Bioreducible Shell-Cross-Linked Hyaluronic Acid Nanoparticles for

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Bioreducible Shell-Cross-Linked Hyaluronic Acid Nanoparticles for Tumor-Targeted Drug Delivery Hwa Seung Han,†,∇ Thavasyappan Thambi,†,∇ Ki Young Choi,‡ Soyoung Son,† Hyewon Ko,§ Min Chang Lee,∥ Dong-Gyu Jo,⊥,§ Yee Soo Chae,# Young Mo Kang,# Jun Young Lee,† and Jae Hyung Park*,†,§ †

School of Chemical Engineering, College of 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 and Technology, SAIHST, Sungkyunkwan University, Suwon 440-746, Republic of Korea ∥ Department of Bionanotechnology, Gachon University, Seongnam 461-701, Republic of Korea ⊥ College of Pharmacy, Sungkyunkwan University, Suwon 440-746, Republic of Korea # School of Medicine, Kyungpook National University, Daegu 700-422, Republic of Korea ‡

S Supporting Information *

ABSTRACT: The major issues of self-assembled nanoparticles as drug carriers for cancer therapy include biostability and tumor-targetability because the premature drug release from and nonspecific accumulation of the drug-loaded nanoparticles may cause undesirable toxicity to normal organs and lower therapeutic efficacy. In this study, we developed robust and tumor-targeted nanocarriers based on an amphiphilic hyaluronic acid (HA)-polycaprolactone (PCL) block copolymer, in which the HA shell was cross-linked via a bioreducible disulfide linkage. Doxorubicin (DOX), chosen as a model anticancer drug, was effectively encapsulated into the nanoparticles with high drug loading efficiency. The DOX-loaded bioreducible HA nanoparticles (DOX-HA-ss-NPs) greatly retarded the drug release under physiological conditions (pH 7.4), whereas the drug release rate was markedly enhanced in the presence of glutathione, a thiol-containing tripeptide capable of reducing disulfide bonds in the cytoplasm. Furthermore, DOX-HA-ss-NPs could effectively deliver the DOX into the nuclei of SCC7 cells in vitro as well as to tumors in vivo after systemic administration into SCC7 tumor-bearing mice, resulting in improved antitumor efficacy in tumor-bearing mice. Overall, it was demonstrated that bioreducible shell-cross-linked nanoparticles could be used as a potential carrier for cancer therapy.

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INTRODUCTION Smart nanocarriers, composed of polymeric self-assemblies, have emerged as promising nanovehicles for tumor-targeted delivery and controlled release of various active agents such as small molecule anticancer drugs, genetic agents, or proteins.1−5 Nanoparticles composed of the amphiphiles have several merits as anticancer drug delivery carriers including enhanced solubility, improved thermodynamic stability, prolonged circulation in the bloodstream, and preferential accumulation into tumor tissue via the enhanced permeation and retention (EPR) effect.6−8 Despite the above advantages, however, clinical results of drug-loaded polymeric self-assemblies have shown limited success, which is possibly due to their instability and undesirable premature drug release in the bloodstream as well as their insufficient targeting into cancer cells. The stability of polymeric self-assemblies in serum is critical for their effective use in drug delivery.9,10 When polymeric nanoparticles are in the physically self-assembled state, they suffer from poor structural integrity under in vivo conditions, primarily due to © XXXX American Chemical Society

the large dilution after intravenous injection and interactions with blood protein components. It was, importantly, reported that a significant amount of drug was released from selfassembled nanoparticles before reaching its intended target site.11−13 This premature drug release may reduce the therapeutic potential of drugs and result in significant side effects. Recently, intraparticular chemical cross-linking of polymeric self-assemblies has emerged as an attractive strategy to elegantly surmount the blood stability issue of the nanoparticles.14−18 In general, cross-linking approaches do not only improve the structural stability of the nanoparticles in the bloodstream, but also enable fine-tuning of drug release in a controlled fashion. However, permanent cross-links may inhibit drug release at the target site, resulting in reduced therapeutic efficacy.19 Thus, Received: August 17, 2014 Revised: December 13, 2014

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Figure 1. Synthetic route for the PDA-conjugated HA-b-PCL copolymer.

carriers for cancer therapy because various cancer cells are known to overexpress the HA receptor, CD44.33−38 In previous studies, we also demonstrated the utility of HA-based nanocarriers for cancer imaging and therapy.39−43 Herein, we describe the synthesis of disulfide cross-linked HA nanoparticles composed of HA-b-poly(caprolactone) (HAb-PCL) for tumor-targeted drug delivery in vivo (Figure 1). The physiochemical characteristics of the cross-linked nanoparticles were determined using 1H NMR, dynamic light scattering (DLS), and transmission electron microscopy (TEM). Doxorubicin (DOX), chosen as a model anticancer drug, was encapsulated into the nanoparticles using an emulsion method. The in vitro release behavior of DOX was evaluated in the presence and absence of GSH. In addition, the in vivo biodistribution of cross-linked nanoparticles was measured using a fluorescence imaging technique in tumorbearing mice. Finally, we examined the therapeutic effect of drug-loaded nanoparticles following their systemic administration into tumor-bearing mice.

cross-linking should be reversed after the target site is reached to enhance the release of active agents. Several degradable linkers including disulfide, ketal, or pH-sensitive ester derivatives have been used for this purpose.20−23 In particular, disulfide cross-linkers that are cleavable in the cytosol have been used extensively based on the finding that glutathione (GSH), a thiol-containing tripeptide capable of reducing disulfide bonds is abundant in the cytoplasm (1−10 mM), whereas it is rarely present in the blood plasma (2−20 μM).24 Furthermore, the concentration of GSH in pathological sites is at least 4-fold higher than that in normal tissues.25 This unique difference in concentration gradient has encouraged researchers to develop GSH-responsive nanoparticles for drug delivery.26−29 The introduction of intraparticular disulfide linkages into polymeric self-assemblies may improve their stability in vivo and allow for controlled release of therapeutic agents at the target site.30 Although the cross-linking approach is promising, for practical applications, nanocarriers should have the ability to specifically target cancer cells. Hyaluronic acid (HA) is an anionic, naturally occurring polysaccharide found in the extracellular constituents of connective tissues.31 Owing to its excellent biocompatibility, HA has been extensively investigated for biomedical and pharmaceutical applications.32 In particular, it has been widely investigated as a targeting constituent of drug

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EXPERIMENTAL PROCEDURES

Materials. Sodium hyaluronate (MW = 12000 Da) was purchased from Lifecore Biomedical, LLC (Chaska, MN, USA). Propargylamine, sodium cyanoborohydride, ε-caprolactone, tin(II) 2-ethylhexanoate, sodium azide (NaN3), 1-hyroxybenzotriazole (HOBt), p-toluenesulB

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Biomacromolecules fonyl chloride (p-TsCl), 1-ethyl-3(3-(dimethylamino)propyl)carbodiimide·hydrochloride (EDC·HCl), GSH, dithiothreitol (DTT), and copper(II) sulfate pentahydrate (CuSO4·5H2O) were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). The near-infrared fluorescence (NIRF) dye Cy5.5 was purchased from Amersham Biosciences (Piscataway, NJ, USA). The water used in the experiments was prepared using an AquaMax-Ultra water purification system (Younglin Co., Anyang, Korea). All other chemicals were of analytical grade and used without further purification. 2-(Pyridyldithio)ethylamine (PDA) and azide-functionalized PCL (PCL-N3) were synthesized as previously described.44,45 Characterization. The chemical structures of the copolymers were characterized using 1H NMR (Varian Unity Inova 500NB, Sparta, NJ, USA) operating at 500 MHz; the samples were dissolved in deuterated dimethyl sulfoxide (DMSO-d6)/D2O. The sizes of the particles were determined at 25 °C using a FPAR-1000 fiber optics particle analyzer (Otsuka Electronics, Osaka, Japan). The particle analyzer used in this study was dynamic light scattering, which is also known as QuasiElastic Light Scattering (QELS). The morphology of the particles was observed using TEM (TEM, Philips CM30) operated at an accelerating voltage of 200 keV. Samples were dispersed in distilled water (1 mg/mL) and dropped on a 200 mesh copper grid. All samples were treated with 1% uranyl acetate for negative staining. The molecular weight of PCL-N3 was measured by gel permeation chromatography (GPC) using a Waters Model 410 equipped with a refractive index detector (Shodex, RI-101) and three Styragel (KF-803, KF-802.5, and KF-802) columns in series. The molecular weight calibration was carried out by using poly(ethylene glycol) standards. The molecular weight and polydispersity of PCL-N3 was found to be 5774 g/mol and 1.18, respectively. Synthesis of PDA-Conjugated HA-b-PCL Block Copolymers. PDA-conjugated HA-b-PCL copolymer was prepared by click chemistry using α-alkyne HA with PDA and PCL-N3, as shown in Figure 1. α-Alkyne HA was synthesized by reductive amination as previously described.44 In brief, HA (0.64 g, 0.053 mmol) and propargylamine (0.29 g, 5.3 mmol) were dissolved in 0.1 M borate buffer (0.4 M NaCl, pH 8.5). Thereafter, sodium cyanoborohydride (0.33 g, 5.3 mmol) was added, and the reaction mixture was allowed to proceed at 50 °C for 120 h (Caution: Sodium cyanoborohydride is toxic and must be used in a fume hood). The resulting solution was dialyzed against distilled water for 3 days using a dialysis tube (MWCO = 1000 Da, Spectrum Laboratories, Inc., CA, USA), followed by lyophilization to obtain αalkyne HA. Disulfide-containing PDA was conjugated to the backbone of αalkyne HA in the presence of EDC and HOBt. In brief, α-alkyne HA (0.28 g, 0.746 mmol) was dissolved in 25 mL of distilled water containing EDC·HCl (0.23 g, 0.298 mmol), HOBt (0.162 g, 1.194 mmol), and PDA (0.067 g, 0.298 mmol). Then, pH of the solution was adjusted to 6.8, and stirred at room temperature for 24 h. The resulting mixture was purified by dialysis (MWCO = 1000 Da) against distilled water for 2 days, followed by lyophilization to obtain PDAconjugated α-alkyne HA. PDA-conjugated HA-b-PCL copolymer was synthesized by Huisgen’s 1,3-dipolar cycloaddition reaction.46 In brief, PCL-N3 (64 mg, 0.01 mmol) and PDA-conjugated α-alkyne HA (200 mg, 0.015 mmol) were dissolved in degassed DMF (5 mg/mL) and deionized water (5 mg/mL), respectively. Then, the solutions were mixed, CuSO4·5H2O (10.60 mg, 0.042 mmol) and sodium ascorbate (8.41 mg, 0.042 mmol) were added, and the mixture was stirred at 45 °C for 2 days. Thereafter, the solution was dialyzed against water (MWCO = 25 000 Da) for 3 days to remove excess HA, followed by lyophilization to obtain PDA-conjugated HA-b-PCL. For in vitro cellular uptake and in vivo animal experiments, HA-b-PCL copolymer was labeled with Cy5.5 (λex = 675 nm, λem = 694 nm) as previously described.39 Cross-Linking of Nanoparticles. The cross-linking of PDAconjugated HA-b-PCL was carried out at room temperature in the presence of a catalytic amount of DTT. Briefly, PDA-conjugated HAb-PCL (0.5 mg/mL) was dissolved in distilled water followed by the addition of a catalytic amount of DTT, and the mixture was stirred at

room temperature for 24 h. Thereafter, the solution was dialyzed against distilled water (MWCO = 3500 Da) for 2 days, followed by lyophilization to obtain cross-linked nanoparticles (HA-ss-NPs). Hereafter, the non-cross-linked PDA-conjugated HA-b-PCL will be denoted as HA-NPs. Stability of Cross-Linked Nanoparticles. The stability of HANPs and HA-ss-NPs was investigated in the presence of sodium dodecyl sulfate (SDS), which acts as a destabilizing agent in aqueous media.47 In brief, the SDS solution (5 mg/mL) was added to an aqueous solution of HA-NPs or HA-ss-NPs (1 mg/mL), and the solution was stirred at room temperature. The scattering light intensity of the nanoparticles in the SDS solution was monitored at predetermined time intervals. In Vitro Release Behavior of DOX-Loaded Nanoparticles. DOX was loaded into the nanoparticles by an emulsion method.48 In brief, DOX·HCl (10 mg) was dissolved in chloroform (1 mL) containing a 3.0 equimolar amount of triethylamine. The resulting solution was added to an aqueous solution (10 mL) of HA-NPs (100 mg) containing a catalytic amount of DTT, leading to the formation of an oil-in-water emulsion. This emulsion was kept in the dark overnight with stirring to allow evaporation of the chloroform. The solution was then dialyzed against an excess amount of distilled water to remove unloaded DOX and byproduct, followed by lyophilization to obtain DOX-loaded cross-linked HA-NPs (DOX-HA-ss-NPs). The loading efficiency and content of DOX in the DOX-HA-ss-NPs were determined using a UV−vis spectrophotometer (Optizen 3220UV, Mecasys Co., Ltd., Daejeon, Korea) at 485 nm. For this experiment, DOX-HA-ss-NPs were dissolved in a DMSO/water (1v/1v) mixture, and a calibration curve was obtained using DMSO/water (1v/1v) solutions with different DOX concentrations. The loading efficiency and loading content of DOX were calculated using the following formulas:

Loading efficiency (%) = (weight of loaded drug/weight of drug in feed) × 100%

Loading content (%) = (weight of loaded drug/weight of polymer) × 100% An identical procedure, without the addition of DTT was used for the preparation of DOX-loaded HA-NPs (DOX-HA-NPs). The stability of DOX-loaded nanoparticles was investigated in a PBS containing 20% fetal bovine serum (FBS) using a DLS. In brief, the DOX-loaded nanoparticles (10 mg) were dispersed in 10 mL of PBS containing 20% FBS at 37 °C. Thereafter, the changes in scattering intensities of the solutions were monitored as a function of time. To observe the in vitro drug release, DOX-loaded nanoparticles (1 mg/mL) were dispersed in PBS (pH 7.4), and the solutions were transferred to cellulose membrane tubes (MWCO = 3500 Da). The dialysis tubes were then immersed in PBS (pH 7.4) with or without 10 mM GSH. Each sample was gently shaken at 100 rpm in a water bath at 37 °C. The medium was replenished at predetermined time intervals, and the DOX concentration was determined using UV−vis spectroscopy at 485 nm. Cytotoxicity and Intracellular Drug Release. SCC7 cell lines obtained from the American Type Culture Collection (Rockville, MD, USA) were cultured in RPMI 1640 medium (Gibco, Grand Island, NY, USA) containing 10% (v/v) FBS and 1% (w/v) penicillinstreptomycin at 37 °C in a humidified 5% CO2-95% air atmosphere. The cells were seeded at a density of 1 × 104 cells/well in 96-well flatbottomed plates. After 1 day of growth, the cells were washed twice with PBS (pH 7.4) and incubated for 24 h with various concentrations of bare nanoparticles. The cells were then washed twice with PBS to remove any remaining polymer, and fresh culture medium was added. Then, 10 μL of cell counting kit-8 (CCK-8) was added to each well, and the cells were incubated for an additional 4 h at 37 °C. Subsequently, the medium was removed, and the cells were dissolved C

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Figure 2. Schematic illustration of (a) the formation of DOX-loaded cross-linked nanoparticles and their GSH-responsive drug release behavior and (b) receptor-mediated internalization of the nanoparticles and subsequent intracellular drug release. in DMSO. The absorbance at 450 nm was measured using a microplate reader (BioTek, Seoul, Korea). To observe the cellular uptake and intracellular drug release behavior of the nanoparticles, cells were incubated for 1 h with Cy5.5labeled nanoparticles and DOX-loaded nanoparticles, respectively. For competitive inhibition studies, the cells were pre-exposed to 2 mL of serum-free medium containing HA (5 mg/mL) for 60 min. The cells were then washed twice with PBS (pH 7.4) and fixed with 4% formaldehyde solution. For nuclear staining, the cells were incubated with 4,6-diamino-2-phenylinodole (DAPI) for 10 min at room temperature and then washed with PBS (pH 7.4). The intracellular localization of DOX was observed using a LSM 510 META NLO confocal laser scanning microscope (CLSM). In Vivo Biodistribution. All experiments with live animals were performed in compliance with relevant laws and institutional guidelines of Sungkyunkwan University, and institutional committees have approved the experiments. To observe the in vivo biodistribution of HA-NPs or HA-ss-NPs, tumor-bearing mice were prepared by injecting a suspension of 1 × 106 SCC7 cells in physiological saline (100 μL) into the subcutaneous dorsa of athymic nude mice (7 weeks old, 20−25 g). Fourteen days after subcutaneous inoculation, Cy5.5labeled nanoparticles were injected into the tail vein of the tumorbearing mice at a dose of 5 mg/kg. For the in vivo study, the fluorescence intensities of the nanoparticles were adjusted to identical fluorescence intensities using a Kodak Image Station 4000MM (New

Haven, CT, USA) equipped with a special C-mount lens and Cy5.5 bandpass emission filter (680−720 nm; Omega Optical). The biodistribution of nanoparticles was evaluated as a function of time using the eXplore Optix system (ART Advanced Research Technologies Inc., Montreal, Canada). Laser power and count time settings were optimized at 15 μW and 0.3 s per point, respectively. Excitation and emission spots were raster-scanned in 1 mm increments over the selected region of interest. A 670 nm pulsed laser diode was used to excite the Cy5.5 molecules. The fluorescence emission at 700 nm was collected and detected through the fast photomultiplier tube (Hammamatsu, Japan) and a time-correlated single photon counting system (Becker and Hickl Gmbh, Berlin, Germany), respectively. The tumor-targeting characteristics of nanoparticles were evaluated by measuring the NIR fluorescence intensity at the tumor site (n = 5 for each group). All the data were calculated using the region of interest (ROI) function of Analysis Workstation software (ART Advanced Research Technologies Inc., Montreal, Canada), and values are presented as means ± SD for groups of five animals. The major organs and tumors were dissected from the SCC7 tumor-bearing mice 24 h after intravenous injection of the Cy5.5labeled nanoparticles. NIRF intensity of dissected organs and tumors were obtained with a 12-bit CCD camera (Kodak Image Station 4000 MM, New Haven, CT) equipped with a special C-mount lens and Cy5.5 bandpass emission filter (680 to 720 nm; Omega Optical). In Vivo Antitumor Efficacy. To evaluate the antitumor efficacy of the nanoparticles, SCC7 tumor-bearing mice were prepared as D

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Figure 3. TEM images of (a) HA-NPs and (b) HA-ss-NPs. (c) The size distribution of HA nanoparticles. (d) Changes in light scattering intensities of HA nanoparticles as a function of time. The error bars in the graph represent standard deviations (n = 3). previously described. Mice were divided into four groups: (i) normal saline, (ii) free DOX at 5 mg/kg, (iii) DOX-HA-NPs at 5 mg DOX/ kg, and (iv) DOX-HA-ss-NPs at 5 mg DOX/kg. When tumors reached 8 mm in diameter, each sample was injected once every 3 days (4 injections per mouse, n = 5 for each group). Tumor volumes were calculated as a × b2/2, were a is the largest and b is the smallest diameter. Statistical Analysis. The statistical significance of differences (p < 0.005) among groups was calculated using one-way ANOVA.

robust blood stability and high targetability to CD44-overexpressing cancer cells. Synthesis and Characterization of PDA-Conjugated HA-b-PCL Block Copolymers. In an attempt to develop robust, tumor-targetable cross-linked nanoparticles, we developed a biocompatible and biodegradable HA-b-PCL diblock copolymer. The HA shell allows the nanoparticle to target the CD44-ligand, facilitating subsequent internalization via receptor-mediated endocytosis. In addition, the HA shell allows the conjugation of the PDA chemical cross-linker, and the hydrophobic PCL enables encapsulation of anticancer drugs. The introduction of DTT into the block copolymers results in cross-linking, which enhances the nanoparticle stability in the extracellular environment. These cross-links can be reduced by the reductive environment of the cytosol, causing an intracellular release of anticancer drug (Figure 2). The block copolymer was synthesized based on click chemistry, a Huisgen 1,3-cycloaddition reaction, which is an emerging and versatile synthetic strategy to prepare conjugates under mild experimental conditions.49−51 The detailed synthetic route used to obtain the PDA-conjugated HA-bPCL copolymer is shown in Figure 1. For the synthesis of polysaccharide-based copolymers, an alkyne group was introduced at the reducing end of HA by reductive amination

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RESULTS AND DISCUSSION In recent years, smart nanocarrier systems have been investigated for targeted cancer therapy. To improve thr safety and therapeutic efficacy of anticancer drugs, numerous tumortargeted nanocarriers were developed.2 For better blood stability of the drug carriers, moreover, cross-linking strategies have been exploited.15 Although these approaches have improved either the tumor-targetability or the blood stability of nanocarriers in vivo, there have been no significant efforts to develop nanocarriers that can meet both requirements of the tumor-specificity and the robust stability in the bloodstream. In this study, an attempt was made to develop a robust synthetic block copolymer (HA-b-PCL) that can be self-assembled into nanoparticles and also can be cross-linked via intraparticular bioreducible disulfide bonds, endowing the nanocarriers with E

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Figure 4. In vitro release behavior of DOX from HA nanoparticles in the presence and absence of GSH: (a) DOX-HA-NPs and (b) DOX-HA-ssNPs. Insets are magnified drug release graphs (0−6 h). The error bars in the graph represent standard deviations (n = 3).

Stability of Cross-Linked Nanoparticles. The stability of the thermodynamically frozen HA-ss-NPs and non-cross-linked HA-NPs was investigated in the presence of SDS, which acts as a destabilizing agent (Figure 3d).47 After the preparation of nanoparticles in aqueous solution (1 mg/mL), each sample was mixed with an aqueous solution of SDS (5 mg/mL), and the scattering intensity was measured as a function of time. For HA-NPs, the scattering light intensity dramatically decreased to 50% over a 3 h incubation period, indicating the SDS induced disintegration of non-cross-linked nanoparticles. Owing to their instability, polydispersity of HA-NPs increased in the presence of SDS as a function of time (Figure S3, Supporting Information). In contrast, the cross-linked HA-ss-NPs showed minimal decreases in scattering intensity, indicating enhanced stability conferred by cross-linking the nanocarriers. As expected, greater than 95% scattering intensity was observed for both nanoparticles in the absence of SDS. Controlled Drug Release of DOX-Loaded Nanoparticles. To assess controlled drug release characteristics, DOX was chosen as a model anticancer agent and was effectively encapsulated into the nanoparticles with high loading efficiency. Table S3 in the Supporting Information shows the physicochemical characteristics of the DOX-loaded nanoparticles prepared by an emulsion method. The loading efficacy of DOX was higher for cross-linked nanoparticles with larger amount of PDA. This result suggests that the highly crosslinked nanoparticles provide the effective diffusion barrier of DOX, thus allowing its high loading efficacy. Before estimating the effect of redox-environment on drug release, we monitored the stability of the DOX-loaded crosslinked and non-cross-linked nanoparticles in serum conditions based on a previously reported procedure.30 As shown in Supporting Information Figure S4, the scattering intensity of cross-linked nanoparticles with a high amount of PDA did not significantly decreased when they were exposed to the serum conditions (20% FBS). The non-cross-linked nanoparticles and cross-linked nanoparticles with a low amount of PDA exhibited remarkable decrease in the scattering intensity. Owing to the better stability and high drug-loading efficiency, we chose HAss-NPs with a DS value of 10.61 for further experiments.

with propargylamine in the presence of sodium cyanoborohydride as a reducing agent. PDA, chosen as the disulfide crosslinker, was conjugated to the backbone of HA through amide bond formation. The degree of substitution (DS), defined as the number of PDA derivatives per 100 HA repeating units, was found to be 10.61%. Then, the PDA-conjugated alkyne HA was reacted with PCL-N3 to obtain block copolymers. The 1H NMR spectrum demonstrated the presence of both HA and PCL, which indicated the successful formation of block copolymers (Figure S1, Supporting Information). The composition of HA and PCL present in the block copolymer was calculated based on the integration ratio of peaks from HA (2.0 ppm) and PCL (3.8 ppm). It was found that an equivalent ratio of HA and PCL was present in the copolymer. The chemical structure of the PDA-conjugated HA-b-PCL copolymer was further confirmed using FT-IR spectra (Figure S2, Supporting Information). The FT-IR spectrum showed the azide peak of PCL-N3 at 2100 cm−1, whereas the HA-b-PCL copolymer exhibited all of the characteristic peaks of both PCL and HA except for the azide peak, which implied the formation of a block copolymer. Owing to its amphiphilicity, the copolymer self-assembled into nanoparticles in an aqueous solution. The size distribution and morphology of the HA nanoparticles are shown in Figures 3a and 3c. As expected, the copolymers formed nanoparticles with a unimodal size distribution. The mean diameter of the HA-NPs was found to be 198 nm (Table S2, Supporting Information). The TEM images showed that the nanoparticles were spherical in shape. Preparation and Characterization of Cross-Linked Nanoparticles. Cross-linking of HA-NPs was achieved by the addition of a catalytic amount of DTT. Nanoparticle crosslinking was confirmed using UV−vis spectroscopy. Disappearance of the characteristic pyridyl disulfide peak at 280 nm and the appearance of a new peak at 343 nm corresponding to pyridine-2-thione indicated the formation of cross-linked nanoparticles (data not shown). After cross-linking, the size of the nanoparticles decreased, which is consistent with the size distribution results (Figure 3b,c). TEM images indicated that the morphology of the nanoparticles was not significantly changed by chemical cross-linking, suggesting that cross-linking did not induce interparticular aggregation. F

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pretreated with an excess amount of free HA. These results indicated that the uptake of Cy5.5-labled nanoparticles into the tumor cells was based on binding of nanoparticles to the HA receptor, CD44. In Vitro Cell Cytotoxicity and Intracellular Drug Release. Figure S5 in the Supporting Information shows the cytotoxic effect of bare nanoparticles on SCC7 cells, as evaluated using the CCK-8 assay. Owing to their biocompatibility, none of the nanoparticles exhibited cytotoxicity to SCC7 cells. In particular, most cells were viable in the presence of up to 75 μg/mL HA-ss-NPs, indicating that the chemical crosslinking did not exhibit cytotoxicity to the cells. In order to assess intracellular drug release, DOX-loaded nanoparticles were exposed to SCC7 and NIH3T3 cells and monitored using CLSM (Figure 6). DOX, chosen as the model

Figure 4 shows the release behavior of DOX from DOX-HAss-NPs and DOX-HA-NPs in the presence and absence of GSH. In the physiological buffer (0 mM GSH), DOX-HA-NPs released 40% of the DOX during the initial 3 h of incubation, after which its release rate steadily increased, implying a burst release by non-cross-linked nanoparticles. In addition, there was no significant change in drug release in the presence of GSH. It should be noted that the release profile in the absence of GSH showed that the HA-ss-NPs efficiently hold most of the drug. On the other hand, in the presence of 10 mM GSH, mimicking the intracellular GSH level, the drug release was facilitated from the DOX-HA-ss-NPs. In particular, greater than 80% of the drug was released within 48 h. The facilitated drug release was primarily due to the cleavage of the disulfide cross-linker disturbing the frozen state of the nanoparticles. From the release experiment, it was found that the HA-ss-NPs efficiently restricted drug release in the physiological solution. However, introduction of GSH to the HA-ss-NPs resulted in degradation of the cross-links, which subsequently triggered drug release. Recently, it was demonstrated that the high stability of the nanoparticles in physiological solution will prolong their circulation in the body.30 Cellular Uptake of Nanoparticles. To observe the in vitro cellular uptake properties, Cy5.5-labeled nanoparticles were exposed to SCC7 cancer cells. As shown in Figure 5,

Figure 6. Confocal microscopic images of SCC7 and NIH3T3 cells incubated with DOX-HA-ss-NPs and DOX-HA-NPs. Each sample was incubated with SCC7 and NIH3T3 cells in a serum-free culture medium for 1 h.

drug, is a fluorescent anticancer drug known to interact with DNA by specific intercalation. When SCC7 cells were treated with DOX-loaded HA nanoparticles, the strong fluorescent signals were observed at the intracellular compartments. This indicates that the nanoparticles were readily internalized into the cancer cells, followed by rapid release of DOX in the presence of GSH at the intracellular level. Interestingly, the fluorescence intensity of DOX remarkably decreased when DOX-loaded nanoparticles were incubated with CD44-negative NIH3T3 cell, implying that the cellular uptake of DOX-HA-ssNPs and DOX-HA-NPs into the SCC7 cells was based on binding of nanoparticles to CD44. In Vivo Biodistribution of Cy5.5-Labeled HA Nanoparticles. In order to verify the tumor-targetability of the HA nanoparticles, their in vivo biodistribution was monitored using a noninvasive fluorescence imaging technique. Although various cross-linked nanoparticles have shown excellent in vitro properties, they often have proven unsatisfactory in vivo because of their instability in the bloodstream and lack of tumor-targetability.52,53 In addition, fluorescent labeling of

Figure 5. Cellular uptake images of Cy5.5-labeled HA-NPs and HA-ssNPs. Each sample was incubated in a serum-free culture medium for 1 h.

irrespective of cross-linked or non-cross-linked nanoparticles, they were efficiently taken up by the cancer cells after 12 h of incubation. This result indicates that cross-linking does not affect the cellular uptake pattern of the nanoparticles. Microscopic images showed strong fluorescence primarily in the cytoplasm, implying the possibility of efficient intracellular delivery of DOX. It should be noted that the cellular uptake of nanoparticles significantly decreased when the cells were G

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Figure 7. In vivo noninvasive fluorescence imaging of HA nanoparticles in tumor-bearing mice. (a) Time-dependent whole body image of athymic nude mice bearing SCC7 tumors after intravenous injection of Cy5.5-labeled HA nanoparticles. (b) Total photon counts in the tumors of panel a were estimated from the fluorescence intensities. (c) Quantification of the ex vivo tumor-targeting characteristics of HA nanoparticles in tumorbearing mice. Error bars in the graph represent the standard deviation for five animals per group.

PEG-based cross-linked block copolymers is often difficult because of the lack of functional groups. The tumortargetability of the nanoparticles developed in this study was assessed by determining their in vivo biodistribution using a real-time NIRF imaging technique following systemic administration of Cy5.5-labeled nanoparticles (200 μL, 5 mg/kg) into the tail vein of SCC7 tumor-bearing mice. Significant NIRF signals were found throughout the bodies of the mice for up to 24 h, regardless of whether or not the nanoparticles were crosslinked, suggesting high tumor-targetability of the HA nanoparticles (Figure 7a). After intravenous injection of the nanoparticles, an increased NIRF signal was observed in the tumor region beginning 1 h postinjection. The tumor location was easily detected as it exhibited maximum NIRF signal, which was maintained for up to 24 h (Figure 7b). In particular, the cross-linked nanoparticles exhibited enhanced fluorescence compared to the non-cross-linked nanoparticles. The enhanced fluorescence intensity from the HA-ss-NPs might be attributed to the improved structural stability, allowing for prolonged circulation in the body.

These results were further confirmed with ex vivo NIRF imaging of tumors (Figure 7c). Tumors were excised after 24 h, and enhanced fluorescence was observed at the tumor site. The stronger fluorescent signals at the tumor site compared to those in other tissues allowed for clear discrimination between subcutaneous tumors and the surrounding normal tissue. This high tumor-targetability of the nanoparticles might be due to a combination of receptor-mediated endocytosis (CD44) as well as the EPR effect. The slightly high fluorescence intensity was also observed from the kidney, implying renal excretion of nanoparticles.54 Very weak intensities were observed in other major organs including the liver, spleen, and heart. These results are consistent with the noninvasive animal imaging findings. Antitumor Activity of Bioreducible Nanoparticles. Preparation of the cross-linked nanoparticles in this study was based on the assumption that they would be rapidly taken up by CD44-positive cancer cells and the disulfide cross-links would be rapidly reduced in the intracellular compartment, resulting in rapid DOX delivery. To determine the antitumor efficacy, we injected saline, free DOX, DOX-HA-NPs, and H

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DOX-HA-ss-NPs into SCC7 tumor-bearing mice (Figure 8). As expected, saline and free DOX exhibited rapid increase in

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ASSOCIATED CONTENT

S Supporting Information *

Preparation procedure for PCL-N3, 1H NMR, FT-IR, cell viability, and physicochemical chararacteristics of the block copolymers. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +82-31-290-7288; Fax: +82-31-299-6857; E-mail: [email protected]. Author Contributions ∇

These authors contributed equally to this paper.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the Korea Healthcare Technology R&D Project (HI14C03810200) and the National R&D Program for Cancer Control (1420040) of the MHW, and the Global Research Laboratory Program (NRF2013K1A1A2A02076442) and the Basic Science Research Programs (20100027955 & 2012012827) of the NRF.

Figure 8. Tumor growth of SCC7 cancer xenografts treated with saline, free DOX, DOX-HA-NPs, and DOX-HA-ss-NPs at a DOX dose of 5 mg/kg. Insets are representative tumor images excised 16 days post-treatment. Error bars in the graph represent the standard deviation for five animals per group. Asterisks (*) denote statistically significant differences (p < 0.05) as determined by one-way ANOVA.

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tumor size as a function of time. In addition, DOX-loaded noncross-linked nanoparticles did not significantly suppress tumor growth. Interestingly, the DOX-HA-ss-NPs treated mice significantly suppressed the tumor growth, suggesting high antitumor efficacy. Sixteen days after injection, the average tumor volume in the DOX-HA-ss-NPs-treated mice had increased relatively slowly compared to that in the other three groups, indicated enhanced anticancer activity of crosslinked nanoparticles. The enhanced in vivo efficacy might be due to the controlled release of DOX at the tumor site as a result of the cross-linked nanoparticles’ improved stability in the bloodstream and efficient tumor-targeting. Overall, our results indicate that HA-ss-NPs are highly effective tumortargetable nanocarriers due to its improved stability in the bloodstream, which effectively prevents an initial burst release, and enhanced tumor-targeting via passive and active targeting mechanisms.

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CONCLUSION In this study, bioreducible and tumor-targetable HA-based cross-linked nanoparticles that could preferentially release anticancer drugs at the intracellular level were developed. The cross-linking of the nanoparticles dramatically minimized the initial burst release of the drug and facilitated drug release in the presence of GSH, mimicking the reductive intracellular environment. The cell experiment showed that these nanoparticles are rapidly taken up by SCC7 cancer cells through their interaction with CD44 receptors on the surface of tumor cells. The tumor-targeting efficiency and therapeutic efficacy of the cross-linked nanoparticles were significantly higher than those of non-cross-linked nanoparticles and free chemotherapeutic drugs. Overall, these results imply that bioreducible HA-ss-NPs may be a useful drug carrier system for DOX. I

DOI: 10.1021/bm5017755 Biomacromolecules XXXX, XXX, XXX−XXX

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DOI: 10.1021/bm5017755 Biomacromolecules XXXX, XXX, XXX−XXX