Article pubs.acs.org/molecularpharmaceutics
Development of in Situ Forming Thermosensitive Hydrogel for Radiotherapy Combined with Chemotherapy in a Mouse Model of Hepatocellular Carcinoma Cheng-Liang Peng,†,‡ Ying-Hsia Shih,†,‡ Kuo-Sheng Liang,† Ping-Fang Chiang,‡ Chung-Hsin Yeh,‡ I-Chang Tang,‡ Cheng-Jung Yao,† Shin-Yi Lee,† Tsai-Yueh Luo,*,‡ and Ming-Jium Shieh*,†,§ †
Institute of Biomedical Engineering, College of Medicine and College of Engineering, National Taiwan University, No. 1, Section 1, Jen-Ai Road, Taipei 100, Taiwan ‡ Isotope Application Division, Institute of Nuclear Energy Research, P.O. Box 3-27, Longtan, Taoyuan 325, Taiwan § Department of Oncology, National Taiwan University Hospital and College of Medicine, Taipei, Taiwan S Supporting Information *
ABSTRACT: This study evaluated a system for local cancer radiotherapy combined with chemotherapy. The delivery system is a thermosensitive hydrogel containing a therapeutic radionuclide (188Re-Tin colloid) and a chemotherapeutic drug (liposomal doxorubicin). The thermosensitive PCL-PEG-PCL copolymer was designed to spontaneously undergo a sol−gel phase transition in response to temperature, remaining liquid at room temperature and rapidly forming a gel at body temperature. A scanning electron microscope was used to observe the microstructure of the fully loaded hydrogel. Release of radionuclide and doxorubicin from the hydrogel was slow, and the system tended to remain stable for at least 10 days. After the intratumoral administration of Lipo-Dox/188Re-Tin hydrogel in mice with hepatocellular carcinoma (HCC), its retention by the tumor, spatiotemporal distribution, and therapeutic effect were evaluated. The residence time in the tumor was significantly longer for 188Re-Tin loaded hydrogel than for Na 188Re perrhenate (Na 188ReO4). The hydrogel after thermal transition kept the radionuclide inside the tumor, whereas free 188Re perrhenate (188ReO4) diffused quickly from the tumor. The tumor growth was more profoundly inhibited by treatment with Lipo-Dox/188Re-Tin hydrogel (with up to 80% regression of well-established tumors on day 32) than treatment with either 188Re-Tin hydrogel or Lipo-Dox hydrogel. Therefore, this injectable and biodegradable hydrogel may offer the advantage of focusing radiotherapy and chemotherapy locally to maximize their effects on hepatocellular carcinoma. KEYWORDS: thermally responsive material, chemotherapy, radiotherapy, drug delivery, hepatocellular carcinoma
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currently under investigation.6,7 Intra-arterial administration of a therapeutic radiopharmaceutical can deliver a tumoricidal dose of radiation without jeopardizing nontumorous liver tissue. However, accurate catheterization is highly dependent on the operator’s skill and special equipment. Most physicians committed to hepatoma treatment are very familiar with local injection therapy. Brachytherapy is the direct implantation of radioactive seeds to enable delivery of the maximum amount of radioactivity to the tumor with minimal dose to surrounding normal structures, thus limiting side effects.8,9 Some tumors are generally treated with brachytherapy including head and neck cancer, uterus cancer, vaginal cancer, cervical cancer, and prostate cancer.
INTRODUCTION
Liver cancer is the sixth-most common cancer worldwide, with the highest incidence rates occurring in Asia and Sub-Saharan Africa.1 The incidence of hepatoma has increased dramatically in Europe and the United States in recent years due to the widespread occurrence of hepatitis C.2,3 With a very poor prognosis, it is the third-most common cause of death from cancer. Survival rates are 3−5% in the United States and developing countries.1 Although surgical excision and ablation methods for focal lesions are usually considered the treatment of choice, the prognosis for hepatocellular carcinoma (HCC) is still dismal for many patients.4 However, the percentage of patients who are suitable for this invasive procedure is only between 20% and 35%.5 Other modalities, such as transcatheter arterial embolization (TAE), chemotherapy, and external radiotherapy, show some efficacy. Alternative therapeutic methods, using lipiodol as a carrier for chemotherapeutic agents or radioisotopes, are © 2013 American Chemical Society
Received: Revised: Accepted: Published: 1854
November 9, 2012 March 8, 2013 March 12, 2013 March 12, 2013 dx.doi.org/10.1021/mp3006424 | Mol. Pharmaceutics 2013, 10, 1854−1864
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Figure 1. Schematic depiction of the chemical structures of triblock copolymer PCL-PEG-PCL and the formation of Lipo-Dox/188Re-Tin colloid hydrogel at different temperatures.
as received from Aldrich (St. Louis, MO). HPLC grade solvents, including methanol, n-hexane, dichloromethane (DCM), acetone, dimethyl sulfoxide (DMSO), and tetrahydrofuran (THF) were purchased from Tedia Inc. (Fairfield, OH, USA). Both DCM and THF were dried over calcium hydride (CaH2) and distilled before use. Stannous (II) octoate (SnOct), 3-caprolactone (CL), and stannous chloride (SnCl2) were purchased from Sigma Aldrich (Milwaukee, WI, USA). The pegylated liposomal doxorubicin (Lipo-Dox) was purchased from TTY BioPharm Co., Ltd. (Taipei, Taiwan). All other chemicals were obtained from other commercial sources. Synthesis of PCL-PEG-PCL Triblock Copolymers. The PCL-PEG-PCL triblock copolymers were prepared by ringopening polymerization of caprolactone in the presence of PEG. Stannous octoate was used as a catalyst (Figure 1).15,16 In particular, to synthesize the PCL-PEG-PCL (CL/EG ratio = 1) triblock copolymer, 2.0 g (2 μmol) of PEG (Mn = 1000) was heated in a neck flask to a final temperature of 120 °C under vacuum to remove the residual water adsorbed to the polymer. After 4 h, 2.0 g (17.5 μmol) of ε-caprolactone and 0.02 g of stannous octoate (0.5 wt % of total reactants) were added to the reaction mixture and stirred at 145 °C for 24 h. The synthesized polymers were recovered by dissolving them in THF and then precipitating them in ice-cooled diethyl ether. The resultant precipitate was filtered and dried at room temperature under vacuum. The molecular weights of the synthesized polymers were characterized by 1H NMR spectroscopy using an Avance-500 MHz FT-NMR spectrometer (Bruker Co., Rheinstetten, Germany) with CDCl3 (deuterated chloroform) as the solvent and gel permeation chromatography (GPC; Waters 510 HPLC pump/410 differential refractometer, Milford, MA) with THF as eluent at a flow rate of 1 mL/min through Styragel HR 1, HR 2, and HR 4 columns (Waters). The thermal stability of the polymers was tested using thermogravimetric analysis (TGA, Perkin-Elmer, Norwalk, CT) at a heating rate of 20 °C/min in a nitrogen atmosphere. The phase transition of PCL-PEG-PCL hydrogel was detected using the test tube-inversion method.17 Gels were defined as nonflowable on test tube inversion for up to 1 min at 4 °C. Each sample at the various concentrations was prepared by dissolution in deionized water at the designated temperature. The hydrous
Hepatocellular carcinoma is currently treated using radioactive seeds of iodine-125 (125I, 150 KeV β− emission, t1/2 = 59.4 days)10 or yttrium-90 (90Y, 2.28 MeV β− emission, t1/2 = 64 h)11,12 placed permanently into the tumor. 125I is used to deliver a low dose of radiation over a period of several months, whereas 90Y has an advantage over 125I in that radiation is delivered rapidly at a very high dose rate, thus avoiding some of the radiological problems associated with 125I. The use of 90 Y-microspheres is considered very promising and has been approved for hepatoma treatment in some countries.12 However, accumulation of 90Y in the skeletal system causing bone marrow depression has been reported.13 These drawbacks limit the clinical value of these two currently available radioisotopes. 188 Re-RC-160 and 188Re sulfide suspension following direct intratumoral injection may also have potential as new clinical agents for cancer treatment.14,15 Rhenium-188 (188Re, t1/2 =16.9 h) is emerging as a promising isotope for clinical use. 188Re has several favorable characteristics, such as high beta-energy emission (2.1 MeV), suitable gamma-ray energy (159 keV), short half-life of 17 h, deep tissue penetration for local treatment (maximum 11 mm, average 3.8 mm),14 and in-house preparation using a 188 W/188Re generator.6 This unique property makes 188Re a suitable radionuclide candidate for both therapeutic and diagnostic purposes. In this study, we evaluated a in situ gel-forming drug delivery system, based on thermosensitive poly(ε-caprolactone)−poly(ethylene glycol)−poly(ε-caprolactone) (PCL-PEG-PCL) hydrogel, containing 188Re-Tin colloid and pegylated liposomal doxorubicin (Lipo-Dox) for local combined radiotherapy and chemotherapy. The prepared PCL-PEG-PCL copolymer was designed to spontaneously undergo a sol−gel phase transition in response to temperature, remaining liquid at room temperature and rapidly forming a gel at around physiological body temperature. The system was used to treat BNL hepatocellular carcinoma in mice, and the efficacy of this treatment was evaluated.
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EXPERIMENTAL SECTION Materials. ε-Caprolactone, stannous octoate, and poly(ethylene glycol) (PEG) (MW = 1000) were used as received from Fluka (Buchs, Switzerland). PEG (MW = 2000) was used 1855
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weight cutoff, 3.5 kDa). The dialysis bags were placed in 50 mL of phosphate-buffered saline (PBS; prewarmed to 37 °C, pH ∼ 7.4) with gentle shaking (300 rpm), and the release media were collected at predetermined time intervals and assayed by a gamma counter (Cobra series model 5003, Packard, CT, USA). All experiments were carried out in triplicate. In vitro release profiles of doxorubicin from liposomal doxorubicin hydrogel were determined as above. A sample of 1 mL of Lipo-Dox (with doxorubicin concentration, 2 mg/mL) or doxorubicin alone (2 mg/mL; used as control) was added to hydrogel solution (30 wt %/vol) and placed in a dialysis bag. The release media were collected and stored at −20 °C after dialysis and before further analysis. The released drug was quantified using high-performance liquid chromatography. All experiments were carried out in triplicate, and all data were expressed as the mean ± SD. Cell Culture. The BNL-Luc cell line, a cancer cell line derived from chemically transformed hepatic epithelial cells of a BALB/c mouse, was used to generate subcutaneous liver tumors. The BNL cells were maintained in a humidified 5% CO2 incubator at 37 °C in Dulbecco’s modified Eagle’s medium (DMEM, Gibco BRL, Gaithersburg, MD, USA) supplemented with 10% heat-activated fetal bovine serum (FBS; Gibco BRL) and 1% antibiotics (Antibiotic-Antimycotic; Gibco BRL). Animal and Implantation of Subcutaneous Liver Tumor. Male BALB/c mice (5−6 weeks old) were purchased from the National Laboratory Animal Center, Taipei, Taiwan. BNL-Luc tumors were initially established by a subcutaneous injection of 106 cells in PBS. Tumor sizes and body weight were measured every 3 days for the duration of the experiment. The tumor volume was calculated as π/6ab2 where a is the length and b is the width of the tumor. Biodistribution of Lipo-Dox/188Re-Tin Colloid Hydrogel. Mice received an intratumoral injection of Lipo-Dox/188ReTin colloid hydrogel (equivalent to 74 MBq of 188Re/0.1 mL), when the tumors reached a volume of 150−200 mm3. At selected time points (1, 4, 24, and 48 h), groups of three mice were sacrificed, and the uptake of radioactivity by the tumor and normal tissues was measured by a gamma counter. Tissue distribution data were expressed as the percentage of injected dose (ID%). In this study, single-photon emission computed tomography (SPECT)/computed tomography (CT) was also performed to evaluate the distribution of 188Re-perrhenate (188ReO4) solution, 188Re-Tin colloid hydrogel, or Lipo-Dox/188Re-Tin colloid hydrogel (equivalent to 74 MBq of 188Re/0.1 mL) in mice. SPECT images and X-ray CT images were acquired using a microSPECT/CT scanner system (XSPECT, Gamma Medica, Northridge, CA, USA). While SPECT imaging using low-energy, high-resolution collimator was taken at 1, 4, 24, and 48 h after intratumoral injection, the mice were immobilized by inhalation of the anesthetic isoflurane (Abbott Laboratories, Kent, England). The SPECT images followed by CT images were acquired (X-ray source: 50 kV, 0.4 mA; 256 projections) with the animal in exactly the same position. COBRA_Exxim software was used for CT image reconstruction, LumaGEM software was used for SPECT image reconstruction, and IDL 6.0 software was used for SPECT/CT imaging fusion. Antitumor Efficacy of the Dual Lipo-Dox/188Re-Tin Colloid Hydrogel. Treatments were started when the tumors reached a volume of 100−150 mm3, which was designated day 0. Mice (n = 6 per group) received one of five treatments, including PBS-control, 188Re-perrhenate (188ReO4) solution,
Table 1. Characteristics of PCL-PEG-PCL Triblock Copolymersa code
PCL/PEGb
Mnc
PCL-PEG-PCLd
PDIe
S−I S−II S−III S−IV S−V S−VI
1000/1000 1250/1000 1500/1000 2500/2000 2700/2000 3000/2000
1634 2170 2502 3614 3742 3810
317-1000-317 585-1000-585 751-1000-751 807-2000-807 871-2000-871 905-2000-905
1.32 1.33 1.27 1.31 1.24 1.32
a Abbreviations: PCL, poly(ε-caprolactone); PEG, poly(ethylene glycol); PDI, polydispersity; 1H-NMR, proton nuclear magnetic resonance; GPC, gel permeation chromatography. bTheoretical value, calculated according to the feed ratio. cCalculated from 1 H NMR. dMolecular weight estimated from 1H NMR. eDetermined by GPC.
samples were incubated in a water bath at 4 °C for 10 min and then slowly heated to the sol−gel phase transition temperature. Preparation of the Liposomal Doxorubicin (Lipo-Dox) and 188Re-Tin Loaded Hydrogel (Lipo-Dox/188Re-Tin Colloid Hydrogel). A highly pure 188Re-perrhenate solution was eluted from an 188W/188Re generator, which was manufactured by Institute of Nuclear Energy Research (INER, Taoyuan, Taiwan). The 188Re-Tin colloid was prepared using a modification of a previously described method.18 In brief, stannous chloride (SnCl2, 5−45 mg) was heated with 188 Re-perrhenate (∼370 MBq/mL) at 50 °C for 5−120 min.19 After cooling, the vials were centrifuged at 3000 rpm for 10 min, the supernatant was removed, and the pellet was resuspended in normal saline or liposomal doxorubicin solution (Lipo-dox, TTY BioPharm, Taipei, Taiwan). Labeling efficiency was determined by chromatography (on instant thin-layer chromatography-silica gel/normal saline plates with silica gel being the stationary phase and saline, the mobile phase), and radioactivity was monitored using a TLC scanner (AR2000, Bioscan, Washington, DC, USA). The particle size and polydispersity of the colloid were measured using dynamic light scattering (Delsa Nano, Beckman Coulter, Fullerton, CA). PCL-PEG-PCL copolymer powder was added to normal saline or liposomal doxorubicin solution containing 188Re-Tin colloid at the designated temperature, cooled to 4 °C, to form a well-mixed 188Re-Tin colloid/liposomal doxorubicin hydrogel complex. The concentration of the hydrogel complex was adjusted to 30 wt %/vol, and then 188Re-Tin was added up to a concentration of ∼370 MBq/mL and liposomal doxorubicin, up to a concentration of ∼2 mg/mL. The phase transition of 188 Re-Tin colloid/liposomal doxorubicin hydrogel was detected using the test tube-inversion method. Microstructure of Lipo-Dox/188Re-Tin Colloid Hydrogel. The surface and cross-sectional views of hydrogel loaded with 188Re-Tin colloid and/or liposomal doxorubicin was examined by scanning electron microscopy (SEM; JSE-6500F, Jeol, Tokyo, Japan). The SEM samples were prepared by depositing hydrogel onto a conductive substrate and freezedrying. Subsequently, the samples were sputter-coated with platinum at 7 mA for 150 s prior to examination. Measurement of in Vitro Release. The profiles of in vitro 188 Re release from the hydrogels was studied at 37 °C using a modified dialysis method20 as follows: 1 mL of 188Re-Tin colloid (∼370 MBq/mL) prepared by reaction with various amounts of stannous chloride (SnCl2) was added to hydrogel solution (30 wt %/vol) and placed in a dialysis bag (molecular 1856
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Figure 2. Preparation and characterization of thermosensitive hydrogel. (A) Gel perfusion chromatography spectra and (B) sol−gel transition diagram of PCL-PEG-PCL triblock copolymers. (C) 1H NMR spectra and (D) thermogravimetric analysis of triblock copolymer S−III.
Lipo-Dox hydrogel, 188Re-Tin colloid hydrogel, and LipoDox/188Re-Tin colloid hydrogel. A total dose of 2 mCi for 188 Re-perrhenate or 188Re-Tin colloid and a dose equivalent to 10 mg/kg of doxorubicin for Lipo-Dox were injected intratumorally. The tumor size and change in body weight of each mouse were recorded. The percentage of tumor growth inhibition (TGI%) was calculated from the relative tumor volume at day 32. The tumor was imaged, detected, and measured using an IVIS imaging system equipped with Living Imaging software (Xenogen, Alameda, CA, USA). Mice were anesthetized with a mixture of oxygen and isoflurane, then intraperitoneally injected with 100 μL of D-luciferin (Xenogen; 30 mg/mL in PBS).
Necropsy and Immunohistochemical Analysis. After the mice were sacrificed, tumors were excised and weighed. For immunohistochemical and H&E staining procedures, the tumor tissue was fixed in formalin, embedded in paraffin, and cut into 5 μm sections. The NADPH-diaphorase staining was carried out to demonstrate the necrosis as described previously with minor modifications.21 The tissue viability was analyzed by reacting the samples for 20 min at room temperature with NADPH-diaphorase reaction solution (10 mL of 10 mmol/L phosphate-buffered saline, pH 7.4, containing 10 mg of NADPH, and 5 mg of nitroblue tetrazolium). The sections were stained immunohistochemically for PCNA (proliferating cell nuclear antigen), treated with 3% hydrogen peroxide for 15 min to quench endogenous 1857
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Figure 3. Scanning electron micrograph of hydrogel. Morphology of (A, D) PCL-PEG-PCL (S−III) hydrogel (30 wt %/vol), (B, E) hydrogel loaded with liposomal doxorubicin, and (C, F) hydrogel loaded with liposomal doxorubicin/188Re-Tin colloid. Magnification: (A−C) 1000×; (D−F) 3500×. The scale bars are 10 and 5 μm at magnifications of 1000× and 3500×, respectively.
Figure 4. (A) In vitro 188Re release profile of 188Re-Tin colloid hydrogel and (B) in vitro doxorubicin release profile of doxorubicin hydrogel and liposomal doxorubicin hydrogel (Lipo-Dox hydrogel) in PBS solution at pH 7.4. The 188Re-Tin colloid (∼370 MBq/mL) prepared by heating with various amounts of stannous chloride (SnCl2) were added to hydrogel solution (30 wt %/vol). Error bars represent the standard deviation (n = 3).
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RESULTS AND DISCUSSION A series of PCL-PEG-PCL triblock copolymers were prepared through ring-opening polymerization of ε-caprolactone in the presence of PEG as a macroinitiator with SnOct as a catalyst (Figure 1). 1H NMR and GPC were used to characterize the chemical structure of PCL-PEG-PCL triblock copolymers. The characteristics of PCL-PEG-PCL copolymers prepared in this study are summarized in Table 1. As shown in Table 1, the triblock copolymers from S−I to S−III belonged to the PEG1000 series, and the others belonged to the PEG-2000 series. The characteristic resonances of both PCL (δHe = 1.38 ppm, δHd = 1.65 ppm, δHc = 2.28 ppm, δHc = 4.07 ppm) and mPEG (δHa =3.39 ppm and δHb = 3.65 ppm) were observed,
peroxidase activity, blocked with 10% normal goat serum for 15 min, rinsed three times with PBS for 2 min, incubated overnight at 4 °C with antibodies specific for PCNA (mouse anti-PCNA, clone PC 10, Sigma, St. Louis, MO), rinsed again with PBS, incubated with biotinylated secondary antibodies for 30 min at room temperature, incubated with an avidin−biotin complex, and visualized by the addition of 3,3′-diaminobenzidine tetrahydrochloride (DAB) chromogen. Immunostaining was carried out using the HistostainPlus Kit (Zymed Laboratories, Inc., San Francisco, CA, USA). The TUNEL assay was carried out using the DeadEnd Colorimetric TUNEL System from Promega (Madison, WI). 1858
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Figure 5. MicroSPECT/CT images in BALB/c mice bearing subcutaneous BNL-Luc murine liver tumor at 1, 4, 24, and 48 h after the intratumoral injection of (A) 188Re-perrhenate (188ReO4) solution and (B) Lipo-Dox/188Re-Tin hydrogel (equivalent to 74 MBq of 188Re/mouse).
in temperature can cause the transition from gel phase to sol phase (turbid sol).22−24 However, copolymers S−I, S−IV, and S−V did not gelate, so they do not appear in the diagram. We chose copolymer S−III as the hydrogel material in future experiments because it gelates at 37 °C at low concentration. The copolymer S−III solution was clear fluid at low temperature and gelated at body temperature to form an in situ gel-forming a controlled drug delivery system. The radio-labeling efficiency of 188Re-Tin colloid showed more than 95% when heated with 188Re-perrhenate (∼370 MBq/mL) at 50 °C for over 10 min (Supporting Information, Figure S1 and Table S1). The average particle size of 188Re-colloid was more than 5 μm as determined by DLS under different reaction conditions (Supporting Information, Table S2). PCL-PEG-PCL hydrogel (S−III, 30 wt %/vol) and hydrogel loaded with 188Re-Tin colloid or liposomal doxorubicin were examined by SEM. The hydrogels were frozen in liquid nitrogen and lyophilized for 72 h before examination. As shown in Figure 3A and C, the PCL-PEG-PCL hydrogel had a porous three-dimensional structure. The average pore size of PCLPEG-PCL hydrogel was 2−5 μm, and the pores were uniformly spherical in shape. By contrast, the cross-sectional views of the pores of liposomal doxorubicin-loaded hydrogels were irregular in shape and size (pleomorphic; Figure 3B,E). However, 188ReTin colloid/liposomal doxorubicin hydrogels had irregularly shaped bulges instead of pores. These results further demonstrated that 188Re-Tin colloid is entrapped in threedimensional structure of hydrogel and fills the pores. The in vitro release of 188Re or doxorubicin was evaluated. The release profiles of 188Re were determined at 37 °C using a modified dialysis method. Figure 4A presents the in vitro release profiles of 188Re from 188Re-Tin colloid hydrogels, which were prepared in the presence of various amounts of stannous chloride (SnCl2). The rate of 188Re release from the hydrogel was inversely related to the concentration of stannous chloride used in the preparation of 188Re-Tin colloid. Less than 15% of the 188Re was released from the 188Re-Tin colloid hydrogel within 60 h when the 188Re-Tin colloid were prepared by heating with more than 35 mg/mL of SnCl2. This suggests that a high concentration of stannous chloride increases the stability
Figure 6. Biodistribution of the Lipo-Dox /188Re-Tin colloid hydrogel at 1, 4, 24, and 48 h after intratumoral injection in BNL liver tumorbearing BALB/c mice.
suggesting the coexistence of three blocks. The molecular weight (Mn,NMR) of PCL was determined by comparing the peak intensities of the methylene protons of the oxyethylene units (δHb) of mPEG to those of the methylene protons (δHf) of PCL (Figure 2C).15 The weights distribution (polydispersity, PDI, Mw/Mn) of copolymers determined by GPC were in the range of 1.27−1.33, respectively. In the PEG-1000 series, the molecular weight (MW) of PCL calculated from NMR was closer to that calculated from the feed ratio than in PEG-2000 series. Figure 2B shows the sol−gel−sol transition phase diagram of PCL-PEG-PCL triblock copolymers in aqueous solutions. Aqueous solutions of copolymers S−II, S−II, and S−VI changed from sol phase (clear sol) to gel phase with increase in temperature when the copolymer concentrations were above the critical gelation concentration (CGC). Gelation seemed to be driven by micelle packing and aggregation. A further increase 1859
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Figure 7. The antitumor effects of Lipo-Dox/188Re-Tin colloid hydrogel. (A) Tumor growth volume (mm3) and (B) weight change (%) of control (■), 188Re perrhenate (⧫), Lipo-Dox hydrogel (●), 188Re-Tin colloid hydrogel (▲), or Lipo-Dox/188Re-Tin colloid hydrogel (▼) were determined in BNL/luc tumor-bearing BALB/c mice. (C) In vivo bioluminescence imaging of BNL/luc tumor bearing BALB/c mice after various treatments on day 21 and day 32.
of 188Re-Tin colloid so that they can be held more readily within hydrogel. Figure 4B shows the profiles of in vitro doxorubicin release from hydrogels. Doxorubicin/hydrogel had a two-phase release profile: a relatively rapid release of doxorubicin after 6 h, followed by a sustained, slower release. Compared with doxorubicin/hydrogel, liposomal doxorubicin loaded hydrogel (Lipo-Dox hydrogel) had a much slower doxorubicin release rate, and the release was sustained for up to 10 days. This sustained release profile of Lipo-Dox hydrogel indicates the potential applicability of liposomal drug/hydrogel as a method for focusing therapeutic drug in the tumor site while minimizing the exposure of healthy tissues. Generally, drug release from hydrogel could be driven by three forces: diffusion, interaction of drugs with hydrogel matrix, and degradation or erosion of the hydrogel.4 As the degradation rate of PCL-PEG-PCL hydrogel in PBS is low, the in vitro drug release behavior of the hydrogel depends mainly
on diffusion and interaction with hydrogel. Since the free form of rhenium-188 and doxorubicin are hydrophilic, they could diffuse out through the pores of the hydrogel in a short time. However, the average particle size of 188Re-colloid (more than 5 μm) resulted in entrapping in the three-dimensional structure of hydrogel and fills the pores. Hence, a low diffusion rate in PBS and a strong interaction with the hydrogel dominated the drug release profile, which resulted in a lower 188Re release rate and a higher residual 188Re content in the hydrogel. For the liposomal doxorubicin (Lipo-DOX) hydrogel, doxorubicin could be released from the hydrogel through processes including: doxorubicin release from liposome, doxorubicin release from hydrogel, and liposome release from hydrogel. Since liposomal doxorubicin release from hydrogel was slow, the release speed of doxorubicin from liposomal doxorubicin (Lipo-DOX) hydrogel was slowed down, and the burst release was reduced compared to doxorubicin hydrogel. The slower release of doxorubicin from liposomal doxorubicin 1860
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hydrogel than 188ReO4 solution. These data clearly establish that retention of the radionuclide was significantly greater after injection with 188Re-colloid hydrogel than after injection of free 188 ReO4 solution, which quickly escaped from the tumor. However, despite the improved tumor response after single treatments, no complete responses were observed in the single treatment groups. In addition, compared with control or single treatments, Lipo-Dox/188Re-colloid hydrogel treatment was responsible for significantly greater tumor growth inhibition (TGI ∼99.8%; Table 2) at day 32 (P < 0.05) and did not cause significant adverse effects, such as weight loss (P > 0.05) (Figure 7B). Therapeutic response monitoring by bioluminescence imaging (Figure 7C) using IVIS imaging system (Xenogen, Alameda, CA, USA) after various treatments on day 32. It has been shown that tumor recurrence after 188Re-colloid hydrogel treatment, through separately illustrating the tumor volume of each mouse (as shown in Figure S3A), which was in agreement with the therapeutic response monitoring by bioluminescence imaging (Figure 7C). In contrast, only one of five mice had tumor recurrence on day 32 as shown in Figure S3B, which was also in agreement with the bioluminescence images shown in Figure 7C. It was found that Lipo-Dox/188Re-colloid hydrogel was the most inhibitory, with up to 80% (4/5) of wellestablished tumors showing complete regression on day 32. No tumors at the original tumor injection site were palpable on days 18−25 in any of the mice, indicating the robustness of the effect of Lipo-Dox/188Re-colloid hydrogel against murine BNL liver tumor. Relative body weight change was utilized as a measure of toxicity (Figure 7B). The body weight of animals in the control and treatment groups was monitored throughout the experimental period; mice that lost >20% of their original body weight were sacrificed. The gradual increase in mean body weight of the control group, reaching approximately 1.3 times the initial mean weight on day 21, was possibly due to the fast tumor growth. The body weight of mice treated with 188ReO4 solution was decreased only 0.8% on day 32 and remained essentially unchanged during the entire observation time (P > 0.05). The body weights of mice treated with Lipo-Dox hydrogel, 188Re-colloid hydrogel, and Lipo-Dox/188Re-colloid hydrogel were increased 19.6%, 8.2%, and 12.7% on day 32, respectively. These increases were not significantly different, suggesting that such treatments were reasonably well-tolerated, efficiently inhibited tumor growth, and were no more toxic than treatment with 188ReO4 solution. To further determine the effect of Lipo-Dox/188Re-Tin colloid hydrogel in vivo, the tumors were subjected to immunohistochemical analysis (Figure 8). Serial sections of tumors that had been treated with Lipo-Dox hydrogel, 188ReTin colloid hydrogel, or Lipo-Dox/188Re-Tin colloid hydrogel were analyzed by hematoxylin and eosin (H&E) staining and nicotinamide-adenine dinucleotide phosphate (NADPH)diaphorase staining and were immunostained for proliferating cell nuclear antigen (PCNA) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). Examination of H&E-stained tissue sections demonstrated differences in tissue morphology between the treatment groups; more necrosis is seen in tumors treated with Lipo-Dox/188Re-Tin colloid hydrogel characterized by alternating irregularly shaped regions of viable cells (purple areas) and necrotic debris (pink areas), which is in good agreement with the loss of NADPHdiaphorase activity (as shown in in Figure 8A). Less necrotic
(Lipo-DOX) hydrogel might be due to the interaction of LipoDOX with hydrogel and collapse of Lipo-DOX instead of diffusion. As shown in Figure 5A, 188Re-perrhenate solution was eliminated from the blood 1 h after intratumoral injection by a combination of glomerular filtration and in part by hepatobiliary elimination. Some residual radioactivity was retained in the injection site up to 1 h, but most 188Re activity could be visualized in the bladder and gallbladder. Most 188Re activity remained in the gallbladder, stomach, and thyroid up to 24 h after intratumoral injection. In contrast, as shown in Figure 5B, 188Re Tin-colloid hydrogel remained within the injection site and little 188Re activity was found in other organ during the 48 h evaluation period. This retention of 188Re radioactivity confirms the potential applicability of hydrogels as radiopharmaceutical carriers to increase the accumulation of therapeutic drug near the tumor site while minimizing the exposure of healthy tissues. As illustrated in Figure 6, after intratumoral injection of 188Re-colloid hydrogel, 188Re remained primarily near the tumor and to a lesser extent in other organs during the 48 h evaluation period. Figure 7 and Table 2 show the antitumor results of LipoDox/188Re-colloid hydrogel injection in a murine BNL liver Table 2. Effect of Lipo-Dox Hydrogel and 188Re-Tin Colloid Hydrogel (Individually or in Combination) on Tumor Growth in a BNL Murine Liver Tumor Model treatment control 188 Re perrhenate Lipo-Dox hydrogel 188 Re-colloid hydrogel Lipo-Dox/188Recolloid hydrogel
doxorubicin dose (mg/kg)
188
Re dose % complete (MBq) % TGIa TGDb responsesc 74
68.7 69.8
3 14
0 0 0
74
94.4
28
0
74
99.8*
>28
75
10
10
a
TGI (tumor growth inhibition) is presented as the percent reduction in the mean tumor volume in experimental groups compared with saline-treated control groups. bTGD (tumor growth delay) is defined as the number of days for the mean tumor volume per group to reach five times the initial volume as compared with the saline-treated control mice. cComplete response was defined as the absence of palpable tumor at the original tumor injection site on day 32. *P < 0.05, as compared with other treatments.
tumor model. BNL solid tumors were measurable 11 days after subcutaneous inoculation of 1 × 106 BNL cells, and treatment was initiated at this time (day 0). The effect of Lipo-Dox/188Re-colloid hydrogel on tumor growth were compared with the respective effects of 188Re perrhenate (188ReO4) solution, Lipo-Dox hydrogel, and 188Recolloid hydrogel. The mean tumor volume in untreated animals on day 4 reached five times the volume on day 0. Progression of tumor growth to this volume was delayed by 188ReO4 solution, Lipo-Dox hydrogel, and 188Re-colloid hydrogel to day 7, 18, and 32, respectively. However, Lipo-Dox/188Re-colloid hydrogel extended the delay in progression of tumor growth to this volume beyond the 32-day evaluation period. After 32 days, the TGI% (tumor growth inhibition %) by 188 ReO4 solution, Lipo-Dox hydrogel, and 188Re-colloid hydrogel was, respectively, 68.7, 69.8, and 94.4 (Table 2). Tumor growth was suppressed to a greater extent by 188Re-colloid 1861
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Figure 8. Histological and immunohistochemical analysis in BNL tumors treated with Lipo-Dox/188Re-Tin colloid hydrogel. (A) Tumor sections were analyzed by hematoxylin and eosin (H&E) staining and NADPH-diaphorase staining. The black dotted squares in the left photographs show the sites of the microphotographs (200×), and triangles show the remaining hydrogel in tumor. On H&E stains, more necrotic (N) tissue (pink areas) on the interior of the tumors was present when the tumors were treated with the combination of Lipo-Dox/188Re-Tin colloid hydrogel, which is in good agreement with the loss of NADPH-diaphorase activity. (B) Immunohistochemical staining of proliferating cell nuclear antigen (PCNA) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) from the blue dotted squares (viable region) in part A. (C) Cellular proliferation was quantified by assessing the number of PCNA-positive cells per field at 200× magnification, and (D) the apoptotic cells were quantified by the TUNEL method at 200× magnification. The results represent the mean ± SD in 10 distinct regions from examining three tumors per group. The star (*) indicates P < 0.01 compared with other treatments.
tumor tissue was induced in Lipo-Dox hydrogel or 188Re-Tin colloid hydrogel-treated tumors. The sections were stained with NADPH-diaphorase staining for the assessment of tissue viability. Necrotic tissue shows loss of NADPH-diaphorase activity.21 The immunohistochemical analysis revealed that tumors treated with Lipo-Dox/188Re-Tin colloid hydrogel had prominent necrosis and vacuolation. Necrotic features caused by the H&E staining and the loss of NADPH-diaphorase activity were observed at the interior of the tumors. The maximum treatable region of Lipo-Dox/188Re-Tin colloid hydrogel appeared to be ∼3−4 mm outside the injection site of hydrogel (as shown in in Figure 8A), which was in agreement with the loss of NADPH-diaphorase activity. These results indicate that irreversible tissue damage (necrosis) mainly in the Lipo-Dox/188Re-Tin colloid hydrogel-treated tumor tissue.
For immunohistochemical analysis, such as PCNA immunostaining (for proliferating cells) and TUNEL staining (for apoptosis), they generally are observed in the viable region of tissue sections (non-necrosis, purple areas in H&E staining). It demonstrated that cell proliferation (as detected by PCNA immunostaining) was significantly decreased in tumors treated with Lipo-Dox/188Re-Tin colloid hydrogel (approximately 25.4 ± 15.3) compared to tumors treated with the Lipo-Dox hydrogel (approximately 88.7 ± 31.5; P < 0.05) and 188Re-Tin colloid hydrogel (approximately 85.6 ± 18.9) as well as control mice (approximately 176.3 ± 21.2; P < 0.05). The number of proliferating (PCNA-positive) cells within the BNL tumors was reduced by treatments with Lipo-Dox hydrogel and 188Re-Tin colloid hydrogel and significantly reduced by treatment with Lipo-Dox/188Re-Tin colloid hydrogel (Figure 8B and C). In addition, apoptotic cells were identified in each group by the TUNEL method. The mean number of apoptotic cells for the 1862
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Lipo-Dox/188Re-Tin colloid hydrogel group (86.2 ± 14.7) was significantly greater than that for the control group (6.2 ± 2.6), Lipo-Dox hydrogel alone group (30.5 ± 6.3), and 188Re-Tin colloid hydrogel alone group (63.8 ± 15.4; P < 0.05; Figure 8B and D). These results suggest that the treatment with LipoDox/188Re-Tin colloid hydrogel acts by greatly reducing the number of proliferating cells and increasing the number of apoptotic cells.
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CONCLUSIONS We have prepared and characterized thermosensitive hydrogels containing 188Re-Tin colloid and liposomal doxorubicin (LipoDox) for local cancer radiotherapy combined with chemotherapy. Scanning electron microscopy demonstrated that 188 Re-Tin colloid is entrapped in the three-dimensional structure of the hydrogel and fills the pores of the hydrogel, resulting in slow radionuclide release. Moreover, Lipo-Dox loaded hydrogel slowly and steadily released doxorubicin. In mice with hepatocellular carcinoma (HCC), the intratumoral residence time was significantly longer for 188Re-Tin colloid hydrogel than for 188Re perrhenate (188ReO4). Compared to rate of free 188Re perrhenate (188ReO4) release, the rate of release of the radionuclide encapsulated by hydrogel and deposited in the tumor was markedly slower. The components in the Lipo-Dox/188Re-Tin colloid hydrogel acted synergistically against tumor growth, that is, Lipo-Dox/188Re-Tin colloid hydrogel had greater effect than either component (188Re-Tin colloid hydrogel or Lipo-Dox hydrogel) individually, resulting in up to 80% complete regression of well-established tumors on day 32. Therefore, Lipo-Dox/188Re-Tin colloid hydrogel potentially offers a more potent multimodalities for the synergistic combination of radiotherapy and chemotherapy with markedly reduced adverse effects in the treatment of hepatocellular carcinoma.
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ASSOCIATED CONTENT
S Supporting Information *
Supplementary radiochemical purity of 188Re-colloids, size distribution of 188Re-colloids, radiochemical purity analysis of 188 Re-colloids by radio-TLC, the morphology of subcutaneously and intratumorally injected hydrogel in vivo, and tumor volume of each mouse treated with 188Re-colloid hydrogel or Lipo-Dox/188Re-colloid hydrogel. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*T.-Y.L.: Tel.: 886-3-4711400 ext 7004. Fax: 886-3-4711416. E-mail:
[email protected]. M.-J.S.: Tel.: 886-2-23123456 ext 67142. Fax: 886-2-23815095. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This research was funded by the Department of Health, Executive Yuan, Taiwan, ROC (DOH101-TD-C-111-001 and DOH101-TD-N-111-009).
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REFERENCES
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