Materials Science and Engineering C 49 (2015) 262–268
Contents lists available at ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Thermogel-mediated sustained drug delivery for in situ malignancy chemotherapy Yanbo Zhang a,b, Jianxun Ding a,⁎, Diankui Sun a, Hai Sun a, Xiuli Zhuang a, Fei Chang b,⁎, Jincheng Wang b,⁎, Xuesi Chen a a b
Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, PR China Department of Orthopedics, The Second Hospital of Jilin University, Changchun 130041, PR China
a r t i c l e
i n f o
Article history: Received 6 October 2014 Received in revised form 12 November 2014 Accepted 6 January 2015 Available online 8 January 2015 Keywords: Biodegradable Chemotherapy In situ administration Sustained drug delivery Thermogel
a b s t r a c t In the past few decades, the in situ sustained drug delivery platforms present fascinating potential in sentinel chemotherapy of various solid tumors. In this work, doxorubicin (DOX), a model antitumor drug, was loaded into the thermogel of poly(lactide-co-glycolide)-block-poly(ethylene glycol)-block-poly(lactide-co-glycolide). The moderate mechanical property of DOX-loaded hydrogel was confirmed by rheological test. In vitro degradation revealed the good biodegradability of thermogel. The DOX-loaded hydrogel exhibited the sustained release profiles up to 30 days without and even with elastase. The improved in vivo tumor inhibition and reduced sideeffects were observed in the DOX-incorporated hydrogel group compared with those in free DOX group. The excellent in vivo results were further confirmed by the histopathological evaluation or terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay. The thermogel with great prospect may be used as an ideal controlled drug delivery platform for the designated and long-term antitumor chemotherapy. © 2015 Elsevier B.V. All rights reserved.
1. Introduction During the past several decades, chemotherapy has been one of the most commonly used therapy approaches to treat various malignant tumors [1,2]. In the course of chemotherapy, some powerful antitumor antibiotics, such as, doxorubicin (DOX), paclitaxel, 10hydroxycamptothecin, and cisplatin, are employed to suppress the proliferation of malignant cells, and then to relieve the pain symptom of patients and improve survival [3–6]. However, the antineoplastics not only kill the diseased cells but also damage the normal cells, which induce serious systemic side effects [7]. For instance, DOX will lead to severe bone marrow suppression and cardiac toxicity [8]. DOX exhibits obvious dose-dependent efficacy, whereas the increase of dose will arouse more severe side effects that limit the wide clinical application [9]. Moreover, as depicted in Scheme 1, the unstable chemical structure of DOX is prone to decompose in the blood circulatory system, which seriously affects the treatment effect [10]. Recently, certain of in situ drug delivery systems in the forms of implants, microspheres, hydrogels, etc. have been developed to enhance the antitumor efficacy and reduce the side effects [11–13]. The drug-
⁎ Corresponding authors. E-mail addresses: [email protected] (J. Ding), [email protected] (F. Chang), [email protected] (J. Wang).
http://dx.doi.org/10.1016/j.msec.2015.01.026 0928-4931/© 2015 Elsevier B.V. All rights reserved.
loaded composites are directly injected into the lesion sites and exert the sustained payload release in a few days to several months [14]. The main advantages of in situ drug delivery platforms are facile implementation, long-lasting drug release, and extensive application [15,16], whereas various deficiencies still exist. In detail, implants are prepared from polymers and drugs via melt technique, which expose the problems of low loading efficiency and heat-induced degeneration of drugs. For instance, the microcapsules in implants containing the drugs that surrounded by films may achieve constant release, while the sudden release occurs once the thin films are broken [17]. In addition, microsphere delivery systems composed of matrix materials and drugs exhibit slowly sustained drug release after administration [18]. However, the complicated preparation processes of microspheres, such as, emulsification and solvent removal, are adverse to the biomedical applications. In recent decades, the in situ forming hydrogel-based drug delivery systems carrying a good prospect catch the eyes of researchers [19]. Among them, thermogels with unique properties show a lot of merits as drug delivery systems: 1) convenient drug encapsulation and local injectable administration; and 2) preparation without using organic solvent and photo irradiation [20,21]. Thermogels possess thermo-sensitive characteristics ascribed to the balance between the hydrophobic and hydrophilic segments [22]. The drugloaded platforms are free-flowing liquid at or below room temperature (i.e., 20 °C), while after in vivo administration, they quickly form into hydrogel at body temperature (i.e., 37 °C) [23]. Moreover,
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2. Materials and methods 2.1. Materials
Scheme 1. Molecular structure of model antitumor drug, i.e., DOX.
thermogels are three-dimensional (3D) networks with porous structures, which exhibit high loading properties, and simultaneously maintain their morphologies and high concentrations of antitumor drugs located in the solid tumors for a long time [24]. Therefore, the antitumor activity will be enhanced, and the side effects will be minimized [25]. Both natural and synthetic polymer-constructed thermogels have been selected as drug delivery platforms [19,26]. Compared with the natural thermogels, the thermogels from the synthesized polymers perform the enhanced mechanical properties and controlled properties [27, 28]. Of them, triblock poly(lactide-co-glycolide)-block-poly(ethylene glycol)-block-poly(lactide-co-glycolide) (PLGA-b-PEG-b-PLGA) copolymer is an excellent material that has many merits including easy preparation, strong dissolving capacity, controlled degradation, and good biocompatibility in pharmaceutical application to provide the sustained drug delivery [29]. In this study, the DOX-loaded hydrogel originated from PLGA-bPEG-b-PLGA (referred as hydrogel + DOX) was exploited as an in situ sustained drug delivery reservoir (Scheme 2). The moderate mechanical properties, sluggish degradation kinetics, and sustained release profiles were revealed. Meanwhile, the lowest tumor volume and reduced visceral tissue toxicities were found in the hydrogel + DOX group. The biodegradable thermogel could be a suitable candidate for the sustained sentinel drug delivery.
PEG (M n = 1500 g mol− 1) was purchased from Sigma-Aldrich (Steinheim, Germany), and triblock PLGA-b-PEG-b-PLGA copolymer (Mn, NMR = 4800 g mol−1, LA/GA = 75:25, mol/mol) was synthesized through the approach reported in our previous works [30–32]. Briefly, the copolymer was synthesized through the ring-opening polymerization of LA and GA with PEG as a macroinitiator and stannous octoate as a catalyst. Doxorubicin hydrochloride (DOX · HCl) was purchased from Beijing HuaFeng United Technology Co., Ltd. (Beijing, P. R. China). Elastase was obtained from Merck Company (Darmstadt, Germany). Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) kit was purchased from Roche Company (Mannheim, Germany). All the other reagents and solvents were purchased from Sigma-Aldrich (Steinheim, Germany) and used without further purification. 2.2. Dynamic mechanical analysis Rheological tests were conducted on a US 302 rheometer (Anton Paar Firma, Graz, Austria). The PLGA-b-PEG-b-PLGA solution (20 wt.%) without or with DOX (6.0 g L−1) in phosphate-buffered saline (PBS) was placed between parallel plates of 25 mm diameter and a gap of 0.5 mm. A layer of oil was dropped around the copolymer samples to prevent the evaporation of water. The data were collected under a controlled strain of 1% and a frequency of 1.0 rad s−1. The heating rate was 0.5 °C min−1. The storage modulus (G′), dissipative modulus (G″), and viscosity (η) were obtained in oscillatory shear flow. 2.3. In vitro DOX loading and release To determine the release profiles, DOX (6.0 g L−1) was directly and adequately dissolved in 0.5 mL of PLGA-b-PEG-b-PLGA copolymer solution of PBS (20 wt.%) at 4 °C within different vials (diameter = 16 mm), and then the laden vials were placed in a water bath at 37 °C for 30 min. Subsequently, 3.0 mL of PBS or PBS containing 0.2 g L−1 elastase, 10.0 mM CaCl2, and 0.2 wt.% NaN3, simulating in vivo microenvironment, were added on top of the thermogel. The vials were placed in
Scheme 2. Schematic presentation of DOX-incorporated hydrogel acted as a sustained drug delivery system for enhanced antitumor efficacy and reduced side effects.
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an incubator chamber at 37 °C with continuous shaking at 70 rpm. At predetermined interval, the whole medium was taken out from the vials, and an equal volume of fresh medium was replenished. The mass of the remaining gel was measured, and the release amount of DOX was tested by an ultraviolet–visible spectroscopy spectrophotometer at a wavelength of 480 nm. 2.4. Cell culture The mouse hepatoma cells (coded as H22) were cultured at 37 °C in a 5% (v/v) carbon dioxide atmosphere in Dulbecco's modified Eagle's medium (Gibco BRL, Grand Island, New York, USA) supplemented with 10% (v/v) fetal bovine serum, penicillin (50 IU mL−1), and streptomycin (50 IU mL−1).
2.7. Histopathological evaluation Hematoxylin and eosin (H&E) staining was used for the histopathological assay. The mice were sacrificed via the dislocation of cervical vertebra on 25 days. Tumors and major organs (i.e., heart, liver, spleen, lung, and kidney) were collected and embedded with paraffin. 6 μm thick transverse sections were cut and then stained with H&E to evaluate the histological alterations by a microscope. The necrotic area was defined as the pink-dyed cell-free region fused from the karyolysis and disintegration of organelle. The relative necrotic area (%) was calculated by Eq. (2). Relative necrotic areað%Þ ¼
necrotic area in tumor section 100: total area in tumor section
ð2Þ
2.5. Animal procedure 2.8. In situ apoptosis detection Kunming mice (male, 25 ± 2 g) were supplied by the Experimental Animal Center of Jilin University. All the experiments on animals were approved by the Animal Care and Use Committee of Jilin University. The animals were raised individually in plastic cages with food and water at a temperature of 25 °C under natural light/dark conditions for 2 weeks before experiment. 2.6. In vivo antitumor assessment The hepatoma model was established in mouse by the subcutaneous injection of 0.1 mL of suspension containing 3.0 × 106 H22 cells in PBS. The treatment was started when the mean tumor volume reached ~ 200 mm3. The mice were divided into four groups (10 mice per group), and 0.1 mL of PBS, PLGA-b-PEG-b-PLGA (hydrogel; 20.0 wt.% copolymer), DOX (30.0 mg kg−1), and DOX-incorporated PLGA-b-PEG-bPLGA solutions in PBS (hydrogel + DOX; 20.0 wt.% copolymer and 30.0 mg kg− 1 DOX) were separately injected into mice by the intratumoral injection. The body weight and tumor volume were recorded for 25 days. Tumor volume was measured by vernier caliper and was calculated by Eq. (1). L S2 3 V mm ¼ : 2
ð1Þ
The TUNEL apoptosis assay was carried out in accordance with the product manual (Roche, Basel, Switzerland). 6 μm thick tumor sections were washed and the nicked DNA ends were labeled by the mixture of enzyme and label solutions. The in situ cell apoptosis of tumor tissues was observed by a confocal laser scanning microscope (CLSM; LSM 700, Carl Zeiss, Jena, Germany). The apoptosis area was defined as the green fluorescence-labeled region originated from the late apoptotic cells. The relative apoptosis rate (%) was calculated by Eq. (3). Relative apoptosis rateð%Þ ¼
apoptosis area in tumor section 100:ð3Þ total area in tumor section
2.9. Statistical analysis All experiments were performed at least three times, and the results were expressed as means ± standard deviation (SD). Statistical significances were analyzed using SPSS (Version 18.0, Chicago, IL, USA). p b 0.05 was considered statistically significant, and p b 0.01 and p b 0.001 were considered highly statistically significant. 3. Results and discussion 3.1. Characterizations of hydrogels without or with DOX
In Eq. (1), L and S (mm) were the largest and smallest diameters of tumor, respectively. Meanwhile, the body weight changes were monitored at each time interval to determine the in vivo security of samples.
Hydrogel + DOX could be prepared through dispersing 6.0 g L−1 of DOX into the aqueous copolymer solution of PBS (20 wt.%) and subsequently elevating temperature. Firstly, the changes of G′ and η versus
Fig. 1. G′ (A) and η (B) of PLGA-b-PEG-b-PLGA solution in PBS (20 wt.%) without or with DOX (6.0 g L−1).
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3.2. In vivo antitumor efficacy
the increase of temperature were determined by rheological tests (Fig. 1). Compared with blank copolymer system, the initial G′ and η of DOX-incorporated platform significantly increased to 2.2 Pa and 2.2 Pa s, respectively, while the maximum of G′ and η decreased. It should be attributed that the amphiphilicity of DOX interfered with the self-assembly of copolymer and the association of micelle [25]. The change trends of rheological results were consistent with the previously reported results [33]. In addition, the G′ of hydrogel + DOX at 37 °C was about 100 Pa, which highlighted the potential of this thermogel as an in situ sustained drug delivery system. The degradation rate is one of the principal influencing factors on drug release behavior [34]. In this work, the degradation rate of hydrogel in PBS with elastase was faster (mass remaining: 37%, v/v) than that in blank PBS (mass remaining: 78%, v/v) at 30 days (Fig. 2). Simultaneously, the DOX release behavior from hydrogel + DOX was investigated in PBS without or with elastase. As shown in Fig. 3, DOX released from laden hydrogel in a controlled and sustained manner without initial burst release [29]. The microchannel formed by the hydrogel dissolution might contribute to the diffusion of DOX release. As expected, the presence of elastase accelerated the DOX release, which was consistent with the degradation profiles and demonstrated the correlation between the degradation and release [34].
Tumor inhibition detection toward tumor-bearing mouse models is the common approach to assess the efficacy of controlled drug delivery systems [35]. In this study, the in vivo antitumor activity assays were performed toward the mice xenografted with H22 tumors. After the tumor volume grew to about 200 mm3, PBS, hydrogel, DOX, or hydrogel + DOX was intratumorally injected one time, respectively. The survival conditions of mice were observed by the mental state and activities. Tumor volumes and body weights were simultaneously recorded. The mice of control groups (i.e., PBS and hydrogel groups) and DOX group showed poorer mental state, sluggish movement, and dull fur in relative to that of hydrogel + DOX group. As shown in Fig. 4, the tumors of control group grew fast and increased to over 1700 mm 3 at 25 days, and there was no statistical difference between PBS and hydrogel groups (p N 0.05). In stark contrast, the tumors in DOX and hydrogel + DOX groups grew slowly, and both of them exhibited significant difference compared with control groups (p b 0.001). It was indicated that DOX inhibited tumor growth effectively. Most importantly, the tumor volumes of hydrogel + DOX group at 25 days were significantly lower than those of DOX group owing to the sustained release of DOX (p b 0.01), which demonstrated the distinct advantage of thermogel as a continuous drug reservoir [19]. The tumor suppression was further confirmed by histopathological and apoptosis assays. The experimental mice were sacrificed on day 25, and the tumors were separated, sliced, and then stained with H&E for histopathological evaluation. H&E staining was defined as the chromatin within the nucleus was stained to be bluish violet by the alkaline hematoxylin, while the cytoplasm and the extracellular matrix were stained to be pink by the acid eosin dyes. As shown in Fig. 5, the PBS and hydrogel groups displayed well-grown tumor cells with large nuclei and a spherical or spindle shape, and small area of necrosis. Both DOX and hydrogel + DOX groups exhibited large areas of hemorrhage and necrosis, and other characteristic pathological features: 1) the morphology of necrotic tissue was not clear; 2) the chromatin was fuzzy or scattered outside cells; 3) the nuclear condensation, fragmentation, or disappearance were observed. It all meant that the DOX formulations, especially the DOX-loaded hydrogel, exhibited the significant antitumor efficacy against hepatic carcinoma in mice. In addition, the relative necrosis areas of control groups were less than that of DOX group (p b 0.01), and significantly lower than that of hydrogel + DOX group (p b 0.001) (Fig. 6A). More interestingly, the hydrogel + DOX group showed more necrosis than that of DOX group (p b 0.01). Simultaneously, the in situ cell apoptosis of tumor after treatment was analyzed by TUNEL assay. Apoptosis activates DNA enzyme leading
Fig. 3. Cumulative release of DOX from DOX-incorporated hydrogel (6.0 g L−1) in PBS without or with elastase at 37 °C. Data were presented as mean ± SD (n = 3).
Fig. 4. In vivo antitumor efficiency after in situ intratumoral injection of PBS, hydrogel, DOX, or hydrogel + DOX. Data were presented as mean ± SD (n = 10; *p b 0.01, **p b 0.001).
Fig. 2. In vitro mass remaining of PLGA-b-PEG-b-PLGA hydrogel (20 wt.%) with DOX (6.0 g L−1) at 37 °C immerged in 3.0 mL of PBS or PBS containing 0.2 g L−1 elastase. Data were presented as mean ± SD (n = 3).
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Fig. 5. H&E and TUNEL evaluations of tumor sections after treatments with PBS, hydrogel, DOX, and hydrogel + DOX. The black arrows pointed to the typical necrotic area. Scale bar, 100 μm.
to the genomic DNA fracture that can be labeled as green fluorescence. The low, moderate, and high fluorescence intensities and areas were observed in control, DOX, and hydrogel + DOX groups via CLSM, respectively, which were further confirmed by the 2.5D TUNEL microimages (Fig. 5). The relative apoptosis rates of all treatment groups with different DOX agents were significantly more than those of control groups (p b 0.001), and the relative apoptosis rate of hydrogel + DOX group was higher than that of DOX group (p b 0.01) (Fig. 6B). The TUNEL results were consistent with those of in vivo tumor inhibition and H&E assays, and these all confirmed the highest antitumor activity of DOX-incorporated hydrogel. 3.3. In vivo security evaluation The in vivo security of antineoplastic agents is another important evaluation index, which is related to the life safety of malignancy patients [36]. In this study, the safety assessments were
performed on the measurements of body weights and the histopathological analyses of visceral organs (i.e., heart, liver, spleen, lung, and kidney). To assess the toxicities of various DOX formulations, the body weights as a key indicator were real-time monitored. As shown in Fig. 7, at the beginning of 3 days after the administrations with different formulations toward hepatic tumors, the body weights in all groups showed growing tendency, while no significant difference was found among various groups (p N 0.05). At the stage of 3 to 9 days, the body weights of DOX group slowly grew down significantly, while those of the other groups continued to grow (p b 0.01). At the stage of 9 to 24 days, the body weights of hydrogel + DOX group were in plateau phase, and there was a significant difference between hydrogel + DOX and DOX groups due to the high toxicity of DOX (p b 0.01). The results revealed that the encapsulation and controlled release of DOX with hydrogel reduced its toxicity and improved its safety.
Fig. 6. Quantifications of relative necrotic area of tumor sections from H&E staining (A) and relative apoptosis rate from TUNEL analyses (B) after in situ administration of PBS, hydrogel, DOX, or hydrogel + DOX. Data were represented mean ± SD (n = 10; *p b 0.01, **p b 0.001).
Y. Zhang et al. / Materials Science and Engineering C 49 (2015) 262–268
Fig. 7. Body weight changes of tumor-bearing mice after in situ administration of PBS, hydrogel, DOX, or hydrogel + DOX. Data were presented as mean ± SD (n = 10; *p b 0.01, **p b 0.001).
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In addition, the pathological assessments of organs were also employed to evaluate the security of different formulations in vivo. The mice were sacrificed and the visceral organs, including the heart, liver, spleen, lung, and kidney, were isolated, sliced, and stained with H&E at 25 days after all treatments. As shown in Fig. 8, different morphologies were revealed in the organ tissues. All the tissue sections in control groups displayed normal morphology. However, there were some reactions to the organ tissues in all the treatment groups with DOX formulations due to the toxicity of DOX: 1) myocardial cells degenerated with scattered bleeding points and local inflammatory cell infiltration; 2) liver cells increased and the cytoplasm revealed pale staining; 3) the spleen showed different levels of congestion and necrosis; 4) lung tissues revealed interstitial edema, hemorrhage, and inflammatory cell infiltration; and 5) the kidney showed renal interstitial hemorrhage and abnormal shape of glomerulus. Fortunately, the hydrogel + DOX group just exhibited mild lesion types in the heart, liver, spleen, lung, and kidney tissues compared with DOX group. The above results further demonstrated that the DOX-incorporated hydrogel exhibited reduced side effects compared with DOX.
Fig. 8. Histopathological analyses of visceral organs on 25 days after in situ intratumoral injection of PBS, hydrogel, DOX, or hydrogel + DOX. Scale bar, 100 μm.
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4. Conclusions The appropriate mechanical strength and biodegradability, and sustained drug release behavior of the PLGA-b-PEG-b-PLGA thermogel highlighted its great potential as a suitable sustained drug delivery platform for in situ administration. Furthermore, the DOX-loaded hydrogel exhibited the enhanced antitumor efficacy and reduced systemic toxicities, which were confirmed by both the apparent data (i.e., the changes of tumor volume and body weight) and the in-depth analysis of the histopathology and immunohistochemistry. The above excellent properties demonstrated that the in situ sustained drug delivery systems exhibit great potential for the chemotherapy of malignancy, especially toward the advanced malignancy, and will maybe attract more and more attentions in the near future. Acknowledgments This work was financially supported by the National Natural Science Foundation of China (Projects 51303174, 81171681, 21104076, 51321062, 51233004, 51390484, 51273196, and 51203153) and the Scientific Development Program of Jilin Province (20140520050JH). References [1] J.M. Kirkwood, A. Tarhini, J.A. Sparano, P. Patel, J.H. Schiller, M.T. Vergo, A.B. Benson III, H. Tawbi, Cancer Treat. Rev. 39 (2013) 27–43. [2] E. Mannion, J.J. Gilmartin, P. Donnellan, M. Keane, D. Waldron, Support Care Cancer 22 (2014) 1417–1428. [3] J. Ding, J. Chen, D. Li, C. Xiao, J. Zhang, C. He, X. Zhuang, X. Chen, J. Mater. Chem. B 1 (2013) 69–81. [4] J. Sakamoto, T. Matsui, Y. Kodera, Gastric Cancer 12 (2009) 69–78. [5] S.M. Arnold, J.J. Rinehart, E. Tsakalozou, J.R. Eckardt, S.Z. Fields, B.J. Shelton, P.A. DeSimone, B.K. Kee, J.A. Moscow, M. Leggas, Clin. Cancer Res. 16 (2010) 673–680. [6] S.Y. Lee, H.S. Park, K.Y. Lee, H.J. Kim, Y.J. Jeon, T.W. Jang, K.H. Lee, Y.C. Kim, K.S. Kim, I.J. Oh, S.Y. Kim, Clin. Lung Cancer 14 (2013) 275–282. [7] B. Guillot, D. Bessis, O. Dereure, Expert Opin. Drug Saf. 3 (2004) 579–587.
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Contents lists available at ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Thermogel-mediated sustained drug delivery for in situ malignancy chemotherapy Yanbo Zhang a,b, Jianxun Ding a,⁎, Diankui Sun a, Hai Sun a, Xiuli Zhuang a, Fei Chang b,⁎, Jincheng Wang b,⁎, Xuesi Chen a a b
Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, PR China Department of Orthopedics, The Second Hospital of Jilin University, Changchun 130041, PR China
a r t i c l e
i n f o
Article history: Received 6 October 2014 Received in revised form 12 November 2014 Accepted 6 January 2015 Available online 8 January 2015 Keywords: Biodegradable Chemotherapy In situ administration Sustained drug delivery Thermogel
a b s t r a c t In the past few decades, the in situ sustained drug delivery platforms present fascinating potential in sentinel chemotherapy of various solid tumors. In this work, doxorubicin (DOX), a model antitumor drug, was loaded into the thermogel of poly(lactide-co-glycolide)-block-poly(ethylene glycol)-block-poly(lactide-co-glycolide). The moderate mechanical property of DOX-loaded hydrogel was confirmed by rheological test. In vitro degradation revealed the good biodegradability of thermogel. The DOX-loaded hydrogel exhibited the sustained release profiles up to 30 days without and even with elastase. The improved in vivo tumor inhibition and reduced sideeffects were observed in the DOX-incorporated hydrogel group compared with those in free DOX group. The excellent in vivo results were further confirmed by the histopathological evaluation or terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay. The thermogel with great prospect may be used as an ideal controlled drug delivery platform for the designated and long-term antitumor chemotherapy. © 2015 Elsevier B.V. All rights reserved.
1. Introduction During the past several decades, chemotherapy has been one of the most commonly used therapy approaches to treat various malignant tumors [1,2]. In the course of chemotherapy, some powerful antitumor antibiotics, such as, doxorubicin (DOX), paclitaxel, 10hydroxycamptothecin, and cisplatin, are employed to suppress the proliferation of malignant cells, and then to relieve the pain symptom of patients and improve survival [3–6]. However, the antineoplastics not only kill the diseased cells but also damage the normal cells, which induce serious systemic side effects [7]. For instance, DOX will lead to severe bone marrow suppression and cardiac toxicity [8]. DOX exhibits obvious dose-dependent efficacy, whereas the increase of dose will arouse more severe side effects that limit the wide clinical application [9]. Moreover, as depicted in Scheme 1, the unstable chemical structure of DOX is prone to decompose in the blood circulatory system, which seriously affects the treatment effect [10]. Recently, certain of in situ drug delivery systems in the forms of implants, microspheres, hydrogels, etc. have been developed to enhance the antitumor efficacy and reduce the side effects [11–13]. The drug-
⁎ Corresponding authors. E-mail addresses: [email protected] (J. Ding), [email protected] (F. Chang), [email protected] (J. Wang).
http://dx.doi.org/10.1016/j.msec.2015.01.026 0928-4931/© 2015 Elsevier B.V. All rights reserved.
loaded composites are directly injected into the lesion sites and exert the sustained payload release in a few days to several months [14]. The main advantages of in situ drug delivery platforms are facile implementation, long-lasting drug release, and extensive application [15,16], whereas various deficiencies still exist. In detail, implants are prepared from polymers and drugs via melt technique, which expose the problems of low loading efficiency and heat-induced degeneration of drugs. For instance, the microcapsules in implants containing the drugs that surrounded by films may achieve constant release, while the sudden release occurs once the thin films are broken [17]. In addition, microsphere delivery systems composed of matrix materials and drugs exhibit slowly sustained drug release after administration [18]. However, the complicated preparation processes of microspheres, such as, emulsification and solvent removal, are adverse to the biomedical applications. In recent decades, the in situ forming hydrogel-based drug delivery systems carrying a good prospect catch the eyes of researchers [19]. Among them, thermogels with unique properties show a lot of merits as drug delivery systems: 1) convenient drug encapsulation and local injectable administration; and 2) preparation without using organic solvent and photo irradiation [20,21]. Thermogels possess thermo-sensitive characteristics ascribed to the balance between the hydrophobic and hydrophilic segments [22]. The drugloaded platforms are free-flowing liquid at or below room temperature (i.e., 20 °C), while after in vivo administration, they quickly form into hydrogel at body temperature (i.e., 37 °C) [23]. Moreover,
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2. Materials and methods 2.1. Materials
Scheme 1. Molecular structure of model antitumor drug, i.e., DOX.
thermogels are three-dimensional (3D) networks with porous structures, which exhibit high loading properties, and simultaneously maintain their morphologies and high concentrations of antitumor drugs located in the solid tumors for a long time [24]. Therefore, the antitumor activity will be enhanced, and the side effects will be minimized [25]. Both natural and synthetic polymer-constructed thermogels have been selected as drug delivery platforms [19,26]. Compared with the natural thermogels, the thermogels from the synthesized polymers perform the enhanced mechanical properties and controlled properties [27, 28]. Of them, triblock poly(lactide-co-glycolide)-block-poly(ethylene glycol)-block-poly(lactide-co-glycolide) (PLGA-b-PEG-b-PLGA) copolymer is an excellent material that has many merits including easy preparation, strong dissolving capacity, controlled degradation, and good biocompatibility in pharmaceutical application to provide the sustained drug delivery [29]. In this study, the DOX-loaded hydrogel originated from PLGA-bPEG-b-PLGA (referred as hydrogel + DOX) was exploited as an in situ sustained drug delivery reservoir (Scheme 2). The moderate mechanical properties, sluggish degradation kinetics, and sustained release profiles were revealed. Meanwhile, the lowest tumor volume and reduced visceral tissue toxicities were found in the hydrogel + DOX group. The biodegradable thermogel could be a suitable candidate for the sustained sentinel drug delivery.
PEG (M n = 1500 g mol− 1) was purchased from Sigma-Aldrich (Steinheim, Germany), and triblock PLGA-b-PEG-b-PLGA copolymer (Mn, NMR = 4800 g mol−1, LA/GA = 75:25, mol/mol) was synthesized through the approach reported in our previous works [30–32]. Briefly, the copolymer was synthesized through the ring-opening polymerization of LA and GA with PEG as a macroinitiator and stannous octoate as a catalyst. Doxorubicin hydrochloride (DOX · HCl) was purchased from Beijing HuaFeng United Technology Co., Ltd. (Beijing, P. R. China). Elastase was obtained from Merck Company (Darmstadt, Germany). Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) kit was purchased from Roche Company (Mannheim, Germany). All the other reagents and solvents were purchased from Sigma-Aldrich (Steinheim, Germany) and used without further purification. 2.2. Dynamic mechanical analysis Rheological tests were conducted on a US 302 rheometer (Anton Paar Firma, Graz, Austria). The PLGA-b-PEG-b-PLGA solution (20 wt.%) without or with DOX (6.0 g L−1) in phosphate-buffered saline (PBS) was placed between parallel plates of 25 mm diameter and a gap of 0.5 mm. A layer of oil was dropped around the copolymer samples to prevent the evaporation of water. The data were collected under a controlled strain of 1% and a frequency of 1.0 rad s−1. The heating rate was 0.5 °C min−1. The storage modulus (G′), dissipative modulus (G″), and viscosity (η) were obtained in oscillatory shear flow. 2.3. In vitro DOX loading and release To determine the release profiles, DOX (6.0 g L−1) was directly and adequately dissolved in 0.5 mL of PLGA-b-PEG-b-PLGA copolymer solution of PBS (20 wt.%) at 4 °C within different vials (diameter = 16 mm), and then the laden vials were placed in a water bath at 37 °C for 30 min. Subsequently, 3.0 mL of PBS or PBS containing 0.2 g L−1 elastase, 10.0 mM CaCl2, and 0.2 wt.% NaN3, simulating in vivo microenvironment, were added on top of the thermogel. The vials were placed in
Scheme 2. Schematic presentation of DOX-incorporated hydrogel acted as a sustained drug delivery system for enhanced antitumor efficacy and reduced side effects.
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an incubator chamber at 37 °C with continuous shaking at 70 rpm. At predetermined interval, the whole medium was taken out from the vials, and an equal volume of fresh medium was replenished. The mass of the remaining gel was measured, and the release amount of DOX was tested by an ultraviolet–visible spectroscopy spectrophotometer at a wavelength of 480 nm. 2.4. Cell culture The mouse hepatoma cells (coded as H22) were cultured at 37 °C in a 5% (v/v) carbon dioxide atmosphere in Dulbecco's modified Eagle's medium (Gibco BRL, Grand Island, New York, USA) supplemented with 10% (v/v) fetal bovine serum, penicillin (50 IU mL−1), and streptomycin (50 IU mL−1).
2.7. Histopathological evaluation Hematoxylin and eosin (H&E) staining was used for the histopathological assay. The mice were sacrificed via the dislocation of cervical vertebra on 25 days. Tumors and major organs (i.e., heart, liver, spleen, lung, and kidney) were collected and embedded with paraffin. 6 μm thick transverse sections were cut and then stained with H&E to evaluate the histological alterations by a microscope. The necrotic area was defined as the pink-dyed cell-free region fused from the karyolysis and disintegration of organelle. The relative necrotic area (%) was calculated by Eq. (2). Relative necrotic areað%Þ ¼
necrotic area in tumor section 100: total area in tumor section
ð2Þ
2.5. Animal procedure 2.8. In situ apoptosis detection Kunming mice (male, 25 ± 2 g) were supplied by the Experimental Animal Center of Jilin University. All the experiments on animals were approved by the Animal Care and Use Committee of Jilin University. The animals were raised individually in plastic cages with food and water at a temperature of 25 °C under natural light/dark conditions for 2 weeks before experiment. 2.6. In vivo antitumor assessment The hepatoma model was established in mouse by the subcutaneous injection of 0.1 mL of suspension containing 3.0 × 106 H22 cells in PBS. The treatment was started when the mean tumor volume reached ~ 200 mm3. The mice were divided into four groups (10 mice per group), and 0.1 mL of PBS, PLGA-b-PEG-b-PLGA (hydrogel; 20.0 wt.% copolymer), DOX (30.0 mg kg−1), and DOX-incorporated PLGA-b-PEG-bPLGA solutions in PBS (hydrogel + DOX; 20.0 wt.% copolymer and 30.0 mg kg− 1 DOX) were separately injected into mice by the intratumoral injection. The body weight and tumor volume were recorded for 25 days. Tumor volume was measured by vernier caliper and was calculated by Eq. (1). L S2 3 V mm ¼ : 2
ð1Þ
The TUNEL apoptosis assay was carried out in accordance with the product manual (Roche, Basel, Switzerland). 6 μm thick tumor sections were washed and the nicked DNA ends were labeled by the mixture of enzyme and label solutions. The in situ cell apoptosis of tumor tissues was observed by a confocal laser scanning microscope (CLSM; LSM 700, Carl Zeiss, Jena, Germany). The apoptosis area was defined as the green fluorescence-labeled region originated from the late apoptotic cells. The relative apoptosis rate (%) was calculated by Eq. (3). Relative apoptosis rateð%Þ ¼
apoptosis area in tumor section 100:ð3Þ total area in tumor section
2.9. Statistical analysis All experiments were performed at least three times, and the results were expressed as means ± standard deviation (SD). Statistical significances were analyzed using SPSS (Version 18.0, Chicago, IL, USA). p b 0.05 was considered statistically significant, and p b 0.01 and p b 0.001 were considered highly statistically significant. 3. Results and discussion 3.1. Characterizations of hydrogels without or with DOX
In Eq. (1), L and S (mm) were the largest and smallest diameters of tumor, respectively. Meanwhile, the body weight changes were monitored at each time interval to determine the in vivo security of samples.
Hydrogel + DOX could be prepared through dispersing 6.0 g L−1 of DOX into the aqueous copolymer solution of PBS (20 wt.%) and subsequently elevating temperature. Firstly, the changes of G′ and η versus
Fig. 1. G′ (A) and η (B) of PLGA-b-PEG-b-PLGA solution in PBS (20 wt.%) without or with DOX (6.0 g L−1).
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3.2. In vivo antitumor efficacy
the increase of temperature were determined by rheological tests (Fig. 1). Compared with blank copolymer system, the initial G′ and η of DOX-incorporated platform significantly increased to 2.2 Pa and 2.2 Pa s, respectively, while the maximum of G′ and η decreased. It should be attributed that the amphiphilicity of DOX interfered with the self-assembly of copolymer and the association of micelle [25]. The change trends of rheological results were consistent with the previously reported results [33]. In addition, the G′ of hydrogel + DOX at 37 °C was about 100 Pa, which highlighted the potential of this thermogel as an in situ sustained drug delivery system. The degradation rate is one of the principal influencing factors on drug release behavior [34]. In this work, the degradation rate of hydrogel in PBS with elastase was faster (mass remaining: 37%, v/v) than that in blank PBS (mass remaining: 78%, v/v) at 30 days (Fig. 2). Simultaneously, the DOX release behavior from hydrogel + DOX was investigated in PBS without or with elastase. As shown in Fig. 3, DOX released from laden hydrogel in a controlled and sustained manner without initial burst release [29]. The microchannel formed by the hydrogel dissolution might contribute to the diffusion of DOX release. As expected, the presence of elastase accelerated the DOX release, which was consistent with the degradation profiles and demonstrated the correlation between the degradation and release [34].
Tumor inhibition detection toward tumor-bearing mouse models is the common approach to assess the efficacy of controlled drug delivery systems [35]. In this study, the in vivo antitumor activity assays were performed toward the mice xenografted with H22 tumors. After the tumor volume grew to about 200 mm3, PBS, hydrogel, DOX, or hydrogel + DOX was intratumorally injected one time, respectively. The survival conditions of mice were observed by the mental state and activities. Tumor volumes and body weights were simultaneously recorded. The mice of control groups (i.e., PBS and hydrogel groups) and DOX group showed poorer mental state, sluggish movement, and dull fur in relative to that of hydrogel + DOX group. As shown in Fig. 4, the tumors of control group grew fast and increased to over 1700 mm 3 at 25 days, and there was no statistical difference between PBS and hydrogel groups (p N 0.05). In stark contrast, the tumors in DOX and hydrogel + DOX groups grew slowly, and both of them exhibited significant difference compared with control groups (p b 0.001). It was indicated that DOX inhibited tumor growth effectively. Most importantly, the tumor volumes of hydrogel + DOX group at 25 days were significantly lower than those of DOX group owing to the sustained release of DOX (p b 0.01), which demonstrated the distinct advantage of thermogel as a continuous drug reservoir [19]. The tumor suppression was further confirmed by histopathological and apoptosis assays. The experimental mice were sacrificed on day 25, and the tumors were separated, sliced, and then stained with H&E for histopathological evaluation. H&E staining was defined as the chromatin within the nucleus was stained to be bluish violet by the alkaline hematoxylin, while the cytoplasm and the extracellular matrix were stained to be pink by the acid eosin dyes. As shown in Fig. 5, the PBS and hydrogel groups displayed well-grown tumor cells with large nuclei and a spherical or spindle shape, and small area of necrosis. Both DOX and hydrogel + DOX groups exhibited large areas of hemorrhage and necrosis, and other characteristic pathological features: 1) the morphology of necrotic tissue was not clear; 2) the chromatin was fuzzy or scattered outside cells; 3) the nuclear condensation, fragmentation, or disappearance were observed. It all meant that the DOX formulations, especially the DOX-loaded hydrogel, exhibited the significant antitumor efficacy against hepatic carcinoma in mice. In addition, the relative necrosis areas of control groups were less than that of DOX group (p b 0.01), and significantly lower than that of hydrogel + DOX group (p b 0.001) (Fig. 6A). More interestingly, the hydrogel + DOX group showed more necrosis than that of DOX group (p b 0.01). Simultaneously, the in situ cell apoptosis of tumor after treatment was analyzed by TUNEL assay. Apoptosis activates DNA enzyme leading
Fig. 3. Cumulative release of DOX from DOX-incorporated hydrogel (6.0 g L−1) in PBS without or with elastase at 37 °C. Data were presented as mean ± SD (n = 3).
Fig. 4. In vivo antitumor efficiency after in situ intratumoral injection of PBS, hydrogel, DOX, or hydrogel + DOX. Data were presented as mean ± SD (n = 10; *p b 0.01, **p b 0.001).
Fig. 2. In vitro mass remaining of PLGA-b-PEG-b-PLGA hydrogel (20 wt.%) with DOX (6.0 g L−1) at 37 °C immerged in 3.0 mL of PBS or PBS containing 0.2 g L−1 elastase. Data were presented as mean ± SD (n = 3).
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Fig. 5. H&E and TUNEL evaluations of tumor sections after treatments with PBS, hydrogel, DOX, and hydrogel + DOX. The black arrows pointed to the typical necrotic area. Scale bar, 100 μm.
to the genomic DNA fracture that can be labeled as green fluorescence. The low, moderate, and high fluorescence intensities and areas were observed in control, DOX, and hydrogel + DOX groups via CLSM, respectively, which were further confirmed by the 2.5D TUNEL microimages (Fig. 5). The relative apoptosis rates of all treatment groups with different DOX agents were significantly more than those of control groups (p b 0.001), and the relative apoptosis rate of hydrogel + DOX group was higher than that of DOX group (p b 0.01) (Fig. 6B). The TUNEL results were consistent with those of in vivo tumor inhibition and H&E assays, and these all confirmed the highest antitumor activity of DOX-incorporated hydrogel. 3.3. In vivo security evaluation The in vivo security of antineoplastic agents is another important evaluation index, which is related to the life safety of malignancy patients [36]. In this study, the safety assessments were
performed on the measurements of body weights and the histopathological analyses of visceral organs (i.e., heart, liver, spleen, lung, and kidney). To assess the toxicities of various DOX formulations, the body weights as a key indicator were real-time monitored. As shown in Fig. 7, at the beginning of 3 days after the administrations with different formulations toward hepatic tumors, the body weights in all groups showed growing tendency, while no significant difference was found among various groups (p N 0.05). At the stage of 3 to 9 days, the body weights of DOX group slowly grew down significantly, while those of the other groups continued to grow (p b 0.01). At the stage of 9 to 24 days, the body weights of hydrogel + DOX group were in plateau phase, and there was a significant difference between hydrogel + DOX and DOX groups due to the high toxicity of DOX (p b 0.01). The results revealed that the encapsulation and controlled release of DOX with hydrogel reduced its toxicity and improved its safety.
Fig. 6. Quantifications of relative necrotic area of tumor sections from H&E staining (A) and relative apoptosis rate from TUNEL analyses (B) after in situ administration of PBS, hydrogel, DOX, or hydrogel + DOX. Data were represented mean ± SD (n = 10; *p b 0.01, **p b 0.001).
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Fig. 7. Body weight changes of tumor-bearing mice after in situ administration of PBS, hydrogel, DOX, or hydrogel + DOX. Data were presented as mean ± SD (n = 10; *p b 0.01, **p b 0.001).
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In addition, the pathological assessments of organs were also employed to evaluate the security of different formulations in vivo. The mice were sacrificed and the visceral organs, including the heart, liver, spleen, lung, and kidney, were isolated, sliced, and stained with H&E at 25 days after all treatments. As shown in Fig. 8, different morphologies were revealed in the organ tissues. All the tissue sections in control groups displayed normal morphology. However, there were some reactions to the organ tissues in all the treatment groups with DOX formulations due to the toxicity of DOX: 1) myocardial cells degenerated with scattered bleeding points and local inflammatory cell infiltration; 2) liver cells increased and the cytoplasm revealed pale staining; 3) the spleen showed different levels of congestion and necrosis; 4) lung tissues revealed interstitial edema, hemorrhage, and inflammatory cell infiltration; and 5) the kidney showed renal interstitial hemorrhage and abnormal shape of glomerulus. Fortunately, the hydrogel + DOX group just exhibited mild lesion types in the heart, liver, spleen, lung, and kidney tissues compared with DOX group. The above results further demonstrated that the DOX-incorporated hydrogel exhibited reduced side effects compared with DOX.
Fig. 8. Histopathological analyses of visceral organs on 25 days after in situ intratumoral injection of PBS, hydrogel, DOX, or hydrogel + DOX. Scale bar, 100 μm.
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