Laponite Nanodisks as an Efficient Platform for Doxorubicin Delivery

Feb 18, 2013 - CQM-Centro de Química da Madeira, Universidade da Madeira, Campus da Penteada, 9000-390 Funchal, Portugal. •S Supporting Information...
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Laponite Nanodisks as an Efficient Platform for Doxorubicin Delivery to Cancer Cells Shige Wang,†,⊥ Yilun Wu,‡,⊥ Rui Guo,‡ Yunpeng Huang,‡ Shihui Wen,‡ Mingwu Shen,‡ Jianhua Wang,*,§ and Xiangyang Shi*,†,‡,∥ †

State Key Laboratory for Modification of Chemical Fibers and Polymer Materials, Donghua University, Shanghai 201620, People’s Republic of China ‡ College of Chemistry, Chemical Engineering and Biotechnology, Donghua University, Shanghai 201620, People’s Republic of China § Department of Biochemistry and Molecular & Cell Biology, School of Medicine, Shanghai Jiao Tong University, Shanghai 200025, People’s Republic of China ∥ CQM-Centro de Química da Madeira, Universidade da Madeira, Campus da Penteada, 9000-390 Funchal, Portugal S Supporting Information *

ABSTRACT: We report a facile approach to using laponite (LAP) nanodisks as a platform for efficient delivery of doxorubicin (DOX) to cancer cells. In this study, DOX was encapsulated into the interlayer space of LAP through an ionic exchange process with an exceptionally high loading efficiency of 98.3 ± 0.77%. The successful DOX loading was extensively characterized via different methods. In vitro drug release study shows that the release of DOX from LAP/DOX nanodisks is pH-dependent, and DOX is released at a quicker rate at acidic pH condition (pH = 5.4) than at physiological pH condition. Importantly, cell viability assay results reveal that LAP/DOX nanodisks display a much higher therapeutic efficacy in inhibiting the growth of a model cancer cell line (human epithelial carcinoma cells, KB cells) than free DOX drug at the same DOX concentration. The enhanced antitumor efficacy is primarily due to the much more cellular uptake of the LAP/DOX nanodisks than that of free DOX, which has been confirmed by confocal laser scanning microscope and flow cytometry analysis. The high DOX payload and enhanced antitumor efficacy render LAP nanodisks as a robust carrier system for different biomedical applications.



INTRODUCTION

Laponite (LAP), a kind of isomorphous substituted smectite clay, is a layered aluminosilicate disk-like clay material.12,13 Because the interlayer space can be used for effective drug encapsulation with high retention capacity, LAP and other smectite clays have been used as drug carriers.14−17 For example, a hydrophobic drug itraconazole (ITA) was able to be incorporated into LAP,14,15 and the release of ITA from ITALAP nanodisks could reach 75% during the first 24 h. Very recently, we used LAP to encapsulate an antibiotic, amoxicillin (AMX), and then incorporated the formed LAP/AMX complexes into electrospun poly(lactic-co-glycolic acid) (PLGA) nanofibers. The formed PLGA/LAP/AMX nanofibers enabled the encapsulated AMX with a sustained release profile and noncompromised antibacterial activity. It is reasonable to hypothesize that the unique disk-like shape with interlayer spacing of LAP may afford its use as an efficient vehicle for DOX delivery to cancer cells. However, there is currently no literature reporting the use of LAP as a carrier system for anticancer drug delivery and cancer therapy applications.17

Doxorubicin (DOX), with a trade name of Adriamycin, is one of the most commonly used chemotherapeutic drugs in antitumor applications.1 However, DOX has turned out to have a strong tendency to bind with cellular macromolecules, which leads to a very low ability for tumor tissue penetration and a low therapeutic index.2 In order to achieve a desired therapeutic efficacy, large dosage or repeated administration is required, which can cause severe side effects to normal tissues.3 To improve the therapeutic efficacy, various nanocarrier systems such as dendrimers,4,5 lipsomes,6 micelles,7 carbon nanotubes,8,9 and inorganic nanoplatelets 10 have been developed for efficient delivery of DOX to cancer cells. The major advantage to using nanocarrier system is that the developed nanocarriers can be efficiently uptaken by tumor cells and be prominently accumulated in the tumor tissues by escaping the excessively leaky microvasculature due to the enhanced permeability and retention (EPR) effect.11 Although the reported nanocarrier systems are able to improve the therapeutic efficacy of DOX, development of a versatile, highpayload, and noncytotoxic drug delivery system with enhanced therapeutic efficacy still remains a great challenge. © 2013 American Chemical Society

Received: November 3, 2012 Revised: February 16, 2013 Published: February 18, 2013 5030

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collected using a Lambda 25 UV−vis spectrometer (PerkinElmer). Samples were dispersed in water at the DOX concentration of 0.5 mg/ mL before measurements. LAP without DOX loading with a concentration of 0.5 mg/mL was also tested. Zeta-potential and dynamic light scattering (DLS) measurements were carried out using a Zetasizer Nano ZS system (Malvern, UK) equipped with a standard 633 nm laser. Samples were dispersed in water with a concentration of 1 mg/mL before measurements. The crystalline structures of LAP, DOX, and LAP/DOX nanodisks were characterized by a Rigaku D/ max-2550 PC XRD system (Rigaku Co., Tokyo, Japan) using Cu Kα radiation with a wavelength of 1.54 Å at 40 kV and 200 mA. The scan was performed from 5° to 60° (2θ). The plane spacing of different diffraction plane (dhkl) can be calculated from Bragg’s law. In Vitro Drug Release. The in vitro release kinetics of DOX from LAP/DOX nanodisks under different pH conditions (pH = 5.4 and pH = 7.4) was monitored using UV−vis spectroscopy. Briefly, LAP/ DOX nanodisks (5 mg) were dispersed into 2 mL of phosphate buffered saline (PBS) solution (pH = 7.4) or 2 mL of acetic acid− sodium acetate buffer solution (pH = 5.4) after 10 s vortexing, immediately placed in a dialysis bag with a molecular weight cutoff of 14 000, and dialyzed against the corresponding buffer solution (8 mL) in a sample vial. All these samples were in triplicate and incubated in a vapor-bathing constant temperature vibrator at 37 °C. At each predetermined time interval, 1 mL of PBS or acetic acid−sodium acetate buffer solution was taken out from each vial, and equal volumes of respective fresh buffer solution were replenished. The released DOX was quantified using a UV−vis spectrophotometer (PerkinElmer Lambda 25) at 480 nm. Cell Cultures and Antitumor Activity Assay. KB cells were continuously cultured in 25 cm2 tissue culture flasks with 5 mL of RPMI-1640 medium containing 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin in a humidified incubator with 5% CO2 at 37 °C. To test the therapeutic efficacy of LAP/DOX nanodisks, cells with a density of 1 × 104 cells per well were seeded into a 96-well plate and cultured overnight to bring the cells to confluence. Then the medium was replaced with fresh medium containing LAP/DOX nanodisks with a DOX concentration of 0, 0.2, 0.4, 0.6, 0.8, and 1.0 μM. For comparison, free DOX with similar concentrations was also used to treat KB cells under similar conditions. After 24 or 48 h incubation, unattached cells were washed out with PBS solution and MTT solution (10 μL) was added to each well. After incubation at 37 °C for 4 h, 100 μL of DMSO was added to dissolve the purple MTT formazan crystal. Then, the plates were read at 570 nm using a Microplate Reader (MK3, Thermo). Mean and standard deviation for the triplicate wells for each sample were reported. After treatment with LAP/DOX nanodisks of varying DOX concentrations for 24 h, the cell morphology was also observed by phase-contrast microscopy (Leica DM IL LED inverted microscope). The magnification was set at 200× for all samples. FCM Analysis. KB cells with a density of 1 × 105 cells/well were seeded in a 6-well tissue culture plate the day before the experiments to bring the cells to confluence. The medium was then replaced with fresh RPMI-1640 medium containing LAP/DOX nanodisks with a DOX concentration of 0.6 μM and cultured for an additional 2 or 4 h. After that, the medium was discarded and cells were rinsed with PBS solution 3 times. Finally, cells in individual wells were trypsinized and suspended in 1 mL of PBS, and the intensity of DOX fluorescence was measured with a FACSCalibur flow cytometer (Becton Dickinson, Mountain View, CA) equipped with a 15 mW, 488 nm, and air-cooled argon ion laser. The fluorescent emissions were collected through a 575 nm band-pass filter and acquired in log mode. For each sample, 1 × 104 cells were counted, and the measurement was repeated for three times. Untreated cells or cells treated with free DOX (0.6 μM) were also tested for comparison. CLSM Imaging. The intracellular DOX uptake was also qualitatively confirmed by CLSM (Carl Zeiss LSM 700, Jena, Germany). Coverslips with a diameter of 14 mm were pretreated with 5% HCl, 30% HNO3, and 75% alcohol and then fixed in a 12-well tissue culture plate. 5 × 104 KB cells were seeded into each well and cultured for about 48 h to allow the KB cells to attach onto the

Herein, in this research, we attempted to use LAP nanodisks as a novel platform to deliver DOX to cancer cells for cancer therapeutics applications (Scheme 1a). The formed LAP/DOX Scheme 1. Schematic Illustration of the Intercalation of DOX into LAP

nanodisks were extensively characterized using UV−vis spectroscopy, Fourier transform infrared (FTIR) spectrometry, and X-ray diffraction (XRD). In vitro drug release was performed under different pH conditions and monitored using UV−vis spectroscopy. Furthermore, in vitro antitumor efficacy of the LAP/DOX nanodisks was assessed and compared with free DOX via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay of a model cancer cell line (human epithelial carcinoma cell line, KB cells). Finally, the intracellular uptake of LAP/DOX nanodisks in vitro was investigated using flow cytometry (FCM) and confocal laser scanning microscope (CLSM). To our knowledge, this is the first report related to the use of LAP nanodisks to efficiently deliver DOX to cancer cells for cancer therapy applications.



EXPERIMENTAL SECTION

Materials. LAP and DOX·HCl (the chemical structure of DOX with a neutral form is shown in Figure S1, Supporting Information) were purchased from Zhejiang Institute of Geologic and Mineral Resources (China) and Beijing Huafeng Pharmaceutical Co., Ltd. (China), respectively. KB cells were obtained from the Institute of Biochemistry and Cell Biology (the Chinese Academy of Sciences, Shanghai, China). Roswell Park Memorial Institute-1640 (RPMI1640) medium, fetal bovine serum (FBS), penicillin, and streptomycin were from Hangzhou Jinuo Biomedical Technology (Hangzhou, China). MTT and Hoechst 33342 were from Sigma. All of the cell culture flasks and plates were from NEST Biotechnology (Shanghai, China). Water used in all experiments was purified using a Milli-Q Plus 185 water purification system (Millipore, Bedford, MA) with resistivity higher than 18 MΩ·cm. Loading of DOX within LAP Nanodisks. LAP was dispersed into water and sonicated using a water bath ultrasonic cleaner (50 W, SK1200H, Shanghai KUDOS Inc., China) at room temperature for 30 min to obtain aqueous solutions with different concentrations (2, 4, 6, and 8 mg/mL). An aqueous solution of DOX (2 mg/mL) was prepared using the same procedure as LAP. Then, equal volumes of LAP and DOX solutions were mixed together and magnetically stirred for 24 h to make the LAP swollen thoroughly and interact with DOX sufficiently. The LAP/DOX nanodisks were obtained by centrifugation (8000 rpm, 5 min) and rinsing with water for 3 times, air-dried, and stored in dark at room temperature before use. To determine the drug loading efficiency, the supernatants after 4 times centrifugation were collected, and the free DOX concentration of the supernatants was quantified using Lambda 25 UV−vis spectrophotometer (PerkinElmer) at 480 nm with the standard absorbance−concentration calibration curve at the same wavelength. The drug loading efficiency was optimized by changing the LAP concentrations when the DOX concentration was fixed at 1 mg/mL. General Characterization Methods. FTIR spectrometry was performed using a Nicolet Nexus 670 FTIR (Nicolet-Thermo) spectrometer. All spectra were recorded using a transmission mode with a wavenumber range of 650−4000 cm−1. UV−vis spectra were 5031

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coverslips. Before CLSM imaging, the KB cells attached onto the coverslips were treated with LAP/DOX nanodisks with a DOX concentration of 0.6 μM or free DOX (0.6 μM) for 4 h. Then the KB cells were fixed with glutaraldehyde (2.5%) for 15 min at 4 °C and counterstained with Hoechst 33342 (1 μg/mL) for 15 min at 37 °C using a standard procedure. Finally, samples were imaged using a 63× oil-immersion objective lens. Statistical Analysis. One-way ANOVA statistical analysis was performed to compare the cell viability and intracellular DOX uptake upon treatment with LAP/DOX nanodisks or free DOX. 0.05 was selected as the significance level, and the data were indicated with (*) for p < 0.05, (**) for p < 0.01, and (***) for p < 0.001.



Figure 1. (a) FTIR and (b) UV−vis spectra of LAP, DOX, and LAP/ DOX nanodisks.

RESULTS AND DISCUSSION Loading of DOX into LAP Nanodisks. LAP is known to be able to form a stable colloidal layered structure in aqueous solution, and the interlayer space of LAP enables drug encapsulation via ionic exchange.18 The high anisotropy of bonding enables the encapsulation of drug molecules within the interlayer spacing of LAP nanodisks. Our previous study has shown that an antibiotic drug AMX can be encapsulated within LAP nanodisks.19 Similarly, in this study we attempted to use LAP nanodisks to encapsulate DOX (Scheme 1). By simply mixing DOX with LAP nanodisks in aqueous solution, we were able to encapsulate DOX molecules within LAP nanodisks. The milky white color of concentrated LAP suspension changes to dark red, indicating the successful loading of DOX within LAP nanodisks (Figure S2). The DOX loading efficiency was optimized by varying the concentration of LAP under the fixed DOX concentration (1 mg/mL). As shown in Table 1, with the

nanodisks can be attributed to the −Si−O− stretching vibration and −OH bending vibration of LAP nanodisks, respectively.19 It is worth noting that some distinctive bands at 1712, 1582, 1412, and 1285 cm−1 associated with DOX are also present in the spectrum of LAP/DOX nanodisks, indicating the successful intercalation of DOX within LAP nanodisks. The LAP/DOX nanodisks were further characterized by UV−vis spectrometry (Figure 1b). Obviously, free DOX and LAP/ DOX show a characteristic absorption peak at around 480 nm. In sharp contrast, DOX-free LAP nanodisks do not show the characteristic absorption peak associated with DOX. Both FTIR and UV−vis spectroscopy data clearly demonstrate the successful encapsulation of DOX within the LAP nanodisks. As reported in the literature,15 the intercalation of drug within the LAP interlayers often results in the enlargement of the LAP interlayer space, which is the key information to disclose the drug loading mechanism. In this context, the crystalline structure of LAP before and after DOX loading was investigated using XRD (Figure 2). For comparison, the

Table 1. Optimization of LAP Concentrations for DOX Encapsulation (the DOX Concentration Was Fixed at 1 mg/ mL) LAP (mg/mL) loading efficiencya (%)

1

2

3

4

72.4 ± 0.89

95.9 ± 0.56

98.3 ± 0.77

98.0 ± 0.08

a Loading efficiency = Mt/M0 × 100%; Mt and M0 stands for the mass of encapsulated DOX and the total DOX used for encapsulation, respectively.

increase of LAP concentration, the DOX loading efficiency increases, and can reach a high loading efficiency of about 98%. Given the remarkable DOX loading efficiency in the studied LAP concentration range as well as the aggregation possibility of LAP in aqueous solution at higher LAP concentrations, we selected 3 mg/mL as the optimized LAP concentration with a DOX loading efficiency of 98.3% for further studies. The successful loading of DOX within LAP nanodisks was characterized using both FTIR and UV−vis spectroscopy (Figure 1). In the FTIR spectra of LAP, free DOX, and LAP/ DOX nanodisks (Figure 1a), free DOX shows its own characteristic bands.20,21 The band at about 3331 cm−1 is due to the overlap of O−H and N−H stretching vibrations. The band located at 2932 cm−1 can be assigned to the C−H stretching vibration. The bending of NH2 on aromatic ring and C−H bond can be observed at 1582 and 1412 cm−1, respectively. The bands located at 1712 and 1530 cm−1 are the characteristic band of CO and amide I stretching vibration, respectively. The bands at 1285 and 1212 cm−1 belong to the C−N stretching vibrations. The peaks located at 1012 and 3440 cm−1 in the spectrum of LAP and LAP/DOX

Figure 2. XRD patterns of LAP (a) and LAP/DOX nanodisks (b).

crystalline structure of free DOX was also given (Figure S3). It is clear that the main diffraction planes of LAP do not have appreciable changes, suggesting that the crystalline structure of LAP remains intact after DOX loading (Figure 2). Some diffraction planes of DOX can also be detected in the 2θ range of 20°−30°, indicative of the successful DOX loading. Detailed diffraction angle and plane spacing data of LAP and LAP/DOX nanodisks derived from XRD analysis are summarized in Table 2. The diffraction angle of (001) plane shifted from 5.98° to 5.06°, and the corresponding plane spacing calculated from Bragg’s law became larger (from 14.95 to 17.67 Å) after DOX encapsulation, primarily due to the fact that the DOX intercalation occurs at the (001) plane via ionic exchange. Diffraction angles and plane spacing of other planes do not show significant changes, further confirming that LAP is able to maintain its original crystalline structure after DOX loading. 5032

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interlayer space. In contrast, under physiological pH condition (pH = 7.4), the DOX·HCl is able to be dissociated to form a hydrophobic neutral molecule. Therefore, the DOX release rate from LAP/DOX is much higher under acidic pH condition than under physiological pH condition. The release of DOX from LAP/DOX nanodisks may also be dependent on the conformation of the LAP/DOX nanodisks dispersed in different pH conditions.22,23 The hydrodynamic sizes of the LAP/DOX nanodisks in both PBS buffer (pH = 7.4) and acetic acid−sodium acetate buffer (pH = 5.4) were analyzed using DLS. We showed that the hydrodynamic size of LAP/DOX was 385 ± 87 nm in acetic acid−sodium acetate buffer solution (pH = 5.4), while the size of the same particles was 969 ± 257 nm when dispersed in PBS (pH = 7.4). In both cases, the size of the particles was larger than that dispersed in water (238.4 ± 14.21 nm, Table 3). This suggests that the LAP/DOX nanodisks form different aggregates under different buffer conditions. The larger aggregates of the LAP/DOX nanodisks in PBS buffer may limit the release of DOX due to the extended drug diffusion distance,24 consequently resulting in slower release in PBS buffer (pH = 7.4) than in acetic acid− sodium acetate buffer (pH = 5.4). Compared with nanohydroxyapatite-based DOX delivery system reported in our previous work,25 which showed a prominent burst release profile with about 25% DOX release at the first 4 h at pH 7.4 and with about 46.9% DOX release at the first 24 h at pH 5.4, the developed LAP-based DOX delivery system is quite promising, because under both pH conditions, no significant burst release at the initial period was observed. The pHdependent DOX release renders the LAP/DOX nanodisks with a promising potential for tumor therapy applications, since the pH of tumor site is lower than that of normal tissues (pH = 5.0−6.0).26 Antitumor Efficacy of LAP/DOX Nanodisks. We next explored the antitumor efficacy of LAP/DOX nanodisks using a model cancer cell line, KB cells. After treatment with LAP/ DOX nanodisks with different DOX concentrations for 24 or 48 h, MTT assay was performed to evaluate the cell viability (Figure 4). It can be seen that LAP/DOX nanodisks are able to

Table 2. Diffraction Angle and Plane Spacing Data of LAP and LAP/DOX Nanodisks from XRD Analysis 2θ peak position (deg)

plane spacing (d, Å)

diffraction plane (hkl)

LAP

LAP/DOX

LAP

LAP/DOX

(001) (02,11) (005) (20,13)

5.98 19.8 29.36 34.46

5.06 19.8 27.06 35.84

14.95 4.54 3.08 2.63

17.67 4.54 3.33 2.54

Overall, the XRD data suggest that the incorporation of DOX within LAP is primarily via ion exchange accompanied by intercalation within the LAP interlayer space. It is also possible that a portion of DOX can be adsorbed onto the LAP surface via hydrogen bonding or electrostatic interaction, which in turn caused an alteration of the LAP surface potential (from −37.9 ± 1.31 mV to −10.8 ± 0.53 mV, Table 3) after DOX loading. It Table 3. Zeta Potential and Hydrodynamic Diameter of LAP and LAP/DOX Nanodisks materials

zeta potential (mV)

hydrodynamic diam (nm)

LAP LAP/DOX

−37.9 ± 1.31 −10.8 ± 0.53

40.7 ± 0.28 238.4 ± 14.21

should be noted that the used LAP nanodisks are not able to form gels in aqueous solution because the LAP concentration used is very low (