Gd(III)-DOTA-modified sonosensitive liposomes for ultrasound-triggered release and MR imaging
© Jung et al.; licensee Springer. 2012
Received: 31 May 2012
Accepted: 14 July 2012
Published: 17 August 2012
Ultrasound-sensitive (sonosensitive) liposomes for tumor targeting have been studied in order to increase the antitumor efficacy of drugs and decrease the associated severe side effects. Liposomal contrast agents having Gd(III) are known as a nano-contrast agent system for the efficient and selective delivery of contrast agents into pathological sites. The objective of this study was to prepare Gd(III)-DOTA-modified sonosensitive liposomes (GdSL), which could deliver a model drug, doxorubicin (DOX), to a specific site and, at the same time, be capable of magnetic resonance (MR) imaging. The GdSL was prepared using synthesized Gd(III)-DOTA-1,2-distearoyl-sn-glycero-3-phosphoethanolamine lipid. Sonosensitivity of GdSL to 20-kHz ultrasound induced 33% to 40% of DOX release. The relaxivities (r1) of GdSL were 6.6 to 7.8 mM−1 s−1, which were higher than that of MR-bester®. Intracellular uptake properties of GdSL were evaluated according to the intensity of ultrasound. Intracellular uptake of DOX for ultrasound-triggered GdSL was higher than that for non-ultrasound-triggered GdSL. The results of our study suggest that the paramagnetic and sonosensitive liposomes, GdSL, may provide a versatile platform for molecular imaging and targeted drug delivery.
Liposomes are spherical vesicles composed of phospholipid bilayer membranes. In the field of targeted drug delivery, liposomes have been extensively studied in an attempt to enhance the therapeutic efficacy of various drugs [1, 2]. Many studies have reported that modification of the surface of liposomes with a hydrophilic moiety such as polyethylene glycol (PEG) can increase the circulation time of the liposomes in the bloodstream due to reduced uptake of the liposomes by the reticuloendothelial system (RES) [3–7]. However, utilization of liposomes as drug-carrying vehicles for intracellular delivery of anticancer drugs loaded in the liposomes is limited due to the lack of specific interaction between liposomal carriers and the target cells . Therefore, to overcome this problem, targeted drug delivery systems such as thermo-, pH-, ultrasound-, and optical-sensitive liposomes have been studied [9, 10]. In particular, ultrasound-sensitive (sonosensitive) liposomes for controlled drug release at the target site have been studied in order to increase the antitumor efficacy of drugs and decrease the associated side effects [11, 12].
Magnetic resonance (MR) is widely used in diagnostic medicine to image pathological areas. Usually, accumulation of contrast agents is essential to achieve successful MR imaging (MRI) and high-resolution images [13, 14]. Most MRI contrast agents are based on either iron oxide particle or gadolinium (III) (Gd(III))-chelated complexes. Gd(III)-based contrast agents have a low r2/r1 ratio and are frequently used to generate positive contrast (increased signal intensity) in T1-weighted images. Recently, various nanoscale carriers such as liposomes, micelles, and polymeric nanoparticles have been modified or incorporated with the MRI contrast agent Gd(III) [14, 15]. Liposomal nanocarriers are able to carry multiple reporter moieties such as peptides and antibodies for the efficient and selective delivery of contrast agents into the pathological sites .
The objective of this study was to develop a novel liposomal carrier that could provide a convenient ultrasonic therapy, such as high- or low-intensity focused ultrasound therapy, by MR image guidance and, moreover, a possibility of ultrasound-mediated targeted drug delivery during ultrasonic therapy. In the current study, we prepared Gd(III)-DOTA-modified sonosensitive liposomes (GdSL), which could deliver doxorubicin (DOX) to a specific site and, at the same time, enhance signal intensity in regions of accumulation on T1-weighted MRI. The GdSL was prepared using synthesized Gd(III)-DOTA-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) lipid. Sonosensitivity and MR properties of the GdSLs with varying lipid ratios were investigated. Furthermore, intracellular uptake property of the GdSL was evaluated according to the intensity of ultrasound.
1,2-Distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-mPEG2000), and cholesterol (CHOL) were purchased from Avanti Polar Lipids Inc. (Alabaster, AL, USA). Doxil® was purchased from ALZA Corporation (Mountain View, CA, USA). Doxorubicin hydrochloride, N,N′-diisopropylethylamine (DiPEA), N,N,N′,N′-tetramethyl-O-(N-succinimidyl) uranium hexafluorophosphate (HSTU), trifluoroacetic acid (TFA), and gadolinium (III) acetate hydrate (Gd(III) (OAc)3) were purchased from Sigma-Aldrich Chemical Co (St. Louis, MO, USA). Fetal bovine serum (FBS), penicillin-streptomycin, paraformaldehyde, and Dulbecco's modified Eagle medium (DMEM) were purchased from Gibco BRL/Life Technologies (New York, NY, USA). Tri-tert-butyl 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate (protected DOTA) was purchased from Tokyo Chemical Industry Corporation (TCI, Tokyo, Japan). All other materials were of analytical grade and used without further purification.
Synthesis of Gd(III)-DOTA-DSPE
Preparation of Gd(III)-DOTA-modified sonosensitive liposomes
Gd(III)-DOTA-modified sonosensitive liposomes (GdSL) and sonosensitive liposomes (SL) without Gd(III)-DOTA-DSPE were prepared by thin-film hydration and sequential extrusion method. The loading of DOX into the aqueous core of the liposomes was carried out using the remote loading method using an ammonium sulfate transmembrane gradient [20, 21]. The lipid compositions and molar ratios of the lipids for the preparation of the liposomes were as follows: (1) GdSL1, Gd(III)-DOTA-DSPE/CHOL/DSPE-mPEG = 31:15:4; (2) GdSL2, Gd(III)-DOTA-DSPE/CHOL/DSPE-mPEG/DSPE = 20:15:4:11; (3) GdSL3, Gd(III)-DOTA-DSPE/CHOL/DSPE-mPEG/DSPE = 10:15:4:21; and (4) SL, CHOL/DSPE-mPEG/DSPE = 15:4:31. The lipids for each liposome formulation were dissolved in 3 ml of chloroform to give 16.67 mM of total lipid concentration (GdSL1, 18.97 mg/ml; GdSL2, 16.97 mg/ml; GdSL3, 15.17 mg/ml; and SL, 13.41 mg/ml) and dried to a thin film using a rotary evaporator. The film was hydrated with 3 ml of 300 mM ammonium sulfate solution, and the liposome suspension was extruded sequentially five times through polycarbonate membrane filters (Whatman, Piscataway, NJ, USA) with a pore size of 200 and 100 nm using a high-pressure extruder (Northern Lipids Inc., Burnaby, Canada). Unloaded ammonium sulfate was removed by dialysis in distilled water for 24 h at 4°C using a cellulose dialysis tube (MWCO, 12,000 to 14,000; Viskase Co., Darien, IL, USA). DOX solution (2 mg/ml) was added to the liposomal solution (1:1, v/v) and incubated for 2 h at 75°C. The mixture was dialyzed for 48 h at 4°C to remove the unloaded DOX. The DOX-loaded liposomes were stored at 4°C until use.
where Ft is the concentration of DOX in the liposomes after the dissolution of DOX-loaded liposomes in an organic solvent mixture consisting of chloroform/methanol (2:1, v/v), and Fi is the initial concentration of DOX. The particle size and zeta potential of the liposomes were measured using an electrophoretic light scattering spectrophotometer (ELS-Z, Otuska Electronics Co., Tokyo, Japan). The amount of chelated Gd(III) was measured using an inductively coupled plasma-atomic emission spectrometer (Ultima-C, Jobin Yvon, Longjumeau, France).
Ultrasound- and temperature-triggered drug release from liposomes
Ultrasound-triggered release of DOX from GdSL and SL was conducted using a 20-kHz ultrasound system (VC 750; Sonic and Materials, Inc, Newtown, CT, USA). The intensity of the generated ultrasound was determined using a calorimetric method [22, 23]. For input power levels of 80, 160, and 240 W, the calculated intensity levels were 14.8, 27.8, and 63.5 W/cm2, respectively. The liposomal solutions were diluted in a ratio of 1:4 (v/v) with PBS (pH 7.4) and exposed to a continuous mode (100% duty cycle) of ultrasound for 1 min at intensity levels of 14.8, 27.8, and 63.5 W/cm2, respectively. During the ultrasound irradiation, the temperature of each sample was controlled to be below 50°C. Temperature-mediated release from GdSL was evaluated using MULTI-BLOK (Lab-Line Instruments, Melrose Park, IL, USA). The liposomal solutions were exposed to 37°C or 50°C for 1 min.
where Ft is the fluorescence intensity of the liposome sample after a given duration (t) of ultrasound irradiation, F0 is the initial background fluorescence of the liposome sample prior to ultrasound irradiation, and Fmax is the fluorescence intensity of DOX in the liposomes after dissolution of DOX-loaded liposomes in an organic solvent mixture consisting of chloroform/methanol (2:1, v/v) . The release test was performed on three independent samples of each liposome.
Morphology of Gd(III)-DOTA-modified sonosensitive liposomes
The morphology of GdSL was observed by cryogenic transmission electron microscopy (cryo-TEM; Tecnai G2 Spirit, FEI Company, Hillsboro, OR, USA). Samples for the cryo-TEM observation were prepared using a controlled-environment vitrification system. Five microliters of the sample were put on a carbon film supported by a copper grid and blotted with filter papers to obtain a thin liquid film on the grid. The sample-loaded grid was quenched in liquid ethane at −180°C and transferred to liquid nitrogen. The acceleration voltage was 80 kV, and the working temperature was −180°C. The images were recorded with a CCD camera (Proscan GmbH, Scheuring, Germany) and an analysis software (Soft Imaging System, GmbH, Munster, Germany) .
The liposomal samples were prepared in the range of 0.05 to 0.40 mM of Gd(III) concentration. The longitudinal relaxation time (T1) of each sample was measured by saturation recovery method using a 4.7-T MR system (Bruker-biospin, Ettlingen, Germany). Relaxivity (r1, in units of mM−1 s−1) was obtained from the slope of the linear fit of the inverse of T1 as a function of Gd(III) concentration. T1-weighted MR images were obtained using a heavily T1-weighted fast spoiled gradient echo sequence. Scans were performed with the following imaging parameters: repetition time (TR) = 8.0, 6.0, 4.0, 2.5, 0.5, 0.2, and 0.07 s; echo time (TE) = 7.8 ms; flip angle (FA) = 180°; field of view (FOV) = 40 × 50 mm2; image matrix = 128 × 128 mm2; and number of signal average = 5.
Intracellular uptake of DOX from ultrasound-triggered liposomes
For the experiments on intracellular uptake of DOX from liposomes, B16F10 murine melanoma cells were cultured in DMEM supplemented with 10% (v/v) heat-inactivated FBS and 10 μl/ml penicillin-streptomycin. The cultures were sustained at 37°C in a humidified incubator containing 5% CO2. The cells were maintained within their exponential growth phase. The intracellular uptake of DOX from liposomes was determined by flow cytometry analysis [24, 26]. B16F10 cells were transferred to 24-well tissue culture plates at a density of 1 × 105 cells/well and incubated for 12 h at 37°C. The liposomal DOX solutions were diluted in a ratio of 1:4 (v/v) with PBS (pH 7.4) just prior to the experiments. The diluted liposomal solutions were irradiated by ultrasound using a 20-kHz ultrasound transducer in a continuous mode (100% duty cycle) at an intensity level of 14.8, 27.8, or 63.5 W/cm2 for 2 min at 37°C. The culture medium was replaced with the ultrasonically irradiated liposomal DOX solution diluted in culture media at a concentration of 15 μg of DOX/ml and then incubated for 45 min. The culture medium was then removed, and each well was washed with PBS (pH 7.4). To fix the cells, 300 μl of paraformaldehyde (5%, v/v) was added to each well. The fluorescence intensities of the sample were determined by flow cytometry with a FACScan (Becton Dickinson, San Jose, CA, USA). Cell-associated DOX was excited with an argon laser (488 nm), and fluorescence was detected at 560 nm. Data of 10,000 gated events were collected and analyzed with the CELL Quest software.
Results and discussion
Physical properties of liposomes
Physical properties of liposomes
Mean particle diameter
Amount of Gd(III)
(molar ratio of lipids)
157.1 ± 8.9
−27.6 ± 4.2
46.6 ± 12.7
4.60 ± 0.16
Gd(III)-DOTA-DSPE/CHOL/DSPE-mPEG2000 = 31:15:4)
156.4 ± 5.1
−21.9 ± 1.0
41.7 ± 9.7
3.17 ± 0.01
Gd(III)-DOTA-DSPE/CHOL/DSPE-mPEG2000/DSPE = 20:15:4:11)
131.4 ± 9.1
−31.6 ± 7.2
62.9 ± 1.5
2.01 ± 0.31
Gd(III)-DOTA-DSPE/CHOL/DSPE-mPEG2000/DSPE = 10:15:4:21)
129.1 ± 6.6
−31.9 ± 2.3
97.5 ± 0.4
(CHOL/DSPE-mPEG2000/DSPE = 15:4:31)
Ultrasound- and temperature-triggered drug release from liposomes
Morphology of ultrasound-irradiated liposomes
Magnetic resonance property of liposomes
MRI is one of the most powerful techniques currently used in medical diagnostics such as tumor detection and vascular imaging. Gd-based complexes, such as Gd(III)-DOTA and Gd(III)-DTPA using paramagnetic material, are known as the most effective T1 agents . The MR images of contrast agents are based on the same principles of nuclear magnetic resonance (NMR). The MR image of contrast agents is related to the relaxation behavior of hydrogen nuclei of water. The principle mechanism for Gd(III)-complexes is due to the interaction of an inner-sphere water molecule with the paramagnetic Gd(III) ion having ninth coordination site, leading to the subsequent magnetic relaxation of the water molecule . The signal intensity of the image is related to the longitudinal relaxation time (T1), and a shortened T1 provides improved images .
Intracellular uptake of DOX released from ultrasound-triggered liposomes
Dual functional Gd(III)-DOTA-modified sonosensitive liposomes were prepared and evaluated for their sonosensitivity, MR properties, and in vitro intracellular uptake. GdSL showed excellent contrast efficiency compared to a commercial contrast agent, MR-bester®, and increased intracellular uptake due to the ultrasound-triggered release of the drug. Therefore, GdSL could deliver drugs to specific sites by ultrasound irradiation and, at the same time, allow MR imaging due to enhanced T1 relaxivity. The results of our study suggest that the novel liposomal carrier may provide a convenient ultrasonic therapy by MR image guidance and, moreover, a possibility of ultrasound-mediated targeted drug delivery during ultrasonic therapy.
This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MEST) (no. 2012-0000101).
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