Co-delivery of doxorubicin and 131I by thermosensitive micellar-hydrogel for enhanced in situ synergetic chemoradiotherapy

Journal of Controlled Release 220 (2015) 456–464

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Co-delivery of doxorubicin and 131I by thermosensitive micellar-hydrogel for enhanced in situ synergetic chemoradiotherapy Pingsheng Huang a,b,1, Yumin Zhang c,1, Weiwei Wang d, Junhui Zhou a,b, Yu Sun a,b, Jinjian Liu c, Deling Kong d, Jianfeng Liu c,⁎, Anjie Dong a,b,⁎⁎ a Department of Polymer Science and Technology and Key Laboratory of Systems Bioengineering of the Ministry of Education, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China b Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin 300072, China c Tianjin Key Laboratory of Radiation Medicine and Molecular Nuclear Medicine, Institute of Radiation Medicine, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300192, China d Institute of Biomedical Engineering, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300192, China

a r t i c l e

i n f o

Article history: Received 1 July 2015 Received in revised form 13 October 2015 Accepted 7 November 2015 Available online 10 November 2015 Keywords: Chemoradiotherapy Micellar hydrogel Doxorubicin 131 I Tumor treatment

a b s t r a c t Combined chemoradiotherapy is potent to defeat malignant tumor. Concurrent delivery of radioisotope with chemotherapeutic drugs, which also act as the radiosensitizer, to tumor tissues by a single vehicle is essential to achieve this objective. To this end, a macroscale injectable and thermosensitive micellar-hydrogel (MHg) depot was constructed by thermo-induced self-aggregation of poly(ε-caprolactone-co-1,4,8-trioxa[4.6]spiro-9undecanone)-poly(ethyleneglycol)-poly(ε-caprolactone-co-1,4,8-trioxa[4.6]spiro-9-undecanone) (PECT) triblock copolymer micelles (Ms), which could not only serve as a micellar drug reservoir to locally deliver concentrated nano chemotherapeutic drugs, but also immobilize radioisotopes at the internal irradiation hot focus. Doxorubicin (DOX) and iodine-131 labeled hyaluronic acid (131I-HA) were used as the model therapeutic agents. The aqueous mixture of drug-loaded PECT micelles and 131I-HA exhibited sol-to-gel transition around body temperature. In vitro drug release study indicated that PECT/DOX Ms were sustainedly shed from the native PECT/ DOX MHg formulation, which could be internalized by tumor cells with rapid intracellular DOX release. This hydrogel formulation demonstrated considerable in vitro antitumor effect as well as remarkable radiosensitization. In vivo subcutaneous injection of PECT MHg demonstrated that 131I isotope was immobilized stably at the injection location and no obvious indication of damage to major organs were observed as indicated by the histopathological analysis. Furthermore, the peritumoral injection of chemo-radiation therapeutic agents-encapsulated MHg formulation on tumor-bearing nude mice resulted in the desired combined treatment effect, which significantly improved the tumor growth inhibition efficiency with minimized drug-associated side effects to major organs. Consequently, such a thermosensitive MHg formulation, which enabled the precise control over the dosage and ratio of combination therapeutic agents to obtain the desired therapeutic effect with a single drug administration and reduced side effects, holds great potential for spatiotemporally delivery of multiple bioactive agents for sustained combination therapy. © 2015 Published by Elsevier B.V.

1. Introduction In present clinical oncology practice, chemoradiotherapy, conducted by concurrent administration of chemotherapy and radiotherapy agents, is an important treatment regimen for many types of cancer, including hepatic, lung, rectal, head and neck, esophageal and cervical

⁎ Corresponding author. ⁎⁎ Correspondence to: A. Dong, Department of Polymer Science and Technology and Key Laboratory of Systems Bioengineering of the Ministry of Education, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China. E-mail addresses: [email protected] (J. Liu), [email protected] (A. Dong). 1 Pingsheng Huang and Yumin Zhang contributed equally to this work.

http://dx.doi.org/10.1016/j.jconrel.2015.11.007 0168-3659/© 2015 Published by Elsevier B.V.

cancers [1–4]. Although external radiation therapy (ERT) offers a relatively well controlled and modulated approach to induce radiation damage, disadvantages including nondistinctive destruction of normal tissue adjacent to tumors as well as in the path of the ion beam, the need of high radiation doses for penetrating tissues with a large field or volume still remain [5,6]. In contrast, the development of nanotechnology significantly promotes the advance of internal radiotherapy (IRT), which demonstrated great potential in improving therapeutic efficiency and reducing the damage to normal tissues. Typically, appropriate β-emitters, mainly including yttrium-90 (90Y), iodine-131 (131I), copper-67 (67Cu), copper-64 (64Cu), rhenium-188 (188Re) and lutetium-177 (177Lu) delivered by radiolabeled antibodies or nanoparticles via intravenous administration induce apoptosis by generation of

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DNA-damaging free radicals through localized decay in target cells [7–9]. However, the radioresistance, which is mainly caused by the accelerated cell repopulation, proficient repair of radiation damage, hypoxia and various molecular inhibitors of cell apoptosis, is one of the major factors that limit the IRT outcome [10,11]. The combination with a radiosensitizer, most of which are clinically used anticancer drugs, has been proved to be a feasible approach to decrease the radioresistance [12–14]. Generally, the construction of multifunctional nanocarriers rendered the possibility of spatiotemporally simultaneous presenting radiosensitizer and radioisotopes [15,16], however, two major challenges existed: (1) co-encapsulation of radioisotopes and radiosensitizers in a precise ratiometric within one multifunctional nanocarrier was difficult due to the significantly different physicochemical characters between radioisotopes and radiosensitizers, such as solubility; (2) unexpected rates and levels of drug bioavailability after intravenous administration due to low targeting efficiency and nonspecific elimination of the nano system [17–24]. Alternatively, in situ formed hydrogels could be employed as a flexible platform for local drug delivery. The thermosensitive PCL-PEG-PCL hydrogel matrix were used to load 188Re–Tin colloid as radionuclide and liposomal doxorubicin for combined chemoradiotherapy in hepatocellular carcinoma mouse model [25]. And this system demonstrated commendable tumor inhibition efficiency in vivo. However, several obstacles including pre-quenching treatment, slow degradation rate and heterogeneous drug encapsulation could possibly restrict its further clinical application [26–29]. Hence, delivery systems, which could contemporaneously deliver radionuclides and radiosensitizers in the tumor tissue, are urgently needed. Herein, highly concentrated drug-loaded micelles solution mixed with 131I labeled HA was peritumorally injected, which then transformed into semi-solid hydrogel due to the thermo-induced selfaggregation of micelles (as shown in Scheme 1). This hydrogel formulation was expected to act as a sustained local delivery depot of nano drugs and radioisotopes. In contrast to conventional i.v. injection of nanoparticulate formulations, this locally administrated system could persistently release the encapsulated therapeutic agents, and improve local drug accumulation and retention, which is anticipated to

Scheme 1. The schematic diagram of the formation of PECT/DOX MHg as the nanodrug and radionuclide reservoir and its following action model of in situ chemoradiotherapy.

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significantly improve the therapeutic efficiency with minimized drugassociated side effects. The model drug, doxorubicin (DOX), a kind of FDA-approved chemotherapeutic drug, exerting radiosensitization by inhibiting the biosynthesis and duplication of DNA, and decreasing the repair of radiation-induced DNA damage [30–32], was encapsulated in PECT micelles (termed as PECT/DOX Ms). 131I-labeled hyaluronic acid (131I-HA) was immobilized as the hot focus due to its proper half-life and straightforward chemical conjugation [33–37]. The preparation of micellar hydrogel (MHg), drug release profile and endocytosis were investigated systematically. The cytotoxicity and radiosensitization were evaluated by CCK-8 and clonogenic survival assay. In addition, in vivo biodistribution of 131I-immobilized MHg was tracked by γ-camera after subcutaneous injection and the histopathological effect of radiation on major organs was evaluated. Subsequently, in vivo antitumor activity was evaluated on HepG2 tumor xenografted femal Balb/c nude mice models. 2. Materials and methods 2.1. Materials Doxorubicin hydrochloride was purchased from Wuhan Hezhong Biochem Co. Ltd. (Wuhan, China). N-(3-Aminopropyl)-imidazole (API), 2, 2, 2-Trifluoroethanol (TFE), dimethyl sulfoxide (DMSO), 4, 6diamidino-2-phenylindole (DAPI), and (5-Dimethylthiazol-2-yl)-2, 5diphenyl tetrazolium bromide (MTT) were purchased from SigmaAldrich (St. Louis MO, USA). N-(3-Dimethylaminopropyl)-Nethylcarbodiimide hydrochloride (EDC), N-Hydroxysuccinimide (NHS), chloramine-T and sodium metabisulfite were purchased from J&K Chemical LTD. Iodine-131 radionuclide, 5 mCi (185 MBq), specific activity: N5 Ci (185 GBq)/mg, 0.1 M NaOH (pH 12–14) 500 mCi/mL was provided by PerkinElmer company. Hyaluronic acid (HA) with molecular weight of 7 KDa was purchased from FuRuiDa pharmaceutical Co. LTD (Shandong, China). 2.2. The synthesis of 131I-labeled hyaluronic acid (131I-HA) 131

I-HA was prepared by a two-step method (shown in Scheme 2). Firstly, HA was conjugated with API through amidation reaction [38,39]. Generally, HA (240 mg, 0.3 mmol) was dissolved in 5 mL anhydrous DMSO for 4 h at 80 °C. The solution was cooled to room temperature, then EDC (118 mg, 0.6 mmol) and NHS (142 mg, 1.2 mmol) were added, which were stirred for another 2 h to activate carboxyl groups. Subsequently, API (77 mg, 0.6 mmol) dissolved in 1 mL anhydrous DMSO was added dropwise, and then the reaction was conducted at RT for another 24 h. The resultant mixture was dialyzed against deionized water for 72 h with a water change frequency of every 12 h. The final product was lyophilized and analyzed by 1H NMR. Subsequently, HA-API was radioiodinated with 131I by chloramine-T method [40–42]. Into a plastic test tube (1 × 10.0 cm) were added successively the HA-API (50 mg) dissolved in 2 mL sodium phosphate buffer (pH = 8.5, 5 mmol), radioactive iodide (10 μL), and chloramine-T solution (2 mg/mL, 25 μL, pH = 8.5). After gentle shaking, the mixture was allowed to stand for 2 min to allow the radioiodination to take place. Then, sodium metabisulfite solution (10 mg/mL, 50 μL, pH = 8.5) was added to stop the radioiodination. The reaction mixture was precipitated against methanol for three times to get the final 131 I-HA. The radiochemical purity was measured by radioactive thin-layer chromatography (TLC) with 2470 Automatic Gamma Counter (PerkinElmer LTD). Detailed procedure: apply 5 μl (1 drop) of the prepared 131 I-HA water solution on the chromatographic paper, which was eluted using a mixture of 20 volumes of water and 80 volumes of methanol along the path of 20 cm. After drying the paper, the distribution of radioactivity was detected by an automatic gamma counter, which was repeated for three

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Scheme 2. Synthetic route of 131I-HA.

times. The radiochemical impurity was identified by the following equation: %peak A ¼

sum of counts measured for the peak A  100% sum of counts for all chromatogram

ð1Þ

In addition, the specific activity was determined by Radioactivity meter (Capintec, CRC-25R). All of the radiological experiments were carried out strictly according to the 449th decree of Radioisotope and Ray Devices Safety and Protective Regulations which was issued by State Council of the People's Republic of China. 2.3. Preparation of DOX-loaded PECT micelles and thermosensitive hydrogels PECT copolymer was synthesized according to our previously reported methods and characterized by 1H NMR, GPC, FT-IR, XRD and DSC (shown in Fig. S1) [43,44]. The DOX-loaded PECT micelles (PECT/ DOX Ms) were prepared by a nanoprecipitation method [45]. Typically, DOX (1 mg) and PECT copolymer (100 mg) were co-dissolved in 4 mL TFE and then the mixture was added dropwise into 20 mL deionized water. After evaporation of TFE, the solution was lyophilized to obtain freeze-dried powder of PECT/DOX Ms. Blank PECT Ms were prepared with the same procedure without the addition of DOX. The size and morphology of blank and DOX-loaded PECT Ms were measured by DLS and TEM, respectively. UV–Vis spectrophotometry was employed to determine the drug loading amount (DLA) and drug loading efficiency (DLE). The freeze-dried powder of PECT Ms or PECT/DOX Ms was redispersed in saline at 25 °C with a concentration of 25% (w/w). In order to verify whether the incorporation of I-HA (without radioactivity) had influence on the gelation property, different amounts of I-HA were added. The thermosensitive assembly behavior was investigated by the tube inversion method and tracing the viscosity change in realtime using a Fluids Rheometer equipment (Stress Tech, Rheological Instruments AB) according to our previous method [46,47]. 2.4. In vitro drug release studies In vitro DOX release was performed according to our previous work [46]. The concentration of DOX in the release medium was determined by UV–Vis at 480 nm according to a calibration curve. Subsequently, the cellular endocytosis of free DOX and PECT/DOX Ms dissociated from hydrogel formulation was evaluated by confocal laser scanning microscopy (CLSM) (TCS SP8, Leica). Culture media were supplemented with 10% heat-inactivated fetal bovine serum (FBS), penicillin (100 U/mL) and streptomycin (100 μg/mL). HepG2 cells were maintained in DMEM/High Glucose media (Hyclone) at 37 °C. Briefly, 500 μL of HepG2 cell suspension was seeded on a confocal microscopic dish at a density of 2 × 105. Then, PECT/DOX Ms solution taken from the release medium of the PECT/DOX MHg formulation was added and the concentration of DOX was maintained at 5 μg/mL. After incubation for 0.5 h or 4 h, cells were washed with PBS for three times and then stained with

DAPI (1 μg/mL in PBS) for 5 min. Cellular endocytosis was examined by CLSM. Cells treated with free DOX were used as the positive control. 2.5. In vitro cytotoxicity analysis and radiosensitization evaluation The cytotoxicity of PECT Ms, I-HA, PECT/DOX Ms and free DOX were evaluated using HepG2 cells by CCK-8 assay. Briefly, cells were seeded in 96-well plates at a density of 5 × 103 cells/well. After incubation for 24 h under standard cell culture conditions, the culture medium was replaced with 100 μL fresh medium containing the above formulations. 48 h later, 10 μL CCK-8 solutions pre-dissolved in phosphate-buffered saline was added to each well. After incubation for another 1 h, the culture medium was cautiously extracted from the well and added to another blank well. Then, the absorbance of each sample wells was measured by a microplate reader (Thermo Scientific Varioskan Flash) at the wavelength of 450 nm. Cells without incubation with any materials and drugs were used as the blank control. The cell viability was expressed as the absorbance ratio between treatment groups and control groups. Subsequently, the radiosensitization of DOX and PECT/DOX Ms was determined by the clonogenic survival assay [48]. Cells (500–4000 per well) were seeded in 6-well plates. After attached, cells were treated with free DOX or PECT/DOX Ms at subtoxic level together with different doses of IR for 48 h. In order to prevent the influence of radiation between different wells, the experiment was carried out by separating the culture plates with lead plate at a thickness of 5 cm. Then the media was replaced with fresh culture media. After incubation for 10 days, cells were fixed with fresh 4.0% paraformaldehyde for 15 min and stained with 0.5% crystal violet for 30 min. All colonies consisted of more than 50 cells were counted. The plating efficiency (PE) and survival fraction (SF) were calculated by the following equations: PE ¼

NO:of colonies formed  100% NO:of cells seeded

ð2Þ

SF ¼

NO:of colonies formed after treatment  100% NO:of cells seeded  PE

ð3Þ

2.6. In vivo animal experiments Normal female ICR mice and HepG2 tumor xenografted bearing femal Balb/c nude mice (6–7 weeks old) were purchased from Vital River Laboratory Animal Technology Co. Ltd., China. All animals were housed individually in plastic cages in a controlled environment with free access to food and water. All the animal experiments were performed in accordance with the protocol approved by Chinese Academy of Medical Sciences. 2.6.1. Local retention of 131I-HA@ PECT/MHg and evaluation of biosafety To investigate the in vivo retention of radioisotopes, 131I-HA@PECT MHg (131I@PECT MHg, 0.3 mCi/300 μL) were injected subcutaneously

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into the normal female ICR mice (n = 15). The gamma images and radio intensity were obtained by the Kodak IS in vivo FX imaging system at given time schedules. Meantime, to investigate the radiation induced damage to major organs, mice were sacrificed (n = 3) and major organs such as heart, liver, spleen, lung, and kidney were harvested immediately. The tissue samples were then dehydrated through a graded series of ethanol solutions. The obtained tissues were fixed with 10% formalin for 1 day at room temperature and embedded in paraffin. The embedded specimens were cut into 5 μm sections onto microscopic slides and stained with hematoxylin and eosin (H&E), which were observed by an optical microscope. Mice without any treatment were taken as control. 2.6.2. Antitumor activity evaluation The in vivo anti-tumor activity was conducted using HepG2 tumor xenografted femal Balb/c nude mice. When the tumor volume reached 100–150 mm3, mice were randomly assigned to one of the following four treatment groups (n = 13): group 1, the untreated control group; group 2, peritumoral administration of PECT/DOX MHg; group 3, peritumoral administration of 131I@PECT MHg; group 4, peritumoral administration of 131I@PECT/DOX MHg. The total dose of DOX administrated was 20 mg/kg per animal and the radiation dose was 0.3 mCi per animal with the injection volume of 200 μL. Tumor volumes were measured using a caliper at designated times and calculated according to the formula (V = L × W2/2), where W is the shorter diameter and L is the longer one. Body weight of each animal was also recorded every two days. At the end of treatment, three mice were sacrificed in each group. The tumor tissues and major organs were dissected and washed in PBS, which were fixed in 4% paraformaldehyde solution overnight. The embedded specimens in paraffin were cut into 5 μm sections and then stained with hematoxylin and eosin (H&E), which were observed using an optical microscope. 2.7. Statistical analysis All data are presented as mean ± standard deviations (SDs). The differences among groups were determined using student′s t-test and p b 0.05 was considered to be statistically significant.

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3. Results and discussion 3.1. Preparation and characterization of 131I-HA 131 I-HA was prepared by the amidation reaction between the amino group on API and the carboxyl groups on HA, and followed by radioiodination with 131I by chloramine-T method [40,42]. The chemical structure and graft ratio of API were determined by 1H NMR measurement using D2O as the solvent. Characteristic signals of API on imidazole ring could be seen clearly among the range of 7.0–8.5 ppm as shown in Fig. 1A. As illustrated in Fig. 1B and C, the result of radioactive thin-layer chromatography (TLC) with automatic gamma counter demonstrated that the labeling efficiency was considerably high and the radiochemical purity was approximately 99%. In addition, the specific activity calculated by a radioactivity meter was 98.5 μCi/mg.

3.2. Preparation and characterization of PECT/DOX Ms and corresponding hydrogels The chemical structure of PECT copolymer was illustrated in Fig. 2A and the characterization results were shown in Fig. S1. PECT/DOX Ms were formed by the co-assembly of drugs and polymers, which were mainly driven by hydrophobic interaction between DOX and hydrophobic segments of amphiphilic PECT copolymer. As shown in Fig. 2B and C, both blank PECT Ms and PECT/DOX Ms exhibited regular spherical shape. Furthermore, the hydration diameter of PECT/DOX Ms was around 120 nm, slightly larger than blank PECT Ms, which could be attributed to the loading of DOX in the hydrophobic core. Subsequently, lyophilized powders of Ms were re-suspended in saline at room temperature with a concentration of 25% (w/w), during which I-HA (without radioactivity) with different weight ratios (0.5%–2% vs. PECT/DOX Ms) were added to verify its influence on the gelation property. The thermo-induced gel formation procedure was tracked by the rheological analysis. As shown in Fig. 2D and E, both solutions of blank PECT Ms and PECT/DOX Ms exhibited similar sol–gel transition, indicating that the encapsulation of DOX in the hydrophobic core has no obvious influence on the gelation behavior. In addition, the introduction of 2% of I-HA did not cause obvious differences of the transition temperature

Fig. 1. Characterization of HA-API by 1H NMR using D2O as the solvent and the radioactive counting of crude (B) and purified (C) 131I-HA product.

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Fig. 2. (A)The chemical structure of triblock copolymer PECT; the diameter and TEM image of (B) blank PECT Ms and (C) PECT/DOX Ms; (D) the optical images of sol or gel states of PECT Ms, PECT/DOX Ms, and I-HA@PECT Ms (2% of I-HA in comparation with PECT); (E) the viscosity variation of PECT Ms, PECT/DOX Ms, and I-HA@PECT/DOX Ms solutions as a function of temperature at a concentration of 25% (w/w).

Fig. 3. (A) DOX release profiles at 37 °C from the hydrogel formulation; (B) particle size and representative TEM image of Ms in the release medium; (C) endocytosis of free DOX and release sample at a drug concentration of 5 μg/mL: for each panel, images from left to right show DOX fluorescence signal in cells (red), cell nuclei stained by DAPI (blue) and overlays of two images. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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and maximum viscosity compared to DOX-loaded PECT hydrogel, indicating that the incorporation of 131I-HA as the internal radiation hot focus was feasible.

3.3. In vitro drug release and intracellular drug distribution It was encouraging that the concentrated PECT/DOX Ms could form hydrogel at body temperature. However, in order to achieve therapeutic goal, DOX should be released to maintain appropriate drug bioavailability. Hence, the DOX release profile from the MHg was studied systematically. As depicted in Fig. 3A, DOX was sustainedly released and complete release was achieved after five weeks, which was in line with our previous reported hydrogel degradation timeframe in vitro [46]. In addition, Ms was detected in the release medium, which almost remained constant size and morphology at different harvest times in comparation with fresh prepared PECT/DOX Ms (shown in Fig. 3B). Therefore, ultrafiltration was adopted to evaluate the amount of free DOX presented in the release medium and in the form of encapsulation in Ms at scheduled test intervals. As shown in Fig. 3A, the portion of DOX existed in the release medium in the form of free molecules contributed to approximate 30% of total accumulative drug release, which was close to the release profile of PECT/DOX Ms being implemented using the dialysis method at pH 7.4 (shown in Fig. S2). Hence, it was inferred that DOX was possibly released in the form of intact PECT/DOX Ms, which was shed from the parent hydrogel formulation. In order to exert therapeutic effects, drugs have to be uptaken by cancer cells. Thus, it is significant for PECT/DOX Ms to be internalized by cancer cells with rapid intracellular drug release. To this end, the cellular uptake and intracellular release behaviors of PECT/DOX Ms were evaluated by fluorescence microscopy imaging on HepG2 cell line. As shown in Fig. 3C, free DOX was quickly internalized by HepG2 cells after incubation for 0.5 h, which mainly localized in the cell nuclei. However, the samples taken from the release media of the hydrogel formulation, exhibited delayed internalization and different intracellular drug distribution. The fluorescence signal of DOX was mainly distributed in the cytoplasm after incubation for 0.5 h and remarkable nuclei accumulation was observed after 4 h, which could be partially attributed to the acidic conditions in the endo/lysosomal compartments facilitating the drug release. It was also consistent with the results of in vitro drug release of PECT/DOX Ms at different pH value (shown in Fig. S2). Furthermore, this distinction in cell internalization and intracellular release patterns further confirmed the existence of PECT/DOX Ms shedding during the drug release from hydrogel.

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Collectively, the hydrogel formulation consistently demonstrated value in providing an effective self-organized reservoir of PECT/DOX Ms for in vivo applications, which not only greatly improved the local drug accumulation and retention in tumor by local injection in avoidance of free drug exposure to normal tissues, but also rendered a convenient pathway for sustained locoregional nanomedicine endocytosis by shedding PECT/DOX Ms. 3.4. In vitro cytotoxicity analysis and radiosensitization evaluation The cell viability of both the PECT Ms and I-HA was more than 90% after incubation for 48 h even at a high concentration, indicating the good cytocompatibility (as shown in Fig. S3). As shown in Fig. 4A, the cell viability was clearly dose-dependent upon the treatment with PECT/DOX Ms or free DOX without the presence of radiation, and the IC50 concentrations were 7.1 μg/mL and 1.32 μg/mL, respectively. Then, the radiosensitization experiments were conducted using clonogenic survival assay, during which the HepG2 cells were treated with PECT/DOX Ms or free DOX at IC50 concentrations for 48 h before radiation with 131I-HA at different radiation dose. As seen from the clonogenic survival fraction depicted in Fig. 4B, the radio-response was significantly enhanced by the pretreatment with both free DOX and PECT/DOX Ms, indicating the radiosensitization activity of DOX. As reported, the therapeutic mechanism of 131I was to cause formation of oxidizing free radicals via water radiolysis through the interaction with surrounding environment, which may then not only react with intracellular macromolecules and alter cellular metabolism but also act directly on DNA and result in single- or double-strand breaks [49]. Synergistically, DOX exerted radiosensitization in the meantime by inhibiting the biosynthesis and duplication of DNA, decreasing the repair of radiation-induced DNA damage [32,50]. 3.5. In vivo animal experiments Biosecurity is always an indispensable concern for any in vivo delivery systems. The biocompatibility of the PECT MHg formulations was evaluated systematically by pathological analysis in our previous work, which was acceptable for use in therapeutic agent delivery systems in vivo [43,51]. Subsequently, the in situ retention of 131I-HA and corresponding biosafety was investigated before evaluating its therapeutic effect against tumor. As shown in Fig. 5A, the 131I@PECT/MHg injected subcutaneously was stably retained without obvious leakage to other areas of normal tissues during the time period of four weeks. And the radioactive signals gradually decayed along with the hydrogel

Fig. 4. (A) Viability of HepG2 cells after incubation with free DOX and PECT/DOX Ms for 48 h; (B) clonogenic survival assays of HepG2 cells treated with radiation 48 h at different dose after pretreatment with free DOX or PECT/DOX Ms. (a = in comparation with 131I-HA radiation, *p b 0.05, **p b 0.01). The data were expressed as mean ± SD, n = 4.

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Fig. 5. (A) γ-images and (B) histological analysis of major organs of mice after injected with 131I@PECT MHg at scheduled days. The nuclei (blue) were stained by hematoxylin, while cytoplasm and extracellular matrix (pink) were stained by eosin. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

degradation. Meanwhile, to investigate the radiation induced damage to major organs, such as heart, liver, spleen, lung and kidney, three mice were sacrificed randomly at each week and tissue slices stained by H&E were subjected to histopathological analysis. As can be seen in Fig. 5B, mice treated with brachyradiation of 131I-HA at a dose of 0.3 mCi revealed no evident changes of tissue pathological structure in comparation with blank controls, indicating that the nonspecific damage of radiation to major organs could be circumvented through the in situ delivery pathway. In order to verify if the merit of PECT MHg acting as not only the reservoir for chemotherapeutic nano agents as well as radiosensitizers but also radionuclide hot focus synergistically, could translate into improved therapeutic outcomes, we assessed the growth of xenografted HepG2 tumors following peritumoral administration of PECT/DOX MHg, 131I@PECT/MHg, or 131I@PECT/DOX MHg. As shown in Fig. 6, administration of all the three therapeutic formulations revealed significant tumor growth inhibition without remarkable fluctuation of body weight in comparation with blank control (p b 0.05). 131I@PECT/DOX MHg led to statistically significant tumor growth delay compared with

single treatment with 131I@PECT MHg, which could attribute to the radiosensitization. In addition, the therapeutic result of 131I@PECT/ DOX MHg was also significantly better than PECT/DOX MHg, indicating that the combined chemoradiotherapy could translate into improved treatment outcomes. Furthermore, the treatment response to tumors was analyzed by pathology analysis. As illustrated in Fig. 6D, for saline-treated group, clear binucleolate cell morphology and abundant chromatin were observed. However, the tumor tissues treated with all MHg formulations showed various degree of necrosis, indicating that all the MHg formulations exhibited antitumor effect. Remarkably, the 131 I@PECT/DOX MHg treated group demonstrated the most distinct damage to tumor tissues, as severe nuclei absence and lack of discernible boundary regions, which was consistent with the results of in vivo tumor growth inhibition study. In addition, mice treated with all three therapeutic groups revealed no significant signal of damage to major organs (shown in Fig. S4). Therefore, these results demonstrated that the peritumoral injection of macroscale MHg enhanced and prolonged the local accumulation and retention of chemoradiotherapeutic agents, which resulted in synergetic treatment outcomes in vivo safely.

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Fig. 6. (A) Relative tumor volumes of different formulations on the HepG2 tumor bearing Balb/c nude mice. (a = in comparison with saline, b = in comparison with PECT/DOX MHg, c = in comparation with 131I@PECT MHg, *p b 0.05, **p b 0.01, ***p b 0.001). (B) Relative body weight change and (C) the survival rate of tumor bearing mice after various formulations were given. Data are presented as the mean ± standard deviation, n = 10. (D) Histopathological analysis of tumors: (a) saline; (b) PECT/DOX MHg; (c) 131I@PECT MHg; (d) 131I@PECT/DOX MHg.

4. Conclusions In this work, a kind of novel macroscale micellar hydrogel was constructed to deliver radionuclides and contemporaneously provide radiosensitizer in the tumor tissue. In vitro and in vivo evaluation demonstrated that the integration of the advantages of nanoparticulate formulation and radionuclide reservoir for IRT translated into improved therapeutic outcomes safely. Such an injectable thermosensitive micellar-hydrogel formulation, which not only enabled the precise control over the dosage and ratio of combination drugs to obtain desired therapeutic effect, but also would improve the patient compliance by avoiding repeated drug administrations, possesses a great potential for spatiotemporally simultaneous delivery of multiple bioactive agents for sustained combination therapy. Acknowledgments This project was supported by the National Natural Science Foundation of China (31470963, 81471727 and 51203189). Outstanding Young Faculty Award of Peking Union Medical College (YR1471), PUMC Youth Fund and the Fundamental Research Funds for the Central Universities (3332015100). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.jconrel.2015.11.007. References [1] T.Y. Seiwert, J.K. Salama, E.E. Vokes, The chemoradiation paradigm in head and neck cancer, Nat. Clin. Pract. Oncol. 4 (2007) 156–171.

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