Microenvironmental Control of MUC1 Aptamer-Guided Acid-Labile

Sep 26, 2017 - Although aptamers are well-known as cell-specific membrane biomarkers for tumor-targeted therapy, it is important to avoid their degrad...
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Microenvironmental Control of MUC1 Aptamer-Guided AcidLabile Nanoconjugate within Injectable Microporous Hydrogels Chenchen Xu, Xiu Han, Yujie Jiang, Shengxiao Yuan, Ziheng Wu, Zhenghong Wu, and Xiaole Qi Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00324 • Publication Date (Web): 26 Sep 2017 Downloaded from http://pubs.acs.org on September 27, 2017

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Bioconjugate Chemistry

Microenvironmental Control of MUC1 AptamerGuided

Acid-Labile

Nanoconjugate

within

Injectable Microporous Hydrogels Chenchen Xu

†, 1

, Xiu Han

†, 1

, Yujie Jiang†, Shengxiao Yuan†, Ziheng Wu‡, Zhenghong Wu†,*,

Xiaole Qi†,* †

Key Laboratory of Modern Chinese Medicines, China Pharmaceutical University, Nanjing

210009, PR China. ‡

Jiangning Campus, High School Affiliated to Nanjing Normal University, Nanjing 211102, PR

China.

*Corresponding author: Zhenghong Wu, Xiaole Qi E-mail address: [email protected]; [email protected] *Corresponding

author

at:

Key

Laboratory

of

Modern

ChinaPharmaceuticalUniversity, Nanjing 210009, PR China Tel: +008615062208341; Fax: +0086-025-83179703

1

Chenchen Xu and Xiu Han contributed equally to this work.

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Chinese

Medicines,

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ABSTRACT Although aptamers are well-known as cell-specific membrane biomarkers for tumor-targeted therapy, it is important to avoid their degradation by nucleases in vivo. In this study, we developed a MUC1 aptamer-doxorubicin nanoconjugate (APT-DOX) through an acid-labile linkage and embedded APT-DOX into a thermosensitive hydrogel for anti-tumor therapy. The hydrogels exhibit a sol-gel transition upon intratumoral injection, resulting in the protection and controlled release control of APT-DOX with the shielding of the gel network. Moreover, the released APT-DOX was prone to be enriched at the tumor cells due to specific intracellular transport by the overexpressing MUC1 protein; however, APT-DOX regained the free DOX form via the rupture of the linkage under tumor cells lysosome acidic conditions and achieved increased concentration in the nucleus for anti-tumor treatment.

KEYWORDS: Aptamer, Doxorubicin, Thermosensitive hydrogel, Intratumoral injection, Tumor therapy

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INTRODUCTION Tumor biological markers are a type of biochemical molecule and are excessively expressed in tumor cells, but in normal cells, these markers are not expressed, or their expression is less than in tumor cells (1, 2). Aptamers are a class of single-stranded oligonucleotides selected by a process known as SELEX (systematic evolution of ligands by exponential enrichment), which are excellent candidates as targeting modifiers for tumor-specific drug delivery due to their high affinity, high specificity and easy modification (3-5). Compared with antibodies, aptamers have such characteristics as higher affinity and specificity, better stability, lower immunogenicity and molecular weight (6, 7). For example, a DNA aptamer MA3 binds to a glycoprotein Mucin 1 with a Kd of 38.3 nM, which is notably greater than the Kd of antibodies. The aptamer MA3 was found to preferentially bind to MUC1-positive but not MUC1-negative cells (8). Moreover, it has also been shown that aptamers can be employed to carry a drug to specific cells by chemical or physical bonding (9). However, unmodified aptamers are unstable and susceptible to the nucleases present in human serum, resulting in rapid blood clearance (10, 11), which challenges their application as highly specific membrane biomarkers for tumor targeted therapy in vivo. As observed with therapeutic nucleic acids, the nuclease degradation is a major issue for aptamers. At present, this issue has been partly addressed by covalently attaching other molecules to protect the aptamers (12). Other studies have also demonstrated that chitosan nanoparticles could protect encapsulated DNA from nuclease degradation (13, 14). It is worth noting that these means inevitably affect the specific targeting capacity of the aptamers. Mucin 1 (MUC1) is a large transmembrane glycoprotein that is highly expressed on the surface of most adenocarcinomas, including ovarian cancer, breast cancer, and lung cancer, with relatively low expression in normal cells (8). The MUC1 aptamer is a 25-base pair DNA aptamer

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that has the capacity to distinguish between MUC1-positive tumor cells and MUC1-negative cells (15). Yan Hu and co-workers formulated an MUC1 aptamer-doxorubicin complex through intercalating doxorubicin into the DNA structure of the MUC1 aptamer (8). Yu-Fen Huang and co-workers covalently linked doxorubicin to the DNA aptamer sgc8c through an acid-labile linkage that can be cleaved inside the acidic endosomal environment (9). However, there are few reports describing the application of the conjugates of aptamers and antitumor drugs in vivo. Thermosensitive hydrogels are a kind of stimuli-responsive hydrogel that can sense the temperature changes in the surrounding environment and then release the loaded drug, which makes them a good drug carrier (16-20). The injectable thermosensitive hydrogel generated from chitosan and β-glycerophosphate (GP) has attracted our attention (21, 22). The system can maintain a stable liquid state at or below room temperature, and when the temperature rises to approximately 37 °C, the system undergoes the sol-geltransition (23, 24). Yingchun Jiang and co-workers developed a chitosan-based thermosensitive hydrogel containing paclitaxel for intratumoral delivery (25). Jiayu Xing and co-workers formulated topotecan hydrochloride liposomes loaded in thermosensitive hydrogels for intratumoral administration (26). Compared with the direct systemic administration of a drug (fast release), in addition to the sustained drug release, the benefits of hydrogels are the high loading and coverage protecting capacity towards encapsulated molecules, which guarantee a prolonged action time between drugs and tumors, improved therapeutic efficiency and reduced toxicity of normal tissue simultaneously. However, these types of hydrogel systems containing drugs or drug carrierslack the specific intracellular uptake for tumor cells. In this study, we developed a microenvironment-responsive MUC1 aptamer-guided acid-labile nanoconjugate (APT-DOX) within microporous hydrogels, which exhibit a sol-gel transition

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upon intratumoral injection, resulting in the protection and controlled release of APT-DOX with the shielding of the gel network. Moreover, the released APT-DOX is intracellularly by the tumor cells’ over-expressing MUC1 protein, and the tumor microenvironment releases free DOX via the rupture of the acid-labile linkage under lysosome acidic conditions to achieve further anti-tumor treatment in the nucleus. The cytotoxicity and cellular uptake efficiency of APT-DOX and free DOX against a human breast tumor cell line (MCF-7) and a human liver tumor cell line (Hep G-2) were compared. Finally, we assessed the in vivo antitumor effect of APT-DOXCS/GP in nude mice bearing the MCF-7 tumor.

RESULTS AND DISCUSSION

MALDI-TOF MS study of APT-DOX

The synthesis of APT-DOX was confirmed by MALDI-TOF mass spectrometry (Figure S1). The molecular weight of APT-DOX is 8,610.95 Da, so the peak at 8,610.95 Da confirmed the synthesis of APT-DOX. The peak at 8,067.43 Da represented the conjugate of APT and a hydrazone without DOX. In addition, the peak at 7,860.2 Da represented the APT.

In vitro cellular uptake studies Fluorescence microscopy and flow cytometry were employed to investigate the in vitro cellular uptake of free DOX and APT-DOX into MUC1-positive (MCF-7) and -negative (Hep G2) cell lines. The results of the fluorescence microscopy showed that the cellular uptake of APTDOX by MCF-7 cells was significantly higher than that of free DOX (Figure 1A, C), but there was no significant difference in the Hep G-2 cells (Figure 1B, D). The results suggested that 5 Environment ACS Paragon Plus

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APT-DOX improved the uptake efficiency in MUC1-positive cell lines, while it did not improve the uptake efficiency in MUC1-negative cell lines. This finding may be observed due to the specific selectivity of the MUC1 transmembrane glycoprotein over-expressed on the surface of MCF-7 cells. A MUC1 aptamer can be combined with the glycoprotein to promote the uptake of DOX into the cell. There is no over-expression of the MUC1 glycoprotein on the surface of Hep G-2, so the cellular uptake of APT-DOX and free DOX were not different. In addition, the results of flow cytometry further proved that the aptamer MUC1 can improve the cell uptake rate by MUC1-positive cells. As presented in Figure 1E, the fluorescent signal produced by APTDOX was significantly higher than that produced by free DOX in MUC1-positive MCF-7 cells. In contrast, as shown in Figure 1F, the fluorescent signals produced by APT-DOX and free DOX in MUC1- negative Hep G-2 were similar.The results further proved that APT-DOX could promote the uptake of DOX by MUC1-positive cells, while it was not effective in the MUC1negative cells.

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Figure 1. In vitro cell uptake by MUC1-positive (MCF-7) and -negative (Hep G-2) cell lines. (A-D) Fluorescence microscopy images of MCF-7 and Hep G-2 cells after incubation with free DOX (A and B) or APT-DOX (C and D) for 2 hours at 0.5 µM with the same exposure of 770 ms. (E and F) Flow cytometry histogram profiles of MCF-7 (E) and Hep G-2 cells (F) after incubation with either free DOX (b) or APT-DOX (c), while (a) is the control signals generated by untreated cells. In vitro cell cytotoxicity assays after MCF-7 cells (G) and Hep G-2 (H) cells were treated with aptamer MUC1, APT-DOX, or free DOX for 2 hours; cells were subsequently grown in fresh medium (10% FBS) for further 48 h. Data are shown as the mean ± SD (n = 3). *indicates p < 0.05 versus free DOX group.

In vitro cytotoxicity study To investigate whether the cytotoxicity to MUC1-positive cells treated with APT-DOX was improved, MTT viability assays were performed on MCF-7 cells and Hep G-2 cells by APTDOX and free DOX. As shown in Figure 1G, compared with the cytotoxicity generated by free DOX, the cytotoxicity produced by APT-DOX against MUC1-positive MCF-7 cells was significantly increased. By contrast, there was no difference between the cytotoxicity produced by APT-DOX and free DOX against MUC1-negative Hep G-2 cells (Figure 1H). In addition, as presented in Figure 1G, H, the aptamer MUC1 was not toxic towards the two cell lines, indicating that the aptamer itself was relatively safe with respect to the cells. The results indicated that the aptamer MUC1 tends to promote cell cytotoxicity in MUC1-positive MCF-7 cells but had no toxicity toward MUC1-negative Hep G-2 cells.

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Preparation of CS/β-GP hydrogels and drug loading The appearance of different hydrogels at ambient temperature or 37 °C was presented in Figure 2. The unloaded hydrogels were transparent and flowing in solution at ambient temperature (Figure 2A), while the hydrogels loaded with APT-DOX were red (Figure 2C). The phase of the hydrogel changed from liquid to gel (Figure 2B, D) in 3 min at 37 °C. Figure 2E indicates that the surface of unloaded CS/β-GP hydrogel is smooth and an irregular porous structure, while the porous structure could be applied to load drugs and control the release rate of loaded drug. Moreover, no obvious differences between the unloaded and loaded CS/β-GP hydrogels (Figure 2F) could be observed, which demonstrated that the loaded APT-DOX had no impact on the structure orthe thermosensitive phase-changing behavior of CS/β-GP hydrogels.

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Figure 2. The appearance of different hydrogels at ambient temperature or 37 °C. (A) Unloaded CS/β-GP hydrogels at ambient temperature; (B) unloaded CS/β-GP hydrogels at 37 °C; (C) CS/ β-GP hydrogels loaded with APT-DOX at ambient temperature; (D) CS/β-GP hydrogels loaded with APT-DOX at 37 °C. The SEM micrographs of unloaded CS/β-GP hydrogels (E) and APTDOX-loaded CS/β-GP hydrogels (F).

Rheological studies As presented in Figure 3, the complex viscosity of CS/β-GP hydrogel is very low in a certain temperature range. When the temperature rises to a certain temperature, the η* will increase obviously. At the same time, the phase of the hydrogel changed from liquid to gel. The gelation temperature of blank CS/β-GP gels, DOX-loaded hydrogels and APT-DOX loaded hydrogels were 29 ± 0.30 (Figure 3A), 32 ± 0.25 (Figure 3B) and 33 ± 0.47 °C (Figure 3C), respectively. Compared with the blank CS/β-GP hydrogel, the gelation temperature of DOX and APT-DOX loaded hydrogels was not significantly changed. All of them were lower than 37 °C (physiological temperature), which ensures the smooth transformation of the hydrogel in the body.

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Figure 3. The sol-gel temperature of (A)blank CS/β-GP solution, (B) DOX-loaded CS/β-GP solution and (C) APT-DOX-loaded CS/β-GP solution. (D) is the in vitro cumulative release of DOX from APT-DOX-loaded CS/β-GP hydrogels at pH 5.5, pH 6.8 or pH 7.4. All data are shown as the mean ± SD, n = 3.

In vitro drug release study To assess the drug release behaviors of APT-DOX-loaded hydrogel in PBS with different pH values (5.5, 6.8, and 7.4), the release of Adriamycin from hydrogel was performed through membranes. As is shown in Figure 3D, hydrogels exhibited sustained drug release for 120 h, and

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with the decrease of pH value of the release medium, the cumulative release of doxorubicin graduallyincreased. After 120 h, the corrosion of the hydrogel skeleton in pH 5.5 PBS was more obvious than in pH 6.8 and 7.4 PBS. This may be due to the distinct degradation of the hydrogel under different acidic microenvironments (27). However, the cumulative release of the drug in the hydrogel in pH 5.5 PBS proved to be less than 80% at 120 h, indicating the remainder of the DOX may be released with the further degradation of the hydrogel matrix. In vivo antitumor effect studies As illustrated in Figure 4A, the average relative nude mouse body weights of in the saline, CS/GP, DOX-CS/GP and APT-DOX-CS/GP groups were 99.26%, 96.84%, 92.67% and 95.95%, respectively. The relative body weight of the DOX-CS/GP group showed a significant difference compared with the saline group (p < 0.05), the relative body weight of the DOX-CS/GP group decreased clearly compared with the APT-DOX-CS/GP group (p < 0.05), and there was no significant difference among the other three groups. Moreover, as shown in Figure 4B, the average relative tumor volumes of saline, CS/GP, DOX-CS/GP and APT-DOX-CS/GP on the sixth day were 140.16%, 137.16%, 112.39% and 107.41%, respectively. It can be seen that the tumor volume of the saline and CS/GP groups grew rapidly due to the absence of DOX, while the tumor growth rate of DOX-CS/GP and APTDOX-CS/GP groups was clearly decreased (p < 0.01). And the relative tumor volume was significantly different between the DOX-CS/GP group and the APT-DOX-CS/GP group (p < 0.05). After been intratumorally injected, the APT-DOX-CS/GP group exhibited a sol-gel transition, resulting in a controlled release of APT-DOX with the shielding of the gel network. As shown in Figure 3D, the release of APT-DOX from CS/β-GP hydrogels was extended for 120 h in a different dissolution medium, indicating that the drug release could also be prolonged

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at the tumor site in vivo. Different from the passive diffusion of free DOX released from the CS/β-GP hydrogels, the intracellular uptake of released APT-DOX has been demonstrated as MUC1-mediated endocytosis. In other words, the APT nanoconjugate increases selectivity of tumor cells to DOX, although the total amount of the released drug may be reduced by the control of the CS/β-GP gels. In addition, the toxicity of DOX-CS/GP had an effect on the body weight of nude mice. The histological changes to the tumors dissected from nude mice bearing MCF-7 tumors treated with saline, CS/GP, DOX-CS/GP and APT-DOX-CS/GP are shown in Figure 4C-F. Compared with the saline and CS/GP groups, both DOX-CS/GP and APT-DOX-CS/GP groups damaged the tumor cells, but the degree of tumor cell damage from the APT-DOX-CS/GP group is more obvious than that of DOX-CS/GP group; from Figure 4C-F, more ruptured tumor cells can be seen. In addition, from the histological analysis in Figure S2, no obvious heart damage was observed in the saline, CS/GP and APT-DOX-CS/GP groups. In contrast, the group treated with DOX-CS/GP displayed critical fragmentation and pathological changes in the heart. In conclusion, the above results suggested that the combination of doxorubicin and aptamer decreased the toxicity of DOX.

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Figure 4. In vivo antitumor effect studies of saline, CS/GP, DOX-CS/GP and APT-DOX-CS/GP were carried out in nude mice with MCF-7 tumors. (A) Relative tumor volume; (B) Relative body weight. (All data are shown as the mean ± SD, n = 6).* indicate p < 0.05, ** indicate p < 0.01 versus saline group. # indicate p < 0.05 versus DOX-CS/GP group. Histological changes of

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tumor dissected from nude mice bearing MCF-7 tumors treated with saline (C), free DOX (D), CS/GP (E) and APT-DOX-CS/GP (F). The tumor was stained with H&E and observed by optical microscopy (Olympus, 400×).

In summary, a MUC1 aptamer-guided acid-labile nanoconjugate-loaded thermosensitive hydrogel was developed for the intratumoral administration of anti-tumor drugs. In this study, the cellular uptake of the nanoconjugate APT-DOX by MCF-7 cells was significantly higher than that of free DOX, which demonstrated that the MUC1 aptamer could be combined with the glycoprotein to promote the uptake of DOX into the cell. Compared with the blank CS/β-GP thermosensitive hydrogel, the gelation temperature of DOX and APT-DOX-loaded hydrogels was not significantly changed, which ensures the smooth transformation of the hydrogel in vivo. Moreover there was significant difference in the relative tumor volume curve between the DOXloaded hydrogel group and the APT-DOX-loaded hydrogel group, and the APT-DOX-loaded hydrogels showed lower toxicity compared with DOX-loaded hydrogel group. These results indicating that the acid-labile bioconjugate could improve the intracellular ability of DOX to tumor cells due to the actively targeting selectivity of of APT, while the CS/GP hydrogel structure could also protect APT-DOX from being degraded. All of these would contribute to the enhanced anti-tumor efficacy of APT-DOX-loaded hydrogel.

EXPERIMENTAL SECTION

Materials and chemicals

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MUC1 aptamer (5’-GCA GTT GAT CCT TTG GAT ACC CTG G-3’) with a 5’-sulfydryl group were synthesized by Takara Bio Co., Ltd. (Dalian, China). The 6-maleimidocaproyl hydrazone derivative prodrug of the anthracycline antibiotic doxorubicin (DOXO-EMCH) was obtained from MedChemexpress, LLC (Monmouth Junction, NJ, USA). Doxorubicin hydrochloride (DOX·HCl) was supplied by Zhejiang Hisun Pharmaceutical Co., Ltd. (Taizhou, China). CS (MW: 100000, degree of deacetylation: more than 90%) was provided by Zhejiang Aoxing Biotechnology Co., Ltd. (Taizhou, China). β-GP was purchased from Alfa Aesar Chemical Co., Ltd. (Shanghai, China). SephadexG-50 was bought from Shanghai WaihingBiochemicals Co., Ltd. (Shanghai, China). Dimethyl sulfoxide (DMSO) was obtained from Lingfeng Chemical Co., Ltd. (Shanghai, China). All other chemicals used were of analytical grade. Synthesis and purification of APT-DOX The synthesis of ATP-DOX conjugates was similar to that of immunoconjugates (28, 29). DOXO-EMCH was dissolved in DMSO with its concentration of 10 mM. The MUC1 aptamer (300 nmol) with a sulfydryl group at the 5’ end was dissolved in PBS (10 mM, pH 7.4). Then, the two solutions were mixed and incubated at 4 °C for 24 h. The product conjugates were purified by a Sephadex G-50 micro-column to remove unconjugated DOX, and the amount of conjugate was measured by HPLC. Finally, the solution was lyophilized for 24 h to obtain APTDOX. The matrix-assisted laser desorption/ionization time of flight mass spectrometry study (MALDI-TOF MS) was performed by MALDI-TOF mass spectrometry system from Sequenom (San Diego, USA) (30). The synthesis of ATP-DOX was confirmed by the appearance of the peak that represented the molecular weight of the product.

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In vitro cellular uptake studies MCF-7 and Hep G-2 cells were seeded into 24-well plates at a density of 5 × 104 cells per well and incubated overnight. Then, the medium was removed, and the cells were treated with DOX (0.5 µM) or APT-DOX (0.5 µM) for 2h. After incubation, the cells were washed twice with PBS and fluorescence microscopy (Olympus IX 53, Osaka, Japan) was used to observe the cells. Flow cytometry (Miltenyi, MACSQuant Analyzer 10, USA) was employed to investigate the cellular uptake of free DOX and APT-DOX. In vitro cytotoxicity study MCF-7 cells were cultured in RPMI 1640 medium (pH 7.4). Hep G-2 cells were cultured in DMEM (pH 7.4) and provided with 10% fetal bovine serum (FBS) containing 50 U/ml penicillin/streptomycin and maintained in 90% relative humidity with 5% atmosphere at 37 °C. An MTT assay was applied to evaluate the cytotoxicity of free MUC1 aptamer, free DOX and APT-DOX against Hep G-2 and MCF-7 cell lines. The cells were seeded in 96-well plates at a density of 4 × 103 cells per well (100 µL) and incubated overnight. After the cells adhered, the upper liquid was discard, 0.5 µM of free MUC1 aptamer, free DOX and APT-DOX (dissolved in 100 µL cell culture medium) were added, and the cells were incubated for 2 h at 37 °C. The cells were then washed twice with PBS, and 200 µL fresh cell culture mediumwas added and the cells were further incubated for 48 h at 37 °C. Twenty microliters of MTT solution (5 mg/mL in PBS) was added to each well, and cells were incubated for 4 h. Then, the medium was removed and 150 µL DMSO was added to each well to dissolve the cells. The plate was agitated for 5 min,the absorbance at 570 nm was read by an Enzyme Immunoassay Instrument (Thermo, 51119000, USA) and the cell viability (%) was confirmed.

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Preparation of CS/β-GP hydrogels and drug loading Briefly, the 2% (w/v) clear CS solution was prepared by dissolving a certain amount of chitosan in 0.1 M acetic acid at room temperature with stirring for 12 h and then stored at 4 °C. The 60% (w/v) β-GP clear solution was prepared by dissolving in water at room temperature. Under the conditions of the ice water bath, β-GP solution was added to the CS solution with a certain volume ratio, and the mixture was continuously stirred for 10 min until it was well mixed and then stored at 4 °C. DOX powder and APT-DOX powder was mixed with unloaded hydrogels in an ice bath under stirring for 30 min, respectively. Thus, the drug-loaded hydrogels were obtained, while the dosages of DOX and APT-DOX loaded in the hydrogels were 0.15 and 2.38 mg/mL, respectively. Measurement of gelation time The gelation time was evaluated using the tube-inverting method (31). Two milliliters of hydrogel sample was placed in a 10 mL test tube, and the test tube was placed in 37 ± 1 °C water bath.The test tube was invertedevery 15 s to check the fluidity of hydrogel. The time at which the liquid is no longer flowing was determined to be the gelation time. Morphological study The morphological study was performed by a Scanning Electron Microscope (HITACHI S3000N, Tokyo, Japan) at a 7.00 kV to obtain the internal spatial structure of the hydrogels (32, 33). The blank hydrogels were prepared,incubated in a 37 ± 0.5 °C water bath for 1 h to gel thoroughly, and then freeze-dried for 48 h. Rheological studies

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The rheology study was performed by Physica MCR 302 (Anton paar, Austria) in triplicate using the temperature sweep test for the definition of the sol–gel temperature. A precise amount of the blank CS/β-GP solution, DOX-loaded CS/β-GP solution and APT-DOX-loaded CS/β-GP solution was put on the test plate. The complex viscosities (η*) of samples at varied temperatures (15-45 °C) were measured under 1Hz and heating rate 1 °C/min. The sol-gel temperature was defined as the temperature at which η* changes significantly (34). In vitro drug release study In this study, a membraneless model was used to study the drug release from hydrogel in vitro. One milliliter APT-DOX-loaded hydrogel solution was added to a 10 mL test tube with a plug and incubated in a water bath at 37 °C for 1 h to be fully gelled. Then, 10 mL phosphate buffered saline (PBS) with different pH values (5.5, 6.8, and 7.4) as release medium was added. These tubes were incubated at 37 °C in a constant-temperature shaker (SHZ-82, Saibole Instrument Co., Ltd., Shanghai) at the oscillation speed of 100 rpm. At scheduled times, all release media were taken out, and the same volume of fresh release medium was added to maintain the total volume. The concentration of DOX in the release medium was measured using SPD-20A Shimadzu instrument (Tokyo, Japan) by high-performance liquid chromatography (HPLC) method. The column was a reversed-phase column (Inertsil ODS-SP, 4.6 mm × 250 mm, i.d. 5 mm, GL Sciences, Tokyo, Japan). The column temperature was 35 °C and the wavelength was 485 nm. A mixture of acetonitrile and buffer (10 mm Tris-HCl, pH 7.4) (30 : 70, v/v) was used as the mobile phase at a flow rate of 1.0 mL/min. Methodological studies, such as linearity, specificity, precision within and between days, were also demonstrated to satisfy the requirements of the methodology. A linear detector response (r = 0.9993) was observed over the concentration range

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of 4.05-40.50 µg/mL (the limitof detection is 0.0135 µg/mL) and blank solvent did not interfere with the determination of DOX. In vivo antitumor effect studies All of the animal experiments were conducted in accordance with guidelines that were evaluated and approved by the Ethics Committee of China Pharmaceutical University, and the humane care of the animals was carried out for these animal studies. BALB/c nude mice (female, 4 weeks old) were supplied by Slrc Laboratory Animal Company and acclimatized for 7 days. To establish the tumor models, a mixture with 0.1 mL of MCF-7 cell suspension (1 × 107 cells/mL) and 0.1 mL Matrigel (BD Biosciences) was injected into the armpit of the mice. Seven days before inoculation, every mouse was injected with estradiol (3 mg/kg) by intramuscular injection, and then every mouse was injected with estradiol (3 mg/kg) every week. When the volume of solid tumors reached 300 mm3, the nude mice were randomly divided into four groups (n = 6): (1) normal saline, (2) DOX-loaded hydrogel, (3) blank hydrogel,and (4) APT-DOX-loaded hydrogel. All the formulations were injected intratumorally at dose of 3 mg/kg every three days for a total of two injections. The body weight and tumor volume of nude mice were measured every day from day 0 to day 6. After 6 days of observation, blood was collected from nude mice, and heparin was added for blood routine examination. Then, the tumors and major organs were isolated, weighed, measured the spleen index, fixed in 10% formalin overnight, and embedded in paraffin for further evaluation. Statistical analysis

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The SPSS15.0 (SPSS, Chicago, IL, USA) software was used for statistical analysis. The obtained data were expressed as the mean value ± standard deviation (SD) of at least performed in triplicate. p < 0.05 was considered to be statistically significant.

ASSOCIATED CONTENT Supporting Information The following files are available free of charge. Figure S1-S2 (PDF) Table S1 (PDF)

AUTHORS’ INFORMATION Corresponding Author *To whom correspondence should be addressed.E-mail addresses: [email protected] (Prof. Zhenghong Wu), [email protected] (Dr. Xiaole Qi). Fax: +0086-025-83179703. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was financially supported by the National Natural Science Foundation of China (No. 81402859).

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Graph of TOC

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