Redox-responsive core-cross-linked mPEGylated starch micelles as nanocarriers for intracellular anticancer drug release

European Polymer Journal 83 (2016) 230–243

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European Polymer Journal journal homepage: www.elsevier.com/locate/europolj

Macromolecular Nanotechnology

Redox-responsive core-cross-linked mPEGylated starch micelles as nanocarriers for intracellular anticancer drug release Can Wu, Jinlong Yang, Xiubin Xu, Chunmei Gao ⇑, Shaoyu Lü, Mingzhu Liu ⇑ Key Laboratory of Nonferrous Metal Chemistry and Resources Utilization of Gansu Province, State Key Laboratory of Applied Organic Chemistry and Department of Chemistry, Lanzhou University, Lanzhou 730000, PR China

a r t i c l e

i n f o

Article history: Received 17 May 2016 Received in revised form 5 August 2016 Accepted 14 August 2016 Available online 17 August 2016 Keywords: Starch Polymeric micelles Redox-responsive Drug delivery

a b s t r a c t Novel redox-responsive core-cross-linked polymers were prepared by cross-linking mPEGylated starch (mPEG-St) with 3,30 -dithiodipropionic acid (DPA), a cross-linker containing a disulfide bond. The structure of the polymer based on starch (mPEG-St-DPA) was characterized using nuclear magnetic resonance (NMR), Fourier transform infrared spectroscopy (FTIR), and Raman spectroscopy. In addition, these polymers could selfassemble into micelles in phosphate-buffered saline (PBS) solution. The size and critical micelle concentration (CMC) of the mPEG-St-DPA micelles decreased with an increase in the degree of cross-linking. Interestingly, the size of the mPEG-St-DPA micelles increased gradually in the presence of 10 mM glutathione (GSH) owing to the cleavage of the disulfide bonds in the micellar core. The mPEG-St-DPA micelles showed good stability, exhibiting slight changes in size after 1000-fold dilution with PBS solution or 10-fold dilution with dimethyl formamide (DMF). The results of a protein adsorption test indicated that the mPEG-St-DPA micelles were hemocompatible. Doxorubicin (DOX), a model anticancer drug, was efficiently loaded into the mPEG-St-DPA micelles. The in vitro release studies revealed that the DOX-loaded mPEG-St-DPA micelles showed enhanced release of DOX in the presence of GSH. An in vitro MTT assay confirmed that the core-cross-linked mPEG-St-DPA micelles were biocompatible with HeLa cells, and the DOX-loaded mPEGSt-DPA micelles displayed higher inhibition of HeLa cell proliferation. These results suggest that the redox-responsive mPEG-St-DPA micelles hold great potential as ideal drug delivery carriers for cancer therapy. Ó 2016 Elsevier Ltd. All rights reserved.

1. Introduction There has been a growing interest in the development of novel drug delivery systems (DDS) to improve therapeutics, and current DDS development is mainly focused on the design and application of drug delivery carriers [1–3]. Common drug carriers include polymeric micelles [4], vesicles [5], nanoparticles [6], and nanohydrogels [7]. Self-assembled polymeric micelles have demonstrated notable and unique properties, for instance improving the solubility of hydrophobic drugs in water, having prolonged in vivo circulation times, and causing increased drug accumulation in target cells owing to the enhanced permeability and retention (EPR) effect, thereby showing much promise as drug delivery systems [8–10]. However, they still have some drawbacks. In particular, stimuli-responsive polymeric micelles released up to 20–30% of the loaded drug ahead ⇑ Corresponding authors. E-mail addresses: [email protected] (C. Gao), [email protected] (M. Liu). http://dx.doi.org/10.1016/j.eurpolymj.2016.08.018 0014-3057/Ó 2016 Elsevier Ltd. All rights reserved.

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of time as a burst, followed by slow release of the remaining drug by diffusion. The existence of the ‘‘burst release” phenomenon leads to drug loss during storage, as well as losses in the blood circulation after administration, decreasing treatment efficacy [11]. Hence, a great deal of effort has been focused on improving the stability of stimuli-responsive micelles, which could provide a safer and more effective platform for cancer therapies [12]. Stimuli-responsive micelles undergo structural changes under specific environmental stimuli, resulting in efficient drug release and decreased multidrug resistance and side effects [4,7]. The most common polymeric micelles are light-, enzyme-, redox-, temperature-, or pH-responsive [4,13–15]. Among the above stimuli-responsive micelles, redox-responsive polymeric micelles have emerged as potential nanocarriers for anticancer drug delivery, especially since the introduction of disulfide bonds into amphiphilic polymers. Disulfide bonds are stable at normal physiological conditions, but they can be reversibly cleaved into free thiols under reductive conditions, such as in the presence of dithiothreitol (DTT) and glutathione (GSH) [16,17]. GSH is a thiol-containing tripeptide, and its intracellular concentration (10 mM) is significantly higher than its extracellular concentration (2 lM) [18,19]. Furthermore, the GSH concentration in tumor cells was shown to be several times higher than that in normal cells [18,20]. These dramatic differences in GSH concentrations make it possible to design and prepare redox-responsive micelles for drug delivery applications. Thus, polymeric micelles containing disulfide bonds have received increasing attention as intracellular anticancer drug delivery systems, since intracellular drug release is facilitated by the cleavage of these disulfide bonds. An ideal drug carrier should not only facilitate controlled drug release in response to environmental stimuli, but also show good stability. In fact, the low in vivo stability of polymeric micelles has been a challenge. Polymeric micelles easily aggregate owing to destabilization and destruction of their structure under conditions of high dilution, high salt concentrations, or high shearing forces, resulting in the loss of encapsulated drug in the blood circulation before reaching the target sites [4,21,22]. To date, much effort has been undertaken to enhance the stability of polymeric micelles; the most promising strategy has been to cross-link the shell or core [11,21,23]. However, cross-linking the shell is not ideal, as it leads to decreased shell fluidity and hydrophilicity, compromising the stealth effect and reducing circulation times [4]. In contrast, cross-linking the core of the polymeric micelles has minimized effects on surface properties and circulatory half-life, while still enhancing stability [4,24,25]. Core-cross-linked redox-responsive polymeric micelles are mainly prepared using crosslinkers containing disulfide bonds. In this study, we attempted to prepare amphiphilic redox-responsive polymers based on starch (mPEG-St-DPA) by covalently cross-linking the main chains of mPEGylated starch (mPEG-St) with the disulfide bond containing the crosslinker 3,30 -dithiodipropionic acid (DPA). DPA is a kind of low molecular weight crosslinker, which contains disulfide bond and the terminal group both active carboxyl. The physiochemical properties of mPEG-St-DPA polymers were characterized by 1H-nuclear magnetic resonance (1H NMR), Fourier transform infrared spectroscopy (FTIR), and Raman spectroscopy. The mPEG-St-DPA polymers self-assembled in phosphate-buffered saline (PBS) solution to form core-cross-linked redoxresponsive polymeric micelles. The mPEG-St-DPA micelles showed good stability after 1000-fold dilution with PBS solution or 10-fold dilution with organic solvent, and presented a good redox response in the presence of GSH. In addition, the results of the protein adsorption test indicated that the mPEG-St-DPA micelles possessed good hemocompatibility. The hydrophobic anticancer drug doxorubicin (DOX) was loaded into core-cross-linked mPEG-St-DPA micelles. In vitro drug release from the DOX-loaded micelles increased in the presence of 10 mM GSH. The in vitro MTT assay demonstrated that mPEG-St-DPA micelles were biocompatible with HeLa cells, while DOX-loaded mPEG-St-DPA micelles inhibited HeLa cell proliferation. Therefore, the core-cross-linked redox-responsive mPEG-St-DPA micelles hold great potential as ideal nanocarriers for intracellular drug delivery. To serve as a control, uncross-linked mPEG-St micelles and mPEG-St-SA micelles cross-linked with suberic acid (SA), a cross-linker with an analogous structure to DPA but without disulfide bonds, were also prepared. 2. Experimental 2.1. Materials Chemical purity grade soluble starch (Mw = 8.8 kDa) was purchased from Zhejiang Linghu Chemical Reagent Factory. The mPEGylated starch (mPEG-St) polymers were prepared as previously reported [26]. 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCHCl), suberic acid (SA), 3,30 -dithiodipropionic acid (DPA), and 4-dimethylaminopyridine (DMAP) were purchased from Aladdin Reagent Inc. (Shanghai, China). Succinic anhydride was purchased from Sinopharm Chemical Reagent Co., Ltd. Pyrene and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were purchased from Sigma-Aldrich. Doxorubicin hydrochloride (DOXHCl) and glutathione (GSH) were purchased from LSB Biotechnology Inc. (Xi’an, China). Bovine serum albumin (BSA) was purchased from Sangon Biotech (Shanghai) Co., Ltd. The reagents were of analytical grade and used without further purification. 2.2. Synthesis of cross-linked mPEGylated starch (mPEG-St-DPA) mPEG-St-DPA polymers were prepared by covalently conjugating DPA onto the main chains of mPEG-St under a nitrogen atmosphere. Typically, mPEG-St polymers (0.12 g, 0.2 mM) were dissolved in 20 mL of DMSO under stirring at 25 °C. DPA at different molar ratios (10, 30, and 50% DPA), EDCHCl (0.2 equiv. DPA), and DMAP (0.2 equiv. DPA) were added to the

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abovementioned DMSO solution. The reaction was allowed to proceed with gentle stirring at 25 °C for 48 h. After completion of the reaction, the mixture was transferred into dialysis bags (Mw - 3.5 kDa) and dialyzed against deionized water for 72 h to remove solvent and unreacted substances. Finally, the solution was filtered and lyophilized to obtain mPEG-St-DPA polymers (yield: 70.2%). The procedure for the preparation of mPEG-St-SA polymers was similar to that of mPEG-St-DPA polymers, but DPA was replaced with SA, and polymers made with a SA molar ratio of 50% were prepared for use as controls. 2.3. Characterization of mPEG-St-DPA polymers The chemical structures of the mPEG-St-DPA polymers were investigated using 1H NMR (ELS-400 MHz, DMSO-d6). To further confirm the structures, the mPEG-St-DPA polymers were pressed with KBr and the FTIR spectra in the range of 4000–400 cm1 and with a resolution 4 cm1 were recorded on a Nicolet 670 FTIR spectrometer. Raman spectroscopy was measured using a SPEX 1403 spectrometer with an excitation wavelength of 785 nm. Using 1H NMR, the degree of DPA cross-linking within mPEG-St-DPA polymers with different molar ratios was calculated based on the integral area ratios of the methylene protons (ACH2ACH2A) in DPA and the C1AH in the main starch. 2.4. Preparation of mPEG-St-DPA micelles The mPEG-St-DPA micelles were prepared by direct dissolution method. Typically, 20 mg of mPEG-St-DPA polymers was dissolved in 20 mL of PBS solution (pH 7.4) and gently stirred at 25 °C for 30 min, followed by sonication at 100 W for 10 min. The sonication was repeated three times to obtain an optically transparent solution. Finally, the mPEG-St-DPA micelles were obtained by passing the solution through a 0.45-lm filter (Millipore), and were stored at room temperature. 2.5. Critical micelle concentration of mPEG-St-DPA micelles The critical micelle concentration (CMC) of mPEG-St-DPA micelles in PBS solution was estimated by the probe fluorescence technique using pyrene as the hydrophobic probe. A series of mPEG-St-DPA solutions, with concentrations ranging from 1  104 to 1.0 mg/mL and containing 6.0  107 mg/mL pyrene, were prepared. The fluorescence spectra of the mPEG-St-DPA solutions were monitored using a fluorescence spectrometer (LS55, Perkin-Elmer). The emission spectra between 350 and 450 nm were recorded at an excitation wavelength of 336 nm, excitation slit of 10 nm, and emission slit of 5 nm. The CMC value was determined by measuring the intensity ratio I385/I374 in the pyrene emission spectrum as a function of mPEG-St-DPA concentration and locating the cross point of the two straight lines. 2.6. Size and morphology of mPEG-St-DPA micelles The size of the mPEG-St-DPA micelles was determined using dynamic light scattering (DLS) at room temperature, on a 90 Plus particle size analyzer (Brookhaven Instruments Corporation), at a 90° scattering angle. The concentration of all sample solutions was 1.0 mg/mL. The morphology of the mPEG-St-DPA micelles was observed by transmission electron microscopy (TEM) (JEM-1200EX/S, Hitachi, Japan) with an accelerating voltage of 200 kV. Prior to measurement, a drop of sample solution (1.0 mg/mL) was placed onto a 300-mesh copper grid coated with carbon and air-dried at room temperature. 2.7. Stability of mPEG-St-DPA micelles The stability of the mPEG-St-DPA micelles in PBS solution (pH 7.4) was investigated. All samples were placed in a shaking bed (HNY-2102C INCUBATOR SHAKER, China) and lightly stirred at 100 rpm, at 37 °C. Their sizes were measured by DLS at 24 h and 48 h. The sizes of uncross-linked mPEG-St and mPEG-St-SA micelles were used as controls. The stability of mPEG-St-DPA micelles and uncross-linked mPEG-St micelles was also investigated at high dilutions and in organic solvents. The PBS solutions of mPEG-St-DPA micelles and mPEG-St micelles (1 mg/mL) were placed in a vial, and diluted 1000-fold using PBS or 10-fold using DMF. Then, the solutions were shaken for 5 min. Finally, the changes in micelle size were observed by DLS. 2.8. Redox-triggered disassembly of mPEG-St-DPA micelles The changes in mPEG-St-DPA micelle size in the presence of 10 mM GSH in PBS solution (pH 7.4) were monitored by DLS in order to confirm a micellar redox-response. Briefly, 16 mL of the mPEG-St-DPA micelle solution at a concentration of 1 mg/mL was degassed with nitrogen for 30 min. Then, 10 mM GSH was added. The solution was mildly stirred in a shaking bed (HNY-2102C INCUBATOR SHAKER, China) at a speed of 150 rpm and temperature of 37 °C for 24 h. Micelle sizes were measured by DLS at different time intervals.

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2.9. Protein adsorption tests Bovine serum albumin (BSA) was used as a model protein to determine the mPEG-St, mPEG-St-DPA, and mPEG-St-SA micelle protein adsorptions. The micelles were incubated with a solution of BSA in PBS solution (pH 7.4), with a final micelle concentration of 0.25 or 0.5 mg/mL, and BSA concentration of 0.5 mg/mL. After incubation in a shaking bed at 150 rpm and 37 °C for 4 h, 5 mL of each sample was withdrawn and centrifuged at 14,000g for 30 min to precipitate the protein-adsorbed micelles. The BSA concentration of the supernatant was determined using UV–vis spectroscopy (Lambda 35, Perkin Elmer) by measuring the maximal absorbance at 280 nm. Then the amount of BSA adsorbed onto the micelles was calculated from a BSA standard calibration curve. mPEG1.9k and PEG25k were used as controls. 2.10. DOX loading and in vitro DOX release DOX was used in loading studies as a model anticancer drug. DOX loading into mPEG-St, mPEG-St-DPA, and mPEG-St-SA micelles was carried out by dialysis method. Typically, the polymers (50 mg), DOXHCl (5 mg), and trimethylamine (10 lL) were mixed in 10 mL of DMSO by stirring at 25 °C for 2 h. Using a dialysis bag (Mw 3.5 kDa), the mixture was dialyzed against deionized water, at room temperature for 24 h, to remove DMSO. The dialysis medium was refreshed four times and the whole procedure was performed in the dark. After dialysis, the solution was filtered and further lyophilized. To determine the drug loading content (DLC) and drug loading efficiency (DLE), a certain amount of DOX-loaded micelles was dissolved in DMSO, and the DOX content was determined by UV–vis spectrophotometer (Lambda 35, Perkin Elmer) at 480 nm, using a standard curve obtained from series of DOX concentrations in DMSO. The DLC and DLE were calculated according to the following equations:

DLCðwt%Þ ¼ ðweight of loaded drug=weight of drug loaded micellesÞ  100% DLEðwt%Þ ¼ ððweight of loaded drugÞ=weight of drug in feedÞ  o 100% The in vitro DOX release profile from DOX-loaded micelles was investigated in PBS solution (pH 7.4) in the presence of absence of 10 mM GSH. Briefly, the weighed freeze-dried DOX-loaded mPEG-St-DPA micelles (final concentration of 1 mg/mL) were suspended in 5 mL of PBS solution containing 10 mM GSH and transferred into a dialysis bag (Mw 3.5 kDa). The dialysis bag was immersed in 100 mL of PBS solution containing 10 mM GSH and gently shaken in a shaking bed at 120 rpm and 37 °C. At predetermined time intervals, 5 mL of dialysate was withdrawn and replaced with the same volume of fresh release medium. The amount of released DOX was determined by UV–vis spectrophotometer at 480 nm. In addition, the DOX release profiles of DOX-loaded cross-linked and uncross-linked micelles in the absence of 10 mM GSH were recorded as controls. 2.11. In vitro cytotoxicity assay The relative cytotoxicity of DOX-free mPEG-St-DPA micelles, DOX-loaded micelles and free DOX in HeLa cells was evaluated in vitro using an MTT assay. HeLa cells were seeded in a 96-well culture plate at a density of 1  104 cells in 200 lL of DMEM supplemented with 10% FBS, and incubated at 37 °C in 5% CO2 for 24 h. After removing the culture medium, the cells were incubated with mPEG-St-DPA micelles, DOX-loaded micelles, and free DOX at 37 °C for 24 h. The concentration of mPEG-St-DPA micelles ranged from 10 to 100 lg/mL. Furthermore, the DOX-loaded micelles were diluted in complete DMEM to obtain final DOX concentrations from 0.5 to 10 lg/mL. Afterwards, the MTT reagent was added into each well and incubated for another 4 h. The culture medium was removed and 150 lL DMSO was added into each well. The absorbance of each well was measured using a microplate reader (VICTOR 1420, Perkin Elmer) at 490 nm. The cell viability (%) was calculated according to the following equation: [(Asample/Acontrol)  100%], where Asample and Acontrol represented the absorbance of the sample well and control well, respectively. The half-maximal inhibitory concentration (IC50) value was determined as the DOX concentration required to reduce the absorbance to 50% of that of the control wells. 3. Results and discussion 3.1. Preparation and characterization of mPEG-St-DPA polymers In order to prepare the amphiphilic redox-responsive mPEG-St-DPA polymers, 3,30 -dithiodipropionic acid (DPA) was used as a disulfide bond-containing cross-linker to covalently cross-link mPEGylated starch (mPEG-St). The synthetic pathway of mPEG-St-DPA polymer is shown in Fig. 1. 3.2. NMR, FTIR, and Raman spectroscopy analyses The chemical structure of mPEG-St-DPA polymers was confirmed by 1H NMR, FTIR, and Raman spectroscopy. Fig. 2 shows the 1H NMR spectra of mPEG-St and mPEG-St-DPA polymers. Compared to the 1H NMR spectrum of mPEG-St polymers (Fig. 2B), the spectrum of mPEG-St-DPA polymers showed two new peaks at 2.89 (i) and 3.01 (h) ppm (Fig. 2A), which can be attributed to the two methylene protons of DPA. In addition, the characteristic peaks of mPEG and starch also

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Fig. 1. The synthetic pathway of mPEG-St-DPA polymer.

Fig. 2. 1H NMR spectra of mPEG-St-DPA (A) and mPEG-St micelles (B) in DMSO-d6.

appeared in the 1H NMR spectrum of mPEG-St-DPA polymers. For example, the four evident peaks located between 4.5 and 5.6 ppm, consisting of C2AOH (a, 5.44 ppm), C3AOH (b, 5.43 ppm), C1AH (c, 5.10 ppm), and C6AOH (d, 4.60 ppm), were assigned to the protons of the glucose unit of starch, while the characteristic peaks appearing at 3.51 ppm

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C. Wu et al. / European Polymer Journal 83 (2016) 230–243 Table 1 The physical characterization of uncrosslinked and crosslinked mPEG-St micelles.

a

Code

Sample

Molar ratioa

Degree of crosslink (%)

S (%)

Size (nm)

CMC (mg/mL)

CCM-0 CCM-1 CCM-2 CCM-3 CCM-4

mPEG23-St mPEG23-St-DPA10 mPEG23-St-DPA12 mPEG23-St-DPA15 mPEG23-St-SA16

0 0.1 0.3 0.5 0.5

0 9.5 11.5 14.5 16.0

0 1.37 1.51 1.64 0

167 130 114 92 96

0.026 0.021 0.012 0.0063 0.0076

Mole ratio of DPA and sugar units of mPEG-St.

Fig. 3. FTIR spectra of mPEG23-St (a), mPEG23-St-DPA10 (b), mPEG23-St-DPA12 (c) and mPEG23-St-DPA15 (d).

(ACH2ACH2A, f, g) and 3.23 ppm (AOACH3, e) were attributed to the methylene protons and terminal methoxyl protons of mPEG, respectively [26,27]. The above data from the 1H NMR spectra indicate the successful cross-linking between DPA and mPEG-St polymers to obtain redox-responsive mPEG-St-DPA polymers containing disulfide bonds. The different degrees of cross-linking in the mPEG-St-DPA polymers were calculated based on 1H NMR results, and these 1H NMR spectra were shown in Fig. S2. According to previous work [26], the degree of substitution of mPEG in mPEG-St polymers was 23. In addition, the degree of cross-linking of mPEG-St in mPEG-St-DPA polymers was calculated by comparing the integrated peak areas at 2.89 (i) and 5.10 (c) ppm, attributed to the methylene protons of DPA and the glucose protons of the starch unit, respectively. In order to prepare mPEG-St-DPA polymers with different degrees of cross-linking, a series of experiments with different ratios of mPEG-St and DPA was designed. The degrees of cross-linking in the mPEG-St-DPA polymers were calculated based on 1H NMR results, and are listed in Table 1. When the feeding amount of DPA in mPEG-St polymers was 10, 30, and 50%, the corresponding degree of cross-linking was 10, 12, and 15, respectively. Cross-linked mPEG-St-SA polymers without redox response were prepared by using SA and used as controls. The FTIR spectra (Fig. 3) also confirmed the chemical structure of mPEG-St-DPA polymers, which showed absorptions at 1732 cm1 assigned to the ester linkages in the polymers and 3409 cm1 attributed to the stretching vibration of the hydroxyl groups in starch backbones [26,27]. It is suggested that mPEG-St polymers were successfully cross-linked with DPA, based on the increased absorption strength at 1732 cm1 and decreased absorption strength at 3409 cm1, with an increase in the feed ratio of DPA to mPEG-St from 10 to 50%. This occurs because the hydroxyl groups of mPEG-St polymer were able to react with carboxyl groups of DPA in the presence of EDC and DMAP, forming more ester linkages and decreasing the amount of starch hydroxyl groups. The absorption peak attributed to the disulfide bonds was not easily detected in the FTIR spectrum because of their weak absorptive intensity. However, the presence of disulfide bonds in mPEG-St-DPA polymers could be confirmed by Raman spectroscopy [11]. As shown in Fig. S1, a new peak at 504 cm1 was clearly established in the Raman spectra after mPEGSt polymers were cross-linked with DPA, which confirmed the existence of disulfide bonds. In addition, elemental analysis was also used to demonstrate the presence of disulfide bonds. As shown in Table 1, by increasing the degree of cross-linking from 9.5 to 14.5, the amount of elemental sulfur in mPEG-St-DPA polymer also increased gradually from 1.37 to 1.64%. 3.3. Self-assembly behaviors of mPEG-St-DPA micelles Amphiphilic polymers spontaneously self-assemble to form micelles with core–shell structures in selective solvents. In this study, redox-responsive mPEG-St-DPA micelles and non-redox-responsive mPEG-St-SA micelles were prepared by direct

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Fig. 4. TEM images of mPEG23-St micelles (A), mPEG23-St-DPA15 micelles (B) and mPEG23-St-DPA15 micelles in the presence of GSH for 2 h (C).

dissolution and simple sonication to obtain optically transparent solutions in PBS (pH 7.4). The formation of core-crosslinked mPEG-St-DPA micelles is attributed to the hydrophilic mPEG outer shell and the hydrophobic starch inner core, cross-linked by DPA. The micelle size is an important parameter for intracellular drug delivery, because small-sized micelles (<200 nm) are favorable to avoid renal excretion and uptake by the reticuloendothelial system; this size is ideal for the EPR effect to take place, leading to accumulation of drug carriers at the site of the tumor and allowing for passive tumor targeting [20,28,29]. In general, cross-linked micelles have a tendency to be smaller than the uncross-linked micelles, owing to the formation of more compact structures [23]. DLS measurements showed that the mean sizes of mPEG-St-DPA micelles with different degrees of cross-linking, in PBS solution (pH 7.4), were smaller in than those of uncross-linked mPEG-St micelles (167 nm). When the degree of cross-linking increased from 9.5 to 14.5, the mean size of mPEG-St-DPA micelles decreased from 130 to 92 nm (Table 1), which indicates the formation of more compact hydrophobic cores due to the cross-linking. In addition, the morphology of mPEG-St micelles and mPEG-St-DPA micelles was studied by TEM. The TEM images (Fig. 4) showed that mPEG23-St micelles and mPEG23-St-DPA15 micelles both were spherical and had a core-shell structure. Furthermore, the TEM measurements also showed that the mPEG23-St-DPA15 micelles were smaller than uncross-linked mPEG23-St micelles. The observed size measurements were smaller with TEM than with DLS owing to the dehydration process used in the TEM sample preparation, which lead to a shrinkage of the mPEG shell [30]. These results indicate that mPEG-St-DPA micelles with sizes close to 100 nm, could be used as ideal nanocarriers for intracellular drug delivery. The CMC of mPEG-St-DPA micelles was confirmed by the fluorescence technique using pyrene as a hydrophobic probe. It is well known that the CMC is an important parameter to verify the self-assembly behavior and physical stability of polymeric micelles. Pyrene has low fluorescence intensity and can react to the polarity of its microenvironment owing to its poor solubility and self-quenching. When pyrene is transferred from a hydrophilic to a hydrophobic microenvironment, the fluorescent intensity ratio increases dramatically. Thus, this change in fluorescence can be monitored at different polymer concentrations to determine the CMC, the polymer concentration at which the hydrophobic portions of the polymers start aggregating to minimize the interaction with water molecules, resulting in the formation of micelles and providing a hydrophobic environment for the pyrene [26,30]. Fig. S3 shows the change in the intensity ratio (I385/I374) of pyrene versus the log of mPEG-St-DPA polymer concentration ranging from 1  104 to 1.0 mg/mL, and the CMC is the corresponding concentration at the crossover point of the two straight lines. As shown in Table 1, when mPEG-St micelles were not cross-linked, the CMC value was 0.026 mg/mL. However, after cross-linking, the CMC value of mPEG-St-DPA and mPEG-St-SA micelles decreased significantly. Moreover, with an increase in the degree of cross-linking from 9.5 to 14.5, the CMC values of mPEG-St-DPA micelle decreased from 0.021 to 6.3  103 mg/mL. The large difference in CMC values is attributed to the much stronger hydrophobic interactions in mPEG-St-DPA micelles because of the formation of more compact cores after cross-linking. It is reported that when the CMC value is below 1  102 mg/mL, micelles have good stability in highly dilute aqueous condition, which is an important feature for drug carriers [30,31]. In this work, the CMC value of mPEG23-St-DPA15 micelles was 6.3  103 mg/mL. Thus, based on the above results, mPEG-St-DPA micelles are stable at low concentrations and could be ideal drug delivery carriers. In addition, it was shown that the cross-linking could enhance the stability of micelles. 3.4. The stability of mPEG-St-DPA micelles One of the most important parameters for ideal intracellular drug carriers is that the polymeric micelles be stable in the blood circulation. This is essential to successfully deliver drugs into the tumor cells by EPR effect, while minimizing losses of encapsulated drug [32]. Thus, to evaluate the stability of mPEG-St-DPA micelles, the changes in mPEG-St-DPA micelle size in mimicked physiological conditions, high dilution conditions, and organic solvents was measured by DLS. The uncross-linked and cross-linked mPEG-St micelles were dispersed in PBS (pH 7.4) and incubated at 37 °C for 48 h. As shown in Fig. 5, it was clearly established that cross-linked mPEG-St micelles showed minimal increases in size after 48 h.

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Fig. 5. Size change of micelles measured by DLS incubated in PBS solution at 37 °C for 48 h. The data are presented as mean ± SD (n = 3).

For example, mPEG23-St-DPA15 micelles showed a slight increase in size from 92 to 133 nm, while mPEG23-St-SA16 micelles increased from 95.68 to 127.51 nm. However, during the same time interval, uncross-linked mPEG-St micelles exhibited a rapid and significant increase in size from 167 to 714 nm. From the above data, it can be deduced that mPEG-St-DPA micelles have a greater stability than uncross-linked mPEG-St micelles. The instability of the uncross-linked mPEG-St micelles can be attributed to the vulnerability of their micellar structure under physiological ionic strength, resulting in easy aggregation of the exposed hydrophobic cores [4,33]. In contrast, mPEG-St-DPA micelles are stable because the cross-linked cores of

Fig. 6. Size change of mPEG23-St and mPEG23-St-DPA15 micelles measured by DLS against dilution with PBS solution (A) and DMF (B).

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mPEG-St-DPA micelles form more compact shell-core structures. In addition, having a hydrophilic mPEG outer shell avoids intermicellar hydrophobic interactions, thus preventing the formation of aggregates. The slight increase in mPEG-St-DPA micelle size was due to swelling. These results indicate that the size of mPEG-St-DPA micelles is suitable for intracellular drug delivery and that these micelles would be stable in blood circulation. The stability of mPEG-St and mPEG-St-DPA micelles in extensively dilute solutions and organic solvent was investigated. The mPEG-St-DPA micelles and mPEG-St micelles were dispersed in PBS solution (pH 7.4), and then diluted 1000-fold using PBS solution or 10-fold using DMF. The change in micelle size was monitored by DLS. Fig. 6A shows the size changes of mPEG23-St and mPEG23-St-DPA15 micelles with 1000-fold PBS dilution. Notably, there was only a slight change in mPEG23-St-DPA15 micelle size. In contrast, uncross-linked mPEG23-St micelles completely disassembled into monomers of sizes below 10 nm [34]. Thus, it is clear that the mPEG-St-DPA micelles were stable in highly dilute conditions. As shown in Fig. 6B, after mPEG23-St-DPA15 micelles were diluted with a 10-fold volume of DMF, their sizes increased slightly from 92 to 136 nm owing to the swelling of their hydrophobic cores [4,35]. However, under the same conditions, the size of mPEG23-St micelles significantly increased from 167 to 286 nm, which can be attributed to the destruction of the core-shell structure, resulting in the dissolution of the cores [4]. These results have demonstrated that mPEG-St-DPA micelles are more stable than mPEG-St micelles, and that mPEG-St-DPA micelles maintain their structural integrity due to the successful ‘‘locking” of the mPEG-St micelle structure by cross-linking with disulfide bonds [36]. 3.5. Redox-triggered disassembly of mPEG-St-DPA micelles Disulfide bonds remain stable under normal physiological conditions, but can easily be cleaved into free thiols in the presence of reductive reagents [16,27]. In this study, core-cross-linked redox-responsive mPEG-St-DPA micelles were obtained through cross-linking mPEG-St polymers with DPA. The disulfide bonds in the inner core makes mPEG-St-DPA micelles responsive to reductive conditions. To investigate the redox-triggered disassembly of mPEG-St-DPA micelles, a 10 mM GSH solution was used to mimic the intracellular reducing microenvironment. The change in the size of mPEG-St-DPA micelles in this solution was determined after 24 h by DLS measurements. As shown in Fig. 7, the sizes of mPEG23-StDPA15 micelles significantly increased from the initial 91 nm to over 1000 nm. This can be attributed to the decrease in the core-cross-linking density, resulting from the cleavage of the disulfide bonds within the micelle inner. This enhances the hydrophobic interactions causing faster micelle aggregation [27,28,37]. Furthermore, the cleavage of the disulfide bonds in the inner core results in amphiphilic mPEG-St micelles with hydrophilic thiol groups, which increases the hydrophilicity of new micelles and could result in swelling [34,38]. In addition, large aggregates with sizes of over 1000 nm were found at 24 h, demonstrating the complete disassembly of mPEG-St-DPA micelles. However, as mentioned above, in the absence of 10 mM GSH, mPEG-St-DPA micelles were stable in PBS solution after 24 h (Fig. 5). Therefore, the size change confirmed the redox-response of mPEG-St-DPA micelles in the presence of 10 mM GSH, and it was indicative that the core-crosslinked redox-responsive mPEG-St-DPA micelles could be suitable for intracellular drug delivery. 3.6. Protein adsorption The hemocompatibility and long-term stability of polymeric micelles are also important parameters of ideal drug carriers, which could effectively minimize non-specific protein adsorption and interactions with blood components [30,39]. In addition, using mPEG as a material for the outer shell of micelles is preferred as it provides a ‘‘stealth coating”. It has been shown that mPEG can prevent recognition by the reticuloendothelial system and prevent protein adsorption, resulting in prolonged blood circulation times [39,40]. To evaluate the hemocompatibility of uncross-linked and cross-linked mPEG-St micelles, the

Fig. 7. Size change of mPEG23-St-DPA15 micelles in PBS solution in response to 10 mM GSH measured by DLS.

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Fig. 8. BSA adsorption on the micelles, mPEG1.9k and PEI25k after incubated at 37 °C with different concentrations for 4 h. The data are presented as mean ± SD (n = 3).

interaction of micelles with a non-specific protein was observed; BSA was selected as a model protein. As shown in Fig. 8, the uncross-linked and cross-linked mPEG-St micelles and mPEG1.9k showed slight BSA adsorption, but PEG25k interacted strongly with BSA. The difference in BSA adsorption is due to the presence of the hydrophilic mPEG shell, which forms a strong hydration layer around the surface of micelles [30]. This was one of the key reasons why the mPEG-St polymer was selected to prepare mPEG-St-DPA micelles. These results demonstrate that mPEG-St-DPA micelles are hemocompatible and can be used as drug delivery carriers. 3.7. In vitro DOX loading and redox-triggered drug release DOX is one of the most effective and common anticancer drugs, which works by intercalating within DNA and inhibiting macromolecular biosynthesis [37]. In this study, to evaluate the potential of mPEG-St-DPA micelle as an ideal drug carrier, DOX was used as a model drug and was easily loaded into the inner core of mPEG-St-DPA micelles by dialysis of polymer/ DOX solutions in DMSO against deionized water (Scheme 1). To serve as controls, DOX was also loaded into mPEG-St and mPEG-St-SA micelles. Considering that micelles with smaller sizes and greater stability are optimal for intracellular drug delivery, mPEG23-St-DPA15 was selected to evaluate the in vitro DOX loading and redox-triggered drug release. The theoretical drug loading content was set at 10 wt%. The drug loading content (DLC) and drug loading efficiency (DLE) are shown in Table 2. The results showed that the DLC of mPEG23-St, mPEG23-St-DPA15, and mPEG23-St-SA16 micelles was 3.46, 5.15, and 4.88% corresponding to a DLE of 35.85, 54.29, and 51.34%, respectively. Therefore, it was determined that DOX could be effectively loaded into the micelles. In addition, according to the above results, cross-linked micelles have increased DLC and DLE, which can be attributed to their more compact cores. Using the dialysis method, the in vitro DOX releasing behavior of redox-responsive mPEG-St-DPA micelles was investigated in PBS solution (pH 7.4), in the presence or absence of 10 mM GSH (Fig. 9). In the absence of GSH, the in vitro DOX releasing behaviors of DOX-loaded micelles were as follows: mPEG23-St micelles > mPEG23-St-DPA15 micelles > mPEG23-St-SA16 micelles, and there were no clear indications of initial burst release. For example, without 10 mM GSH, 37% of the loaded DOX was released from mPEG23-St micelles by 72 h, but only 23% and 11% of the DOX was released from mPEG23-St-DPA15 and mPEG23-St-SA16 micelles, respectively. The low percentages of release from the cross-linked micelles could be explained by their compact core-shell structure. The interaction between the drug and hydrophobic core was stronger than that between the drug and the solvent. However, it is interesting to note that in the presence of 10 mM GSH, DOX release from DOX-loaded mPEG23-St-DPA15 micelles was noticeably faster than in the absence of GSH, demonstrating that GSH could accelerate DOX release markedly. For instance, more than 50% of the DOX was released within 27 h, which was higher than the release at 72 h in the absence of GSH, and the cumulative release from DOX-loaded mPEG23-St-DPA15 micelles was up to 70% by 72 h. The rapid DOX release was due to the cleavage of the disulfide bonds in the mPEG-St-DPA micelles, resulting in the alteration of the three-dimensional structure of the micelles into loose and instable structures [41], as illustrated in Scheme 1. Based on the above results, the disulfide bonds in mPEG-St-DPA micelles can be reduced in the presence of GSH, which accelerates DOX release. Furthermore, it has been confirmed that cross-linking of mPEG-St-DPA micelles decreases encapsulated drug loss in circulation [35]. Thus, the core-cross-linked redox-responsive mPEG-St-DPA micelles are promising drug carriers.

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Scheme 1. Schematic illustration of core-crosslinked redox-responsive mPEG-St-DPA micelles for DOX loading and intracellular DOX release triggered by GSH.

Table 2 Characterization of DOX-loaded micelles and IC50 values against HeLa cells. Sample

Feed ratio (wt%)

DLC (%)

DLE (%)

IC50 (lg/mL)

mPEG23-St mPEG23-St-DPA15 mPEG23-St-SA16

10 10 10

3.46 5.15 4.88

35.85 54.29 51.34

4.42 2.64 5.38

Fig. 9. DOX release from DOX-loaded micelles in PBS solution with or without 10 mM GSH at 37 °C. The data are presented as mean ± SD (n = 3).

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3.8. In vitro cytotoxicity assay In order to be an ideal drug carrier, polymeric micelles must demonstrate biocompatibility. Thus, it was necessary to evaluate the cell cytotoxicity of DOX-free micelles and DOX-loaded micelles against HeLa cells by MTT assay. In this study, cell viability was examined by incubating HeLa cells with different concentrations of mPEG-St-DPA micelles and DOX-loaded micelles for 24 h. As shown in Fig. 10A, the viability of HeLa cells incubated with mPEG23-St-DPA15 micelles for 24 h remained high, 94% compared to the blank control, at micelle concentrations up to 100 lg/mL. Therefore, it can be said that HeLa cells were not influenced by the addition of mPEG23-St-DPA15 micelles at any concentration, and that these micelles are biocompatible with HeLa cells. As such, they are promising as ideal drug carriers for biomedical application. The in vitro anticancer activity of DOX-loaded micelles and free DOX against HeLa cells were also determined by MTT assay. Fig. 10B shows the inhibition of HeLa cell growth in the presence of DOX-loaded uncross-linked and cross-linked mPEG-St micelles, as well as free DOX, for 24 h. There was a clear dose-dependent increase in cytotoxicity with DOXloaded micelles. Table 2 summarizes the IC50 values of DOX-loaded micelles and free DOX. The IC50 values of DOX-loaded mPEG-St-DPA, mPEG-St, and mPEG-St-SA micelles were 2.64, 4.42, and 5.38 lg/mL, respectively. Compared with free DOX, DOX-loaded micelles had lower cytotoxicity for the same DOX dose. DOX is a small molecule, which can be rapidly transported into cells by passive diffusion and interact with DNA [28]. However, DOX-loaded micelles must first be internalized by endocytosis, and then DOX is gradually released, which delays the interaction with DNA and resulted in lower anticancer efficiency [31]. However, in comparison with non-redox-responsive mPEG23-St-SA16 micelles and mPEG23-St micelles, redox-responsive mPEG23-St-DPA15 micelles displayed significantly higher cytotoxicity, due to the cleavage of the disulfide bonds in the intracellular environment, resulting in a much faster DOX release from the these micelles. In addition, these results are in accordance with the previous in vitro experiments on DOX release behaviors, presented above. Thus, it can be hypothesized that the DOX-loaded mPEG23-St-DPA15 micelles were easily internalized into tumor cells through

Fig. 10. Cytotoxicity study of mPEG23-St-DPA15 micelles (A) and DOX-loaded micelles and free DOX (B) against HeLa cells after incubation for 24 h. The data are presented as mean ± SD (n = 5).

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endocytosis and the loaded DOX was rapidly released in the presence of GSH and diffused into the nuclei, resulting in an increase in anticancer efficiency, as shown in Scheme 1. 4. Conclusion In this work, an amphiphilic redox-responsive mPEG-St-DPA polymer was successfully prepared by covalently crosslinking mPEG-St polymers with DPA, a cross-linker containing a disulfide bond. In PBS solution, this mPEG-St-DPA polymer could self-assemble into core-cross-linked micelles with a spherical morphology. As the degree of cross-linking in mPEG-St-DPA micelles increased, the size of the micelles gradually decreased. In addition, mPEG-St-DPA micelles exhibited good stability, excellent hemocompatibility, and high resistance to non-specific protein adsorption. DOX could be efficiently loaded into micelles, and compared to mPEG-St micelles, mPEG-St-DPA micelles showed higher drug loading capacity. In vitro DOX release behavior revealed that DOX-loaded mPEG-St-DPA micelles could rapidly release DOX via cleavage of the disulfide bonds in the presence of 10 mM GSH. The results of MTT assays confirmed that the mPEG-St-DPA micelles were biocompatible, and that DOX-loaded mPEG-St-DPA micelles displayed a significantly greater cytotoxicity against HeLa cells compared to DOX-loaded mPEG-St and mPEG-St-SA micelles. Therefore, core-cross-linked redox-responsive mPEG-St-DPA micelles show great potential as carriers for intracellular anticancer drug delivery. Acknowledgments The authors gratefully acknowledge the financial support of the National Natural Science Foundation of China (Grant No. 51273086), Special Doctorial Program Fund from the Ministry of Education of China (Grant No. 20130211110017) and the Fundamental Research Funds for the Central Universities (Grant No. lzujbky-2015-198, lzujbky-2015-26). Appendix A. 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