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

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 re- sonance (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 apop- tosis 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.

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-im- munogenicity 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 hya-luronan-b-poly(γ-benzyl glutamate) block copolymers shown pronounced antitumor ability against MCF-7 cells and C6 glioma cells over- expressing CD44 glycoprotein in their cells surface after loading DOX [4,5]. Furthermore, amphiphilic diblock copolymers of hyaluronan-b- poly(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 in- tracellular 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 HA- PCL nanoparticles have been developed as high performance drug de- livery system to improve antitumor efficacy [12]. Tumor targeted dis- ulfide 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 in- troduced into stimuli-responsive carriers, including fluorescent probe magnetic nanoparticles, quantum dots and so on [14]. Magnetic re- sonance 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 con- jugated 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-re- sponsive 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 nano-particles 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 ex- hibit a high affinity to CD44 rector, which over-expressed on the surface of many tumor cells, and lead to high levels of cellular drug accumu- lation. On the other hand, disulfide bond linked HA-SS-PCL nano- particles possessed reductant-triggered doXorubicin release ability

2.Materials and methods
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 dihy- drochloride were all purchased from Aladdin Chemical Company and used as received. Fetal bovine serum (FBS), trypsin-ethylenediamine- tetraacetic acid (Trypsin-EDTA) and Dulbecco’s modified Eagle medium (DMEM) were purchased from Gibco-BRL (Canada). 3-(4, 5- Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) was purchased from Invitrogen Corporation (Washington, USA). 4′,6-Diamidino-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 ethy- lamine (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-alkyne- SS-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 (eN-HCH2CH2e), 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 approXi- mately 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;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 po- tential as a multifunctional tumor-targeting nanocarrier that combines MRI and drug delivery functions.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). Reductive amination between HA and 1-azido-3-aminopropane with sodium cyanoborohydride (NaBH3CN) as the reducing agent was car- ried 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 solu- bilized in 20 mL of acetate buffer (pH 5.6) and 0.55 g of 1-azido-3- aminopropane (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 reac-nanoparticles were precipitated in excess diethyl ether and separated as a dry product. The dry magnetic particles were suspended in an an- hydrous DMSO (5 mL) and stored at room temperature for further use.

2.6. Characterization
FTIR spectra were obtained from a Perkin-Elmer Paragon1000 spectrometer using the KBr disk method in the transmission mode. 1Htion
miXtures Bruker spectrometer at 500 NMR. The 1H NMR spectra of α-alkyne-SS- PCL were measured in deuterated chloroform (CDCl3-d) or DMSO‑d6. The chemical structure of HA-SS-PCL was obtained by dissolving HA-

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 anhy-
drous 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 approXi- mately 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).

2.5.Preparation of hydrophobic SPIO nanoparticles
Hydrophobic SPIO NPs were synthesized following a previously described procedure. In brief, iron (III) acetylacetonate (1 g) was dis- solved 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 200SMGoniometer 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.

2.7.Micelle formation and the critical micellar concentration
The HA-SS-PCL micelles were prepared by dissolving 10 mg of HA- SS-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 from1.0 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 mea- surement. 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 copolymer concentrations. The CMC value was taken from the inter- section 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 re- move the DMSO and un-encapsulated DOX and SPIO at room tem- perature. 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:DLC (%) =weight of loaded drug× 100% 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 absor- bency 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 tempera-
ture. The cell apoptosis rates were evaluated using a flow cytometer.

2.11. Cellular uptake studies
The cellular uptakes of DOX and SPIO were revealed by using flow SPIO amount in the micelles was determined from Atomic absorp- tion 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% 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 fol- lowed by washing with PBS twice. HepG2 cells were incubated with fresh culture media containing free DOX or DOX/SPIO-loaded micelles weight of SPIO loaded micelles

2.9.In vitro DOX release(2)(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 tryp- sinized, collected and resuspended in 0.3 mL PBS and analyzed by flow cytometry.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/SPIO-
loaded micelles at different DOX concentrations (0–10 μg/mL DOX).

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% for-
maldehyde. After that, the cell nuclei were stained with DAPI and washed with cold PBS to remove the excess DAPI. Finally, a Leica, TCS- SP2 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 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, α- alkyne-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 at4.12–4.01 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 fol- lowing the procedure reported by LecommandouX et al. with a re- ductive amination reaction, which is a versatile method to introduce functional groups to the reducing end of polysaccharides [4]. The tar- geted di-block polymers were then synthesized by the click cycloaddition between the HA-N3 and the α-alkyne-SS-PCL in DMSO. Fig. S3
shows the FTIR spectrum of HA-N3, where the typical vibration ab- sorption 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 HA- SS-PCL copolymer did not show the characteristic peak of the azide group at 2106 cm−1, which implied that the excess HA-N3 was com- pletely 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].

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 self- assemble 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 con- centration at room temperature. Obviously, it was found that the fluorescence intensity ratio (I337/I335) stayed stable at the low con- centrations, while it gradually increased with an increase in the copo- lymer 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-SS- PCLs 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.
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).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 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 environ- ment, thus, leading to faster drug release. Therefore, the reduction-re- sponsive behavior of the micelles was studied under reductive condi- tions mimicking the intracellular environment. As shown in Fig. 2E, both the HA-PCL and the HA-SS-PCL micellar solutions showed a ty- pical Tyndall phenomenon (laser beam passes through the solution) under non-reductive conditions. The reduction-insensitive HA-PCL mi- cellar solution still showed the Tyndall phenomenon after adding 10 mM GSH. However, the HA-SS-PCL micellar solution became opaque

Fig. 3. In vitro DOX release profiles from reduction-sensitive HA-SS-PCL mi- celles under various GSH concentrations at 37 °C.
under the same reductive environment, indicating that larger hydro- phobic 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-SS- PCL 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 reductant- triggered 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-re- ductive 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-SS- PCL70 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 to- wards 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-SS- PCL70 and DOX/SPIO-loaded HA-PCL towards HepG2 cells were in- vestigated by flow cytometry at a DOX dosage of 10 μg/mL (Fig. 5). Due to the different cellular uptake mechanisms, the free DOX group in- duced 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/SPIO- loaded HA-SS-PCL70 micelles than that of DOX-loaded HA-PCL micelle was probably caused by the faster reduction-triggered DOX release.

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 con- centrations after 48 h.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 in- ternalized in HepG2 cell via a receptor mediated mechanism between HA and CD44 [4,29]. Notably, HepG2 cells treated with reduction- sensitive theranostic micelles shown stronger DOX fluorescence in- tensity 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-SS- PCL micelles owing to disulfide cleavage [30]. Hence, HA-SS-PCL mi- celles 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 in- cubated 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.

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 T2- weighted 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 reduction- sensitive HA-SS-PCL micelles have potential as a multifunctional na- nocarrier for tumor diagnosis and treatment.

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.

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.

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 HA- SS-PCL70 micelles. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Multifunctional nanocarriers based on disulfide bonds linked HA- SS-PCL block copolymers were developed for tumor diagnosis and treatment. These copolymers exhibited tumor targeting ability, redoXresponsiveness 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, con- focal laser scanning microscopy image and flow cytometric results, the DOX-loaded HA-SS-PCL Oxiglutatione micelles exhibited higher cellular uptake and cytotoXicity to HepG2 cells than the reduction-insensitive HA-PCL mi- celles. 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 na- nocarriers for hepatic carcinoma diagnosis and chemotherapy.