Magnetic liposomes for colorectal cancer cells therapy by high-frequency magnetic field treatment
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In this study, we developed the cancer treatment through the combination of chemotherapy and thermotherapy using doxorubicin-loaded magnetic liposomes. The citric acid-coated magnetic nanoparticles (CAMNP, ca. 10 nm) and doxorubicin were encapsulated into the liposome (HSPC/DSPE/cholesterol = 12.5:1:8.25) by rotary evaporation and ultrasonication process. The resultant magnetic liposomes (ca. 90 to 130 nm) were subject to characterization including transmission electron microscopy (TEM), dynamic light scattering (DLS), X-ray diffraction (XRD), zeta potential, Fourier transform infrared (FTIR) spectrophotometer, and fluorescence microscope. In vitro cytotoxicity of the drug carrier platform was investigated through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay using L-929 cells, as the mammalian cell model. In vitro cytotoxicity and hyperthermia (inductive heating) studies were evaluated against colorectal cancer (CT-26 cells) with high-frequency magnetic field (HFMF) exposure. MTT assay revealed that these drug carriers exhibited no cytotoxicity against L-929 cells, suggesting excellent biocompatibility. When the magnetic liposomes with 1 μM doxorubicin was used to treat CT-26 cells in combination with HFMF exposure, approximately 56% cells were killed and found to be more effective than either hyperthermia or chemotherapy treatment individually. Therefore, these results show that the synergistic effects between chemotherapy (drug-controlled release) and hyperthermia increase the capability to kill cancer cells.
KeywordsLiposomes Magnetic nanoparticle Colorectal cancer High-frequency magnetic field Drug controlled release
Development of smart materials that could response to the environmental stimuli is gaining importance over the past decade in the drug delivery system . Remote control drug carrier behavior has been regarded as a function that could enhance the efficacy and efficiency of drug delivery to the target sites [2, 3]. Among several drug carrier candidates, liposomes exhibit a number of excellences. Liposomes are synthetic lipid bilayer with enclosed structure up to several hundred nanometers in diameter. It is highly biocompatible and biodegradable and can encapsulate both hydrophilic and hydrophobic pharmaceutical agents and protect them from the inactivating effect of external condition. Moreover, liposomes provide a unique characteristic to deliver pharmaceuticals into cells or even inside individual compartments [4, 5, 6, 7]. Recently, researchers have developed novel liposomes to provide a smart treatment in human body which can undergo the releasing of encapsulated contents as the response to the environmental stimuli like temperature , pH , light , ultrasound [10, 11], magnetic field [12, 13], and so on. These specific environment stimuli are used as the driving force for triggered drug release based on the interaction between the stimuli and liposomes. Among the aforementioned stimuli, magnetic-triggered system has become one of the most potential strategies as the release and targeting stimuli.
Recently, iron oxide (Fe3O4)-based magnetic nanoparticles (MNP) has aroused great interest in magnetic-based releasing system in biomedical applications due to their physical properties and biocompatible nature [14, 15]. MNP for biomedical applications should be hydrophilic and stable in water . Aqueous colloidal dispersions of MNP hold great potential for use in a variety of novel and existing bioprocesses because of the compatibility of the aqueous medium with biosystem [17, 18]. A number of surfactants or compounds have been reported as a stabilizing agent of MNP. Among of them, citric acid (CA) is frequently used to obtain an aqueous stable dispersion of MNP [17, 19, 20, 21, 22, 23]. Targeting drug delivery system can be divided into two general categories: passive and active. MNP provided the main advantages of both types of targeting: as with passive targeting, modification of the nanoparticle surface is not necessary, and like active targeting, they can be directed to the site of interest . This is due to the fact that MNP respond strongly to the magnetic fields, and magnetic fields can penetrate human tissue without impediment . Furthermore, MNP accumulated in a tumor can act as hyperthermia-inducing agents, using the exothermic properties derived from hysteresis and/or Néel relaxation losses by application of high-frequency magnetic field (HFMF) to raise the local temperature around the cells [16, 23, 25]. Moreover, MNP have been used as a part of integrating system with liposomes termed as magnetic liposomes or magnetoliposomes [24, 25, 26].
HFMF is one of the most promising external stimulus because it is less invasive than other methods and has a high permeability in relation to the human body and can be operated remotely. HFMF can depress the drug-drug carrier interaction and accelerate diffusion . Drug release rate is significantly enhanced in the presence of a magnetic field because of the pulsatile mechanical deformation that generates compressive and tensile stresses. Moreover, HFMF-triggered drug delivery system utilized the collapse or the volume transition of drug carriers then induce the drug release . Previous study confirmed that the combination of dual-functional (magnetic and thermal) drug carriers upon the exposure to the HFMF upon a short time could lead to heating effect and a rapid release, hence could enhance the wide application for biomedical applications .
In the present work, we reported the preparation and evaluation of doxorubicin-loaded magnetic liposomes for colorectal cancer cells (CT-26 cells) treatment. The resultant of doxorubicin-loaded magnetic liposomes were subject to characterization including transmission electron microscopy (TEM), dynamic light scattering (DLS), X-ray diffraction (XRD), zeta potential, Fourier transform infrared (FTIR) spectrophotometer, and fluorescence microscopy. Biocompatibility was evaluated with mammalian cells (L-929 fibroblast). Further, the efficacy of this drug carrier against CT-26 cells with HFMF exposure was conducted to define the efficacy of this treatment.
Synthesis of aqueous stable CAMNP
The coprecipitation method was used for the synthesis of citric acid-coated magnetic nanoparticles as the method described previously with minor modification [14, 18, 19]. Briefly, FeCl3.6H2O and FeCl2.4H2O (molar ratio of 2:1) were mixed using double-distilled water and stirred at 1,000 rpm in a three-necked flask. The temperature of the solution was increased to 80°C in N2 atmosphere and kept for 30 min. Further, 20 mL of ammonia solution was added while mixing. The mixture was allowed to complete the magnetite formation for 30 min; afterwards, 0.5 g/mL CA was added, and the reaction temperature was increased to 90°C and kept for 1 h with continuous stirring. The black precipitates were harvested by cooling the reaction mixture to the room temperature, and then the particles were allowed to settle down with the help of a magnet. Further, these magnetic nanoparticles were washed carefully using double-distilled water. The products were dried under vacuum at room temperature and termed as citric acid-coated magnetic nanoparticles (CAMNP).
Preparation of liposomes and DOX-loaded magnetic liposomes
Liposomes composed of fully hydrogenated soy phosphatidylcholine (HSPC)/1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE)/cholesterol at mole ratios of 12.5:1:8.25 were prepared via conventional thin film hydration technique as method described previously with minor modification [29, 30]. Briefly HSPC, DSPE, and cholesterol were dissolved in chloroform and ethanol (3:1 v/v). The mixture was transferred into a 300-mL round bottom flask and placed to rotary evaporation system (N-1200 series, Eyela®, Tokyo Rikakikai Co., Ltd., Tokyo, Japan) for the elimination of any traces and residual of organic solvents. Further, a thin dry lipid film would form on the wall of the round bottom flask. This thin dry lipid film was kept in rotary evaporation system for 6 h to ensure complete removal of organic solvents. Hydration process of the dry lipid film was accomplished by adding the phosphate buffer solution (PBS) of pH 7.4, which resulted in liposome suspension. In addition, CAMNP and doxorubicin (DOX)/CAMNP were introduced in the hydration process to obtain magnetic liposomes and DOX-loaded magnetic liposomes, respectively. This suspension was subjected to a bath-type sonicator (Ultrasonic Cleaner, Kudos, Shanghai, China) for harvesting the liposomes. Afterward, the liposomes were homogenized using ultrasonicator (Probe-type sonicator, VCX 750, Vibra-Cell™, SONICS®, Sonics and Materials, Inc., Newton, CT, USA) at 24 W for 10 min. The suspension was then centrifuged at 1,000 × g for 15 min, which precipitated unincorporated magnetic particles at the bottom of the tube and retained the drug-containing magnetic liposomes in the supernatant . Further, the suspension was dialyzed to remove unincorporated DOX . Then, the suspension was extruded through a 0.22-μm filter for sterilization and to reduce the size. Furthermore, the resulting suspensions of liposomes were then preserved at 4°C prior to characterizations.
Particle size was measured using a DLS spectrophotometry (Horiba Instrument, Horiba, Kyoto, Japan) with helium-neon laser with wavelength of 633 nm, scattering angle of 90°, and refractive index of 1.33 at 25°C. The zeta potential was determined through electrophoretic mobility measurement (Horiba Instrument) with the following specifications: a dispersion medium viscosity of 0.894 mPa.s, a refractive index of 1.33, and temperature of 25°C. The structure and morphology of liposomes and doxorubicin liposomes were examined using transmission electron microscope (TEM-7650, Hitachi, Chiyoda-ku, Japan) at an acceleration voltage of 75 kV. Phosphotungstic acid (PTA) 1% w/v was used as the staining agent. For CAMNP and DOX-loaded magnetic liposomes, TEM was conducted without using PTA. XRD was conducted using a D2 Phaser BRUKER X-ray powder diffractometer (Bruker AXS, Inc., Madison, WI, USA) to evaluate the crystallographic characteristics by scanning dried powder in the 2θ range of 20° to 70° with Cu Kα-1 (1.5406 Å) radiation. The infrared spectra were recorded using a FTIR-460 PLUS (Jasco Co. Ltd., Tokyo, Japan) to determine the major characteristic functional groups. Briefly, the sample was mixed with KBr pellet, and the mixtures were pressed into a pellet form prior to characterization. The incorporation of DOX into the drug carrier was visualized using fluorescence microscope (Olympus, Shinjuku-ku, Japan) equipped with a × 40 objective. Fluorescence images were obtained with a fluorescence microscope (Olympus) at wavelengths of 490 (excitation) and 590 nm (emission). The amount of DOX encapsulated into the liposomes or DOX-loaded magnetic liposomes was determined by a fluorescence spectrophotometer at wavelength of 490 nm for excitation and 590 nm for emission, after lysis of liposomes with a sufficient amount of acetonitrile . The encapsulation efficiency (EE) was calculated by the amount of drug encapsulated/initial drug loading × 100%.
Hyperthermia experiment was performed as the method reported previously with minor modification . Adiabatic condition is assumed, where the initial temperature of the sample must be equal to the temperature of the surrounding medium . Therefore, the temperature of the surrounding medium was kept constant using a heated chamber at 37°C then the initial temperature was 37°C for all of the measurements. Briefly, CAMNP and or DOX-loaded magnetic liposomes suspension were subjected to the center of copper coil in the HFMF apparatus for 10 min. The change of the temperature was recorded every 2 min by an alcohol thermometer. Each experiment was performed three times.
In vitro drug release test
In vitro drug release test was conducted to evaluate the release behavior of DOX from drug carriers during the exposure of HFMF. Before conducting drug release test, the system was first purified using centrifugation in order to remove the unincorporated magnetic nanoparticles . Briefly, doxorubicin liposomes and DOX-loaded magnetic liposomes were placed into glass tube which contained the release solution (pH 4) and placed under HFMF for 10 min. About 1 mL of drug was taken from the test tube during the test for 2 min first and continued 2 min until 10 min during HFMF exposure. The quantity of DOX was quantified using UV-visible spectrophotometer at wavelength of 480 nm.
where D and R t represent the initial amount of drug loaded and the cumulative amount of drug released at time t, respectively.
Mouse fibroblasts (L-929 cells) were obtained from ATCC CRL-1503TM. Dulbecco's modified Eagle's medium high glucose (DMEM), trypsin, dimethylsulfoxide (DMSO), trypan blue, and 3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) powder were purchased from Sigma Aldrich, St. Louis, MO, USA. Fetal bovine serum (FBS) was purchased from BD Biosciences, San Jose, CA, USA. Fibroblasts cells (L929) were cultured using DMEM containing 10 vol.% FBS and 1 vol.% antibiotic antimycotic solution. The cultures were incubated under saturated humid conditions at 37°C with 5% CO2. The medium was changed every day until reaching approximately 70% to 80% confluency. Mouse colon carcinoma cell line (CT-26) was obtained from Bioresource Collection and Research Centre (BCRC, Hsinchu, Taiwan). The controlled cell (CT-26-Ctrl) was cotransfected with pLKO-AS3W-neo plasmid and packaging plasmid and infected with virus in order to grow up the tumor cell. After antibiotic selection for 2 weeks, the CT-26-Ctrl was obtained. CT-26 cells were cultured using DMEM containing 10 vol.% FBS and 1 vol.% antibiotic antimycotic solution. The cultures were incubated under saturated humid conditions at 37°C with 5% CO2. The medium was changed every day until reaching approximately 70% to 80% confluency.
MTT cytotoxicity analysis
where Ac is the absorbance of each system in each day and A0 is the absorbance of the control on day 0.
In vitro evaluation of chemotherapy and hyperthermia studies
In vitro evaluation of chemotherapy and hyperthermia was conducted using drug carriers against CT-26 cells with HFMF exposure. Briefly, CT-26 cells were incubated with liposomes, doxorubicin liposomes, magnetic liposomes, or DOX-loaded magnetic liposomes (DOX concentration: 1 μM) in a 15-mL polypropylene tube which is positioned in the center of a copper coil in the HFMF system. The cells were exposed to the HFMF for 10 min. After the treatment, the cells were washed and seeded in a 96-well plate at a density of 10,000 cells per well and were incubated for another 24, 48, and 72 h. Furthermore, the MTT assay was conducted as aforementioned method and was taken at 570 nm using an ELISA reader (Sunrise, Tecan).
Results and discussion
Formulation of liposomes containing doxorubicin and magnetic nanoparticles has been developed as a part in the integrating treatment of chemotherapy and hyperthermia with the introduction of high-frequency magnetic field system. Cytotoxicity screening of blank drug carriers against mammalian cells, L-929 fibroblast cells, showed that these drug carriers exhibited no cytotoxicity against cellular system which further emphasized these drug carriers as a potential candidate for biocompatible materials. However, the combination between DOX and magnetic liposomes (drug concentration 1 μM) and high-frequency magnetic field treatment synergistically would increase the cytotoxicity effects to kill colorectal cancer cells. Eventually, these results confirmed the potential application of this drug carrier for cancer treatment through combination between chemotherapy (drug-controlled release) and hyperthermia (inductive heating).
AH is a PhD student at National Taiwan University of Science and Technology. LYH is a postdoctoral fellow at National Taiwan University of Science and Technology. MCY holds a professor position at National Taiwan University of Science and Technology. TYL holds an assistant professor position at Ming Chi University of Technology. SCT is a researcher at National Yang-Ming University. CYY holds an assistant professor position at National Yang-Ming University. CYK is a PhD student at National Taiwan University. TYC and HMZ are undergraduate students at Ming Chi University of Technology. WNL is a postdoctoral fellow at National Yang-Ming University. CHL holds a professor position at National Yang-Ming University.
This work was financially supported by National Science Council of Taiwan (NSC 102-2321-B-131-001 and MOST 103-2221-E-131-019).
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