Article pubs.acs.org/molecularpharmaceutics
Heparin-Conjugated Pluronic Nanogels as Multi-Drug Nanocarriers for Combination Chemotherapy Yoon Ki Joung,†,⊥,§ Ji Young Jang,‡,§ Jong Hoon Choi,† Dong Keun Han,⊥ and Ki Dong Park*,† †
Department of Molecular Science and Technology, Ajou University, 5 Wonchon, Yeongtong, Suwon, 443-749, Republic of Korea Tumor Immunity Medical Research Center, Cancer Research Institute, Seoul National University College of Medicine, 28 Yongon-dong, Jongno-gu, Seoul 110-799, Republic of Korea ⊥ Center for Biomaterials, Biomedical Research Institute, Korea Institute of Science and Technology, Hwarangno 14-gil 5, Seongbuk-gu, Seoul 136-791, Republic of Korea ‡
ABSTRACT: Combination chemotherapy using more than two therapeutic agents with different modes of action is a promising strategy that can be used to enhance the therapeutic efficacy of cancer treatment, even though it is a complicated treatment modality. The aim of this study was to investigate how a novel multidrug nanocarrier is effective for combination chemotherapy in vitro and, more specifically, whether combined agents with different modes of action and physicochemical properties show synergistic cytotoxicity with the use of this nanocarrier. A heparin−Pluronic (Hep−Pr) nanogel encapsulating both paclitaxel and DNase was shown to be efficient for intracellular delivery with respect to size, encapsulation efficiency, and intracellular uptake/fates. As a result of these properties, a Hep−Pr nanogel combined with paclitaxel and DNase exhibited a dose-dependent synergistic cytotoxicity compared to single drug and free-drug treatments, whose combination indices were 0.93 and 0.45 at higher concentrations (250 and 500 μg/mL). Therefore, Hep−Pr nanogels have the potential to deliver multitherapeutic agents with different characteristics and thereby enhance the therapeutic efficacy of combination cancer chemotherapy. KEYWORDS: heparin, Pluronic, combination chemotherapy, DNase, paclitaxel, nanocarrier
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INTRODUCTION Over the past five decades, cancer chemotherapy has been intensively researched, but it requires complicated treatments.1 Combination chemotherapy has been highlighted as an alternative strategy because of the finding that chemotherapy drugs are more effective when given in combination.2 The rationale for combination chemotherapy is to use drugs that work by different mechanisms of action and thereby decrease the likelihood that resistant cancer cells will develop. When drugs with different effects are combined, each drug can be used at its optimal dose without intolerable side effects and ultimately result in synergistic cytotoxicity in vitro and in vivo.3 Among many potent anticancer agents with different modes of action, there are many examples of their combination. For instance, paclitaxel is an emerged anticancer drug that is effective against a broad range of tumor types, including breast, ovarian, lung, head, and neck cancers, other malignancies that are refractory to conventional chemotherapy, including previously treated lymphoma and small-cell lung cancers and esophageal, gastric endometrial, bladder, and germ cell tumors, and even AIDS-associated Kaposi’s sarcoma. The mode of action of paclitaxel is known to interfere with the normal function of microtubule breakdown. Consequently, paclitaxel arrests tumor function by having the opposite effect: it hyperstabilizes their structure. This destroys the cell’s ability to use its cytoskeleton in a flexible manner.4,5 In addition, © 2012 American Chemical Society
deoxyribonuclease (DNase) is an enzyme that catalyzes the hydrolytic cleavage of phosphodiester linkages in the DNA backbone and is a type of nuclease.6 A wide variety of DNases is known, and they differ in their substrate specificities, chemical mechanisms, and biological functions. Some DNases cleave only residues at the ends of DNA molecules (exoDNases, which are a type of exonuclease). Others cleave anywhere along the chain (endoDNases, which are a subset of endonucleases). Some are fairly indiscriminate about the DNA sequence at which they cut, while others, including restriction enzymes, are very sequence-specific. Some cleave only double-stranded DNA, others are specific for single-stranded molecules, and still others are active toward both. Over the last several decades, there have been many clinical and preclinical trials of combination chemotherapies against various cancers, along with analytical studies of their synergistic effects.7−11 However, most of these studies were limited to nonformulated forms without drug carriers or the use of similar drug classes. A few examples of other combinations were reported, such as epidermal growth factor (EGF)-conjugated micelles loaded with ellipticine, the combination of doxorubicin Received: Revised: Accepted: Published: 685
August 30, 2012 November 30, 2012 December 13, 2012 December 13, 2012 dx.doi.org/10.1021/mp300480v | Mol. Pharmaceutics 2013, 10, 685−693
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thromboplastin time (aPTT).12,13 For confocal microscopic analysis, FITC was conjugated to the Hep−Pr conjugate according to our previous method.23 Preparation of Hep−Pr Nanogels with Drugs. The paclitaxel and/or DNase-loaded Hep−Pr nanogels and Pluronic micelles (control sample) were prepared by a direct dissolution method. Briefly, 100 mg of the Hep−Pr conjugate or Pluronic was dissolved in 50 mL of deionized water below 4 °C. To the solution, various concentrations of paclitaxel (0.5, 1, and 3 mg/ 1 mL of ethanol) or DNase (1, 5, and 20 mg/1 mL of deionized water) were added and stirred at room temperature for 24 h. The product solution was filtered through a membrane (pore size = 0.45 μm) in order to remove unloaded paclitaxel. In order to remove unloaded DNase, a poly(ethylene glycol) solution (1 wt %) was added and filtered out. The filtrate was dispersed at 37 °C for 10 min by applying ultrasound (Branson 5510 sonicator), which was followed by lyophilization. The yield of the Hep−Pr nanogel was about 80%. The critical micelle concentration (CMC) in the aqueous solution was measured by fluorescence spectroscopy (FP− 6500, JASCO, Inc., Japan) using pyrene as a probe.14 Determination of Drug Loading Efficiency. In order to estimate the loading efficiency of paclitaxel into the Hep−Pr nanogel and Pluronic micelle, an extraction method was carried out.15 Paclitaxel-loaded Hep−Pr nanogels and Pluronic micelles were dissolved in deionized water at 4 °C, and methylene chloride was added. After stirring vigorously, paclitaxel was extracted to the organic phase, and the solvent was evaporated. The amount of paclitaxel in the dried samples was measured by using high-performance liquid chromatography (HPLC; JASCO, Inc.). The samples and standard solutions were injected into a reverse-phase column (Kromasil KR100 5C18, 250 mm × 4.6 mm, Sweden). The mobile phase of acetonitrile/ water (50/50, w/w) was eluted at a rate of 1.0 mL/min and detected at a wavelength of 227 nm. The efficiency of DNase encapsulation was determined by using a Micro BCA protein assay kit (Pierce Protein Research Products, Rockford, IL, USA). DNase-loaded Hep−Pr nanogels were dissolved in deionized water, which was followed by adding a Micro BCA reagent mixture (A/B/C = 25:24:1) and incubating for 2 h at 37 °C. The optical densities of the solutions were then detected at 630 nm using a microplate reader. Dynamic Light Scattering and ξ-Potential Measurements. The size of Hep−Pr nanogels in aqueous solution was determined by a dynamic light scattering (DLS) particle analyzer with autosampler (DLS, FPAR-1000, Photal, Otsuka Electronics Co., Ltd., Osaka, Japan) that was equipped with a He−Ne laser beam at 633 nm (scattering angle = 90°) at 37 °C in triplicate. Hep−Pr nanogels and Pluronic micelles were dissolved in deionized water at 37 °C. All of the samples were then filtered through a membrane (0.45 μm) and dispersed under ultrasound for 10 min prior to the measurements. The acquisition time was 3 min, and the repeating time was 10 times. The surface charge of the Hep−Pr nanogels was measured using an electrophoretic light scattering spectrophotometer equipped with a 10 mW He−Ne laser (632.8 nm) (ELS-8000, Otsuka Electronics Co., Ltd.). All samples were prepared at 37 °C. Transmission Electron Microscopy. The surface morphology and size of the Hep−Pr nanogels were observed by transmission electron microscopy (TEM; JEOL 300 kV, JEOL Ltd., Tokyo, Japan). A drop of sample solution (1 mg/mL) was placed on a carbon−copper TEM grid (300-mesh, Ted Pella
and anti-EGF antibodies, and the combination of green tea catechins and anticancer drugs.7−9 Therefore, it may be meaningful to develop a well-formulated form of combined drugs from different species and modes of action for enhanced anticancer efficacy. To the best of our knowledge, a formulation consisting of a combination of a hydrophobic agent and a hydrophilic protein (a nuclease) using a nanocarrier has not been investigated. The current study describes a novel platform of a multidrug nanocarrier that enables paclitaxel and DNase to be delivered to the intracellular sites of a cancer cell with each agent acting as well as the combination treatment with these cytotoxic agents. The multidrug nanocarrier was a nanosized gel-like structure, called a nanogel, that was composed of heparin and Pluronic and that possessed both hydrogel-like and micellar properties. Our goal was to investigate whether the nanocarriers showed a synergistic cytotoxicity in combination with these agents that had different modes of action, and these findings could suggest novel multidrug nanocarriers and efficient strategies for combination chemotherapy.
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EXPERIMENTAL SECTION Materials. Unfractionated heparin (165 units/mg) was obtained from Acros (Pittsburgh, PA, USA). Pluronic F127 (Mn = 12,600) was purchased from BASF Company Ltd. (Seoul, Korea). 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), N-hydroxysuccinimide (NHS), succinic anhydride, 4-morpholineethanesulfonic acid (MES), and 4-dimethylaminopyridine (DMAP) were purchased from SigmaAldrich Co. (St. Louis, MO, USA). Triethylamine (TEA) was purchased from Kanto Chemical Co., Inc. (Tokyo, Japan). Paclitaxel was supplied from Samyang Corporation (Seoul, Korea). DNase and fluorescein isothiocyanate (FITC) were purchased from Sigma-Aldrich Co. All other chemicals were of analytical grade and used without further purification. Synthesis of the Heparin−Pluronic Conjugate. Carboxylated Pluronic was synthesized according to our previous reports.10 In brief, 35 g (2.778 mmol) of Pluronic was dissolved in 350 mL of dioxane. Then, 0.336 g (3.336 mmol) of succinic anhydride was added to the solution, which was followed by the addition and stirring of 0.339 g (2.778 mmol) of DMAP and 0.281 g (2.778 mmol) of TEA, which was then dissolved in dioxane (25 mL) under a nitrogen atmosphere at room temperature for 24 h. After filtering, the solution was precipitated in an excess amount of diethyl ether and dried under vacuum for 2 days. The heparin−Pluronic (Hep−Pr) conjugate was synthesized in the following steps. Carboxylated Pluronic (6.25 g) was dissolved in 100 mL of 0.1 M MES buffer, and EDC and NHS were added to the solution as coupling agents. Then, the heparin solution (1 g) that was dissolved in 100 mL of dioxane was poured into the mixture and stirred at room temperature for 24 h. The solution was filtered and dialyzed against deionized water for 3 days (molecular weight cutoff, 50,000) and lyophilized. The Hep− Pr conjugate was analyzed using Fourier Transform infrared spectroscopy (FTIR; Nicolet Magma-IR 550, USA) and 1H nuclear magnetic resonance (NMR; NMR−400, Varian, USA). The heparin content in the conjugate was determined using a toluidine blue chromogenic assay.11 A thermogravimetric analysis (TGA, Q50, TA Instruments, USA) was conducted in order to analyze the composition of the Hep−Pr conjugate. The biological activity of the Hep−Pr conjugate was measured by an anti-Factor Xa assay and by the activated partial 686
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Inc., Redding, CA, USA). After air drying, a drop of phosphotungstic acid (1 mg/mL) was added onto the grid as a staining solution and air-dried again. DNase Stability Assay. In order to estimate the conformational stability of DNase, circular dichroism (CD, JASCO 810W, Japan) was conducted with a quartz cell with a path length of 1 mm. Intact DNase (10 μg/mL) and encapsulated DNase (10 μg/mL) solutions were prepared in PBS buffer. CD spectra were recorded at 25 °C under optimized conditions and consisted of 10 scans between 190 and 260 nm using 1.0 nm bandwidth, 10 nm/min scanning rate, 0.2 nm wavelength gap, and 2 s time constant. The CD band intensity was expressed as molar ellipticity in degrees·cm2·dmol−1. Cell Culture. A human cervical cancer cell line (HeLa cells) was purchased from the American Type Culture Collection ATCC (Manassas, VA, USA) and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) that was supplemented with 10% fetal calf serum, 100 units/mL penicillin, and 100 μg/ mL streptomycin (Gibco, Invitrogen Corporation, Carlsbad, CA, USA) in a humidified 5% CO2/95% air atmosphere at 37 °C. Flow Cytometric Analysis of Cellular Uptake. HeLa cells were seeded at a density of 5 × 104 cells/well in a 24-well plate and grown for 24 h. Various concentrations of FITC− Hep−Pr nanogels were added to the media without any transfection agent, and the cells were incubated for various times. The adhered cells were trypsinized, washed with PBS, and centrifuged, which was followed by resuspension of the pellet in PBS for fluorescent-activated cell sorter (FACS; Becton Dickinson FACSCalibur, BD Biosciences) analysis. Confocal Laser Scanning Microscopy. HeLa cells were seeded at a density of 5 × 104 cells/well in a 24-well plate on a glass coverslip the day before their use, and they were preincubated in serum-free DMEM for 30 min at 37 °C prior to the treatment of FITC−Hep−Pr nanogels. Cells were incubated together with 50 g/mL of FITC−Hep−Pr nanogels for a predetermined time interval at 37 °C. After stopping cellular uptake by adding three volumes of ice-cooled PBS, cells were washed twice with cold PBS, fixed with 2% paraformaldehyde (PFA) in PBS for 10 min at room temperature, and then maintained and permeabilized with Perm-buffer (1% bovine serum albumin, 0.1% saponine, and 0.1% sodium azide in PBS) for 10 min at room temperature. Cells on a coverslip were mounted in a Vectashield antifade mounting medium (Vector Laboratories, Inc., Burlingame, CA, USA), observed with a confocal microscope (Zeiss LSM 510 laser), and analyzed with Image Analysis software (Carl Zeiss LSM). Cytotoxicity Test. HeLa cells were seeded at a density of 5 × 103 cells/well in 96-well plates and cultured overnight. A series of concentrations of the Hep−Pr nanogels were added to the cultured cells. Cell viability was evaluated using a colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-based cell growth determination kit (Sigma-Aldrich Co.). Evaluation of the Combination Effect. In order to evaluate the combination effect of paclitaxel and DNase with each different anticancer action, the combination index (CI) was utilized by partly modifying a method used in other studies.12,16,17 The CI was determined with the following equation that was selected by plotting cytotoxicity versus drug concentration.
CI =
VC1 V V V + C2 + C1 C2 VS1 VS2 VS1VS2
where VS1 and VS2 indicate the cell viability after treatment with each drug separately, and VC1 and VC2 are the values of the treatments of the drugs in combination. The definition of CI values are as follows: CI > 1.5, antagonistic; CI = 1.1−1.5, moderate antagonistic; CI = 0.9−1.1, additive; CI = 0.9−0.5, synergistic; and CI < 0.5, strongly synergistic. Statistical Analysis. All data were obtained from groups with over three samples and were presented as the mean value ± standard deviation (SD). Student’s t-test was used to measure the statistical significance of the data, and p values of 0.93 > 0.45 in the order of increasing treatment dose, indicating that the combination effect becomes higher with increases in the treatment dose (Table 1). This tendency may be explained with some assumptions. The synergistic effect may be derived from the different modes of action of the drugs. The drug resistance of cancer cells might be overcome by the two different mechanisms of the drugs. For instance, it can be supposed that DNase will supplement the growth inhibitory effects of paclitaxel, while the effect of paclitaxel is limited by drug efflux or insufficient drug doses. Although both paclitaxel and DNase are simultaneously internalized into a cancer cell, the releasing time or site can be different from each other, and this is supported by the fact that DNase has a stronger affinity for the Hep−Pr structure than paclitaxel. The more sustained release of DNase is beneficial for the efficacy of the enhanced combination because the target site of DNase is the nucleus. Considering the results that were obtained in this study, it is thought that heparin may play a critical role in enhancing the combination effect. The conjugation of heparin to Pluronic enabled the DNase to be encapsulated with high efficiency, as well as to transport it to the appropriate sites of action (i.e., the nucleus), while paclitaxel was affected in a similar mode with a micellar form. In addition to this, heparin seemed to contribute to an enhancement of the cellular uptake and wide dispersion of the whole cellular regions containing nuclei. These facts are supported by our suggestion that heparin was likely to be related to its cellular uptake mechanism and interactions with chromatins in nuclear membranes.24,25 Ordinarily, it is not easy to translate an in vitro evaluation that resulted in synergistic effects to an in vivo application because of very complex criteria, such as pharmacokinetics and biodistribution. However, our previous in vivo results suggest that Hep−Pr nanogels with drug combination will be effective, even when applied to in vivo cancer chemotherapy.26
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CONCLUSIONS
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AUTHOR INFORMATION
§
These authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (2012-046885) and Priority Research Center Program through the National Research Foundation of Korea (NRF), Ministry of Education, Science and Technology (20120006887).
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A novel self-assembled Hep−Pr nanogel was prepared as multidrug nanocarriers that enabled the simultaneous delivery of a hydrophobic drug and a protein, and this methodology can be employed in combination chemotherapy. Consequently, the Hep−Pr nanogels loaded with the combined agents (paclitaxel and DNase) were found to have synergistic growth inhibitory effects that acted in a dose-dependent manner, compared to single agent and carrier-free treatments. The enhanced efficacy was supported by robust evidence that includes high encapsulation efficiency, different modes of action of the cytotoxic agents, and intracellular and nuclear uptake behaviors. Finally, the combined delivery of chemotherapeutic agents with different characteristics using simple multidrug nanocarriers may be a promising strategy for combination chemotherapy for enhanced cancer chemotherapy.
Corresponding Author
*Tel: +82-31-219-1846. Fax: +82-31-219-1592. E-mail: kdp@ ajou.ac.kr. 692
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