Intracellular delivery and antitumor effects of a redox-responsive polymeric paclitaxel conjugate based on hyaluronic acid

Acta Biomaterialia xxx (2015) xxx–xxx

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Intracellular delivery and antitumor effects of a redox-responsive polymeric paclitaxel conjugate based on hyaluronic acid Shaoping Yin a, Jue Huai a, Xi Chen a, Yong Yang b, Xinxin Zhang c, Yong Gan c, Guangji Wang d, Xiaochen Gu e, Juan Li a,⇑ a

Department of Pharmaceutics, China Pharmaceutical University, Nanjing 210009, China Center for New Drug Safety Evaluation and Research, China Pharmaceutical University, Nanjing 210009, China Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Science, Shanghai 201203, China d Center of Pharmacokinetics, Key Laboratory of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University, Nanjing 210009, China e College of Pharmacy, University of Manitoba, Winnipeg, MB R3E 0T5, Canada b c

a r t i c l e

i n f o

Article history: Received 29 April 2015 Received in revised form 17 July 2015 Accepted 19 August 2015 Available online xxxx Keywords: Redox-response HA-ss-PTX conjugates Cancer-targeted therapy Intracellular release CD44

a b s t r a c t Polymer–drug conjugates have demonstrated application potentials in optimizing chemotherapeutics. In this study a new bioconjugate, HA-ss-PTX, was designed and synthesized with cooperative dual characteristics of active tumor targeting and selective intracellular drug release. Paclitaxel (PTX) was covalently attached to hyaluronic acid (HA) with various sizes (MW 9.5, 35, 770 kDa); a cross-linker containing disulfide bond was also used to shield drug leakage in blood circulation and to achieve rapid drug release in tumor cells in response to glutathione. Incorporation of HA to the conjugate enhanced the capabilities of drug loading, intracellular endocytosis and tumor targeting of micelles in comparison to mPEG. HA molecular weight showed significant effect on properties and antitumor efficacy of the synthesized conjugates. Intracellular uptake of HA-ss-PTX toward MCF-7 cells was mediated by CD44-caveolae-mediated endocytosis. Compared to Taxol and mPEG-ss-PTX, HA9.5-ss-PTX demonstrated improved tumor growth inhibition in vivo with a TIR of 83.27 ± 5.20%. It was concluded that HA9.5-ss-PTX achieved rapid intracellular release of PTX and enhanced its therapeutic efficacy, thus providing a platform for specific drug targeting and controlled intracellular release in chemotherapeutics. Statement of Significance Polymer–drug conjugates, promising nanomedicines, still face some technical challenges including a lack of specific targeting and rapid intracellular drug release at the target site. In this manuscript we designed and constructed a novel bioconjugate HA-ss-PTX, which possessed coordinated dual characteristics of active tumor targeting and selective intracellular drug release. Redox-responsive disulfide bond was introduced to the conjugate to shield drug leakage in blood circulation and to achieve rapid drug release at tumor site in response to reductant like glutathione. Paclitaxel was selected as a model drug to be covalently attached to hyaluronic acid (HA) with various sizes to elucidate the structure–activity relationship and to address whether HA could substitute PEG as a carrier for polymeric conjugates. Based on a series of in vitro and in vivo experiments, HA-ss-PTX performed well in drug loading, cellular internalization, tumor targeting by entering tumor cells via CD44-caveolae-mediated endocytosis and rapidly release drug at target in the presence of GSH. One of the key issues in clinical oncology is to enhance drug delivery efficacy while minimizing side effects. The study indicated that this new polymeric conjugate system would be useful in delivering anticancer agents to improve therapeutic efficacy and to minimize adverse effects, thus providing a platform for specific drug targeting and controlled intracellular release in chemotherapeutics. Ó 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

⇑ Corresponding author at: Department of Pharmaceutics, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, China. E-mail address: [email protected] (J. Li). http://dx.doi.org/10.1016/j.actbio.2015.08.029 1742-7061/Ó 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Please cite this article in press as: S. Yin et al., Intracellular delivery and antitumor effects of a redox-responsive polymeric paclitaxel conjugate based on hyaluronic acid, Acta Biomater. (2015), http://dx.doi.org/10.1016/j.actbio.2015.08.029

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1. Introduction Nanomedicine plays an important role in improving biological efficacy of the conventional small-molecule chemotherapeutics [1–3]. Polymer–drug conjugate, also known as polymeric prodrug, has demonstrated significant potentials in nanomedicine for their delivery of active agents to the targets, by controlling drug release with minimal burst effect during transition from the blood circulation to the extracellular tumor matrix [4]. Several PEGylated enzymes and cytokines have been successfully developed and approved for clinical applications; polyglutamic acid-palitaxel has also shown particular potential in Phase III clinical trial in women with non-small-cell lung cancer [5,6]. Nevertheless developing such a drug delivery system still faces technical challenges including a lack of specific drug targeting and rapid intracellular drug release at the target site [7]. Introduction of various biological ligands or antibodies into novel drug delivery system has been proven beneficial for selective delivery of anticancer compounds to tumor cells [8]. Hyaluronic acid (HA) has recently received extensive attention for its high affinity to CD44 [9,10]; it also possesses numerous desirable physicochemical and biological properties such as biocompatibility, biodegradability and non-immunogenicity [11] for drug delivery applications. HA-based conjugates are capable of simultaneous passive targeting of solid tumors via enhanced permeation and retention (EPR) effect and active targeting of CD44-bearing cancer cells without additional targeting ligands [12]. HA-paclitaxel conjugates have shown improvement of drug efficacy in standard chemotherapeutics in brain metastases of breast cancer (HA MW 3–5 kDa) [13], in orthotopic OSC-19-luciferase and HN5 xenograft models (HA MW 35 kDa) [14], and in mice bearing ovarian cancer (HA MW 200 kDa) [15]. Despite the promising results in tumor targeting, it was unclear how variability of HA molecular weight had influenced the tumor-targeting ability; HA of low molecular weight (LMW HA) behaved quite differently than HA of high molecular weight (HMW HA) in CD44 binding affinities (MW 5– 8 < 10–12 < 175–350 kDa). In addition, nanocarrier of HMW HA displayed faster clearance than that of LMW HA [16]. Therefore, it would be important to elucidate the structure–activity relationship between HA molecular weight and HA-modified conjugates. To achieve rapid intracellular release of anticancer drug, nanocarriers should become adaptive to tumor microenvironment (e.g., pH, temperature, redox) timely and appropriately to exemplify their unique advantages in applications [17–19]. It is noteworthy that concentration of reducing glutathione (GSH) in cytosol is 100–1000 times higher than that of other body fluids, and that tumor tissues are highly hypoxic with at least 4-fold higher than the normal tissues [20,21]. Delivery vehicles with disulfide functionality thus are cleaved in the presence of reducing agents including L-cysteine and glutathione (GSH) [22,23] efficiently. As a result, several glutathione-responsive polymeric conjugates including CPT-SS-PEG-SS-CPT [4] and H-shaped PEGylated methotrexate conjugates [24] have been developed for selective drug release. In this study, the concept of redox potential for anticancer drug paclitaxel (PTX) was designed and confirmed, the structure–activity relationship between HA molecular weight and HA-modified bioconjugates was investigated. mPEG-ss-PTX was also synthesized as a control to address whether or not hydrophilic polymer HA could substitute PEG as a carrier for polymeric bioconjugates. Scheme 1 depicts the hypothesis and mechanism of specific drug targeting and delivery, i.e., HA-ss-PTX is to be specifically transferred to the tumor site by the EPR effect and absorbed by tumor cells via CD44-mediated endocytosis. Upon entering the cells, the

disulfide bond would be disrupted, releasing PTX rapidly from the micelles. A series of in vitro and in vivo experiments were subsequently carried out to evaluate the potential of this novel delivery system in tumor therapy. 2. Materials and methods 2.1. Materials and reagents Sodium hyaluronate (MW 9.5 kDa, 35 kDa and 770 kDa) was purchased from Freda Biochem Co., Ltd. (Jinan, Shandong, China). Monomethoxy poly(ethylene glycol) (mPEG, MW 2 kDa), pyrene and thiazolylblue tetrazolium bromide (MTT) were obtained from Sigma–Aldrich Co. (St. Louis, MO, USA). 1-Ethyl-3 (3dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS), 4-dimethylaminopyridine (DMAP), 3,30 -dithiodipropionic acid (DPA), dithiothreitol (DTT) and glutathione (GSH) were purchased from Aladdin Reagent Database Inc. (Shanghai, China). Paclitaxel (PTX) was purchased from Shanghai Zhongxi Sunve Pharmaceutical Co., Ltd. (Shanghai, China). Coumarin-6 (C6) and 40 ,6-diamidino-2-phenylindole (DAPI) were purchased from Shanghai H-Y Biological Technology Co., Ltd. (Shanghai, China) and Beyotime Institute of Biotechnology Co., Ltd. (Shanghai, China), respectively. RPMI1640 media, fetal bovine serum (FBS) and tyrisin were purchased from GIBCO Co., Ltd. (Grand Island, NY, USA). Near-infrared dye DiR was provided by Beijing Fanbo Science and Technology Co., Ltd. (Beijing, China). Purified deionized water was obtained from a Milli-QÒ Plus System (Billerica, MA, USA). All other chemicals were of chromatographic or analytical grades and used without further purification. 2.2. Synthesis and preparation 2.2.1. Synthesis of adipic dihydrazido-functionalized HA (HA-ADH) Raw sodium hyaluronate was purified and desalted prior to use. Briefly, 1.0 g sodium hyaluronate was dissolved in 20 mL deionized water, and the pH adjusted to 3.5. The liquid was incubated overnight, dialyzed (MWCO 3.5 kDa), and then lyophilized. HA-ADH was prepared by acylation process between ADH and HA [25,26]. In a representative example, 2.5 mmol ADH was added to 0.5 mmol HA in deionized water and the pH adjusted to 5.0. Subsequently, 0.25 mmol EDC was dissolved in a small amount of water and added to the solution under agitation. The pH of the mixture was readjusted to 5.0. After 4 h at room temperature, the reaction was discontinued by raising pH to 7.0. The reactant was purified by successive dialysis (MWCO 3.5 kDa) against 0.1 M NaCl, 25% (v/v) ethanol solution and deionized water. Then the solution was lyophilized and stored at 4 °C for further use. A series of HA-ADH with various carboxyl substitutes was sequentially prepared by adjusting the ratios of HA, ADH and EDC. 2.2.2. Synthesis of DPA-functionalized PTX (PTX-ss-DPA) DPA was used as a donor to introduce disulfide bond to PTX in preparing PTX-ss-DPA. Fig. S1 illustrates the synthesis pathway. In brief, 1.5 mmol DMAP and EDC were added to equivalent DPA in 20 mL CH2Cl2 under nitrogen and agitation at 0 °C. The reaction continued for 0.5 h to activate the carboxyl group of DPA. 0.5 mmol PTX in CH2Cl2 was added drop-wise to the mixture, and the reaction continued for 24 h at room temperature. Esterification process was monitored by thin layer chromatography. The product was washed with 0.01 M HCl twice and deionized water three times, dried and concentrated under vacuum. Final residue was dissolved in CH2Cl2 and purified by silica gel column chromatography with CH2Cl2:CH3OH = 40:1 (Rf = 0.31) to yield PTX-ss-DPA.

Please cite this article in press as: S. Yin et al., Intracellular delivery and antitumor effects of a redox-responsive polymeric paclitaxel conjugate based on hyaluronic acid, Acta Biomater. (2015), http://dx.doi.org/10.1016/j.actbio.2015.08.029

S. Yin et al. / Acta Biomaterialia xxx (2015) xxx–xxx

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Scheme 1. Illustration of redox-responsive, biodegradable molecule HA-ss-PTX for efficient intracellular delivery of PTX. Drug delivery includes steps of intravenous injection, EPR effect, CD44-mediated cellular internalization, and cytoplasmic PTX release trigged by GSH.

2.2.3. Synthesis of HA-ss-PTX Briefly, 0.25 mmol PTX-ss-DPA was dissolved in 15 mL anhydrous dimethylformamide (DMF) and mixed with two times EDC and NHS for activation. 0.5 mmol HA-ADH in formamide was added to the mixture drop-wise, and the solution stirred vigorously under nitrogen for 48 h at 40 °C. For purification, the solvent was extensively dialyzed (MWCO 3.5 kDa) in 50% (v/v) ethanol solution for 24 h and deionized water for 48 h. The product was lyophilized and stored at 4 °C for further use. By changing the molar ratio of HA-ADH to PTX-ss-DPA, a series of HA-ss-PTX conjugates with different PTX contents was synthesized.

2.2.4. Preparation of HA-ss-PTX and mPEG-ss-PTX micelles HA-ss-PTX micelles were prepared by a simple sonication-dialy sis-lyophilization method. Briefly, 30 mg of HA-ss-PTX was dissolved in 3 mL DMF and the solution was sonicated at 100 W with a probe-type ultrasonicator for 10 min in an ice bath, followed by dialysis (MWCO 3.5 kDa) against an excessive amount of deionized water for 12 h. Then the solution was filtered through a 0.45 lm

membrane and lyophilized. Synthesis and preparation of mPEGss-PTX are shown in Supporting Information. 2.3. Characterization of conjugates and micelles 2.3.1. Structural identification FT-IR spectra of the products were recorded on a Bruker FT-IR spectrometer (Tensor 27, Bruker, Germany). 1H NMR spectra were recorded on a Bruker Avance 500 spectrometer (Billerica, MA, USA) operating at 500 MHz. HA-ss-PTX was dissolved in DMSO; PTX and mPEG-ss-PTX were dissolved in CDCl3. Substitution degree of ADH was determined from ratio of methylene hydrogen to acetyl methyl proton, as measured by 1H NMR. Using Ellman’s assay (DTNB) and L-cysteine as the standard, the amount of thiol groups in the backbone of HA after the disulfide linkage in HA-ss-PTX and mPEG-ss-PTX was confirmed [27]. PTX contents of HA-ss-PTX and mPEG-ss-PTX were measured by UV–Vis spectroscopy at 227 nm [28]. Structure of HA-ss-PTX and mPEG-ss-PTX was further identified by 13C NMR spectra on a Bruker Avance 500 spectrometer operating at 300 MHz.

Please cite this article in press as: S. Yin et al., Intracellular delivery and antitumor effects of a redox-responsive polymeric paclitaxel conjugate based on hyaluronic acid, Acta Biomater. (2015), http://dx.doi.org/10.1016/j.actbio.2015.08.029

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Degradation mechanism, critical micelle concentrations (CMCs), morphology, size distribution, zeta potential, and DTT-induced disassembly of micelles are shown in Supporting Information. 2.3.2. In vitro release of PTX Lyophilized HA-ss-PTX and mPEG-ss-PTX micelles were suspended in distilled water to obtain an isotonic solution, 0.5 mL sample was transferred to a dialysis bag (MWCO 1 kDa) and immersed in PBS (pH 7.4) containing 0.1% (w/v) Tween 80. Various concentrations of GSH (2 lM, 10 mM and 40 mM) were added to PBS; release medium at pH 5.0, 40 mM GSH was also tested. Study samples were incubated at 150 rpm and 37 °C, and release medium collected at different time intervals and determined by HPLC. 2.4. Cellular studies MCF-7 cells were kindly donated by Shanghai Institute of Material Medica (Shanghai, China), and B16F10 and VERO cells were presented by China Pharmaceutical University Center for Safety and Evaluation of New Drugs (Nanjing, Jiangsu, China). All cell lines were maintained in RPMI 1640, supplemented with 10% inactivated fetal bovine serum (FBS), 100 U/mL penicillin and 100 mg/ mL streptomycin. Cells were cultured under 5% CO2 and 37 °C. 2.4.1. Intracellular distribution of HA-ss-PTX micelles Confocal laser scanning microscopy (CLSM) was used to evaluate intracellular distribution of coumarin-6 (C6) labeled micelles in MCF-7 cells. Cells were inoculated onto glass bottom culture dishes for 24 h at 37 °C. Fresh medium containing micelles loaded with C6 (100 ng/mL) was added, measurement was taken at 2 h, 4 h and 8 h. Cells were fixed with 4% paraformaldehyde for 15 min, and washed thrice with PBS. The nuclei were stained with DAPI for 15 min, followed by PBS washing another three times. 2.4.2. Cellular uptake of HA-ss-PTX micelles In vitro cellular uptake of C6 labeled HA-ss-PTX micelles was determined by flow cytometry (FCM). MCF-7 cells were separately seeded at 300,000 cells per dish for 24 h at 37 °C prior to the study. Cells were then incubated in medium containing C6-loaded micelles for 4 h. At the end of incubation, dishes was rinsed with PBS thrice, harvested by trypsinization, and collected in PBS to quantify fluorescent signals of C6. 2.4.3. Cellular uptake mechanism of HA-ss-PTX micelles The expression of CD44 in MCF-7 cells is high [29], but in VERO is very low [30]. Free HA polymer (9.5 kDa, 10 mg/mL) pretreated MCF-7 cells for 2 h and VERO cells were incubated with C6labeled micelles for 4 h to confirm CD44-mediated uptake. CLSM, Fluorescence microscopy and flow cytometry were conducted. 2.4.4. Mechanism of endocytosis Nanoparticles with particle size ranging 20 nm to 1 lm are internalized via several pathways, including clathrin-mediated endocytosis, caveolar-mediated endocytosis, macropinocytosis, and clathrin-independent and caveolin-independent endocytosis [31]. To study the endocytosis route of HA-ss-PTX, MCF-7 cells were pre-incubated individually with different endocytosis inhibitors, i.e., chlorpromazine hydrochloride (CH, 10 mg/mL), nystatin (NY, 25 lg/mL) and colchicine (CL, 40 lg/mL) for 30 min. C6labeled HA-ss-PTX micelles were then added to each well and incubated for another 4 h. Cellular uptake was determined by flow cytometry. At the same time, viability of MCF-7 cells treated with different inhibitors for 4 h was detected. All measurements were performed in triplicate.

2.4.5. Mechanism of drug release Mechanism of drug release was performed by using CLSM to monitor intracellular drug release behavior and a reported method [32] to determine concentration of intracellular GSH. MCF-7 cells were seeded onto glass bottom culture dishes and cultured for 24 h. Fresh medium containing 0, 10 or 40 mM GSH was added and incubated for 2 h. The cells were then incubated with C6labeled HA9.5-ss-PTX micelles for another 60 min. Finally the cells were stained and observed with CLSM. The intracellular GSH level was determined and expressed as the percentage of control.

2.5. In vivo animal studies 2.5.1. Animals and tumor xenograft models Male BALB/c nude mice (4–5 weeks, 18–20 g) used for the study were purchased from Nanjing KeyHEN Biotech. Co., Ltd. (Jiangsu, China). The study protocol was designed in accordance to guidelines approved by the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The animals were provided with autoclaved chow and water ad libitum. To induce solid tumor, 1 mL murine hepatic carcinoma cells (Heps, 1  108 cells in 1 mL PBS) was inoculated subcutaneously in the right armpit region of the animals. A Vernier caliper was used to measure tumor size, and tumor volume (mm3) was calculated as a2  b/2, where a was the minor axis and b the major axis.

2.5.2. Biodistribution of micelles in tumor-bearing mice Once tumor mass reached 500 mm3, the tumor-bearing mice were injected via tail vein at a dose of 5 mg/kg PTX with the following agents, free DiR diluted in Cremophor EL/ethanol/5% of glucose (1:1:9, v/v/v), DiR-labeled HA35-ss-PTX, HA9.5-ss-PTX and mPEGss-PTX micelles. A non-invasive near infrared optical imaging system was used to observe biodistribution and tumor accumulation of the preparations in mice at 0.5, 2, 4, 8, 12 and 24 h postinjection. Visualization was collected at excitation of 730 nm and emission of 790 nm, and images were analyzed by a Kodak Molecular Imaging Software. The study animals were sacrificed after living imaging; tumors were excised and analyzed using the same system.

2.5.3. In vivo antitumor efficacy Study animals were randomly divided into five groups with six mice in each group. The treatment preparations were Saline, Taxol, mPEG-ss-PTX, HA35-ss-PTX or HA9.5-ss-PTX at a dose of 7.5 mg/ kg, respectively; intravenous injection through the tail vein continued for 15 doses on every second day. The tumor volume was measured daily, while animal behavior and body weight were also monitored to assess dosing safety and study survival rate. At the end of the study, tumor tissue, and vital organs including heart, liver, spleen, lung, and kidney were harvested, weighed, and stained with haematoxylin and eosin (H&E) for pathological evaluation.

2.6. Data analysis Each experiment was performed in triplicate with at least three independent samples. Data sets were evaluated using t-test or One-Way Analysis of Variance (ANOVA) followed by Tukey’s post hoc test, and expressed as mean ± SD. Statistically significant difference was set at p < 0.05.

Please cite this article in press as: S. Yin et al., Intracellular delivery and antitumor effects of a redox-responsive polymeric paclitaxel conjugate based on hyaluronic acid, Acta Biomater. (2015), http://dx.doi.org/10.1016/j.actbio.2015.08.029

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3. Results and discussion 3.1. Synthesis and characterization of HA-ss-PTX and mPEG-ss-PTX Use of HA was intended to improve circulation time, biocompatibility, non-immunogenicity, drug solubility, and specific tumor targeting of the final preparations [33,34]. HA with molecular weight of 9.5 kDa, 35 kDa and 770 kDa was selected respectively in the experiment to prepare HA-ss-PTX, in order to study the structure–activity relationship between HA size and performance of the conjugate. Drug loading of HA770-ss-PTX was far below that of HA9.5-ss-PTX and HA35-ss-PTX. Furthermore, particle size of HA770-ss-PTX was larger than 500 nm, a property quite different from the other two conjugates. According to previous studies, nanocarriers of high MW HA displayed faster clearance than those of low MW HA or PEGylated vectors [16]; HA of low molecular weight stimulated intracellular signaling [35]; HA oligomers of 10–100 kDa was reported to inhibit growth and metastasis of various tumors, due to apoptosis of cancer cells by disruption of the

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binding between matrix-HA and receptors, downregulation of cell survival mechanisms such as PI 3-kinase/Akt signaling pathway [36]. Consequently HA770-ss-PTX was not selected for further evaluation even though it had improved CD44 binding affinity. From design perspective, PTX without a thiol moiety required chemical modification to produce prodrug containing disulfide bond [8]. DPA was utilized as a donor of disulfide bond to modify PTX, due to its high reactivity with the hydroxyl groups. Upon in contact with high concentration of reducing agents in site, disulfide bonding could be readily cleaved to release PTX molecule without compromising drug activity [37]. C-20 of PTX rather than C-70 was modified, since this modification was easy without affecting 70 -OH position. Structures of HA-ss-PTX were confirmed by 1H NMR, 13C NMR and FT-IR spectra. HA-ss-PTX showed prominent aromatic protons at d = 7.28, 7.41, 7.52, and 8.15 ppm corresponding to benzene rings for PTX, glycoside protons at d = 3.28–4.91 ppm corresponding to repeating chain for HA (Fig. 1A). Due to conformational change, it was difficult to detect specific glycoside protons. How-

Fig. 1. Characterization of PTX, HA-ss-PTX or mPEG-ss-PTX. (A) 1H NMR spectra. (B)

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C NMR spectra. (C) FTIR spectra. (D) HPLC analysis.

Please cite this article in press as: S. Yin et al., Intracellular delivery and antitumor effects of a redox-responsive polymeric paclitaxel conjugate based on hyaluronic acid, Acta Biomater. (2015), http://dx.doi.org/10.1016/j.actbio.2015.08.029

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high viscosity, the higher HA molecular weight, the larger the particle size is. HA-ss-PTX micelles were spherical and uniform in size; zeta potential of the tested HA-ss-PTX micelles was approximately 20 mV, providing sufficient repelling force among particles for improved physical stability (Fig. S5D).

ever, HA-ss-PTX conjugates demonstrated methyl peak of acetoamide group for HA at d = 2.09 ppm. Signals in HA-ss-PTX 13C NMR spectrum were assigned as follows (Fig. 1B): 7.2 ppm (C-19, PTX), 43.6 ppm (C-15, PTX), 57.4–59.2 ppm (C-8 and C-30 , PTX), 62.1–79.8 ppm (glycoside of C-2, C-3, C-4, C-5 and C-6, HA), 81.9–84.6 ppm (C-4 and C-5, PTX), 101.8–103.2 ppm (glycoside of C-1, HA), 128.3–137.6 ppm (benzene, PTX), 204.1 ppm (C-9, PTX). As shown in Fig. 1C, HA-ss-PTX polymers showed characteristic bands of HA at 1154, 1087, 1041 and 947 cm1, while bonds at 710 cm1 and 1493 cm1 were associated with substituted benzene of PTX. The results suggested that PTX was indeed grafted to HA chain. Characterization of mPEG-ss-PTX is shown in Supporting Information. As shown in Fig. 1D, PTX and HA-ss-PTX exhibited monodisperse peak at elution time of 8.72 and 2.02 min, respectively. When HA-ss-PTX was incubated with DTT for 2 h, the peak of HA-ss-PTX apparently lowered, a new peak belonged to PTX appeared at elution time of 8.70 min, indicating rapid release of active PTX through cleavage of the disulfide linkage. Conversion for HA-ssPTX conjugate was proposed as Fig. S2 shown, the disulfide bond of the conjugate was cleaved under the function of a reducing agent to generate a sulfhydryl group; the resultant free thiol group would undergo an intramolecular nucleophilic acyl substitution on the ester moiety to release PTX molecule in its original active form [38]. Table 1 lists degree of substitution in prepared conjugates calculated by Ellman’s Method. The results suggested successful synthesis of HA-ss-PTX with no cross-linking reaction during modification of HA with ADH and HA-ADH with PTX-ss-DPA. The highest PTX loading of HA-ss-PTX was 26.7%, measured by UV– Vis spectroscopy, which was considerably higher than that of mPEG-ss-PTX (7.2%), a desirable character in drug loading over PEGylated conjugate. At this loading level, solubility of HA-ssPTX was smaller than 0.1 mg/mL; therefore HA-ss-PTX with 18% drug loading was selected for further experiments. HA-ss-PTX revealed CMC values in the range of 66.7–1.3 mg/L and mPEG-ssPTX showed a CMC value of 20.9 mg/L, which would guarantee that self-assembled micelles retain their original morphology in vivo under highly diluted conditions before reaching targeting site.

3.3. Reduction-triggered disassembly of micelles Disulfide bonds are dynamic and reversible to oxidation and reduction conditions [39]; redox-responsive micelles are hence to undergo shell-shedding, core/membrane aggregation, and rapid drug release in response to a reducing condition [40]. HA-ss-PTX and mPEG-ss-PTX micelles exhibited significant increase in particle size over the time with DTT, which might be caused by cleavage of the disulfide linkage in micelles (Figs. 2A and S6). In comparison particle size of micelles was essentially unchanged without DTT. It was reasonable to predict that the disulfide-containing micelles were responsive to the reducing agent DTT. 3.4. Redox-responsive release of PTX Drug carriers capable of fast releasing payload at focused site tend to produce improved therapeutic efficacy [21]; rearrangement of micelles under tumor-relevant reductive condition in this study was hypothesized to accelerate PTX release in tumor cells. In the presence of 10 mM GSH, substantially higher than GSH concentration in extracellular environment [41], drug release from micelles accelerated obviously. When GSH concentration was increased to 40 mM, comparable to reported intracellular GSH level in tumor cells [42], PTX release was further accelerated, with over 74.9%, 89.5% and 86.2% of PTX released in 96 h for mPEG-ssPTX, HA35-ss-PTX and HA9.5-ss-PTX micelles, respectively (Figs. S7, S8 and 2B). Attributed to specific reticular formation of HA and linear structure of mPEG, HA-ss-PTX was more stable than mPEG-ss-PTX in the absence of GSH, whereas faster PTX release could be achieved within high level GSH. Tumors often possess acidic conditions that could be utilized for facilitating specific drug delivery [43]. Drug release from HA-ss-PTX was higher at pH 5.0 than at pH 7.4, in common with the report that HA would adopt stiff helical configuration with a coil structure of large hydrodynamic volume in physiological solution to form a barrier against rapid drug release [44].

3.2. Preparation and characterization of HA-ss-PTX and mPEG-ss-PTX micelles HA-ss-PTX containing intervening disulfide bonds selfassembled into shell-shedding micelles in aqueous medium (Fig. S3). This was a simple yet effective platform to trigger drug release inside tumor cells. Fig. S4 illustrates the preparation procedures of HA-ss-PTX micelles. Both HA-ss-PTX and mPEG-ss-PTX were able to form micelles with average particle size lower than 200 nm and narrow polydispersity index (Fig. S5A–C). Because of

3.5. Internalization and intracellular drug release from micelles To illustrate the effect of HA shielding on cellular uptake, coumarin-6 (C6) was utilized as a fluorescent probe for confocal laser scanning microscopy, and Figs. 3 and S9 shows the serial images of the test samples. Fluorescence intensity of C6 in cytoplasm increased over the incubation time, and reached the stron-

Table 1 Characteristics of HA-ss-PTX and mPEG-ss-PTX conjugates with different drug loading. Prodruga HA9.5-ss-PTX-1 HA35-ss-PTX-1 HA9.5-ss-PTX-2 HA35-ss-PTX-2 HA9.5-ss-PTX-3 HA35-ss-PTX-3 mPEG-ss-PTX a b c d e f

Feed ratiob 1:1 3:2 4:1 1:1

DSc (%) 18.4 18.2 11.1 10.6 4.2 4.1 –

Solubility Partially Partially Yes Yes Yes Yes Yes

d

PTX loadinge (%)

CMC (mg/L)

Particle size (nm)

PDIf (l2/C2)

26.7 26.4 18.6 18.5 8.8 8.7 7.2

– – 1.3 3.3 21.7 66.7 20.9

– – 183.5 ± 5.1 197.1 ± 5.3 218.3 ± 6.7 226.9 ± 8.4 172.4 ± 5.9

– – 0.129 0.206 0.187 0.211 0.192

Molecular weight of HA in polymers: 9.5 kDa or 35 kDa. Molar ratio of ADH in HA-ADH or mPEG to PTX-ss-DPA. Degree of substitution of –SH calculated by Ellman’s method. Limited solubility; only soluble below 0.1 mg/mL due to high loading of PTX. Drug loading of conjugates calculated by UV–Vis method. Polydispersity index of conjugates.

Please cite this article in press as: S. Yin et al., Intracellular delivery and antitumor effects of a redox-responsive polymeric paclitaxel conjugate based on hyaluronic acid, Acta Biomater. (2015), http://dx.doi.org/10.1016/j.actbio.2015.08.029

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Fig. 2. Characterization of HA9.5-ss-PTX micelles. (A) Time-dependent size changes in the absence or presence of 40 mM DTT in PBS buffer (pH 7.4) at 37 °C determined by DLS measurements. (B) In vitro PTX release in the absence or presence of 2 lM, 10 mM or 40 mM at pH 7.4 or 5.0. Data are presented as mean ± SD (n = 3).

Fig. 3. CLSM images of MCF-7 cells after incubation with C6-labeled HA35-ss-PTX and HA9.5-ss-PTX micelles for 2 h, 4 h and 8 h. Nuclei of cells stained blue by DAPI, green fluorescence from C6-labeled micelles distributed in cytoplasm, and the overlay of two images. Scale bars 30 lm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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gest at 8 h, demonstrating clear time-dependent cellular uptake and effective drug delivery to cytoplasm. Compared to mPEG-ssPTX, HA-ss-PTX displayed stronger fluorescence in tumor cells for the same interval, attributed to HA receptor-mediated endocytosis of the micelles [45,46]. The molecular weight of HA apparently played a crucial role in CD44-mediated cell signaling. HA of HMW was capable of binding to multiple CD44, leading to CD44 inactivation and decrease in cellular uptake [47]. Signals introduced by monovalent binding of LMW HA were different from those by multivalent binding of HMW HA to multiple receptors; for instance, HA containing 6–20 saccharide units could activate cell-signaling pathways and induce expression of various cytokines like TNF-a and IGF-1 [48]. In this study, HA9.5-ss-PTX exhibited higher fluorescence intensity than HA35-ss-PTX, confirmed the above conclusion. It was observed from merged channel that nuclei were surrounded closely by micelles internalized into cells in all test times; this would be beneficial for PTX to act on microtubule system and subsequently to inhibit cell division and proliferation. Flow cytometry was utilized to verify cellular accumulation of the developed micelles in MCF-7 cells, and Fig. S10 shows the results. Cellular uptake of HA9.5-ss-PTX and HA35-ss-PTX micelles amounted to 4.1 and 3.0 times higher than that of mPEG-ss-PTX micelles, demonstrating the role of molecular weight in cellular uptake of HA shielding micelles. Results from FCM assay were consistent with those of CLSM uptake assay, confirming that HA-ssPTX micelles were efficiently internalized in MCF-7 cells. Cellular uptake and intracellular drug release were qualitatively analyzed by CLSM and quantified by FCM to further decipher mechanism of cellular uptake of the designed micelles, following incubation of C6-labeled HA-ss-PTX and mPEG-ss-PTX micelles with HA (10 mg/mL) for 2 h [49]. It was noteworthy that fluorescence intensity of HA9.5-ss-PTX in MCF-7 cells with HA was significantly lower than that without HA polymers (Fig. 4A). However, no fluorescence decrease was observed in mPEG-ss-PTX treated with HA. The fluorescence intensity of HA9.5-ss-PTX in VERO cells was very low. Similar results were also obtained from fluorescence microscopy analysis (Fig. S11). HA binds a domain expressed in all CD44 isoforms and it is located on CD44 most exposed region. Therefore, when free soluble HA was added, it competitively binds to CD44, which simultaneously blocked all the receptors expressed on the cell surface [47]. Indeed, FL1-H value for HA9.5-ss-PTX in MCF-7 cells was 1.9 times of that in HA pretreated MCF-7 cells (Fig. 4B). The presence of free HA significantly reduced the fluorescence of HA9.5-ss-PTX micelles, whereas it exerted no effect on mPEG-ss-PTX micelles, suggesting that CD44-mediated endocytosis pathway was important in efficient intracellular delivery of HA-ss-PTX micelles. FL1-H value for HA9.5-ss-PTX in VERO cells was significantly lower than that in MCF-7 cells, which was consistent with the expression of CD44 in the two cell types. Cellular uptake of copolymer micelles may involve numerous forms of endocytosis. Fig. 5A shows that cell viabilities of CH, NY and CL were 98.9%, 99.4% and 98.7% respectively, indicating that the inhibitors were nontoxic to MCF-7 cells. As shown in Fig. 5B, the inhibition of macropinocytosis pathway did not alter the cellular uptake of HA9.5-ss-PTX. This was consistent with previous conclusion that the macropinocytosis pathway was not closely involved in the internalization of micelles with size about 200 nm [48]. In contrast, inhibitions of caveolae-mediated endocytosis with NY and clathrin-mediated endocytosis with CH reduced the cellular uptake of HA9.5-ss-PTX micelles by 54.6% and 31.2%. Thus caveolae-mediated endocytosis played an important role in the uptake of HA9.5-ss-PTX micelles while a minor fraction of the micelles could be internalized via clathrin-mediated pathway. Accelerated drug release at target site is necessary for the effective inhibition to cancer cell growth. Cellular uptake and intracel-

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Fig. 4. Subcellular distribution and cellular uptake of HA (10 mg/mL, 2 h) pretreated MCF-7 cells or VERO cells incubation with mPEG-ss-PTX or HA9.5-ss-PTX micelles for 4 h. (A) CLSM images. Scale bars 30 lm. (B) Flow cytometry of cellular uptake. (t-test, *p < 0.05 vs. MCF-7 cells without HA pretreated).

Fig. 5. Endocytosis mechanism of HA9.5-ss-PTX micelles. (A) Viability of MCF-7 cells treated with different inhibitors. (B) Effects of inhibitors on endocytosis in MCF-7 cells (ANOVA, *p < 0.01 vs. control, n = 3).

lular drug release of HA9.5-ss-PTX micelles at different GSH levels was monitored by confocal laser scanning microscopy and the changes of intracellular GSH levels. MCF-7 cells were pre-treated with 10 or 40 mM GSH for 2 h to up-regulate the intracellular GSH concentration (Fig. S12). Fluorescence observed in cancer cells heightened as the pre-treated GSH concentration increased, indicating that HA9.5-ss-PTX micelles were capable of efficient cellular uptake and drug release. (Fig. 6A). As shown in Fig. 6B, the intracellular GSH levels of MCF cells pre-treated with 0, 10 and 40 mM GSH were reduced by 61.2%, 55.7% and 41.3% respectively after treated with HA9.5-ss-PTX micelles in comparison to the control, indicating GSH consumption in the degradation of the micelles. These results confirmed that the higher intracellular GSH concentration could promote the degradation of the disulfide bond and accelerate the drug release from HA9.5-ss-PTX micelles.

Fluorescence in liver would gradually decrease, while in tumor site steadily increased over time, cumulating to the strongest 8 h after the injection for HA-ss-PTX micelles (Fig. 7A). In contrast, fluorescence from control group decreased over the time and became negligible 8 h post injection. Compared to mPEG micelles, HA micelles exhibited much higher fluorescence signal in tumor region at all time points, which might be assigned to the mediated effect of HA located in micelle surface. It was apparent that highly permeable vascular structure of neoplasm could lead to passive accumulation of nanosized vehicles by EPR effect [53]. As expected, tumor accumulation of micelle groups was much higher than control group. HA9.5-ss-PTX showed the highest uptake in tumor, 1.38 times higher than HA35-ss-PTX and 2.80 times higher than mPEG-ss-PTX (Fig. 7B and C). This suggested that the tumor targeting efficiency of HA9.5-ss-PTX was superior to that of HA35-ss-PTX and mPEG-ss-PTX.

3.6. In vivo biodistribution and tumor targeting 3.7. In vivo efficacy of redox-sensitive micelles Xenograft tumors in nude mice can form the construction of a capillary network to allow the accumulation of micelles in tumors via EPR effect [50]. Real-time biodistribution and tumor targeting efficiency of micelles were monitored in xenograft nude mice using non-invasive near infrared optical imaging technique [51]. Considerable fluorescence was detected in liver for HA-ss-PTX 0.5 h after intravenous administration, indicating rapid distribution and clearance of some micelles through hepatobiliary excretion [52].

In vivo efficacy was evaluated in Heps tumor bearing mice, using Saline as negative control and Taxol as positive control. Significant delays in tumor growth were observed in all treatment groups compared to the Saline group after the third administration (Fig. 8A). Micelles exhibited improved tumor suppression in comparison to Taxol after 9 days, attributed to EPR effects and redoxresponsive drug release. At the end of the test, HA9.5-ss-PTX

Please cite this article in press as: S. Yin et al., Intracellular delivery and antitumor effects of a redox-responsive polymeric paclitaxel conjugate based on hyaluronic acid, Acta Biomater. (2015), http://dx.doi.org/10.1016/j.actbio.2015.08.029

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Fig. 6. Cellular uptake and drug release property of HA9.5-ss-PTX micelles in cells. (A) CLSM images of the subcellular distribution and cellular uptake in the presence of 0, 10, or 40 mM GSH in MCF-7 cells for 60 min. (B) Intracellular GSH level of MCF-7 cells treated with HA9.5-ss-PTX (ANOVA, *p < 0.01 vs. control, n = 3).

Fig. 7. In vivo disposition of DiR-loaded micelles in tumor-bearing mice. (A) NIRF images of DiR solution and mPEG-ss-PTX, HA35-ss-PTX and HA9.5-ss-PTX micelles at different study intervals. (B) Ex vivo fluorescence images of tissue samples collected 24 h post-injection. (C) Comparison of NIRF uptake in ex vivo tissue samples collected 24 h post-injection. Uptake expressed as photo flux per mm2 of tumor. (ANOVA, *p < 0.05 and **p < 0.01 vs. DiR solution, n = 6).

Please cite this article in press as: S. Yin et al., Intracellular delivery and antitumor effects of a redox-responsive polymeric paclitaxel conjugate based on hyaluronic acid, Acta Biomater. (2015), http://dx.doi.org/10.1016/j.actbio.2015.08.029

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Fig. 8. In vivo antitumor efficacy of test micelles in comparison to Saline (negative control) and Taxol (positive control) in Heps tumor-bearing mice. (A) Tumor growth curves. (B) Photographs of tumors from each treatment group excised on day 30. (C) Body weight change curves. (D) Survival curves. (Injection times are indicated by arrows, mean ± SD, n = 6, ANOVA, *p < 0.05 and **p < 0.01 significant difference from Saline, +p < 0.05 and ++p < 0.01 significant difference from Taxol).

Fig. 9. Pathological examination of various tissue samples from Heps tumor-bearing mice treat with Saline, Taxol, mPEG-ss-PTX, HA35-ss-PTX and HA9.5-ss-PTX micelles collected on day 30 (400). Black arrows indicate examples of positive tumor suppression with H&E staining.

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micelles possessed the smallest average tumor weight and tumor volume (Figs. S13, 8B). Inhibitory rate of mPEG-ss-PTX, HA35-ssPTX and HA9.5-ss-PTX calculated from tumor weight was 51.38 ± 9.34%, 68.66 ± 6.76% and 83.27 ± 5.20%, respectively (Fig. S14), suggesting that HA9.5-ss-PTX improved the antitumor efficacy. Animals in micelles groups did not show weight loss for the duration of the study, while those treated with Saline or Taxol showed body weight loss (Fig. 8C). Mice treated with Saline died in 29 days, 5 of 6 mice in Taxol group died in 30 days. On contrary, no mice died in HA9.5-ss-PTX group in 30 days, and only 1 animal died in HA35-ss-PTX group in 30 days (Fig. 8D). The median survival time of HA micelles exceeded 30 days and was longer than all other study groups, indicating a satisfactory safety profile for HA-based micelles. Pathological changes were evaluated using H&E staining assay to further verify antitumor efficacy and safety of the micelles. Compared to Saline and Taxol, micelle groups did not induce inflammatory response, cell degeneration or necrosis in major organs, suggesting reduced toxicity of PTX from the polymeric micelles (Fig. 9). On the other hand, large amount of severe apoptotic cells, hyperplasia of fibrous tissue, phenomenon of inflammatory, and cell infiltration were observed in tumor tissues from HA9.5-ssPTX group. The results hence suggested that HA-ss-PTX micelles exhibited satisfactory tumor suppression and reduced adverse drug effects, in inconsistent with that HA shielding prolongs the circulation time and actively targeted to CD44-overexpressed tumor sites [54].

4. Conclusions In summary, bioconjugate HA-ss-PTX was designed and tested for its dual functionality in specific tumor targeting and rapid intracellular drug release. Compared to mPEG-ss-PTX, HA-ss-PTX showed better drug loading, cellular internalization, and tumor targeting capability by entering tumor cells via CD44-caveolaemediated endocytosis and rapidly release drug at target in the presence of GSH. The results indicating that HA was applicable to serve as a substitute for PEG in the construction of polymeric conjugates. One of the key issues in clinical oncology is to enhance drug delivery efficacy while minimizing side effects of the anticancer compounds. This study was believed to provide opportunities for further exploration of intelligent drug delivery systems for future clinical applications. Acknowledgments The research was financially supported by Grants from the National Natural Science Foundation of China (No. 81373363), the National Major Scientific and Technological Special Project for ‘‘Significant New Drugs Development” during the Twelfth Five-year Plan Period (2015ZX09501001), the Fundamental Research Funds for the Central Universities (No. PT2014YX0085), the Huahai Pharmaceutical Postgraduate Innovation Fund (No. CX14B-001HH), and Advantages of Disciplines in Colleges and Universities in Jiangsu Province Construction Grant Program.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.actbio.2015.08. 029.

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