Reduction sensitive hyaluronan-SS-poly(ε-caprolactone) block copolymers as theranostic nanocarriers for tumor diagnosis and treatment

Materials Science & Engineering C 98 (2019) 9–18

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Reduction sensitive hyaluronan-SS-poly(ε-caprolactone) block copolymers as theranostic nanocarriers for tumor diagnosis and treatment

T

Huikang Yanga,b,1, Nianhua Wanga,1, Lei Moa,b,1, Mei Wua,b, Ruimeng Yanga,b, Xiangdong Xua,b, ⁎ ⁎⁎ Yugang Huangc, Jiantao Lind, Li-Ming Zhange, , Xinqing Jianga,b, a

Department of Radiology, Guangzhou First People's Hospital, Guangzhou Medical University, Guangzhou 510180, China Department of Radiology, Guangzhou First People's Hospital, School of Medicine, South China University of Technology, Guangzhou, Guangdong 510640, China c School of Pharmaceutical Sciences, Guangzhou Medical University, Guangzhou 511436, China d Dongguan Scientific Research Center, Guangdong Medical University, Dongguan 523808, China e School of Materials Science and Engineering, Sun Yat-sen University, Guangzhou 510275, China b

A R T I C LE I N FO

A B S T R A C T

Keywords: Reduction-sensitive Hyaluronan Theranostic nanocarriers Drug delivery SPIO MR imaging

Tumor-targeted multifunctional nanocarriers play an important role in tumor diagnosis and treatment. Herein, disulfide bonds linked amphiphilic hyaluronan-SS-poly(ε-caprolactone) diblock copolymers (HA-SS-PCL) were synthesized and studied as theranostic nanocarriers for tumor diagnosis and treatment. The chemical structure of HA-SS-PCL was confirmed by Fourier transform infrared spectroscopy (FTIR) and proton nuclear magnetic resonance (1H NMR). The self-assembling behavior of the HA-SS-PCL into GSH-responsive micelles and their degradation were characterized by fluorescence spectroscopy, dynamic light scattering (DLS) and transmission electron microscopy (TEM). Theranostic nanocarriers encapsulating doxorubicin (DOX) and superparamagnetic iron oxide (SPIO) were formed via a dialysis. In vitro drug release results suggested that the HA-SS-PCL micelles possessed reductant-triggered doxorubicin release ability, which was confirmed by 100% of DOX release from HA-SS-PCL micelles within 12 h under 10 mM of glutathione (GSH), whereas about 40% of DOX was released under non-reductive condition within 24 h. Both flow cytometry and confocal laser scanning microscopy (CLSM) analysis revealed that the HA-SS-PCL micelles loaded with DOX were internalized in HepG2 cell via a receptor mediated mechanism between hyaluronan and the CD44 receptor. Furthermore, the MTT assay and cell apoptosis analysis revealed that the DOX-loaded HA-SS-PCL micelles exhibited pronounced antitumor ability towards HepG2 cells compared with that of the reduction-insensitive HA-PCL micelles at the same DOX dosage. The r2 relaxivity value of the DOX/SPIO loaded HA-SS-PCL micelles was up to 221.2 mM−1 s−1 (Fe). Thus, the obtained HA-SS-PCL block copolymers demonstrate promising potential as tumor targeting theranostic nanocarriers in the field of tumor diagnosis and treatment.

1. Introduction Chemotherapy is a common approach for treatment of various types of solid tumors. Significant challenges in chemotherapy include the poor water solubility, lack of tumor selectivity and high toxicities of drugs towards healthy cells [1]. Thus, many drug delivery systems, such as liposomes, polymeric micelles and hybrid nanoparticles have been designed to overcome these drawbacks. Among which, self-assembled polymeric micelles obtained from amphiphilic copolymers exhibited several unique features, including prolonged drug blood circulation times, favorable bio-distributions, enhanced therapeutic effects and

reduced systemic side effects [2,3]. Hyaluronan (HA) is a natural hydrophilic polysaccharide that not only displays biodegradability, biocompatibility and non-immunogenicity but also is a major ligand for the adhesion receptor of CD44, which is over-expressed on the surface of many tumor cells. Ligand-receptor interaction between HA and CD44 have been explored for targeting delivery of anticancer drugs to CD44-overexpressing tumor cells. For example, self-assembled polymersomes based on hyaluronan-b-poly(γ-benzyl glutamate) block copolymers shown pronounced antitumor ability against MCF-7 cells and C6 glioma cells overexpressing CD44 glycoprotein in their cells surface after loading DOX

⁎

Corresponding author. Correspondence to: X. Jiang, Department of Radiology, Guangzhou First People's Hospital, Guangzhou Medical University, Guangzhou 510180, China. E-mail addresses: [email protected] (Y. Huang), [email protected] (L.-M. Zhang), [email protected] (X. Jiang). 1 These authors contributed equally to this work. ⁎⁎

https://doi.org/10.1016/j.msec.2018.12.132 Received 27 February 2018; Received in revised form 6 December 2018; Accepted 28 December 2018 Available online 29 December 2018 0928-4931/ © 2018 Published by Elsevier B.V.

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2. Materials and methods

[4,5]. Furthermore, amphiphilic diblock copolymers of hyaluronan-bpoly(lactic acid) and hyaluronan-b-poly(ε-caprolactone) were also prepared and employed as nanocarriers for drug and dye delivery [6,7]. To overcome the inadequate drug release in tumor cells, various stimuli-responsive drug-delivery systems were developed to exhibit fast drug release behaviors at the target sites at a high level, such as systems that could respond to changes in temperature, pH, reducing agents and enzymes [8–10]. As a biological reducing agent, glutathione (GSH) is capable to clave the disulfide bonds and lead to the deformation of disulfide-based micelles. It is reported that GSH concentration in the tumor tissues is at least 4-fold higher compared to normal tissues. Therefore, disulfide linked reduction-sensitive polymeric micelles have received considerable attention as drug delivery systems because they can maintain high plasma stability during blood circulation and rapidly release their therapeutic payload upon the reduction responsive intracellular microenvironments in the tumor cells, which could not only significantly enhance inhibition efficacy but also reduce systemic toxicity [11]. One such example is bioreducible shell cross-linked HAPCL nanoparticles have been developed as high performance drug delivery system to improve antitumor efficacy [12]. Tumor targeted disulfide bonds linked HA/Camptothecin micellar prodrug HA-g-SS-CPT has also been employed to treat tumor. The results showed that the micellar prodrug improved the tumor accumulation of CPT and led to strong inhibition of tumor growth and metastasis in the lungs and liver [13]. To obtain real-time information on the drug bio-distributions, quantitative determination of drug intracellular uptake and tumor treatment effects, some imaging molecules and nanoparticles were introduced into stimuli-responsive carriers, including fluorescent probe magnetic nanoparticles, quantum dots and so on [14]. Magnetic resonance imaging (MRI) is a powerful diagnostic tool, which is capable to determine drug bio-distribution and quantify the drug release in deep tissues through a non-invasive manner [15,16]. For example, Daniela et al. reported that SPIO loaded hyaluronan polymeric micelles not only exhibited selective cytotoxicity towards colon adenocarcinoma but also accumulated in vivo in tumors [17]. Doxorubicin-hyaluronan conjugated super-paramagnetic iron oxide nanoparticles were developed as tumor-targeting multifunctional theranostic nanocarriers for breast cancer chemotherapy and diagnosis [18]. Fu et al. developed pH-responsive theranostic nanocarriers loaded with SPIO and DOX for MRI diagnosis and chemotherapy of hepatocellular carcinoma [19]. Hence, stimuli-responsive nanocarriers based on HA derivatives have great potential as theranostic platforms for drug delivery and MRI. In this work, we reported on multifunctional theranostic nanoparticles self-assembled from HA-SS-PCL block copolymers for efficient hepatoma-targeting DOX delivery and MRI contrast enhancement agents. On one hand, theranostic nanoparticles bearing a HA shell exhibit a high affinity to CD44 rector, which over-expressed on the surface of many tumor cells, and lead to high levels of cellular drug accumulation. On the other hand, disulfide bond linked HA-SS-PCL nanoparticles possessed reductant-triggered doxorubicin release ability under high GSH level. Furthermore, DOX and SPIO were encapsulated into the core of the micelles thereby making targeted cancer diagnosis and treatment possible. The self-assembly behavior, drug and SPIO loading capacity, reduce-responsiveness of the HA-SS-PCL micelles were fully studied. The intracellular uptake and 110 internalization of the micelles in HepG2 cells were confirmed using flow cytometry, confocal laser scanning microscopy (CLSM) and Prussian blue stain. In addition, in vitro cell cytotoxicity the, apoptosis and MR relaxivity were also studied. The obtained HA-SS-PCL polymers show promising potential as a multifunctional tumor-targeting nanocarrier that combines MRI and drug delivery functions.

2.1. Materials Hyaluronan acid (HA) with a molecular weight of 8.5 kDa, was obtained from the C.P. Freda Pharmaceutical Co. Ltd. (Shangdong, China) as sodium salts. ε-Caprolactone was purchased from Alfa Aesar and was stirred overnight over CaH2, follow by distillation prior to use. Tetrahydrofuran (THF, 99.5%) and toluene (99.5%) were refluxed and distilled over sodium benzophenone until a purple color was obtained. Copper sulfate (CuSO4·5H2O), tin (II) 2-ethylhexanoate (Sn(Oct)2, 95%), sodium ascorbate (99%) and 1-dodecanol were purchased from Alfa Aesar. Doxorubicin hydrochloride (DOX·HCl), triethylamine (TEA), glutathione (GSH), sodium azide (NaN3, 99%) and cystamine dihydrochloride were all purchased from Aladdin Chemical Company and used as received. Fetal bovine serum (FBS), trypsin-ethylenediaminetetraacetic acid (Trypsin-EDTA) and Dulbecco's modified Eagle medium (DMEM) were purchased from Gibco-BRL (Canada). 3-(4, 5Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) was purchased from Invitrogen Corporation (Washington, USA). 4′,6Diamidino-2-phenylindole (DAPI) was purchased from the Beyotime Institute of Biotechnology (China). All other reagents were of analytical grade and used without further purification. The human hepatocellular liver carcinoma (HepG2) cell line was purchased from the Animal Centre of the Sun Yat-sen University (Guangzhou, China). 2.2. Synthesis of disulfide-containing α-alkyne-PCL via ring-opening polymerization Disulfide-containing α-alkyne-PCL (α-alkyne-SS-PCL) was prepared via the ring opening polymerization (ROP) of ε-CL using (propargyl carbamate) ethyl dithio ethylamine (PPA-Cyst) as the initiator and Sn (Oct)2 as the catalyst (Scheme 1A) as reported by Hazer [20]. Typically, under an argon atmosphere, (propargyl carbamate) ethyl dithio ethylamine (PPA-Cyst), (0.028 g, 0.15 mmol), Sn(Oct)2 (15 mg, 3 × 10−2 mmol), ε-CL (1.28 g, 11.5 mmol), and 5.0 mL of dry toluene were added into a round-bottom flask. The glass flask was sealed and placed in an oil bath at 110 °C. After 24 h, the reaction was terminated by rapidly cooling the glass flask to room temperature. The crude product was precipitated in an excess of cold methanol. The α-alkyneSS-PCL was filtered off and dried in vacuum (1.24 g, yield: 95.2%, Mn,GPC = 6.4 kDa, PDI = 1.26). 1H NMR (500.10 MHz, CDCl3, 298 K), δ, ppm: 4.69 (HC^CCH2Oe), 4.12–4.01 (eCOOCH2CH2e), 3.64 (eNHCH2CH2e), 2.81–2.79 (eCH2SSCH2e), 2.36–2.24 (eCH2CH2COOe) and 1.73–1.58 (eCOOCH2CH2CH2CH2CH2e) and 1.41–1.32 (eCOOCH2CH2CH2CH2CH2e) of the PCL chain. FTIR (cm−1): 2950, 1733, 1654, 1563, 1454. According to the 1H NMR analysis, the degree of polymerization (DP) of the obtained α-alkyne-SS-PCL was approximately 70. Therefore, the polymer was denoted as α-alkyne-SS-PCL70. Furthermore, α-alkyne-SS-PCL90 (yield: 96%; Mn,GPC = 8.7 kDa; PDI = 1.40) and α-alkyne-SS-PCL140 (yield: 98%; Mn,GPC = 16.3 kDa; PDI = 1.50) were also synthesized. 2.3. Preparation of α-azido-HA by reductive amination 1-Azido-3-aminopropane was synthesized according to a previous report. Briefly, 3-chloropropylamine hydrochloride (8.0 g, 61.0 mmol), sodium azide (12 g, 183.0 mmol, 3 equiv) and water (60 mL) were added into a round bottom flask (100 mL) and then heated at 80 °C for 24 h. The solution was concentrated and alkalized with KOH (8.0 g) in order to extract the organic phase with dichloromethane (50 mL, three times). The organic phase was separated, combined, and then dried with MgSO4. After the organic solution was removed an oil liquid was obtained and further purified by reduced pressure distillation. Yield: 4 g (65%). 1H NMR (500.10 MHz, CDCl3, 298 K), δ, ppm: 1.10 (eNH2), 1.71 (eCH2eCH2eCH2N3), 2.77 (eCH2eNH2), 3.33 (eCH2eN3). 10

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Scheme 1. Synthesis of HA-SS-PCL block copolymers.

nanoparticles were precipitated in excess diethyl ether and separated as a dry product. The dry magnetic particles were suspended in an anhydrous DMSO (5 mL) and stored at room temperature for further use.

Reductive amination between HA and 1-azido-3-aminopropane with sodium cyanoborohydride (NaBH3CN) as the reducing agent was carried out to introduce an azido group at the end of HA, as shown in Scheme 1B. In detail, sodium hyaluronan (2 g, 0.10 mmol) was solubilized in 20 mL of acetate buffer (pH 5.6) and 0.55 g of 1-azido-3aminopropane (100 equiv) was added under magnetic stirring. Then, 0.64 g of NaCNBH3 (100 equiv) was added, and 20 mg of NaCNBH3 was added every day. The reaction lasted for one week at 50 °C. The reaction mixtures were dialyzed against deionized water (MWCO = 1000 Da) for 3 days. The dialysate was lyophilized, and thus, a white powder (1.74 g, 87%) was obtained.

2.6. Characterization FTIR spectra were obtained from a Perkin-Elmer Paragon1000 spectrometer using the KBr disk method in the transmission mode. 1H NMR spectra and 2D DOSY NMR experiments were recorded on a Bruker spectrometer at 500 NMR. The 1H NMR spectra of α-alkyne-SSPCL were measured in deuterated chloroform (CDCl3-d) or DMSO‑d6. The chemical structure of HA-SS-PCL was obtained by dissolving HASS-PCL into deuterated dimethyl sulfoxide (DMSO‑d6). A BI200SMGoniometer particle size analyzer from Brookhaven Instruments Corporation was applied to measure the size and size distribution of the HA-SS-PCL micelles. A Waters 1515 gel permeation chromatograph (GPC) was used to determine the number average molecular weight (Mn) and polydispersity index (Mw/Mn) of the PCL. TEM images of the HA-SS-PCL micelles were recorded by transmission electron microscopy (JEM2010) operated at 80 kV.

2.4. Synthesis of the HA-SS-PCL As illustrated in Scheme 1C, α-alkyne-SS-PCL70 (0.50 g, 78 μmol, 1 equiv), α-azido-HA (1.32 g, 156 μmol, 2 equiv) and 30 mL of anhydrous DMSO were charged into a round-bottom flask under an argon atmosphere. After that, CuSO4·5H2O (15 mg) and sodium ascorbate (30 mg) were then added and further bubbled with argon for approximately 15 min. The flask was sealed and placed in an oil bath at 50 °C for 3 days. The crude reaction solution was dialyzed (MWCO = 50,000 Da) against water containing 5% edetate disodium (EDTA-2Na) and pure water for 3 days to remove the excess HA-N3. The dialysate was collected and lyophilized, and a white solid powder (1.04 g, 92%) was finally obtained (Scheme 2).

2.7. Micelle formation and the critical micellar concentration The HA-SS-PCL micelles were prepared by dissolving 10 mg of HASS-PCL in 2.0 mL of DMSO at 50 °C. The solution was added dropwise into 5 mL of deionized water under stirring. DMSO was removed by extensive dialyzed against deionized water (MWCO = 6000 Da) for 2 days. The critical micelle concentration (CMC) of HA-SS-PCL was determined by the fluorescence probe technique using pyrene as a fluorescence probe on a fluorescence spectrometer (RF-5301PC Shimadzu). In brief, aliquots of pyrene stock solution (6 × 10−5 M in acetone, 50 μL) were added to 10 mL volumetric flasks, and acetone was allowed to evaporate. Then block copolymer solutions in the range from 1.0 to 1.0 × 10−4 mg/mL were added to the vials, respectively, and the final concentration of pyrene was 6 × 10−7 M in water. The combined solutions of pyrene and block copolymer were kept on a shaker at 37 °C to reach the solubilization equilibrium in dark for 24 h before measurement. The excitation spectra of block copolymer/pyrene solutions were scanned from 300 to 350 nm at room temperature, with an emission wavelength of 373 nm and a bandwidth of 5 nm. The intensity ratios of I337 to I335 were plotted as a function of logarithm of block

2.5. Preparation of hydrophobic SPIO nanoparticles Hydrophobic SPIO NPs were synthesized following a previously described procedure. In brief, iron (III) acetylacetonate (1 g) was dissolved in benzyl alcohol (15 mL), and the reaction mixture was heated to reflux (200 °C) for 7 h under a flow of argon. Afterwards, the mixture was cooled to room temperature and poured into cooled ethanol, and the precipitate was collected via centrifugation. Fe3O4 nanoparticles were redispersed into chloroform, and then, APTS (50 μL) was added and kept stirring for 1 h. Excess APTES was removed by pouring the mixture into the cooled methanol, and then, amino-functionalised magnetic nanoparticles (MNP-APTS) were collected and redispersed in anhydrous chloroform. The MNP-APTS suspension (5 mL, 15 mg/mL) and Lys (Z)-NCA (200 mg) were added into a Schlenk tube, and stirred for 72 h under an argon atmosphere at room temperature. The magnetic 11

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Scheme 2. Preparation of theranostic nanocarriers for chemotherapy and MR imaging.

The cells were then washed with PBS 3 times, and then, each well was combined with 20 μL of MTT solution in PBS (5 mg/mL) and incubated for another 4 h at 37 °C. The medium was removed completely, and the formazan crystals were dissolved in 150 μL DMSO. Finally, the absorbency of the resulting solution at 490 nm was measured by a microplate reader. For apoptosis assay, HepG2 cells were seeded in 6-well plates at a density of 1 × 106 cells per well and treated with free DOX or DOX/ SPIO-loaded micelles (10 μg/mL DOX) at 37 °C for 24 h. After that, the cells were trypsinized, collected and resuspended in 200 μL binding buffer. The dead or apoptosis cells were stained by adding 5 μL DAPI solutions and 5 μL Annexin V-fluorescein isothiocyanate (FITC) into the cell suspension, following by incubation for 15 min at room temperature. The cell apoptosis rates were evaluated using a flow cytometer.

copolymer concentrations. The CMC value was taken from the intersection of the tangent to the curve at the inflection with the horizontal tangent through the points at low concentrations. 2.8. Preparation of DOX and SPIO co-loaded micelles Theranostic nanocarriers loaded with DOX and SPIO were obtained via dialysis method. 10 mg HA-SS-PCL, hydrophobic SPIO (1.5 mg), DOX·HCl (2.0 mg) and TEA (0.4 mg) were dissolved in 2.0 mL warm DMSO. The above solution was added dropwise into 5 mL of deionized water under moderate stirring. Afterwards, the mixed solution was dialyzed against deionized water (MWCO = 6000 Da) for 3 days to remove the DMSO and un-encapsulated DOX and SPIO at room temperature. DOX amount in the micelles was determined by a Shimadzu UV-3150 UV–vis spectrometer at 485 nm. The DOX loading content was calculated according to the following equation:

weight of loaded drug DLC (%) = × 100% weight of drug loaded micelles

2.11. Cellular uptake studies The cellular uptakes of DOX and SPIO were revealed by using flow cytometry, confocal laser scanning microscopy (CLSM) and Prussian blue staining. For flow cytometric analysis, HepG2 cells were seeded in 6-well plates at a density of 1 × 106 cells per well at 37 °C with a 5% CO2 atmosphere overnight. Then, the culture media was removed followed by washing with PBS twice. HepG2 cells were incubated with fresh culture media containing free DOX or DOX/SPIO-loaded micelles (DOX concentration: 10 mg/mL) at 37 °C for 4 h. Cells cultured without any DOX were used as a control. After 4 h, the culture medium was removed and washed with PBS thrice. After that, the cells were trypsinized, collected and resuspended in 0.3 mL PBS and analyzed by flow cytometry. Cellular uptake of DOX was revealed by confocal laser scanning microscopy. HepG2 cells were seeded on microscope slides in a 6-well plate at a density of 1 × 106 cells per well. After 24 h of incubation, the cells were cultured with free DOX or DOX/SPIO loaded micelles (equivalent DOX concentration: 10 μg/mL) for another 4 h. Then, the cells were washed three times with PBS and fixed with 4% formaldehyde. After that, the cell nuclei were stained with DAPI and washed with cold PBS to remove the excess DAPI. Finally, a Leica, TCSSP2 confocal laser scanning microscope was applied to observe the cells. Cellular uptake of SPIO was revealed by Prussian blue staining. HepG2 cells (1 × 106) were seeded on 6-well plates and incubated with SPIO-loaded micelles in culture media at 37 °C for an additional 4 h. Afterwards, the cells were washed three times with PBS and fixed with 4% formaldehyde for 10 min. Then, the cells were stained with 2 mL Prussian blue solution for 30 min. After washing with PBS, the cells were dyed with nuclear fast red for 5 min. An Olympus BX51 optical

(1)

SPIO amount in the micelles was determined from Atomic absorption spectrophotometer (AAS) at 248.3 nm which belongs to the specific absorption wavelength of Fe. The SPIO loading content was calculated according to the following equation:

SLC (%) =

weight of loaded SPIO × 100% weight of SPIO loaded micelles

(2)

2.9. In vitro DOX release In vitro DOX release was carried out via a dialysis method. In brief, 3.0 mL DOX-loaded micelles (1.0 mg/mL) was sealed in a dialysis bag (MWCO = 6000 Da) and incubated in 27 mL PBS containing 0 or 10 mM GSH at 37 °C. At predetermined time intervals, 3.0 mL release medium was withdrawn and replenished with 3.0 mL fresh medium. The DOX concentration was determined using a Shimadzu UV-3150 UV–vis spectrometer at 485 nm. 2.10. Cell cytotoxicity assays and apoptosis The cytotoxicities of free DOX and DOX-loaded micelles towards HepG2 cells were revealed by MTT assays. HepG2 cells were seeded at a density of 1 × 104 cells per well in a 96-well plate with 200 μL of DMEM for 24 h. The culture media was removed, and the cells were washed with PBS two times. HepG2 cells were incubated for another 48 h with 200 μL culture media containing free DOX or DOX/SPIOloaded micelles at different DOX concentrations (0–10 μg/mL DOX). 12

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shows the FTIR spectrum of HA-N3, where the typical vibration absorption peak of the azide group was seen at 2106 cm−1. As shown in Fig. 1c, after the click reaction, the FTIR spectrum of the resulting HASS-PCL copolymer did not show the characteristic peak of the azide group at 2106 cm−1, which implied that the excess HA-N3 was completely removed. Moreover, the 1H NMR spectrum of the HA-SS-PCL90 is given in Fig. 1, where the proton assignments attributed to the PCL block and the HA block are also made. The proton signals belonging to the HA block were found at 2.90–4.77 ppm, but methylene proton signals of the PCL block were found at 4.12–4.01, 2.36–2.24, 1.73–1.58 ppm and 1.41–1.32 ppm, respectively. Especially, a new proton signal at 7.46 ppm showed the existence of the resulting structure of a triazole ring after the click reaction. Fig. S6 shows the DOSY NMR spectrum of HA-SS-PCL recorded in DMSO. Similar diffusion patterns for HA and PCL blocks were observed which confirmed the formation of HA-SS-PCL blocks copolymers [21].

microscope was operated to record the Prussian blue stained images. 2.12. Relaxivity measurement For SPIO loaded HA-SS-PCL micelles, T2 relaxivities were measured with a Siemens 3.0 T clinical MRI scanner. Transverse relaxation times were acquired using the following parameters: TR, 1000 ms; TE, 13.8/ 27.6/41.4/55.2/69.0 ms; flip angle, 180°; slice thickness, 3.0 mm; and matrix, 444 × 448. The relaxivity value r2 was calculated through the linear fitting of the relaxation rate (s−1) vs iron concentration (mM). 3. Results and discussion 3.1. Preparation and characterization of HA-SS-PCL As shown in Scheme 1, the HA-SS-PCL was synthesized via a click conjugation method between α-alkyne-SS-PCL and α-azido-HA. First, αalkyne-SS-PCL was prepared via the ROP of ε-CL initiated by PPA-Cyst. In this work, three α-alkyne-SS-PCLs with difference molecular weights were synthesized. The chemical structures of α-alkyne-SS-PCLs were characterized by 1H NMR. The proton signals belonging to the PCL block and the initiator are all assigned in Fig. S1. In addition, the degree of polymerization (DP) of the α-alkyne-SS-PCLs was calculated by comparing the integration area attributed to protons of the PCL at 4.12–4.01 ppm to that of the initiator at 4.69 ppm. The obtained DP values of the PCL were 70, 90 and 140, respectively. GPC analysis was also applied to reveal the molecular weight and PDI. As shown in Fig. S2, the GPC curves showed a mono-modal and symmetric elution peaks for all of the PCLs with PDI values of 1.20, 1.40 and 1.50, respectively. Second, the azido-terminated HA (HA-N3) was synthesized following the procedure reported by Lecommandoux et al. with a reductive amination reaction, which is a versatile method to introduce functional groups to the reducing end of polysaccharides [4]. The targeted di-block polymers were then synthesized by the click cycloaddition between the HA-N3 and the α-alkyne-SS-PCL in DMSO. Fig. S3

3.2. Preparation and characterization of HA-SS-PCL micelles The chemical composition of HA-SS-PCL copolymers includes a hydrophilic HA and hydrophobic PCL, and thus, HA-SS-PCL can selfassemble into micelles in water. The critical micelle concentration (CMC) is an important index for micellar stability. In our work, the CMC values of the HA-SS-PCL copolymers were measured through a pyrene fluorescence probe method [22]. Fig. 2A shows the relationship of the fluorescence intensity ratios (I337/I335) with the HA-SS-PCL90 concentration at room temperature. Obviously, it was found that the fluorescence intensity ratio (I337/I335) stayed stable at the low concentrations, while it gradually increased with an increase in the copolymer concentration. Pyrene aggregates into the hydrophobic core of the micelles via hydrophobic interactions, leading to the fast increase of the fluorescence intensity ratio [23]. The CMC values were measured by the interception of two straight lines. The CMC values for all the HA-SSPCLs are listed in Table 1, where the HA-SS-PCL with longer PCL blocks had smaller CMC values. The low CMC values demonstrated that HA-

Fig. 1. 1H NMR spectrum of the HA-SS-PCL90 in d-DMSO. 13

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(E)

Fig. 2. (A) Plot of fluorescence intensity ratio of I337/I335 versus HA-SS-PCL90 concentration, (B) size distribution of HA-SS-PCL90 micelles determined by DLS, (C) plot of decay frequency, Γ, versus the square of the scattering vector q2 for HA-SS-PCL90 micelles at the test angles ranging from 40 to 130°, (D) morphology of blank HA-SS-PCL90 micelles revealed by TEM; (E) optical photographs for (a) HA-PCL and (b) HA-SS-PCL90 micelles respond to GSH (10 mM).

same time, because Fig. 2D confirmed the well-defined spherical-like structures with a size of approximately 100 nm. Disulfide bonds linked block copolymer micelles can act as smart drug delivery systems, due to their ability to maintain stable in the blood circulation but cleave under the intracellular reductive environment, thus, leading to faster drug release. Therefore, the reduction-responsive behavior of the micelles was studied under reductive conditions mimicking the intracellular environment. As shown in Fig. 2E, both the HA-PCL and the HA-SS-PCL micellar solutions showed a typical Tyndall phenomenon (laser beam passes through the solution) under non-reductive conditions. The reduction-insensitive HA-PCL micellar solution still showed the Tyndall phenomenon after adding 10 mM GSH. However, the HA-SS-PCL micellar solution became opaque

SS-PCL could form aggregates at lower concentrations in aqueous medium better than lower molecular weight surfactants. The average size is an important parameter of nanoparticles in aqueous solution used for drug delivery. According to the DLS results in Table 1 and Fig. 2B, the hydrodynamic diameters of the HA-SS-PCL micelles were dependant on the length of the PCL block. The average diameter increased from 83 to 193 nm when the DP was increased from 70 to 140. The morphology of the self-assembled micelles could be estimated from multi-angle DLS measurement from 40° to 130°, as shown in Fig. 2C. A linear variation between the relaxation frequency (Γ) and squared scattering vector q2 was passed through the origin, indicating that HA-SS-PCL90 self-assembled into spherical micelles [24]. TEM images of the HA-SS-PCL90 micelles supported this result at the

Table 1 Characteristics of blank micelles and DOX/SPIO loaded micelles.a Sample code

HA-SS-PCL70 HA-SS-PCL90 HA-SS-PCL140 a b

Blank micelles

DOX/SPIO-loaded micelles

Diameter (nm)a

PDIa

CMC (mg/mL)b

Diameter (nm)a

PDIa

DLC (%)

SLC (%)

83.5 ± 2.59 126.9 ± 2.68 192.5 ± 5.71

0.23 ± 0.03 0.15 ± 0.02 0.10 ± 0.06

0.012 0.0088 0.0033

120.8 ± 1.7 150.1 ± 2.6 236.4 ± 3.5

0.12 ± 0.01 0.15 ± 0.03 0.16 ± 0.05

12.1 11.3 12.5

11.4 12.3 11.8

Determined by dynamic light scattering (DLS); micelles concentration were 1.00 mg/mL. Obtained from pyrene-based fluorescent spectrometry. 14

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Free DOX HA-SS-PCL70

80

Cell viability(%)

HA-PCL 60 40 20 0

0.1

0.25

0.5

1.0

2.5

5.0

DOXconcentration(μg/mL)

10

Fig. 4. Viability of HepG2 cells incubated with free DOX, DOX/SPIO-loaded HA-SS-PCL70 and DOX/SPIO-loaded HA-PCL micelles at different DOX concentrations after 48 h.

Fig. 3. In vitro DOX release profiles from reduction-sensitive HA-SS-PCL micelles under various GSH concentrations at 37 °C.

Cellular uptake and intracellular release of DOX in the HepG2 cells were further studied by confocal laser scanning microscopy (CLSM, Fig. 6). Strong DOX fluorescence was observed inside the cells after incubation with free DOX and DOX/SPIO-loaded micelles for 4 h, which indicated that the DOX was transported into the cells. HepG2 cells treated with free DOX exhibited stronger intracellular fluorescence than the cells incubated with DOX/SPIO loaded micelles. Moreover, free DOX was mainly located in the cell nucleus, while DOX delivered by HA-based micelles was detected in cytoplasm and nucleus. This might be explained by the face that free DOX diffused easily into cells, while the DOX/SPIO-loaded HA-SS-PCL and the HA-PCL micelles were internalized in HepG2 cell via a receptor mediated mechanism between HA and CD44 [4,29]. Notably, HepG2 cells treated with reductionsensitive theranostic micelles shown stronger DOX fluorescence intensity than non-reduction-sensitive control groups possibly due to that GSH-triggered releases from disulfide bonds linked theranostic micelles. Quantitative determination the intracellular uptake of DOX was further conducted using flow cytometric analysis. As shown in Fig. 7A, HepG2 cells treated with the free DOX showed the highest fluorescence among these three systems. The flow cytometric analysis was consistent with the CLSM observations. Moreover, DOX/SPIO loaded HA-SS-PCL micelles shown higher DOX fluorescence than the DOX/SPIO loaded HA-PCL micelles, a result of GSH accelerated DOX release from HA-SSPCL micelles owing to disulfide cleavage [30]. Hence, HA-SS-PCL micelles could be utilized as a smart drug delivery carrier that can release rapidly the payload under intracellular reductive microenvironment. Cellular uptake of SPIO-loaded HA-SS-PCL micelles was evaluated by Prussian blue staining, as shown in Fig. 7B and C. HepG2 cells incubated with SPIO-loaded HA-SS-PCL micelles showed remarkable blue spots inside the cells (Fig. 7C). Meanwhile, no obvious blue stains were observed in the control group (Fig. 7B). In this Prussian blue staining, the presence of ferric ions led to a characteristic blue colors [31]. These results confirmed that SPIO was efficient on the uptake within HepG2 cells.

under the same reductive environment, indicating that larger hydrophobic aggregates appeared, and thus the presence of GSH resulted in the detachment of the disulfide bonds in HA-SS-PCL. Our results were the same as other previous reports [10]. 3.3. Reduction triggered drug release Herein, reduction-sensitive nanocarriers prepared from the HA-SSPCL block copolymers were developed for drug delivery. In this work, doxorubicin (DOX) and SPIO were co-loaded into the micelles by a dialysis method. DOX loading contents (DLC %) and SPIO loading contents (SLC %) are listed in the Table 1, where both the DOX and the SPIO had a loading efficiency above 10%. The in vitro DOX release experiments were performed in the presence or absence of 10 mM of GSH in PBS at a pH of 7.4, which are displayed in Fig. 3. Drug release results revealed that the HA-SS-PCL micelles possessed a reductanttriggered DOX release ability, which was confirmed by the 100% DOX release from HA-SS-PCL micelles within 12 h under 10 mM of GSH, whereas approximately 40% of DOX was released under a non-reductive condition within 24. The faster DOX releases was likely due to the degradation of the disulfide linkages in micelles [25,26]. 3.4. Cytotoxicity and cellular uptake in vitro The in vitro cell cytotoxicity for free DOX, DOX/SPIO-loaded HA-SSPCL70 and DOX/SPIO-loaded HA-PCL micelles towards HepG2 cells with various DOX concentrations were evaluated using MTT assay (shown in Fig. 4). Compared with the DOX/SPIO-loaded HA-SS-PCL70 and HA-PCL micelles, free DOX exhibited the highest cytotoxicity towards HepG2 cells at equal DOX dosages. It was reported that the free DOX diffused into the cells, but the DOX-loaded micelles entered the cells via a receptor-mediated mechanism [27]. Notably, the disulfide bonds linked SPIO/DOX-loaded micelles of HA-SS-PCL showed a higher antitumor efficacy than the DOX/SPIO-loaded HA-PCL micelles, which is likely due to the degradation of disulfide bonds under a high GSH concentration, which leads to a faster DOX release [28]. The apoptotic activities of free DOX, DOX/SPIO-loaded HA-SSPCL70 and DOX/SPIO-loaded HA-PCL towards HepG2 cells were investigated by flow cytometry at a DOX dosage of 10 μg/mL (Fig. 5). Due to the different cellular uptake mechanisms, the free DOX group induced 37.57% cell apoptosis which was higher than that of the DOX/ SPIO-loaded HA-SS-PCL70 (28.88%) and the DOX/SPIO-loaded HA-PCL micelles (22.81%). The higher apoptotic activity of the DOX/SPIOloaded HA-SS-PCL70 micelles than that of DOX-loaded HA-PCL micelle was probably caused by the faster reduction-triggered DOX release.

3.5. MRI contrast measurement As a widely used T2 negative contrast agent, SPIO nanoparticles have the ability to shorten the transverse relaxation time. From the T2weighted images (Fig. 8A), it was shown that a graduated darkening appeared with increasing Fe concentrations. As shown in Fig. 8B, r2 was approximately 221.2 Fe mM−1 s−1, which was obtained by the linear fitting of the 1/relaxation time (1/T2, s−1) as a function of the iron concentration (mM). All of the above results suggested that reductionsensitive HA-SS-PCL micelles have potential as a multifunctional nanocarrier for tumor diagnosis and treatment. 15

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Control

Free DOX

Q1QQ

Q2

Q1

Q3

Q4

Q3

Q2

Q1

Q4

Q3

DOX/HA-SS-PCL Q1

16.27%

12.61%

Q3

20.53%

17.04%

Q2

Q4

DOX/HA-PCL

8.34%

14.47%

Q2

Q4

Fig. 5. Apoptosis analysis by flow cytometry after HepG2 cells incubated with free DOX, DOX loaded HA-SS-PCL micelles and DOX loaded HA-SS-PCL70, respectively.

DOX

DAPI

Merge

(A)

(a)

(b) (B)

(C)

(c) Fig. 7. (A) Flow cytometric profiles of HepG2 cells incubated with (a) free DOX, (b) DOX/SPIO-loaded HA-SS-PCL70 and (c) DOX/SPIO-loaded HA-PCL micelles for 4 h at 37 °C; Prussian blue stains of (B) HepG2 cells cultured with only culture media and (C) HepG2 cells culture media containing SPIO loaded HASS-PCL70 micelles. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 6. Confocal laser scanning microscopy images (scale bars: 200 μm) of HepG2 cells incubated with (a) free DOX, (b) DOX/SPIO-loaded HA-SS-PCL70 and (c) DOX/SPIO-loaded HA-PCL micelles for 4 h at 37 °C.

4. Conclusions Multifunctional nanocarriers based on disulfide bonds linked HASS-PCL block copolymers were developed for tumor diagnosis and treatment. These copolymers exhibited tumor targeting ability, redox16

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Fig. 8. (A) T2-weighted MRI images of DOX/SPIO-loaded HA-SS-PCL70 micelles, (B) T2 relaxation rates (s−1) as a function of Fe concentrations (mM).

responsiveness and a high loading capacity for DOX and SPIO. A faster DOX released from the HA-SS-PCL micelles was observed in response to 10 mM GSH. According to the MTT assay, cell apoptosis analysis, confocal laser scanning microscopy image and flow cytometric results, the DOX-loaded HA-SS-PCL micelles exhibited higher cellular uptake and cytotoxicity to HepG2 cells than the reduction-insensitive HA-PCL micelles. After loading the SPIO, HA-SS-PCL micelles could improve the MRI sensitivity and relaxivity. CD44 is upregulated in hepatic cellular carcinoma (HCC) cell lines and tumors, hence, HA-SS-PCL micelles have great potential as active targeting reduction-sensitive theranostic nanocarriers for hepatic carcinoma diagnosis and chemotherapy.

[8]

[9] [10]

[11]

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Acknowledgements

[13]

This work was supported by the National Natural Science Foundation of China (81571665, 51403043), the Natural Science Foundation of Guangdong Province (2014A030313647), the Science and Technology Plan Foundation of Guangzhou (201510010263, 1563000477, 201510010086, 201607010038), the Social Development Fund of Guangdong Province (2014A020212031, 2014A020212030). The authors thank Dr Wei Li (Instrumental Analysis & Research Center, Sun Yat-sen University, Guangzhou 510275, China) for his assistance in 2D DOSY NMR experiments.

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