HAMs and DOX-HAMs in PBS solution containing SDS, which is a strong micelle-disrupting agent. For the DOX-HAMs, the scattering intensity was dramatically decreased to 43% after 2 h, which resulted from the SDS-induced dissociation of the self-assembled structure. On the other hand, the DOX-CC-HAMs consistently maintained an intensity of over 86% of the initial scattering intensity after 3 h, which indicates thermodynamically frozen assembly

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Please cite this article as: H.S. Han, et al., Bioreducible core-crosslinked hyaluronic acid micelle for targeted cancer therapy, J. Control. Release (2014), http://dx.doi.org/10.1016/j.jconrel.2014.12.032

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Besides the biodistribution and tumor targeting attributes of the car- 446 riers, we further demonstrated in vivo pharmacokinetics and tumor ac- 447 cumulation profiles of the active drug, DOX molecules loaded in CC- 448

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3.4. In vivo biodistribution of CC-HAMs

To evaluate the in vivo biodistribution and tumor-targetability of CCHAMs, we used the NIRF animal imaging system. Time-dependent biodistribution of the nanoparticles was observed after intravenous administration of Cy5.5-labeled-CC-HAMs. As shown in Fig. 4a, considerable fluorescence signals from both nanoparticles were initially detected throughout the body, indicating the prolonged circulation of nanoparticles. Interestingly, in the case of Cy5.5-labeled-HAMs, the fluorescence signal gradually decreased over 9 h and rapidly became negligible after 12 h. On the other hand, the CC-HAMs exhibited a strong fluorescence signal in the body for up to 1 day, implying prolonged circulation of CC-HAMs with stabilized core-crosslinked structures compared with HAMs (Fig. 4b). To estimate the tumor specificity of the CC-HAMs, the major organs were sacrificed 2 days post-injection. Ex vivo images of each organ's fluorescence distribution indicated that CC-HAMs exhibited high tumor accumulation and slow excretion, compared to HAMs (Fig. 4c). These strong fluorescence signals of CC-HAMs in tumor tissue were 2-fold brighter than the signals of the HAMs (Fig. 4d). This phenomenon can be due to the improved structural stability of CC-HAMs under physiological conditions that can facilitate tumor accumulation of PMs with the intact nanostructure.

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To further investigate the effect of environmental GSH levels on the drug release, DOX-release profiles were investigated after DOX-loaded PMs incubated in PBS solution at 37 °C in the presence or absence of GSH as well as into the tumor lysate. First, DOX release profiles were determined as a function of GSH concentration (0–20 mM) by monitoring fluorescence quenching and recovery behavior. Fluorescence signals of DOX are significantly quenched when DOX molecules are compactly encapsulated in the PMs. With the DOX released, however, the fluorescence signals can be recovered. As shown in Fig. 3a, fluorescence signals from DOX-CC-HAMs were highly quenched after 30 min incubation in PBS buffer without GSH. In particular, the fluorescence signals from DOX-CC-HAMs were more than 3-fold lower compared to those from non-crosslinked DOX-HAMs, indicating more robust DOX-loading in CC-HAMs than that in HAMs. However, after incubation in PBS buffer containing GSH, fluorescence signals from DOX-CC-HAMs were sharply amplified. The signal amplification is correlated with the concentration of GSH, clearly showing GSH-responsive DOX release features of DOXCC-HAMs. On the other hand, the fluorescence signals from DOX were not significantly changed with the GSH concentration changed, implying GSH-independent and passive DOX release from DOX-HAMs. In addition, the drug release from DOX-CC-HAMs was assessed by comparison with that of DOX-HAMs after DOX-loaded PMs were incubated in the tumor lysate to estimate their drug release behavior in the tumor tissue (Fig. 3b). The fluorescence signals from DOX released from DOX-CC-HAMs were almost 2-fold higher than those from DOXHAMs after 30 min incubation in the tumor extract. The results confirmed that the high GSH level in tumor tissue expedites DOX release from DOX-CC-HAMs but does not considerably affect the DOX release from DOX-HAMs. Second, the GSH-responsive DOX release profiles were also determined as a function of time by a dialysis method. As confirmed by the fluorescence imaging method, the release of DOX from DOX-CC-HAMs was inefficient and slow in the absence of GSH, (GSH 0 mM) in which only 36% of DOX was released in 2 days in comparison with DOX-

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HAMs (Fig. 3c). This indicated that core-crosslink effectively inhibited DOX release as a diffusion barrier, resulting in a lower release rate and amount. However, in the presence of high GSH concentration (GSH 10 mM), the release rate and amount of DOX from CC-HAMs increased significantly and more than 90% of loaded DOX was release during 12 h. These results show that the cleavage of the disulfide linkage in PMs no longer acts as a diffusion barrier, resulting in accelerated DOX release. Since tumor cells have higher GSH level in the tumor cytosol than normal cells, DOX-CC-HAMs demonstrated the possibility of rapid drug release for improved therapeutic effects in the target cells. Therefore, the DOX-CC-HAM is a promising drug carrier for targeted tumor treatment over the DOX-HAM without core cross-linking.

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structure (Fig. 2d). This tendency was consistent with the results that we observed in the PBS solution containing FBS. These results demonstrate that DOX-CC-HAMs can remain in their assembled structure under harsh conditions. Therefore, DOX-CC-HAMs may serve as a robust drug carrier.

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Fig. 3. In vitro GSH-triggered release of DOX from CC-HAM and HAM. Fluorescent images and quantification of DOX-CC-HAMs and DOX-HAMs after incubation with different concentrations of GSH in PBS buffer (pH = 7.4, 37 °C) (a) or with SCC7 tumor lysate (b) for 30 min. (c) Release profiles of DOX from CC-HAM and HAM in the absence or the presence of GSH. The error bars in the graph represent standard deviations (n = 5). Asterisks (*) denote statistically significant differences (*p b 0.05) calculated by one-way ANOVA test.

Please cite this article as: H.S. Han, et al., Bioreducible core-crosslinked hyaluronic acid micelle for targeted cancer therapy, J. Control. Release (2014), http://dx.doi.org/10.1016/j.jconrel.2014.12.032

H.S. Han et al. / Journal of Controlled Release xxx (2014) xxx–xxx

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SD rats than that of free DOX or DOX-HAMs, respectively (Fig. 5a). The extended blood circulation based on the robust nanostructure of DOXCC-HAMs, moreover, facilitated their passive tumor accumulation through the leaky blood vessels in tumor tissues after systemic administration of DOX-CC-HAMs into the SCC7 tumor bearing mice (Fig. 5b). The active interaction of the surface HA on PMs with CD44 receptors

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HAM, DOX in HAM or free DOX. As shown in Fig. 5a, DOX loaded in CCHAM showed prolonged circulation in the bloodstream, which can be resulted from the robust nanostructure of core-crosslinked PMs as we confirmed in in vitro drug release tests and in vivo biodistribution studies. The plasma concentration of DOX was over 30-fold or 2-fold higher in the plasma 12 h after an intravenous injection of DOX-CC-HAMs into

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Fig. 4. (a) In vivo fluorescence images of Cy5.5-labeled CC-HAMs and HAMs; white circles indicate tumor sites. (b) Fluorescence intensities at the tumor site. (c) Ex vivo fluorescence images of tumors and organs 24 h post-injection. (d) Fluorescence intensities of tumors and organs. Asterisks (*) denote statistically significant differences (*p b 0.05) calculated by one-way ANOVA test.

Fig. 5. (a) In vivo pharmacokinetics of DOX in blood after DOX-CC-HAM, DOX-HAM or free DOX was intravenously injected at 5 mg/kg of DOX into Sprague–Dawley rats through the tail vein (n = 5). (b) Tumor tissue distribution of DOX 12 h after DOX-CC-HAM, DOX-HAM or free DOX was systemically administrated at 5 mg/kg of DOX into SCC7 tumor-bearing nude mice (n = 5). Asterisks (*) denote statistically significant differences (*p b 0.05) calculated by one-way ANOVA test.

Please cite this article as: H.S. Han, et al., Bioreducible core-crosslinked hyaluronic acid micelle for targeted cancer therapy, J. Control. Release (2014), http://dx.doi.org/10.1016/j.jconrel.2014.12.032

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We finally assessed the anti-tumor therapeutic efficacy of DOX-CCHAMs and DOX-HAMs as tumor-targeting drug carriers. We prepared SCC7 tumor-bearing mouse models. After injection of DOX-CC-HAMs, DOX-HAMs, free DOX and saline via the tail vein, the tumor volume of mice was measured for 12 days (Fig. 6a). As expected, saline treatment did not show any regression effect on the tumor growth. Free DOX treatment slightly inhibited the tumor growth to some extent in the initial days, but the therapeutic activity of free DOX was much lower than DOX-CC-HAMs and DOX-HAMs. In the case of DOX-CC-HAMs the final tumor volume of DOX-CC-HAM-treated mice was 1500 cm3, which was only 60% and 42.61% of that of DOX-HAMs and free DOX-treated mice, respectively. These results demonstrate that the DOX-CC-HAM group exhibited the highest anti-tumor therapeutic efficacy among the groups given that DOX-CC-HAMs could inhibit premature drug release with the improved stability during blood circulation, target tumor and unload drug payloads in tumor cells. Therefore, we could conclude that the DOX-CC-HAM is very stable in the bloodstream given its robust core-crosslinking, and thus can effectively deliver the DOX into the tumor cells, but it can unload DOX in the tumor cells with the micellar structure dissociated in response to GSH at the intracellular level.

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4. Conclusion

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In this study, we developed a robust core-crosslinked hyaluronic acid micelle (CC-HAM) via a facile method of disulfide bond formation. The CC-HAM showed enhanced in vivo tumor-targetability and therapeutic efficacy compared to the non-crosslinked HAMs, which can be attributed to the improved blood stability of CC-HAM upon systemic administration in vivo. The robust CC-HAM could also safely secure an anticancer drug, DOX in the crosslinked core while circulating in the

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blood and unload it into tumor cells in response to intracellular GSH, resulting in enhanced antitumor efficacy compared to free DOX or HAMs. Overall, these results suggest that the bioreducible, corecrosslinked HAM is a promising drug delivery carrier with improved stability and tumor targetability for high therapeutic activity on tumor.

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on tumor cells could also help the tumor targeting and cellular uptake of DOX loaded in CC-HAMs, resulting in over 4-fold and 2-fold higher tumor concentration of DOX in CC-HAMs than that of free DOX or DOX in HAMs, respectively (Fig. 5b). The active tumor targeting behaviors of CC-HAM will be further examined in the future study.

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Fig. 6. In vivo antitumor efficacies. (a) Tumor volume after intravenous injection of DOX-CC-HAM, DOX-HAM, free DOX and saline at 5 mg/kg of DOX into SCC7 tumor-bearing nude mice. (b) Weight of tumors collected 12 days after injection. The error bars represent standard deviation (n = 5). Asterisks (*) denote statistically significant differences (*p b 0.05) calculated by one-way ANOVA test.

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This work was financially supported by the National R&D Program 502 for Cancer Control (1420040) of MHW and the Basic Science Research 503 Programs (20100027955 & 2012012827) of NRF, Republic of Korea. 504 Appendix A. Supplementary data

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