Acta Biomaterialia 10 (2014) 1280–1291

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A tumoral acidic pH-responsive drug delivery system based on a novel photosensitizer (fullerene) for in vitro and in vivo chemo-photodynamic therapy Jinjin Shi 1, Yan Liu 1, Lei Wang, Jun Gao, Jing Zhang, Xiaoyuan Yu, Rou Ma, Ruiyuan Liu, Zhenzhong Zhang ⇑ School of Pharmaceutical Sciences, Zhengzhou University, 100 Kexue Avenue, Zhengzhou 450001, People’s Republic of China

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Article history: Received 22 April 2013 Received in revised form 8 October 2013 Accepted 30 October 2013 Available online 6 November 2013 Keywords: Fullerene pH-responsive Nanocarrier Photodynamic therapy Synergistic effect

a b s t r a c t Fullerene has shown great potential both in drug delivery and photodynamic therapy. Herein, we developed a doxorubicin (DOX)-loaded poly(ethyleneimine) (PEI) derivatized fullerene (C60–PEI–DOX) to facilitate combined chemotherapy and photodynamic therapy in one system, and DOX was covalently conjugated onto C60–PEI by the pH-sensitive hydrazone linkage. The release profiles of DOX from C60–PEI–DOX showed a strong dependence on the environmental pH value. The biodistributions of C60–PEI–DOX were investigated by injecting CdSe/ZnS (Qds) labeled conjugates (C60–PEI–DOX/Qds) into tumor-bearing mice. C60–PEI–DOX/Qds showed a higher tumor targeting efficiency compared with Qds alone. Compared with free DOX in an in vivo murine tumor model, C60–PEI–DOX afforded higher antitumor efficacy without obvious toxic effects to normal organs owing to its good tumor targeting efficacy and the 2.4-fold greater amount of DOX released in the tumor than in the normal tissues. C60–PEI–DOX also showed high antitumor efficacy during photodynamic therapy. The ability of C60–PEI–DOX nanoparticles to combine local specific chemotherapy with external photodynamic therapy significantly improved the therapeutic efficacy of the cancer treatment, the combined treatment demonstrating a synergistic effect. These results suggest that C60–PEI–DOX may be promising for high treatment efficacy with minimal side effects in future therapy. Ó 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction The ultimate goal of cancer therapeutics is to increase the survival time and the quality of life of the patient by reducing the unintended harmful side effects [1]. The most common cancer treatments are chemotherapy, radiation and surgery [2], with chemotherapy being the major treatment modality. Many therapeutic anticancer drugs, while pharmacologically effective in cancer treatment, are limited in their clinical applications due to their serious toxicities [3]. For example, doxorubicin (DOX) is one of the most effective drugs against a wide range of cancers. However, its clinical use is limited by severe side effects, such as cardiotoxicity and acquired drug resistance [4], which may be the cause of cancer treatment failure [5]. To overcome these obstacles, in recent years, with the development of cancer nanotechnology, researchers have focused on developing nanoscale anticancer drug carriers to improve therapeutic efficacy as well as reduce unwanted side effects [6–8]. Fullerene (C60), an allotrope of carbon, is a nanoscale carbon material with unique photochemical, electrochemical and ⇑ Corresponding author. Tel.: +86 371 67781910; fax: +86 371 67781908. 1

E-mail address: [email protected] (Z. Zhang). These authors contributed equally to this work.

physical properties and, with its low systemic toxicity, shows tremendous promise for target-specific delivery of drugs in the body. However, its inherent hydrophobicity limits its use in biology, so the employment of fullerenes for drug delivery is still at an early stage of development [9]. To date, there are only a few reports about fullerene derivatives being used for the delivery of anticancer drugs [10,11]. Recently, we reported a polyethyleneimined fullerene (C60–PEI) that was modified with folic acid (FA) through an amide linker, then docetaxel was successfully encapsulated within the C60–PEI–FA nanoparticles, and this C60-based drug delivery system afforded higher antitumor efficacy without obvious toxic effects to normal organs owing to its prolonged blood circulation and the 7.5-fold higher DTX uptake in the tumor [12]. Another great advantage of fullerene is its potential in photodynamic therapy (PDT) [13,14]. PDT had been applied in the treatment of a number of malignant tumors, employing the activation of tumor-localizing photosensitizers (PS) by visible light to produce reactive oxygen species (ROS), leading to a cytotoxic effect [15]. The absorption of visible light combined with an efficient intersystem crossing to a long-lived triplet state caused fullerenes to generate ROS upon illumination and make fullerenes PS [16,17]. Due to the enormous PDT potential of fullerene, in recent years there has been much interest in studying the biological activity

1742-7061/$ - see front matter Ó 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.actbio.2013.10.037

J. Shi et al. / Acta Biomaterialia 10 (2014) 1280–1291

of fullerenes with a view to using them in medicine [18]; for example, malonic acid derivatives of C60 show obvious photosensitivity and significant cytotoxicity in in vitro experiments [19]. Sugar derivatives of C60 with high solubility can cause the death of cancer cells exposed to visible light [20]. In this work, PDT was also conducted in order to obtain a synergistic effect to B16-F10 cells and malignant tumor in C57 mice models by use of the C60-based drug delivery system. The metabolic profile of tumors is different within the interstitial matrix, where poor oxygen perfusion causes elevated levels of lactic acid and a reduction in pH from 7.40 to about 6.00 [21,22]. These variations in the pH of tumor tissues can be strategies for pH-sensitive drug delivery at local microenvironments due not only to the decrease in cytotoxicity but also the increased efficacy of chemotherapy. Over the past decade, pH-responsive biomaterial studies have attracted much attention. Recently, Nobusawa and co-workers [23] have developed a new pH-responsive smart carrier of C60 which was prepared with 6-amino-c-cyclodextrin (ACD) for photodynamic therapy. In that study C60 was rapidly released from its inclusion complex with ACD at pH 6.7, due to electrostatic repulsion between ammonium groups, followed by rapid C60 release at slightly acidic cancer cell surfaces. Guo et al. [24] demonstrated that super-paramagnetic nanocarriers with folatemediated and pH-responsive targeting properties had obvious antitumor efficacy for HeLa cells. The hyaluronic acid–DOX nanoconjugate exhibits a sustained release characteristic, reduces toxicity and inhibits breast cancer progression in xenografts of human breast cancer, leading to an increased survival rate [2]. In general, a well-designed pH-responsive nanocarrier should have a high loading efficiency and long-term stability in the circulatory system, and should respond rapidly to an acidic pH stimulus and consequently release the drug in the pathological area [25]. For pH-sensitive nanocarrier–anticancer drug conjugates, various synthetic approaches have been adopted, including spacers such as acid-labile linkages between the nanoscale materials and the drug. The hydrazone bond is such an acid-labile linkage. It can quickly hydrolyze in an acidic environment (pH < 7), such as tumor tissue, but is stable in a neutral environment (pH 7.4), such as blood [26]. The hydrazone bond has been widely used in pH-sensitive drug delivery. In this study, DOX was attached to C60 via hydrolytically degradable pH-sensitive hydrazone bonds to obtain a controlledrelease DOX delivery system. In the current study, a new DOX delivery system with pH sensitivity and photodynamic therapy was designed, synthesized, characterized and explored for its potential applications both in drug delivery and in photodynamic therapy. The drug delivery system was based on C60–PEI, which has high aqueous solubility and a neutral pH, and is accessible for further modification. PEI grafted onto the surface of C60 has a dendritic structure with a large number of NH2 groups [12], and the antitumor drug can link to these groups to a very high loading capacity. Furthermore, the dendritic structure PEI can spread the antitumor drug throughout the whole drug delivery system and obstruct the release of drug, thereby reducing the burst release. DOX, which has been used extensively in the treatment of various cancers, was employed as the model drug and linked to PEI via two steps. First, an intermediate, carboxyl phenylhydrazine, was reacted with carboxyl to form amide bonds and linked to the termini of branched PEI via primary amine bonds, then DOX was linked to the carboxyl phenylhydrazine via hydrolytically degradable pH-sensitive hydrazone bonds. The DOX release rate and efficiency could be controlled by hydrazone bonds by changing the pH. Herein, a controlled-release DOX drug delivery system (C60–PEI–DOX) with photodynamic therapeutic activity (Fig. 1) was developed and characterized by transmission electron microscopy (TEM) and dynamic light scattering (DLS), and its DOX release efficiency or its treatment effect was examined

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Fig. 1. Scheme of drug loading, pH-dependent released from C60–PEI–DOX and photodynamic therapy.

using mouse melanoma B16-F10 cells and melanoma tumor-bearing mice models. 2. Materials and methods 2.1. Materials Fullerene (C60, purity >95%) was purchased from Henan Fengyuan Chemicals Co. Ltd. Doxorubicin (DOX, 20120503, purity >98%) was obtained from Beijing Yi-He Biotech Co. Ltd. Aziridine, ethylenediamine (H2NCH2CH2NH2), carboxyl phenylhydrazine (HBA), N-(3-dimethylamino propyl-N0-ethylcar-bodiimide) hydrochloride (EDCHCl) and dimethyl sulfoxide (DMSO) were obtained from Sigma–Aldrich Co. LLC. Sulforhodamine B (SRB), RPMI 1640 cell culture medium, penicillin, streptomycin, fetal bovine serum (FBS) and heparin sodium were bought from Gibco Invitrogen. Quantum dots (CdSe/ZnS, Qds) were supplied by WuHan Jiayuan Quantum Dots Co. Ltd. Other reagents were acquired from China National Medicine Corporation Ltd. The dialysis bags (MWCO = 10,000) were from Spectrum Laboratories Inc. 2.2. Synthesis of C60–PEI–DOX C60–PEI was synthesized according to the procedure of our previous study [12]. In brief, ethylenediamine (40 ml), dissolved in an ethanol–water mixture (ethanol:water = 5:1, 96 ml), was added dropwise to a stirring dry toluene solution (200 ml) containing C60 (200 mg) and NaOH (2.0 g). After stirring at room temperature under the protection of Ar for 7 days, the mixture was evaporated and dried in a vacuum at 60 °C for 24 h, then purified by repeated rinsing with an ethanol–water mixture (ethanol:water = 6:1) and filtration. The resulting solid product was dried in a vacuum at 60 °C for 24 h. The above product (100 mg), concentrated HCl (20 ll) and aziridine (1.0 ml) were added to dichloromethane (60 ml), stirred and refluxed at 40 °C under the protection of Ar for 24 h. C60–PEI was obtained by filtering and washing, in order, in CH2Cl2 10 times, methanol twice and deionized water several times. DOX was then attached to the C60–PEI through a hydrazone bond via the following steps. C60–PEI (50 mg) was suspended with HBA (50 mg) in a pH 7.4 phosphate-buffered saline (PBS) solution (50 ml), then EDCHCl (80 mg) and N-hydroxysuccinimide (35 mg) were added. The mixture was allowed to react at room temperature for 48 h, after which the conjugate was precipitated using 200 ml of anhydrous ethanol, then the precipitated was purified by repeated rinsing with ethanol and filtrations to remove the

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unreacted reagents to obtain C60–PEI–HBA complex. The resulting solid products were dried in a vacuum at 60 °C for 24 h. The above product (50 mg) and DOX (100 mg) were added to DMSO (50 ml), stirred at room temperature in the dark for 24 h, then dialyzed with a membrane (MWCO = 10,000, Spectrum Laboratories Inc.) for 3 days to remove free DOX and DMSO. Finally, the solution was freeze-dried to obtain C60–PEI–DOX as a fine powder. The amount of DOX loaded onto C60–PEI was measured at 490 nm using an ultraviolet–visible (UV-Vis) spectrometer (Lambda 35, Perkin-Elmer, USA). 2.3. Characterization DLS (Zetasizer Nano ZS-90, Malvern, UK) and TEM (Tecnai G2 20, FEI, HK) were used to characterize the particle size, zeta potential and morphology of C60–PEI–DOX. The optical properties of C60–PEI and C60–PEI–DOX were characterized using a UV-Vis spectrometer. Fourier transform infrared (FTIR) spectra were recorded with a Nicolet iS10 IR spectrophotometer (Thermo, USA). 2.4. Evaluation of pH sensitivity The release studies were performed in a glass beaker at 37 °C in acetate buffer (pH 5.5) and phosphate buffer (pH 7.4) solutions. First, 20 mg of C60–PEI–DOX was dispersed in 5 ml of water and placed in a dialysis bag. The dialysis bag was then immersed in 45 ml of the release medium and kept in a horizontal laboratory shaker at a constant temperature and with constant stirring (100 rpm). Samples (2 ml) were periodically removed and the volume of each sample was replaced by the same volume of fresh medium. The amount of DOX released was analyzed with a spectrophotometer at 490 nm. The drug release studies were performed in triplicate for each sample. 2.5. Cell culture, cytotoxicity and phototoxicity assay The B16-F10 mice melanoma cell line was obtained from the Chinese Academy of Sciences Cell Bank (Catalog No. TCM36). Cells were cultured in normal Dulbecco’s modified Eagle’s medium (DMEM) with 10% FBS and 1% penicillin/streptomycin in 5% CO2 and 95% air at 37 °C in a humidified incubator. The cytotoxicity of C60–PEI–DOX to B16-F10 cells was assessed by using the standard SRB assay. The B16-F10 cells were cultured and lifted as described above before seeding (1  104) into 96-well plates and incubating for 24 h. The medium was then replaced with fresh medium containing various concentrations of free DOX, C60–PEI and C60–PEI–DOX for 24 h. The cells were irradiated or not with a 532 nm laser (diode laser, CW, Changchun Laser Research Center), with a power density of 100 mW cm2 for 5 min. 2.6. Cellular uptake The cellular uptake experiments were performed using flow cytometry and fluorescence microscopy. For the flow cytometry, B16-F10 cells were seeded at 5  104 cells per well in 6-well plates. The cells were then treated with free DOX (10 lg ml1) or C60– PEI–DOX (DOX concentration: 10 lg ml1 and C60–PEI concentration: 11.2 lg ml1) for 1 and 2 h. At specific time points, cell medium was removed and cells were washed with PBS, then trypsinized and resuspended in fresh DMEM culture medium. After adjusting the cell density to 1  106 cells ml1, the samples were analyzed by flow cytometry (Epics XL, Coulter, USA). For fluorescence microscopy studies, B16-F10 cells were seeded at 5  104 cells per well on glass cover slips in 6-well plates. When cells reached 70% confluence, they were treated with DOX (10 lg ml1) and C60–PEI–DOX (DOX concentration: 10 lg ml1

and C60–PEI concentration: 11.2 lg ml1) for 2 h. At specific time points, cell medium was removed and cells were washed with PBS, followed by soaking in 4% polymerisatum for 15 min and washing with deionized water. The cells were imaged with a fluorescence microscope (LSM 510, Zeiss, Germany, excitation wavelength: 633 nm). 2.7. Determination of intracellular ROS B16-F10 cells were incubated with C60–PEI–DOX (DOX concentration: 5 lg ml1 and C60–PEI concentration: 5.6 lg ml1) for 24 h, then 100 mW cm2 of irradiation was delivered by a 532 nm laser for 5 min. After that, 5-(and-6)-chloromethyl-20 -70 dichlorodihydrofluoresceindiacetate (5 lg ml1, CM-H2DCFDA; Molecular Probes, Invitrogen) in complete medium was added to incubate at 37 °C. After incubation for 30 min in the dark, cells were washed with PBS. The cells were imaged with the fluorescence microscope at an excitation wavelength of 488 nm. 2.8. Detection of DNA fragmentation B16-F10 cells were incubated with C60–PEI–DOX (DOX concentration: 5 lg ml1 and C60–PEI concentration: 5.6 lg ml1) for 24 h, then 100 mW cm2 was delivered by the 532 nm laser for 5 min. Next, the cells were collected and resuspended in prewarmed low-melting-point agarose (LMA), before 100 ll of the cell LMA suspension was placed on a slide that had been pre-coated with normal-melting-point agarose and lysed in cold lysis solution (Beijing CoWin Bioscience Co., Ltd.) for 2 h. After lysis, the samples were electrolyted for 25 min. Neutralization buffer was next added to the samples at room temperature in the dark for 45 min, before the samples were dried at room temperature and stained in the dark with 2% ethidium bromide. Finally, the samples were observed by the fluorescence microscope at an excitation wavelength of 633 nm. 2.9. Xenograft tumor mouse model All animal experiments were performed under a protocol approved by Henan laboratory animal committee. Mice melanoma tumor models were generated by subcutaneous injection of 1  106 B16-F10 cells in 0.1 ml saline into the right shoulder of female C57 mice (18–22 g, Henan Laboratory Animal Center). The mice were used when the tumor volume reached 60–100 mm3 (4 days after tumor inoculation). 2.10. In vivo antitumor effect The mice were divided into five groups (six mice per group), minimizing the differences in weights and tumor sizes in each group. The mice were administered with (a) saline (0.1 ml), (b) C60–PEI/532 nm laser (5.6 mg kg1), (c) DOX (5 mg kg1), (d) C60–PEI–DOX (DOX dose: 5 mg kg1, C60–PEI dose: 5.6 mg kg1) and (e) C60–PEI–DOX/532 nm laser (DOX dose: 5 mg kg1, C60– PEI dose: 5.6 mg kg1) in saline, injected intravenously via the tail vein every 2 days, then the tumor regions were irradiated at 100 mW cm2 for 5 min with the 532 nm laser at 3 h post-injection. The mice were observed daily for clinical symptoms and the tumor sizes were measured by a caliper every other day and calculated as volume = (tumor length)  (tumor width)2/2. After treatment for 10 days, the mice were killed to collect the heart, liver, spleen, lung, kidney and tumor. The collected tissues were soaked in 10% formalin solution and embedded with paraffin for hematoxylin and eosin (H&E) staining. Morphological changes were observed under the fluorescence microscope.

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2.11. DOX release from C60–PEI–DOX in different tissues The tumor-bearing mice were fasted but had access to water ad libitum for 12 h before being killed. Their main organs (heart, liver, spleen, lung, kidney and tumor) and blood were collected separately and weighed, then saline was added to each sample (saline weight:organ weight = 1:1) and homogenized. C60–PEI–DOX (4 mg) was dispersed in 1 ml of water and placed in a dialysis bag. The dialysis bag was then immersed in 9 ml of the homogenates of the different organs and kept in a horizontal laboratory shaker that was maintained a constant temperature with constant stirring (100 rpm). After 48 h, 500 ll of homogenate was removed, added to a chloroform–methanol mixture (chloroform:methanol = 4:1, 2 ml) and mixed by vortex. After centrifugation at 4000 rpm for 20 min, the chloroform layers were collected and the concentrations of DOX were determined by high-performance liquid chromatography (1100 Agilent, USA) under the following conditions: an Eclipse XDB-C18 column (150 mm  4.6 mm, 5 lm); mobile phase methanol:0.01 mol ml1 potassium dihydrogen phosphate:glacial acetic acid (68:31.5:0.5); column temperature 30 °C; flow rate 1.0 ml min1; injection volume 40 ll; and fluorescence excitation wavelength (480 nm) and emission wavelength (550 nm) [27]. The studies were performed in triplicate for each sample. 2.12. Biodistribution studies Qds (CdSe/ZnS, amino-modified, 10 nm, excitation wavelength: 633 nm, 1.5 lM, 200 ll) were added to C60–PEI–DOX nannosuspension (C60–PEI–DOX dose: 1 mg ml1, 3 ml) and stirred at room temperature in the dark for 12 h. The resulting product (C60–PEI– DOX–Qds) was obtained by repeating rinsing with deionized water and filtration. A 0.2 ml volume of Qds or C60–PEI–DOX–Qds (with the same dose of Qds, confirmed by fluorescence spectra) was intravenously injected into tumor-bearing mice (three mice per group). After injection for 3 h, the mice were killed to collect the heart, liver, spleen, lung, kidney and tumor, then the collected tissues were made into frozen sections and imaged with the fluorescence microscope at an excitation wavelength of 633 nm. 2.13. Statistical analysis The quantitative data are expressed as mean ± SD and statistically analyzed using Student’s t-test. p values of

A tumoral acidic pH-responsive drug delivery system based on a novel photosensitizer (fullerene) for in vitro and in vivo chemo-photodynamic therapy.

Fullerene has shown great potential both in drug delivery and photodynamic therapy. Herein, we developed a doxorubicin (DOX)-loaded poly(ethyleneimine...
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