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Estimating the Delivery Efficiency of Drug-Loaded Microbubbles in Cancer Cells with Ultrasound and Bioluminescence Imaging
Ultrasound in Med. & Biol., Vol. 38, No. 11, pp. 1938–1948, 2012 Copyright Ó 2012 World Federation for Ultrasound in Medicine & Biology Printed in the USA. All rights reserved 0301-5629/$ - see front matter
http://dx.doi.org/10.1016/j.ultrasmedbio.2012.07.013
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Original Contribution ESTIMATING THE DELIVERY EFFICIENCY OF DRUG-LOADED MICROBUBBLES IN CANCER CELLS WITH ULTRASOUND AND BIOLUMINESCENCE IMAGING AI-HO LIAO,* YING-KAI LI,* WEI-JIUNN LEE,y MING-FANG WU,z HAO-LI LIU,x and MIN-LIANG KUOy y
* Graduate Institute of Biomedical Engineering, National Taiwan University of Science and Technology, Taipei, Taiwan; Institute of Toxicology College of Medicine, National Taiwan University, Taipei, Taiwan; z Medicine of Animal Medicine Center, National Taiwan University, Taipei, Taiwan; and x Department of Electrical Engineering, Chang Gung University, Tao-Yuan, Taiwan (Received 26 June 2012; revised 17 July 2012; in final form 18 July 2012)
Abstract—The application of drug-loaded microbubbles (MBs) in combination with ultrasound (US), which results in an increase in capillary permeability at the site of US-sonication-induced MB destruction, may be an efficient method of localized drug delivery. This study investigated the mechanism underlying the US-mediated release of luciferin-loaded MBs through the blood vessels to targeted cells using an in vivo bioluminescence imaging (BLI) system. The luciferin-loaded MBs comprised an albumin shell with a diameter of 1234 ± 394 nm (mean ± SD) and contained 2.48 3 109 bubbles/mL; within each MB, the concentration of encapsulated luciferin was 1.48 3 10210 mg/bubble. The loading efficiency of luciferin in MBs was only about 19.8%, while maintaining both the bioluminescence and acoustic properties. In vitro and in vivo BLI experiments were performed to evaluate the US-mediated release of luciferin-loaded MBs. For in vitro results, the increase in light emission of luciferinloaded albumin-shelled MBs after destruction via US sonication (6.24 ± 0.72 3 107 photons/s) was significantly higher than that in the luciferin-loaded albumin-shelled MBs (3.11 ± 0.33 3 107 photons/s) (p , 0.05). The efficiency of the US-mediated release of luciferin-loaded MBs in 4T1-luc2 tumor-bearing mice was also estimated. The signal intensity of the tumor with US destruction at 3 W/cm2 for 30 s was significantly higher than without US destruction at 3 (p 5 0.025), 5 (p 5 0.013), 7 (p 5 0.012) and 10 (p 5 0.032) min after injecting luciferinloaded albumin-shelled MBs. The delivery efficiency was, thus, improved with US-mediated release, allowing reduction of the total injection dose of luciferin. (E-mail: [email protected]) Ó 2012 World Federation for Ultrasound in Medicine & Biology. Key Words: Luciferin-loaded microbubbles, High-frequency ultrasound, In vivo bioluminescence imaging system, Luciferase-expressing breast cancer cell.
INTRODUCTION
somes (Lentacker et al. 2010). A major drawback of coinjecting drugs and MBs is that drugs can still extravasate and accumulate in tissues other than those targeted (i.e., not exposed to US). Furthermore, drug molecules may become incorporated within the thick polymer shell or inside the gas core (or multiple cores) of thickshelled MBs (Hernot and Klibanov 2008). It has been demonstrated that the release of drugs from the shells of polymer MBs embedded Fe3O4 nanoparticles can be controlled by sonoporation (Yang et al. 2011). Paclitaxel-loaded polymer MBs triggered with focused US have the potential to provide a targeted and sustained delivery of the drug to tumors (Cochran et al. 2011). For lipid MBs, the drug could be attached to the external surface of the monolayer by covalent or noncovalent bonds, or incorporated into liposomes or
Ultrasound (US)-mediated gene- and drug-delivery methods have been proposed as targeting tools in recent years. In combination with drug-loaded microbubbles (MBs), local drug concentrations can be increased by taking advantage of US-targeted drug release and the transiently increased capillary permeability at the site of MB destruction (Chen et al. 2011; Lin et al. 2010; Maruyama et al. 2007). Most of the previous reports have focused on US-assisted intracellular delivery of the free drug or drugs encapsulated in micelles or lipoAddress correspondence to: Ai-Ho Liao, Ph.D., Graduate Institute of Biomedical Engineering, National Taiwan University of Science and Technology, TR-916, #43, Sec. 4, Keelung Rd, Taipei, Taiwan 10607, ROC. E-mail: [email protected] 1938
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nanoparticles that are then associated with the bubble surface (Lum et al. 2006). A recent study found that the US-targeted destruction of drug-loaded MBs led to a 12-fold higher tissue concentration of the drug and a significantly lower tumor growth in the target tumor compared with the contralateral control tumor (Tinkov et al. 2010a, b). However, the mechanism underlying the US-mediated release of drug-loaded MBs through the blood vessels to targeted cells is unclear and not easy to control. The dynamic mechanisms that lead to the destruction of MBs are not well understood because the process is often complicated and difficult to quantify (Liu et al. 2006). Recently, poly butyl cyanoacrylate based hard-shell, VEGFR2-targeted and fluorophore loaded MBs were shown to be highly suitable for image-guided drug targeting to tumor blood vessels and to release significant amounts of drugs upon exposure to high-mechanical index destructive US pulses (Fokong et al. 2012). In addition, developing ultrasound-modulated fluorescence tomography has been proposed and demonstrated (Yuan 2009). The US-mediated release of fluorescent drugs loaded MBs could be imaged noninvasively. This situation prompted the present study to ascertain the mechanism underlying the US-mediated release of the drug from drug-loaded MBs using a new model with a method that combines therapeutic US, a high-frequency US imaging system, and an in vivo bioluminescence imaging (BLI) system. The US-assisted intracellular delivery was performed with luciferin-loaded albuminshelled MBs targeting luciferase-gene-transfected breast cancer cells in mice. The efficiency of the US-mediated release of luciferin-loaded MBs in tumor-bearing mice was investigated. BLI has evolved as a highly sensitive and powerful technique for the noninvasive, cost-effective and realtime monitoring of sophisticated biologic processes in intact animals (Kheirolomoom et al. 2010). In vivo BLI requires delivery of D-luciferin, which is rapidly cleared from the blood circulation and enters the target cells. Since luciferin can penetrate cell membranes, it allows the transformed cells to be monitored for luciferase activity (McElroy and Strehler 1954). Luciferase enzymes emit light (bioluminescence) by catalyzing the oxidation of the substrate luciferin to oxyluciferin in an oxygen- and adenosine-triphosphate-dependent process (Chen et al. 2006). Luciferase has previously served as the reporter gene to assess gene or luciferin substrate delivery in cancer research (Contag and Ross 2002). MBs with an albumin shell, such as OptisonÔ (GE Healthcare, East Norwalk, CT, USA), can persist in the bloodstream for a sufficiently long time for imaging because the denatured albumin shell and core gas have low solubility in blood (Cohen et al. 1998). Albuminbased MBs consisting of perfluorocarbon gas surrounded by a human serum albumin (HSA)-based shell have been
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used clinically to enhance the contrast in US imaging (Abdelmoneim et al. 2010). In addition, albuminshelled MBs incorporated with targeting ligands are used as a US contrast agent (Cohen et al. 1998) for local inflammation and angiogenesis detection (Korpanty et al. 2005). During the formation of air-filled albumin-shelled MBs, the sonicator horn is placed at the air–water interface (Gedanken 2009; Suslick et al. 1994). It has recently been demonstrated that if the starting solution of the native bovine serum albumin (BSA) protein contains an antibiotic drug, 3 min of sonication forms a BSA microsphere encapsulating up to 90% of the drug in the proteinaceous microsphere (Grinberg et al. 2007). A recent study of drug-loaded albumin-shelled microparticles revealed that peak concentrations of drugs were reduced in the serum, liver, spleen and brain (Lee et al. 2011). In the present study, the treatment drug, luciferin, was encapsulated in the albumin-shelled MBs. The luciferin-loaded albumin-shelled MBs were produced by sonicating a solution containing albumin, luciferin and perfluorocarbon gas. However, the D-luciferin can be stored at room temperature only for a short period of time. The sonication has been shown to increase the temperature of samples and may affect the structure of D-luciferin. Based on in vitro and in vivo experiments, the aim of this study was to elucidate the feasibility and efficacy of US-mediated intracellular drug delivery of the novel MBs in a murine mammary tumor model. First, the luciferinloaded albumin-shelled MBs were produced and the loading efficiency of luciferin was measured. The luciferin-loaded albumin-shelled MBs were then characterized in vitro and in vivo with the aid of a US imaging system and BLI. In addition, the efficiency of USmediated local drug delivery with luciferin-loaded albumin-shelled MBs was evaluated in transgenic mouse models expressing the luciferase reporter gene with BLI. MATERIALS AND METHODS Production of luciferin-loaded albumin-shelled MBs Luciferin-loaded albumin-shelled MBs were prepared according to the procedure used in our previous study (Liao et al. 2008, 2012; Wang et al. 2012). An albumin solution (210 mg/mL albumin; Octapharma, Vienna, Austria) was treated with 20 mg/mL luciferin solution (Caliper Life Sciences, Hopkinton, MA, USA) and then stirred for 24 h at 4 C. Luciferin-loaded albuminshelled MBs were generated by sonicating 5 mL of the solution containing 70 mg of albumin, 6.6 mg of luciferin and perfluorocarbon gas in phosphate-buffered saline (PBS; pH 7.4) using a US sonicator (Branson, Danbury, CT, USA) for 2 min. In previous studies, the airfilled and most nonaqueous liquid-filled proteinaceous
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microspheres synthesized with a high-intensity US probe were found to be less dense than water (Suslick et al. 1994). In the present study, the luciferin-loaded albuminshelled MBs were centrifuged (1200 rpm, relative centrifugal force 5 128.6 g) and then washed three times to ensure that any free luciferin was removed. The number
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against which to measure the absorption peak and the corresponding concentration of luciferin in the luciferinloaded albumin-shelled MBs. Triplicate measurements were performed for luciferin at each concentration (80, 160, 320, 640 and 1280 mg/L). The loading efficiency was determined as (Kheirolomoom et al. 2010).
Loading efficiency ð%Þ 5 100 3 ðluciferin loaded into MBsÞ=ðtotal luciferin addedÞ:
of MBs in the solution was measured with a MultiSizer III device (Beckman Coulter, Fullerton, CA, USA) with a 30-mm-aperture probe, the measurement boundaries of which were 0.6–20 mm. The size distributions in suspension were measured by dynamic light scattering (DLS; Nanoparticle Analyzer; Horiba, Kyoto, Japan). Luciferin-loaded albumin-shelled MBs were filtered with a 5-mm syringe filter (Sartorius, Goettingen, Germany) and cross-linked using 2.5% glutaraldehyde. The glutaraldehyde was used in biological electron microscopy as a fixative. The morphology of the luciferin-loaded albumin-shelled MBs was studied using transmission electron microscopy (TEM, JEM-2000 EXII; JEOL, Tokyo, Japan) at an accelerating voltage of 75 kV. In vitro high-frequency US imaging of luciferin-loaded albumin-shelled MBs High-frequency US imaging was performed using a US animal-imaging system (Prospect; S-Sharp Corporation, Taiwan) at a frequency of 40 MHz with a transducer (acoustic pressure: 0.59 MPa [MI 5 0.09]) with a diameter of 7 mm and a fixed focus at 12 mm. A 2% agarose phantom was constructed with a 2 3 2 3 20-mm3 chamber at its center to load the luciferin-loaded albumin-shelled MBs. The loaded phantom was then sonicated by the 1-MHz US transducer of the sonoporation gene transfection system (ST 2000V; NepaGene, Ichikawa, Japan) at successive acoustic pressures of 1 W/cm2 for 1 min (1w1min), 2 W/cm2 for 1 min (2w1min) and 3 W/cm2 for 1 min (3w1min). The duty cycle was set at 10% and a 6-mm-diameter transducer was used. Measurements of luciferin loaded into albumin-shelled MBs The level of luciferin in vitro was quantified by measuring the absorbance of luciferin at 330 nm using a microplate spectrophotometer (ELISA reader; Epoch, Biotek, Winooski, VT, USA). Sample volumes of 2 mL were added to the spectrophotometer microplate. The luciferin calibration curve served as the standard curve
In vitro BLI of luciferin-loaded albumin-shelled MBs The 4T1-luc2 murine mammary tumor cell line (Caliper Life Sciences, Hopkinton, MA, USA) was used; this cell line expresses the luciferase gene, which serves as an optical indicator of gene expression or tumorigenesis in vivo. 4T1-luc2 cells (passage 4) were grown in high-glucose Roswell Park Memorial Institute (RPMI) 1640 medium (ATCC, Manassas, VA, USA). The medium was supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA) without antibiotics. For the in vitro luciferase assay (n 5 3), 4T1-luc2 cells were plated on black-walled 24-well plates at an initial concentration of 1 3 105 cells/well. Cells were grown overnight with RPMI 1640 growth medium. After 24 h, the regular medium was replaced with D-luciferin (150 mg/mL), MBs (2 3 107/mL), luciferin-loaded albumin-shelled MBs (2 3 107/mL), and luciferin-loaded albumin-shelled MBs (2 3 107/mL) after US disruption of the containing medium. Bioluminescence images were obtained using an in vivo imaging system (Xenogen IVIS-200; Caliper Life Sciences, Alameda, CA, USA) immediately after adding the substrate to the cells. The light output and bioluminescence signals were quantified using an imaging system. Human breast cancer orthotopic model 4T1-luc2 cells were cultured at 37 C in an incubator containing 5% CO2 in humidified air. Tumor cells were harvested for passages or injections by washing the monolayer with PBS, followed by a brief incubation in 0.25% trypsin and 0.02% ethylenediaminetetraacetic acid (EDTA) to detach the cells. The trypsin and EDTA were removed from the cell suspension before it was injected into the mice. Then, 0.1 mL of the cell suspension (containing 1 3 105 tumor cells in PBS) was injected via a 27-gauge needle orthotopically into the abdominal mammary fat pads of female BALB/c mice. All animal experiments were performed using an animal care protocol approved by the National Animal Center of Taiwan. All mice were maintained according to the regulations of National Taiwan University
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Hospital. The tumor diameters along three directions were measured with calipers, with the volume calculated using the ellipsoid formula. Most of the tumors had grown to 100–150 mm3 at approximately 10 days after inoculation. In vivo high-frequency US imaging of luciferin-loaded albumin-shelled MBs 4T1-luc2 tumor-bearing female BALB/c mice (n 5 3) were kept anesthetized with 2% isoflurane in oxygen at 2 l/min on a scanning stage. The hair on the skin over the tumor was clipped and acoustic gel was applied to the tumor. A commercial small-animal US imaging system (Vevo 2100; VisualSonics, Toronto, Canada) was used. The array transducer had a central frequency of 21 MHz, with axial and lateral resolutions of 75 and 165 mm, respectively. The focal length was 8 mm and acoustic pressure was 0.92 MPa (MI 5 0.2), respectively. Real-time imaging was performed at a frame rate of 30 Hz (corresponding to a temporal resolution of 33 ms). Two-dimensional B-mode image planes were acquired with optimization of the gain and the timegain compensation settings, which were kept constant throughout the experiments. High-frequency US imaging was applied to the entire tumor of each mouse. After a radiologist identified the tumor lesions, about 3–4 3 108 luciferin-loaded albumin-shelled MBs (0.15–0.2 mL) were injected through the lateral tail vein, and post-contrast-injection US imaging of the tumor lesions was immediately performed for 30 min. The region-of-interest (ROI) was drawn over the whole tumor in a two-dimensional imaging plane by a radiologist, and the average pre- and postcontrast image intensities were measured in B-mode images. In vivo BLI of luciferin-loaded albumin-shelled MBs 4T1-luc2 tumor-bearing female BALB/c mice were kept anesthetized with 2% isoflurane in oxygen at 2 l/min. BLI of the tumor-bearing mice was performed with and without US sonication after injecting 3–4 3 108 luciferin-loaded albumin-shelled MBs (0.15–0.2 mL) using the Xenogen IVIS-200 imaging system. The duration of the injection was approximately 30 s, with simultaneous US sonication of the tumor. The 1-MHz transducer of the sonoporation gene transfection system (ST 2000V; NepaGene) produced an acoustic pressure of 1 W/cm2 for 30 s (1w30s), 2 W/cm2 for 30 s (2w30s), 3 W/cm2 for 30 s (3w30s) or 4 W/cm2 for 30 s (4w30s) (n 5 4 for each condition). Photon emissions were measured, and the obtained grayscale photographic images and bioluminescence color image were superimposed using Living Image software (Caliper Life Sciences). The size of the manually selected ROI
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was kept constant and the intensity was recorded as the average radiance (in photons/s/cm2/sr) within an ROI. Tumor histochemistry The mice with and without US-mediated release of luciferin-loaded MBs were euthanized after performing BLI and the tumors were removed en bloc and fixed in 10% formalin. Sections were prepared for lightmicroscopy evaluation. They were stained with hematoxylin and eosin (Sigma-Aldrich, Saint Louis, MO, USA) to enable assessment of the overall tumor morphology and regional viability; sections were viewed at a magnification of 3400 (Primo Star; Zeiss, Jena, Germany). Histologic evaluation was performed by an individual who was blinded to the experimental condition. Statistical analysis The obtained data were analyzed statistically using Student’s t-test. A probability value of p , 0.05 was considered indicative of a significant difference. Data are presented as mean 6 SD values. RESULTS Production of luciferin-loaded albumin-shelled MBs The size distribution and concentration of the MBs in suspension were measured by DLS (Fig. 1a) and an electrical sensing zone (ESZ) (Fig. 1b), respectively. The concentration of luciferin-loaded albumin-shelled MBs (from the ESZ system) was 2.48 6 0.05 3 109/mL for a size range of 0.8–4 mm (Fig. 1b) and their mean diameter was 1591 nm (Fig. 1a). The absorbance spectrum of the HSA solution, shells of MBs, luciferin, luciferin-loaded albumin-shelled MBs after US sonication (destruction) and normal saline solution are shown in Figure 2a. HSA and albumin-shelled MBs absorb light at 280 nm, whereas luciferin and luciferin-loaded albumin-shelled MBs absorb light at 330 nm. Figure 2b shows the calibration curve of luciferin at various concentrations. The concentration of luciferin encapsulated in the MBs was 1.48 3 10210 mg/bubble. The loading efficiency of luciferin in MBs was 19.8% (n 5 5). Figure 3 shows TEM images of an albumin-shelled MB and a luciferin-loaded albumin-shelled MB. The measured thickness of the MBs ranged from 30 to 60 nm (Fig. 3a), while that of the luciferin-loaded albumin-shelled MBs ranged from 60 to 95 nm (Fig. 3b). The MBs appeared brighter than the luciferin-loaded albumin-shelled MBs on TEM images. In vitro high-frequency US imaging of luciferin-loaded albumin-shelled MBs US images of luciferin-loaded albumin-shelled MBs without and with US sonication at 1w1min, 2w1min, and
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Fig. 1. Size (a) and concentration (b) distributions of the luciferin-loaded albumin-shelled MBs (Lu_MB) in suspension as measured by dynamic light scattering (DLS) and the electrical sensing zone (ESZ).
3w1min are shown in Figure 4a–d, respectively. The image intensities of the luciferin-loaded albumin-shelled MBs in these conditions were 33.4 6 0.01, 19.8 6 0.32, 14.1 6 0.09 and 12.6 6 0.08 dB, respectively, indicating that the luciferin-loaded albumin-shelled MBs were destroyed after being sonicated with US (p , 0.001; Fig. 4e). In vitro BLI of luciferin-loaded albumin-shelled MBs To demonstrate the in vitro activity of light emission, the 4T1-luc2 cells were inoculated on 24-well plates and incubated for 24 h at 37 C in RPMI medium. RPMI (Fig. 5a), D-luciferin (Fig. 5b), albumin-shelled MBs (Fig. 5c), albumin-shelled MBs after US destruction (Fig. 5d), luciferin-loaded albumin-shelled MBs (Fig. 5e)
and luciferin-loaded albumin-shelled MBs after US destruction (Fig. 5f) were added to each well and luminescence was counted by a BLI light-detection system and quantified with Living Image software (Fig. 5g; n 5 5). The color bar indicates the signal intensity, with red and blue representing high and low bioluminescence signals, respectively. The increase in light emission of luciferin-loaded albumin-shelled MBs after destruction via US sonication at 1 W/cm2 for 1 min (6.24 6 0.72 3 107 photons/s, p , 0.05; Fig. 5g) was significantly higher than that in the luciferin-loaded albuminshelled MBs (3.11 6 0.33 3 107 photons/s, p , 0.05; Fig. 5g). The light emissions of luciferin-loaded albuminshelled MBs and luciferin-loaded albumin-shelled MBs after US destruction were significantly higher than those of albumin-shelled MBs (0.36 6 0.026 3 107 photons/s,
Fig. 2. (a) Absorbance spectrum of human serum albumin (HSA) and shells of MBs, luciferin, and luciferin-loaded albumin-shelled MBs after US destruction in normal saline solution. (b) Calibration curve of luciferin at various concentrations. OD 5 optical density.
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Fig. 3. TEM images of an albumin-shelled MB (a) and a luciferin-loaded albumin-shelled MB (b).
p , 0.05; Fig. 5g) and albumin-shelled MBs after US destruction (0.77 6 0.039 3 107 photons/s, p , 0.05; Fig. 5g). In vivo high-frequency US imaging of luciferin-loaded albumin-shelled MBs For 4T1-luc2 tumors, the average image intensity was significantly higher after injecting luciferin-loaded albumin-shelled MBs: 0.54 6 0.12 and 8.87 6 0.53 inten-
sity units (n 5 3) before and after injecting luciferin-loaded albumin-shelled MBs, respectively. A precontrast image of the tumor and the differences in video intensity from subtraction of the postcontrast image (green) on grayscale images are shown in Figure 6a and b, respectively. In vivo BLI of luciferin-loaded albumin-shelled MBs Typical in vivo BLI images of BALB/c mice bearing 4T1-luc2 xenografts 5 min after an intravenous
Fig. 4. In vitro US images of luciferin-loaded albumin-shelled MBs before (a) and after (b) sonication at 1 W/cm2 for 1 min (1w1min), (c) 2 W/cm2 for 1 min (2w1min), and (d) 3 W/cm2 for 1 min (3w1min). The signal intensities in panels a–d are quantified in (e). The signal intensity of luciferin-loaded albumin-shelled MBs decreased significantly after being sonicated (*p , 0.001).
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Fig. 5. In vitro BLI of 4T1-luc2 cells. Determination of light emissions using the IVIS-200 system. Light emissions induced by Roswell Park Memorial Institute (RPMI) medium (a), the addition of D-luciferin to the medium (b), albumin-shelled MBs (c), albumin-shelled MBs after US destruction (d), luciferin-loaded albumin-shelled MBs (e), and luciferin-loaded albumin-shelled MBs after US destruction (f). Photons were collected for 5 s. (g) Quantification of light emissions for wells A–F using Living Image software. The light emissions were significantly higher for luciferin-loaded albumin-shelled MBs and luciferin-loaded albumin-shelled MBs after US destruction than for albumin-shelled MBs and albumin-shelled MBs after US destruction (*p , 0.05). In addition, the increase in light emission was significantly higher for luciferin-loaded albumin-shelled MBs after US destruction than for luciferin-loaded albumin-shelled MBs (**p , 0.05). Lu MBs 5 luciferin-loaded albumin-shelled MBs.
injection of luciferin-loaded albumin-shelled MBs are shown in Figure 7. All amplitudes shown in the figure are normalized to the 1-min amplitude for each mouse. The BLI images of control (150 mg/kg luciferin via injection into the peritoneal cavity), luciferin-loaded albumin-shelled MBs without US destruction and luciferin-loaded albumin-shelled MBs with US-
sonication-induced destruction at 1w30s, 2w30s, 3w30s and 4w30s in a breast tumor are shown in Figure 7a–f, respectively. The light emissions of luciferin without and with US destruction at 3, 5, 7 and 10 min after injecting luciferin-loaded albumin-shelled MBs are shown in Figure 7g. The destruction of MBs increased proportionally with the US power (Fig. 4). However, for in vivo
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Fig. 6. Transverse color-coded US images of a subcutaneous 4T1-luc2 breast tumor in the same BALB/c mouse before (a) and after (b) intravenous administration of luciferin-loaded albumin-shelled MBs. The intensity of images was higher at 5 min after the administration of luciferin-loaded albumin-shelled MBs (b) than in precontrast images (a).
imaging, the light emissions of luciferin-loaded albuminshelled MBs (Fig. 7g) were significantly higher in breast tumors sonicated at 3w30s than without US destruction at 3 (p 5 0.025), 5 (p 5 0.013), 7 (p 5 0.012) and 10
min (p 5 0.032) postinjection. Relative to no US sonication, the light emissions of luciferin-loaded albuminshelled MBs were significantly higher at 3 min after US sonication at 1w30s (p 5 0.026), at 7 min after US
Fig. 7. Luminescence emissions from BALB/c mice 5 min after injections of luciferin-loaded albumin-shelled MBs. (a) BLI of the tumor was administered luciferin via injection into the peritoneal cavity. BLI of the tumor without (b) and with US sonication (destruction) were at 1 W/cm2 for 30 s (1w30s) (c), 2 W/cm2 for 30 s (2w30s) (d), 3 W/cm for 30 s (3w30s) (e), and 4 W/cm2 for 30 s (4w30s) (f). (g) Quantification of the light emission by the tumors shown in images a–f, and at 3, 7 and 10 min after injecting luciferin-loaded albumin-shelled MBs, was achieved using Living Image software (*p , 0.05).
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Fig. 8. Light microscopy of tumor sections at a magnification of 3400. Histologic analysis after hematoxylin and eosin staining showing tumor tissue from a control specimen (a) and from a specimen after injecting luciferin-loaded albuminshelled MBs and with US sonication (destruction) at 3 W/cm2 for 30 s (b). (a) Intact live tumor tissues in the control group grew in a solid pattern without obvious vascular rupture or necrosis. (b) Intercellular edema (arrow) and multiple cyst formation (arrowhead) were commonly observed after US treatment.
sonication at 2w30s (p 5 0.039) and at 3 min after US sonication at 4w30s (p 5 0.049). Tumor histochemistry Examples of light-microscope images of tumor specimens from the control group and the luciferinloaded albumin-shelled MBs US sonicated at 3w30s group are shown in Figure 8. It was found that intact live tumor tissues in the sham group grew in a solid pattern with few vacuolated cells and without obvious vascular rupture and necrosis (Fig. 8a). Vacuolated cells, intercellular edema and multiple cyst formation were commonly observed after US treatment (Fig. 8b). DISCUSSION It is well known that US sonication of liquids produces both emulsification and cavitation (Suslick et al. 1994). Proteinaceous microspheres are formed as a result of the creation of S–S bonds between the cysteine residues of the protein in an oxidation reaction, with an HO2 radical forming in the sonochemical process (Suslick et al. 1994). These bonds are formed around a micrometer-sized gas bubble or a nonaqueous droplet (Grinberg et al. 2007). The maximum drug-loading capacity in BSA microspheres was found to exceed 90% (Grinberg et al. 2007). In the present study, the loading efficiency of luciferin in MBs was only about 19.8%. The amount of drug loaded into the MBs was lower since otherwise the structure of luciferin may have been destroyed during the formation process of luciferin-loaded MBs and this would affect the quantification of luciferin by absorbance measurements at 330 nm using a microplate spectrophotometer. In vitro imaging assays revealed that the luciferinloaded albumin-shelled MBs can enhance US imaging and increase the signal intensity of BLI after albuminshelled MB US destruction in 4T1-luc2 murine
mammary tumor cells. Treatment of cells with MBs and MBs US destruction leads to an increase in signal intensity compared with the RPMI only group, while these MBs do not contain luciferin. Moreover, in a phantom study, 62.3%-luciferin-loaded MBs were destroyed by US sonication at 3 W/cm2 for 1 min. For in vivo highfrequency US imaging, the mean video intensity of the tumor after injecting luciferin-loaded MBs increased by 16.4-fold. The luciferin-loaded albumin-shelled MBs were used as a US contrast agent for both the in vitro and in vivo experiments in this study. For the in vivo BLI assay, the signal intensity of the tumors was significantly higher for US sonication at 3 W/cm2 for 30 s than without US destruction at 3, 5, 7 and 10 min after injecting luciferin-loaded albumin-shelled MBs (p , 0.05). However, under other US conditions, the differences in intensities were not statistically significant at each measurement time point of luciferin release (p 5 0.0620.30). The lower intensity US did not result in statistically changes in luciferase expression because the energy did not reach the threshold to affect the luciferin-loaded albumin-shelled MBs for cavitation and to increase the vessel permeability. Increases in the intensity of US did not appear to be proportional to the increased signal intensity seen on BLI. This discrepancy might be related to the presence of vessel injury in the tumor. Moreover, the image intensity of luciferinloaded albumin-shelled MBs without US destruction increased obviously, which indicates the leakage of luciferin from the MBs in vivo. US-mediated MB drug delivery is currently used preclinically to provide targeted local drug therapy via the application of directed US. This method is worth investigating for the local, focused delivery of anticancer drugs to tumor cells. The US-targeted destruction of doxorubicin-loaded MBs leads to a 12-fold higher tissue concentration of doxorubicin and a significantly lower tumor growth in the target tumor compared with the
Efficiency of drug-loaded microbubbles in cancer cells d A.-H. LIAO et al.
contralateral control tumor (Tinkov et al. 2010a). However, invasive methods are used to measure drug concentrations—the organs must be removed after the mice are sacrificed. It is difficult to measure the concentration of drugs in the cells after MB destruction due to the drugs released from the blood vessels penetrating the cells, which cannot be observed in a US imaging system. In the present study, BLI signals were 1.7-fold higher with US sonication (3 W/cm2 for 30 s at 10 min postinjection) than without US after injecting luciferin-loaded albuminshelled MBs. Typically, 150 mg/kg luciferin is administered via injection into the peritoneal cavity for BLI (Lim et al. 2009). The US-mediated release of luciferinloaded MBs resulted in only 3 mg/kg luciferin needing to be injected through the lateral tail vein of the mice. The improved delivery efficiency with US-mediated release could reduce the total injection dose of luciferin. Moreover, we were able to evaluate the efficiency of US-mediated local drug delivery with luciferin-loaded albumin-shelled MBs noninvasively in a transgenic mouse model expressing the luciferase reporter gene. The treatment drug could be combined with luciferin for US-mediated MB drug delivery, and the resulting signals observed using BLI. CONCLUSIONS In the present study, the US contrast agent D-luciferin was encapsulated into albumin-shelled MBs that were subsequently destroyed using a therapeutic US system. The number of photons in the US-mediated release of luciferin-loaded MBs increased markedly in both the in vitro and the in vivo BLI images. The luciferin encapsulated within the MBs was released into the tumor vessels and entered the 4T1-luc2 tumor cells in small animals. Moreover, noninvasive evaluation of the efficiency of US-mediated local drug delivery with luciferin-loaded albumin-shelled MBs demonstrated an improved technique, in that the total injection dose of luciferin could be reduced under targeted US sonication conditions. Acknowledgments—The authors thank the S-Sharp Corporation in Taiwan for kindly providing the Prospect US imaging system. This work was supported in part by the National Taiwan University Center for Genomic Medicine and grant nos. NSC 99-2218-E-011-031 and NSC 100-2628-E-011-015-MY3 from the National Science Council of Taiwan.
REFERENCES Abdelmoneim SS, Bernier M, Scott CG, Dhoble A, Ness SA, Hagen ME, Moir S, McCully RB, Pellikka PA, Mulvagh SL. Safety of contrast agent use during stress echocardiography in patients with elevated right ventricular systolic pressure: A cohort study. Circ Cardiovasc Imaging 2010;3:240–248.
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Chen X, Zhang X, Larson CS, Baker MS, Kaufman DB. In vivo bioluminescence imaging of transplanted islets and early detection of graft rejection. Transplantation 2006;81:1421–1427. Chen YC, Jiang LP, Liu NX, Wang ZH, Hong K, Zhang QP. P85, Optison microbubbles and ultrasound cooperate in mediating plasmid DNA transfection in mouse skeletal muscles in vivo. Ultrason Sonochem 2011;18:513–519. Cochran MC, Eisenbrey J, Ouma RO, Soulen M, Wheatley MA. Doxorubicin and paclitaxel loaded microbubbles for ultrasound triggered drug delivery. Int J Pharm 2011;414:161–170. Cohen JL, Cheirif J, Segar DS, Gillam LD, Gottdiener JS, Hausnerova E, Bruns DE. Improved left ventricular endocardial border delineation and opacification with OPTISON (FS069), a new echocardiographic contrast agent. Results of a phase III Multicenter Trial. J Am Coll Cardiol 1998;32:746–752. Contag CH, Ross BD. It’s not just about anatomy: In vivo bioluminescence imaging as an eyepiece into biology. J Magn Reson Imaging 2002;16:378–387. Fokong S, Theek B, Wu Z, Koczera P, Appold L, Jorge S, Resch-Genger U, van Zandvoort M, Storm G, Kiessling F, Lammers T. Image-guided, targeted and triggered drug delivery to tumors using polymer-based microbubbles. J Control Release 2012, in press, Epub ahead of print May 9. http://dx.doi.org/10. 1016/j.jconrel.2012.05.007. Gedanken A. Preparation and properties of proteinaceous microspheres made sonochemically. Chemistry 2009;14:3840–3853. Grinberg O, Hayun M, Sredni B, Gedanken A. Characterization and activity of sonochemically-prepared BSA microspheres containing Taxol–An anticancer drug. Ultrason Sonochem 2007;14:661–666. Hernot S, Klibanov AL. Microbubbles in ultrasound-triggered drug and gene delivery. Adv Drug Deliv Rev 2008;60:1153–1166. Kheirolomoom A, Kruse DE, Qin S, Watson KE, Lai CY, Young LJ, Cardiff RD, Ferrara KW. Enhanced in vivo bioluminescence imaging using liposomal luciferin delivery system. J Control Release 2010;141:128–136. Korpanty G, Grayburn PA, Shohet RV, Brekken RA. Targeting vascular endothelium with avidin microbubbles. Ultrasound Med Biol 2005; 31:1279–1283. Lee KN, Ye Y, Carr JH, Karem K, D’Souza MJ. Formulation, pharmacokinetics and biodistribution of Ofloxacin-loaded albumin microparticles and nanoparticles. J Microencapsul 2011;28:363–369. Lentacker I, Geers B, Demeester J, De Smedt SC, Sanders NN. Design and evaluation of doxorubicin-containing microbubbles for ultrasound-triggered doxorubicin delivery: Cytotoxicity and mechanisms involved. Mol Ther 2010;18:101–108. Liao AH, Cheng YC, Weng CH, Tsai TF, Lin WH, Yeh SH, Yeh WC, Li PC. Characterization of malignant focal liver lesions with contrast-enhanced 40 MHz ultrasound imaging in hepatitis B virus X transgenic mice: A feasibility study. Ultrason Imaging 2008;30: 203–216. Liao AH, Liu HL, Su CH, Hua MY, Yang HW, Weng YT, Hsu PH, Huang SM, Wu SY, Wang HE, Yen TC, Li PC. Paramagnetic perfluorocarbon-filled albumin-(Gd-DTPA) microbubbles for the induction of focused-ultrasound-induced blood-brain barrier opening and concurrent MR and ultrasound imaging. Phys Med Biol 2012;57:2787–2802. Lim E, Modi KD, Kim J. In vivo bioluminescent imaging of mammary tumors using IVIS spectrum. J Vis Exp 2009;26:1210. Lin CY, Liu TM, Chen CY, Huang YL, Huang WK, Sun CK, Chang FH, Lin WL. Quantitative and qualitative investigation into the impact of focused ultrasound with microbubbles on the triggered release of nanoparticles from vasculature in mouse tumors. J Control Release 2010;146:291–298. Liu Y, Miyoshi H, Nakamura M. Encapsulated ultrasound microbubbles: Therapeutic application in drug/gene delivery. J Control Release 2006;114:89–99. Lum AF, Borden MA, Dayton PA, Kruse DE, Simon SI, Ferrara KW. Ultrasound radiation force enables targeted deposition of model drug carriers loaded on microbubbles. J Control Release 2006; 111:128–134.
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Maruyama K, Suzuki R, Takizawa T, Utoguchi N, Negishi Y. Drug and gene delivery by ‘‘bubble liposomes’’ and ultrasound. Yakugaku Zasshi 2007;127:781–787. McElroy WD, Strehler BL. Bioluminescence. Bacteriol Rev 1954;18: 177–194. Suslick KS, Grinstaff MW, Kolbeck KJ, Wong M. Characterization of sonochemically prepared proteinaceous microspheres. Ultrason Sonochem 1994;1:S65–S68. Tinkov S, Coester C, Serba S, Geis NA, Katus HA, Winter G, Bekeredjian R. New doxorubicin-loaded phospholipid microbubbles for targeted tumor therapy: In vivo characterization. J. Control. Release 2010a;148(3):368–372.
Volume 38, Number 11, 2012 Tinkov S, Winter G, Coester C, Bekeredjian R. New doxorubicinloaded phospholipid microbubbles for targeted tumor therapy: Part I–Formulation development and in-vitro characterization. J. Control. Release 2010b;143:143–150. Wang YH, Liao AH, Chen JH, Wang CR, Li PC. Photoacoustic/ultrasound dual-modality contrast agent and its application to thermotherapy. J Biomed Opt 2012;17:045001. Yang F, Zhang M, He W, Chen P, Cai X, Yang L, Gu N, Wu J. Controlled release of Fe3O4 nanoparticles in encapsulated microbubbles to tumor cells via sonoporation and associated cellular bioeffects. Small 2011;7:902–910. Yuan B. Ultrasound-modulated fluorescence based on a fluorophorequencher-labeled microbubble system. J Biomed Opt 2009;14:024043.
http://dx.doi.org/10.1016/j.ultrasmedbio.2012.07.013
d
Original Contribution ESTIMATING THE DELIVERY EFFICIENCY OF DRUG-LOADED MICROBUBBLES IN CANCER CELLS WITH ULTRASOUND AND BIOLUMINESCENCE IMAGING AI-HO LIAO,* YING-KAI LI,* WEI-JIUNN LEE,y MING-FANG WU,z HAO-LI LIU,x and MIN-LIANG KUOy y
* Graduate Institute of Biomedical Engineering, National Taiwan University of Science and Technology, Taipei, Taiwan; Institute of Toxicology College of Medicine, National Taiwan University, Taipei, Taiwan; z Medicine of Animal Medicine Center, National Taiwan University, Taipei, Taiwan; and x Department of Electrical Engineering, Chang Gung University, Tao-Yuan, Taiwan (Received 26 June 2012; revised 17 July 2012; in final form 18 July 2012)
Abstract—The application of drug-loaded microbubbles (MBs) in combination with ultrasound (US), which results in an increase in capillary permeability at the site of US-sonication-induced MB destruction, may be an efficient method of localized drug delivery. This study investigated the mechanism underlying the US-mediated release of luciferin-loaded MBs through the blood vessels to targeted cells using an in vivo bioluminescence imaging (BLI) system. The luciferin-loaded MBs comprised an albumin shell with a diameter of 1234 ± 394 nm (mean ± SD) and contained 2.48 3 109 bubbles/mL; within each MB, the concentration of encapsulated luciferin was 1.48 3 10210 mg/bubble. The loading efficiency of luciferin in MBs was only about 19.8%, while maintaining both the bioluminescence and acoustic properties. In vitro and in vivo BLI experiments were performed to evaluate the US-mediated release of luciferin-loaded MBs. For in vitro results, the increase in light emission of luciferinloaded albumin-shelled MBs after destruction via US sonication (6.24 ± 0.72 3 107 photons/s) was significantly higher than that in the luciferin-loaded albumin-shelled MBs (3.11 ± 0.33 3 107 photons/s) (p , 0.05). The efficiency of the US-mediated release of luciferin-loaded MBs in 4T1-luc2 tumor-bearing mice was also estimated. The signal intensity of the tumor with US destruction at 3 W/cm2 for 30 s was significantly higher than without US destruction at 3 (p 5 0.025), 5 (p 5 0.013), 7 (p 5 0.012) and 10 (p 5 0.032) min after injecting luciferinloaded albumin-shelled MBs. The delivery efficiency was, thus, improved with US-mediated release, allowing reduction of the total injection dose of luciferin. (E-mail: [email protected]) Ó 2012 World Federation for Ultrasound in Medicine & Biology. Key Words: Luciferin-loaded microbubbles, High-frequency ultrasound, In vivo bioluminescence imaging system, Luciferase-expressing breast cancer cell.
INTRODUCTION
somes (Lentacker et al. 2010). A major drawback of coinjecting drugs and MBs is that drugs can still extravasate and accumulate in tissues other than those targeted (i.e., not exposed to US). Furthermore, drug molecules may become incorporated within the thick polymer shell or inside the gas core (or multiple cores) of thickshelled MBs (Hernot and Klibanov 2008). It has been demonstrated that the release of drugs from the shells of polymer MBs embedded Fe3O4 nanoparticles can be controlled by sonoporation (Yang et al. 2011). Paclitaxel-loaded polymer MBs triggered with focused US have the potential to provide a targeted and sustained delivery of the drug to tumors (Cochran et al. 2011). For lipid MBs, the drug could be attached to the external surface of the monolayer by covalent or noncovalent bonds, or incorporated into liposomes or
Ultrasound (US)-mediated gene- and drug-delivery methods have been proposed as targeting tools in recent years. In combination with drug-loaded microbubbles (MBs), local drug concentrations can be increased by taking advantage of US-targeted drug release and the transiently increased capillary permeability at the site of MB destruction (Chen et al. 2011; Lin et al. 2010; Maruyama et al. 2007). Most of the previous reports have focused on US-assisted intracellular delivery of the free drug or drugs encapsulated in micelles or lipoAddress correspondence to: Ai-Ho Liao, Ph.D., Graduate Institute of Biomedical Engineering, National Taiwan University of Science and Technology, TR-916, #43, Sec. 4, Keelung Rd, Taipei, Taiwan 10607, ROC. E-mail: [email protected] 1938
Efficiency of drug-loaded microbubbles in cancer cells d A.-H. LIAO et al.
nanoparticles that are then associated with the bubble surface (Lum et al. 2006). A recent study found that the US-targeted destruction of drug-loaded MBs led to a 12-fold higher tissue concentration of the drug and a significantly lower tumor growth in the target tumor compared with the contralateral control tumor (Tinkov et al. 2010a, b). However, the mechanism underlying the US-mediated release of drug-loaded MBs through the blood vessels to targeted cells is unclear and not easy to control. The dynamic mechanisms that lead to the destruction of MBs are not well understood because the process is often complicated and difficult to quantify (Liu et al. 2006). Recently, poly butyl cyanoacrylate based hard-shell, VEGFR2-targeted and fluorophore loaded MBs were shown to be highly suitable for image-guided drug targeting to tumor blood vessels and to release significant amounts of drugs upon exposure to high-mechanical index destructive US pulses (Fokong et al. 2012). In addition, developing ultrasound-modulated fluorescence tomography has been proposed and demonstrated (Yuan 2009). The US-mediated release of fluorescent drugs loaded MBs could be imaged noninvasively. This situation prompted the present study to ascertain the mechanism underlying the US-mediated release of the drug from drug-loaded MBs using a new model with a method that combines therapeutic US, a high-frequency US imaging system, and an in vivo bioluminescence imaging (BLI) system. The US-assisted intracellular delivery was performed with luciferin-loaded albuminshelled MBs targeting luciferase-gene-transfected breast cancer cells in mice. The efficiency of the US-mediated release of luciferin-loaded MBs in tumor-bearing mice was investigated. BLI has evolved as a highly sensitive and powerful technique for the noninvasive, cost-effective and realtime monitoring of sophisticated biologic processes in intact animals (Kheirolomoom et al. 2010). In vivo BLI requires delivery of D-luciferin, which is rapidly cleared from the blood circulation and enters the target cells. Since luciferin can penetrate cell membranes, it allows the transformed cells to be monitored for luciferase activity (McElroy and Strehler 1954). Luciferase enzymes emit light (bioluminescence) by catalyzing the oxidation of the substrate luciferin to oxyluciferin in an oxygen- and adenosine-triphosphate-dependent process (Chen et al. 2006). Luciferase has previously served as the reporter gene to assess gene or luciferin substrate delivery in cancer research (Contag and Ross 2002). MBs with an albumin shell, such as OptisonÔ (GE Healthcare, East Norwalk, CT, USA), can persist in the bloodstream for a sufficiently long time for imaging because the denatured albumin shell and core gas have low solubility in blood (Cohen et al. 1998). Albuminbased MBs consisting of perfluorocarbon gas surrounded by a human serum albumin (HSA)-based shell have been
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used clinically to enhance the contrast in US imaging (Abdelmoneim et al. 2010). In addition, albuminshelled MBs incorporated with targeting ligands are used as a US contrast agent (Cohen et al. 1998) for local inflammation and angiogenesis detection (Korpanty et al. 2005). During the formation of air-filled albumin-shelled MBs, the sonicator horn is placed at the air–water interface (Gedanken 2009; Suslick et al. 1994). It has recently been demonstrated that if the starting solution of the native bovine serum albumin (BSA) protein contains an antibiotic drug, 3 min of sonication forms a BSA microsphere encapsulating up to 90% of the drug in the proteinaceous microsphere (Grinberg et al. 2007). A recent study of drug-loaded albumin-shelled microparticles revealed that peak concentrations of drugs were reduced in the serum, liver, spleen and brain (Lee et al. 2011). In the present study, the treatment drug, luciferin, was encapsulated in the albumin-shelled MBs. The luciferin-loaded albumin-shelled MBs were produced by sonicating a solution containing albumin, luciferin and perfluorocarbon gas. However, the D-luciferin can be stored at room temperature only for a short period of time. The sonication has been shown to increase the temperature of samples and may affect the structure of D-luciferin. Based on in vitro and in vivo experiments, the aim of this study was to elucidate the feasibility and efficacy of US-mediated intracellular drug delivery of the novel MBs in a murine mammary tumor model. First, the luciferinloaded albumin-shelled MBs were produced and the loading efficiency of luciferin was measured. The luciferin-loaded albumin-shelled MBs were then characterized in vitro and in vivo with the aid of a US imaging system and BLI. In addition, the efficiency of USmediated local drug delivery with luciferin-loaded albumin-shelled MBs was evaluated in transgenic mouse models expressing the luciferase reporter gene with BLI. MATERIALS AND METHODS Production of luciferin-loaded albumin-shelled MBs Luciferin-loaded albumin-shelled MBs were prepared according to the procedure used in our previous study (Liao et al. 2008, 2012; Wang et al. 2012). An albumin solution (210 mg/mL albumin; Octapharma, Vienna, Austria) was treated with 20 mg/mL luciferin solution (Caliper Life Sciences, Hopkinton, MA, USA) and then stirred for 24 h at 4 C. Luciferin-loaded albuminshelled MBs were generated by sonicating 5 mL of the solution containing 70 mg of albumin, 6.6 mg of luciferin and perfluorocarbon gas in phosphate-buffered saline (PBS; pH 7.4) using a US sonicator (Branson, Danbury, CT, USA) for 2 min. In previous studies, the airfilled and most nonaqueous liquid-filled proteinaceous
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microspheres synthesized with a high-intensity US probe were found to be less dense than water (Suslick et al. 1994). In the present study, the luciferin-loaded albuminshelled MBs were centrifuged (1200 rpm, relative centrifugal force 5 128.6 g) and then washed three times to ensure that any free luciferin was removed. The number
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against which to measure the absorption peak and the corresponding concentration of luciferin in the luciferinloaded albumin-shelled MBs. Triplicate measurements were performed for luciferin at each concentration (80, 160, 320, 640 and 1280 mg/L). The loading efficiency was determined as (Kheirolomoom et al. 2010).
Loading efficiency ð%Þ 5 100 3 ðluciferin loaded into MBsÞ=ðtotal luciferin addedÞ:
of MBs in the solution was measured with a MultiSizer III device (Beckman Coulter, Fullerton, CA, USA) with a 30-mm-aperture probe, the measurement boundaries of which were 0.6–20 mm. The size distributions in suspension were measured by dynamic light scattering (DLS; Nanoparticle Analyzer; Horiba, Kyoto, Japan). Luciferin-loaded albumin-shelled MBs were filtered with a 5-mm syringe filter (Sartorius, Goettingen, Germany) and cross-linked using 2.5% glutaraldehyde. The glutaraldehyde was used in biological electron microscopy as a fixative. The morphology of the luciferin-loaded albumin-shelled MBs was studied using transmission electron microscopy (TEM, JEM-2000 EXII; JEOL, Tokyo, Japan) at an accelerating voltage of 75 kV. In vitro high-frequency US imaging of luciferin-loaded albumin-shelled MBs High-frequency US imaging was performed using a US animal-imaging system (Prospect; S-Sharp Corporation, Taiwan) at a frequency of 40 MHz with a transducer (acoustic pressure: 0.59 MPa [MI 5 0.09]) with a diameter of 7 mm and a fixed focus at 12 mm. A 2% agarose phantom was constructed with a 2 3 2 3 20-mm3 chamber at its center to load the luciferin-loaded albumin-shelled MBs. The loaded phantom was then sonicated by the 1-MHz US transducer of the sonoporation gene transfection system (ST 2000V; NepaGene, Ichikawa, Japan) at successive acoustic pressures of 1 W/cm2 for 1 min (1w1min), 2 W/cm2 for 1 min (2w1min) and 3 W/cm2 for 1 min (3w1min). The duty cycle was set at 10% and a 6-mm-diameter transducer was used. Measurements of luciferin loaded into albumin-shelled MBs The level of luciferin in vitro was quantified by measuring the absorbance of luciferin at 330 nm using a microplate spectrophotometer (ELISA reader; Epoch, Biotek, Winooski, VT, USA). Sample volumes of 2 mL were added to the spectrophotometer microplate. The luciferin calibration curve served as the standard curve
In vitro BLI of luciferin-loaded albumin-shelled MBs The 4T1-luc2 murine mammary tumor cell line (Caliper Life Sciences, Hopkinton, MA, USA) was used; this cell line expresses the luciferase gene, which serves as an optical indicator of gene expression or tumorigenesis in vivo. 4T1-luc2 cells (passage 4) were grown in high-glucose Roswell Park Memorial Institute (RPMI) 1640 medium (ATCC, Manassas, VA, USA). The medium was supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA) without antibiotics. For the in vitro luciferase assay (n 5 3), 4T1-luc2 cells were plated on black-walled 24-well plates at an initial concentration of 1 3 105 cells/well. Cells were grown overnight with RPMI 1640 growth medium. After 24 h, the regular medium was replaced with D-luciferin (150 mg/mL), MBs (2 3 107/mL), luciferin-loaded albumin-shelled MBs (2 3 107/mL), and luciferin-loaded albumin-shelled MBs (2 3 107/mL) after US disruption of the containing medium. Bioluminescence images were obtained using an in vivo imaging system (Xenogen IVIS-200; Caliper Life Sciences, Alameda, CA, USA) immediately after adding the substrate to the cells. The light output and bioluminescence signals were quantified using an imaging system. Human breast cancer orthotopic model 4T1-luc2 cells were cultured at 37 C in an incubator containing 5% CO2 in humidified air. Tumor cells were harvested for passages or injections by washing the monolayer with PBS, followed by a brief incubation in 0.25% trypsin and 0.02% ethylenediaminetetraacetic acid (EDTA) to detach the cells. The trypsin and EDTA were removed from the cell suspension before it was injected into the mice. Then, 0.1 mL of the cell suspension (containing 1 3 105 tumor cells in PBS) was injected via a 27-gauge needle orthotopically into the abdominal mammary fat pads of female BALB/c mice. All animal experiments were performed using an animal care protocol approved by the National Animal Center of Taiwan. All mice were maintained according to the regulations of National Taiwan University
Efficiency of drug-loaded microbubbles in cancer cells d A.-H. LIAO et al.
Hospital. The tumor diameters along three directions were measured with calipers, with the volume calculated using the ellipsoid formula. Most of the tumors had grown to 100–150 mm3 at approximately 10 days after inoculation. In vivo high-frequency US imaging of luciferin-loaded albumin-shelled MBs 4T1-luc2 tumor-bearing female BALB/c mice (n 5 3) were kept anesthetized with 2% isoflurane in oxygen at 2 l/min on a scanning stage. The hair on the skin over the tumor was clipped and acoustic gel was applied to the tumor. A commercial small-animal US imaging system (Vevo 2100; VisualSonics, Toronto, Canada) was used. The array transducer had a central frequency of 21 MHz, with axial and lateral resolutions of 75 and 165 mm, respectively. The focal length was 8 mm and acoustic pressure was 0.92 MPa (MI 5 0.2), respectively. Real-time imaging was performed at a frame rate of 30 Hz (corresponding to a temporal resolution of 33 ms). Two-dimensional B-mode image planes were acquired with optimization of the gain and the timegain compensation settings, which were kept constant throughout the experiments. High-frequency US imaging was applied to the entire tumor of each mouse. After a radiologist identified the tumor lesions, about 3–4 3 108 luciferin-loaded albumin-shelled MBs (0.15–0.2 mL) were injected through the lateral tail vein, and post-contrast-injection US imaging of the tumor lesions was immediately performed for 30 min. The region-of-interest (ROI) was drawn over the whole tumor in a two-dimensional imaging plane by a radiologist, and the average pre- and postcontrast image intensities were measured in B-mode images. In vivo BLI of luciferin-loaded albumin-shelled MBs 4T1-luc2 tumor-bearing female BALB/c mice were kept anesthetized with 2% isoflurane in oxygen at 2 l/min. BLI of the tumor-bearing mice was performed with and without US sonication after injecting 3–4 3 108 luciferin-loaded albumin-shelled MBs (0.15–0.2 mL) using the Xenogen IVIS-200 imaging system. The duration of the injection was approximately 30 s, with simultaneous US sonication of the tumor. The 1-MHz transducer of the sonoporation gene transfection system (ST 2000V; NepaGene) produced an acoustic pressure of 1 W/cm2 for 30 s (1w30s), 2 W/cm2 for 30 s (2w30s), 3 W/cm2 for 30 s (3w30s) or 4 W/cm2 for 30 s (4w30s) (n 5 4 for each condition). Photon emissions were measured, and the obtained grayscale photographic images and bioluminescence color image were superimposed using Living Image software (Caliper Life Sciences). The size of the manually selected ROI
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was kept constant and the intensity was recorded as the average radiance (in photons/s/cm2/sr) within an ROI. Tumor histochemistry The mice with and without US-mediated release of luciferin-loaded MBs were euthanized after performing BLI and the tumors were removed en bloc and fixed in 10% formalin. Sections were prepared for lightmicroscopy evaluation. They were stained with hematoxylin and eosin (Sigma-Aldrich, Saint Louis, MO, USA) to enable assessment of the overall tumor morphology and regional viability; sections were viewed at a magnification of 3400 (Primo Star; Zeiss, Jena, Germany). Histologic evaluation was performed by an individual who was blinded to the experimental condition. Statistical analysis The obtained data were analyzed statistically using Student’s t-test. A probability value of p , 0.05 was considered indicative of a significant difference. Data are presented as mean 6 SD values. RESULTS Production of luciferin-loaded albumin-shelled MBs The size distribution and concentration of the MBs in suspension were measured by DLS (Fig. 1a) and an electrical sensing zone (ESZ) (Fig. 1b), respectively. The concentration of luciferin-loaded albumin-shelled MBs (from the ESZ system) was 2.48 6 0.05 3 109/mL for a size range of 0.8–4 mm (Fig. 1b) and their mean diameter was 1591 nm (Fig. 1a). The absorbance spectrum of the HSA solution, shells of MBs, luciferin, luciferin-loaded albumin-shelled MBs after US sonication (destruction) and normal saline solution are shown in Figure 2a. HSA and albumin-shelled MBs absorb light at 280 nm, whereas luciferin and luciferin-loaded albumin-shelled MBs absorb light at 330 nm. Figure 2b shows the calibration curve of luciferin at various concentrations. The concentration of luciferin encapsulated in the MBs was 1.48 3 10210 mg/bubble. The loading efficiency of luciferin in MBs was 19.8% (n 5 5). Figure 3 shows TEM images of an albumin-shelled MB and a luciferin-loaded albumin-shelled MB. The measured thickness of the MBs ranged from 30 to 60 nm (Fig. 3a), while that of the luciferin-loaded albumin-shelled MBs ranged from 60 to 95 nm (Fig. 3b). The MBs appeared brighter than the luciferin-loaded albumin-shelled MBs on TEM images. In vitro high-frequency US imaging of luciferin-loaded albumin-shelled MBs US images of luciferin-loaded albumin-shelled MBs without and with US sonication at 1w1min, 2w1min, and
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Fig. 1. Size (a) and concentration (b) distributions of the luciferin-loaded albumin-shelled MBs (Lu_MB) in suspension as measured by dynamic light scattering (DLS) and the electrical sensing zone (ESZ).
3w1min are shown in Figure 4a–d, respectively. The image intensities of the luciferin-loaded albumin-shelled MBs in these conditions were 33.4 6 0.01, 19.8 6 0.32, 14.1 6 0.09 and 12.6 6 0.08 dB, respectively, indicating that the luciferin-loaded albumin-shelled MBs were destroyed after being sonicated with US (p , 0.001; Fig. 4e). In vitro BLI of luciferin-loaded albumin-shelled MBs To demonstrate the in vitro activity of light emission, the 4T1-luc2 cells were inoculated on 24-well plates and incubated for 24 h at 37 C in RPMI medium. RPMI (Fig. 5a), D-luciferin (Fig. 5b), albumin-shelled MBs (Fig. 5c), albumin-shelled MBs after US destruction (Fig. 5d), luciferin-loaded albumin-shelled MBs (Fig. 5e)
and luciferin-loaded albumin-shelled MBs after US destruction (Fig. 5f) were added to each well and luminescence was counted by a BLI light-detection system and quantified with Living Image software (Fig. 5g; n 5 5). The color bar indicates the signal intensity, with red and blue representing high and low bioluminescence signals, respectively. The increase in light emission of luciferin-loaded albumin-shelled MBs after destruction via US sonication at 1 W/cm2 for 1 min (6.24 6 0.72 3 107 photons/s, p , 0.05; Fig. 5g) was significantly higher than that in the luciferin-loaded albuminshelled MBs (3.11 6 0.33 3 107 photons/s, p , 0.05; Fig. 5g). The light emissions of luciferin-loaded albuminshelled MBs and luciferin-loaded albumin-shelled MBs after US destruction were significantly higher than those of albumin-shelled MBs (0.36 6 0.026 3 107 photons/s,
Fig. 2. (a) Absorbance spectrum of human serum albumin (HSA) and shells of MBs, luciferin, and luciferin-loaded albumin-shelled MBs after US destruction in normal saline solution. (b) Calibration curve of luciferin at various concentrations. OD 5 optical density.
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Fig. 3. TEM images of an albumin-shelled MB (a) and a luciferin-loaded albumin-shelled MB (b).
p , 0.05; Fig. 5g) and albumin-shelled MBs after US destruction (0.77 6 0.039 3 107 photons/s, p , 0.05; Fig. 5g). In vivo high-frequency US imaging of luciferin-loaded albumin-shelled MBs For 4T1-luc2 tumors, the average image intensity was significantly higher after injecting luciferin-loaded albumin-shelled MBs: 0.54 6 0.12 and 8.87 6 0.53 inten-
sity units (n 5 3) before and after injecting luciferin-loaded albumin-shelled MBs, respectively. A precontrast image of the tumor and the differences in video intensity from subtraction of the postcontrast image (green) on grayscale images are shown in Figure 6a and b, respectively. In vivo BLI of luciferin-loaded albumin-shelled MBs Typical in vivo BLI images of BALB/c mice bearing 4T1-luc2 xenografts 5 min after an intravenous
Fig. 4. In vitro US images of luciferin-loaded albumin-shelled MBs before (a) and after (b) sonication at 1 W/cm2 for 1 min (1w1min), (c) 2 W/cm2 for 1 min (2w1min), and (d) 3 W/cm2 for 1 min (3w1min). The signal intensities in panels a–d are quantified in (e). The signal intensity of luciferin-loaded albumin-shelled MBs decreased significantly after being sonicated (*p , 0.001).
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Fig. 5. In vitro BLI of 4T1-luc2 cells. Determination of light emissions using the IVIS-200 system. Light emissions induced by Roswell Park Memorial Institute (RPMI) medium (a), the addition of D-luciferin to the medium (b), albumin-shelled MBs (c), albumin-shelled MBs after US destruction (d), luciferin-loaded albumin-shelled MBs (e), and luciferin-loaded albumin-shelled MBs after US destruction (f). Photons were collected for 5 s. (g) Quantification of light emissions for wells A–F using Living Image software. The light emissions were significantly higher for luciferin-loaded albumin-shelled MBs and luciferin-loaded albumin-shelled MBs after US destruction than for albumin-shelled MBs and albumin-shelled MBs after US destruction (*p , 0.05). In addition, the increase in light emission was significantly higher for luciferin-loaded albumin-shelled MBs after US destruction than for luciferin-loaded albumin-shelled MBs (**p , 0.05). Lu MBs 5 luciferin-loaded albumin-shelled MBs.
injection of luciferin-loaded albumin-shelled MBs are shown in Figure 7. All amplitudes shown in the figure are normalized to the 1-min amplitude for each mouse. The BLI images of control (150 mg/kg luciferin via injection into the peritoneal cavity), luciferin-loaded albumin-shelled MBs without US destruction and luciferin-loaded albumin-shelled MBs with US-
sonication-induced destruction at 1w30s, 2w30s, 3w30s and 4w30s in a breast tumor are shown in Figure 7a–f, respectively. The light emissions of luciferin without and with US destruction at 3, 5, 7 and 10 min after injecting luciferin-loaded albumin-shelled MBs are shown in Figure 7g. The destruction of MBs increased proportionally with the US power (Fig. 4). However, for in vivo
Efficiency of drug-loaded microbubbles in cancer cells d A.-H. LIAO et al.
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Fig. 6. Transverse color-coded US images of a subcutaneous 4T1-luc2 breast tumor in the same BALB/c mouse before (a) and after (b) intravenous administration of luciferin-loaded albumin-shelled MBs. The intensity of images was higher at 5 min after the administration of luciferin-loaded albumin-shelled MBs (b) than in precontrast images (a).
imaging, the light emissions of luciferin-loaded albuminshelled MBs (Fig. 7g) were significantly higher in breast tumors sonicated at 3w30s than without US destruction at 3 (p 5 0.025), 5 (p 5 0.013), 7 (p 5 0.012) and 10
min (p 5 0.032) postinjection. Relative to no US sonication, the light emissions of luciferin-loaded albuminshelled MBs were significantly higher at 3 min after US sonication at 1w30s (p 5 0.026), at 7 min after US
Fig. 7. Luminescence emissions from BALB/c mice 5 min after injections of luciferin-loaded albumin-shelled MBs. (a) BLI of the tumor was administered luciferin via injection into the peritoneal cavity. BLI of the tumor without (b) and with US sonication (destruction) were at 1 W/cm2 for 30 s (1w30s) (c), 2 W/cm2 for 30 s (2w30s) (d), 3 W/cm for 30 s (3w30s) (e), and 4 W/cm2 for 30 s (4w30s) (f). (g) Quantification of the light emission by the tumors shown in images a–f, and at 3, 7 and 10 min after injecting luciferin-loaded albumin-shelled MBs, was achieved using Living Image software (*p , 0.05).
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Fig. 8. Light microscopy of tumor sections at a magnification of 3400. Histologic analysis after hematoxylin and eosin staining showing tumor tissue from a control specimen (a) and from a specimen after injecting luciferin-loaded albuminshelled MBs and with US sonication (destruction) at 3 W/cm2 for 30 s (b). (a) Intact live tumor tissues in the control group grew in a solid pattern without obvious vascular rupture or necrosis. (b) Intercellular edema (arrow) and multiple cyst formation (arrowhead) were commonly observed after US treatment.
sonication at 2w30s (p 5 0.039) and at 3 min after US sonication at 4w30s (p 5 0.049). Tumor histochemistry Examples of light-microscope images of tumor specimens from the control group and the luciferinloaded albumin-shelled MBs US sonicated at 3w30s group are shown in Figure 8. It was found that intact live tumor tissues in the sham group grew in a solid pattern with few vacuolated cells and without obvious vascular rupture and necrosis (Fig. 8a). Vacuolated cells, intercellular edema and multiple cyst formation were commonly observed after US treatment (Fig. 8b). DISCUSSION It is well known that US sonication of liquids produces both emulsification and cavitation (Suslick et al. 1994). Proteinaceous microspheres are formed as a result of the creation of S–S bonds between the cysteine residues of the protein in an oxidation reaction, with an HO2 radical forming in the sonochemical process (Suslick et al. 1994). These bonds are formed around a micrometer-sized gas bubble or a nonaqueous droplet (Grinberg et al. 2007). The maximum drug-loading capacity in BSA microspheres was found to exceed 90% (Grinberg et al. 2007). In the present study, the loading efficiency of luciferin in MBs was only about 19.8%. The amount of drug loaded into the MBs was lower since otherwise the structure of luciferin may have been destroyed during the formation process of luciferin-loaded MBs and this would affect the quantification of luciferin by absorbance measurements at 330 nm using a microplate spectrophotometer. In vitro imaging assays revealed that the luciferinloaded albumin-shelled MBs can enhance US imaging and increase the signal intensity of BLI after albuminshelled MB US destruction in 4T1-luc2 murine
mammary tumor cells. Treatment of cells with MBs and MBs US destruction leads to an increase in signal intensity compared with the RPMI only group, while these MBs do not contain luciferin. Moreover, in a phantom study, 62.3%-luciferin-loaded MBs were destroyed by US sonication at 3 W/cm2 for 1 min. For in vivo highfrequency US imaging, the mean video intensity of the tumor after injecting luciferin-loaded MBs increased by 16.4-fold. The luciferin-loaded albumin-shelled MBs were used as a US contrast agent for both the in vitro and in vivo experiments in this study. For the in vivo BLI assay, the signal intensity of the tumors was significantly higher for US sonication at 3 W/cm2 for 30 s than without US destruction at 3, 5, 7 and 10 min after injecting luciferin-loaded albumin-shelled MBs (p , 0.05). However, under other US conditions, the differences in intensities were not statistically significant at each measurement time point of luciferin release (p 5 0.0620.30). The lower intensity US did not result in statistically changes in luciferase expression because the energy did not reach the threshold to affect the luciferin-loaded albumin-shelled MBs for cavitation and to increase the vessel permeability. Increases in the intensity of US did not appear to be proportional to the increased signal intensity seen on BLI. This discrepancy might be related to the presence of vessel injury in the tumor. Moreover, the image intensity of luciferinloaded albumin-shelled MBs without US destruction increased obviously, which indicates the leakage of luciferin from the MBs in vivo. US-mediated MB drug delivery is currently used preclinically to provide targeted local drug therapy via the application of directed US. This method is worth investigating for the local, focused delivery of anticancer drugs to tumor cells. The US-targeted destruction of doxorubicin-loaded MBs leads to a 12-fold higher tissue concentration of doxorubicin and a significantly lower tumor growth in the target tumor compared with the
Efficiency of drug-loaded microbubbles in cancer cells d A.-H. LIAO et al.
contralateral control tumor (Tinkov et al. 2010a). However, invasive methods are used to measure drug concentrations—the organs must be removed after the mice are sacrificed. It is difficult to measure the concentration of drugs in the cells after MB destruction due to the drugs released from the blood vessels penetrating the cells, which cannot be observed in a US imaging system. In the present study, BLI signals were 1.7-fold higher with US sonication (3 W/cm2 for 30 s at 10 min postinjection) than without US after injecting luciferin-loaded albuminshelled MBs. Typically, 150 mg/kg luciferin is administered via injection into the peritoneal cavity for BLI (Lim et al. 2009). The US-mediated release of luciferinloaded MBs resulted in only 3 mg/kg luciferin needing to be injected through the lateral tail vein of the mice. The improved delivery efficiency with US-mediated release could reduce the total injection dose of luciferin. Moreover, we were able to evaluate the efficiency of US-mediated local drug delivery with luciferin-loaded albumin-shelled MBs noninvasively in a transgenic mouse model expressing the luciferase reporter gene. The treatment drug could be combined with luciferin for US-mediated MB drug delivery, and the resulting signals observed using BLI. CONCLUSIONS In the present study, the US contrast agent D-luciferin was encapsulated into albumin-shelled MBs that were subsequently destroyed using a therapeutic US system. The number of photons in the US-mediated release of luciferin-loaded MBs increased markedly in both the in vitro and the in vivo BLI images. The luciferin encapsulated within the MBs was released into the tumor vessels and entered the 4T1-luc2 tumor cells in small animals. Moreover, noninvasive evaluation of the efficiency of US-mediated local drug delivery with luciferin-loaded albumin-shelled MBs demonstrated an improved technique, in that the total injection dose of luciferin could be reduced under targeted US sonication conditions. Acknowledgments—The authors thank the S-Sharp Corporation in Taiwan for kindly providing the Prospect US imaging system. This work was supported in part by the National Taiwan University Center for Genomic Medicine and grant nos. NSC 99-2218-E-011-031 and NSC 100-2628-E-011-015-MY3 from the National Science Council of Taiwan.
REFERENCES Abdelmoneim SS, Bernier M, Scott CG, Dhoble A, Ness SA, Hagen ME, Moir S, McCully RB, Pellikka PA, Mulvagh SL. Safety of contrast agent use during stress echocardiography in patients with elevated right ventricular systolic pressure: A cohort study. Circ Cardiovasc Imaging 2010;3:240–248.
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Chen X, Zhang X, Larson CS, Baker MS, Kaufman DB. In vivo bioluminescence imaging of transplanted islets and early detection of graft rejection. Transplantation 2006;81:1421–1427. Chen YC, Jiang LP, Liu NX, Wang ZH, Hong K, Zhang QP. P85, Optison microbubbles and ultrasound cooperate in mediating plasmid DNA transfection in mouse skeletal muscles in vivo. Ultrason Sonochem 2011;18:513–519. Cochran MC, Eisenbrey J, Ouma RO, Soulen M, Wheatley MA. Doxorubicin and paclitaxel loaded microbubbles for ultrasound triggered drug delivery. Int J Pharm 2011;414:161–170. Cohen JL, Cheirif J, Segar DS, Gillam LD, Gottdiener JS, Hausnerova E, Bruns DE. Improved left ventricular endocardial border delineation and opacification with OPTISON (FS069), a new echocardiographic contrast agent. Results of a phase III Multicenter Trial. J Am Coll Cardiol 1998;32:746–752. Contag CH, Ross BD. It’s not just about anatomy: In vivo bioluminescence imaging as an eyepiece into biology. J Magn Reson Imaging 2002;16:378–387. Fokong S, Theek B, Wu Z, Koczera P, Appold L, Jorge S, Resch-Genger U, van Zandvoort M, Storm G, Kiessling F, Lammers T. Image-guided, targeted and triggered drug delivery to tumors using polymer-based microbubbles. J Control Release 2012, in press, Epub ahead of print May 9. http://dx.doi.org/10. 1016/j.jconrel.2012.05.007. Gedanken A. Preparation and properties of proteinaceous microspheres made sonochemically. Chemistry 2009;14:3840–3853. Grinberg O, Hayun M, Sredni B, Gedanken A. Characterization and activity of sonochemically-prepared BSA microspheres containing Taxol–An anticancer drug. Ultrason Sonochem 2007;14:661–666. Hernot S, Klibanov AL. Microbubbles in ultrasound-triggered drug and gene delivery. Adv Drug Deliv Rev 2008;60:1153–1166. Kheirolomoom A, Kruse DE, Qin S, Watson KE, Lai CY, Young LJ, Cardiff RD, Ferrara KW. Enhanced in vivo bioluminescence imaging using liposomal luciferin delivery system. J Control Release 2010;141:128–136. Korpanty G, Grayburn PA, Shohet RV, Brekken RA. Targeting vascular endothelium with avidin microbubbles. Ultrasound Med Biol 2005; 31:1279–1283. Lee KN, Ye Y, Carr JH, Karem K, D’Souza MJ. Formulation, pharmacokinetics and biodistribution of Ofloxacin-loaded albumin microparticles and nanoparticles. J Microencapsul 2011;28:363–369. Lentacker I, Geers B, Demeester J, De Smedt SC, Sanders NN. Design and evaluation of doxorubicin-containing microbubbles for ultrasound-triggered doxorubicin delivery: Cytotoxicity and mechanisms involved. Mol Ther 2010;18:101–108. Liao AH, Cheng YC, Weng CH, Tsai TF, Lin WH, Yeh SH, Yeh WC, Li PC. Characterization of malignant focal liver lesions with contrast-enhanced 40 MHz ultrasound imaging in hepatitis B virus X transgenic mice: A feasibility study. Ultrason Imaging 2008;30: 203–216. Liao AH, Liu HL, Su CH, Hua MY, Yang HW, Weng YT, Hsu PH, Huang SM, Wu SY, Wang HE, Yen TC, Li PC. Paramagnetic perfluorocarbon-filled albumin-(Gd-DTPA) microbubbles for the induction of focused-ultrasound-induced blood-brain barrier opening and concurrent MR and ultrasound imaging. Phys Med Biol 2012;57:2787–2802. Lim E, Modi KD, Kim J. In vivo bioluminescent imaging of mammary tumors using IVIS spectrum. J Vis Exp 2009;26:1210. Lin CY, Liu TM, Chen CY, Huang YL, Huang WK, Sun CK, Chang FH, Lin WL. Quantitative and qualitative investigation into the impact of focused ultrasound with microbubbles on the triggered release of nanoparticles from vasculature in mouse tumors. J Control Release 2010;146:291–298. Liu Y, Miyoshi H, Nakamura M. Encapsulated ultrasound microbubbles: Therapeutic application in drug/gene delivery. J Control Release 2006;114:89–99. Lum AF, Borden MA, Dayton PA, Kruse DE, Simon SI, Ferrara KW. Ultrasound radiation force enables targeted deposition of model drug carriers loaded on microbubbles. J Control Release 2006; 111:128–134.
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Ultrasound in Medicine and Biology
Maruyama K, Suzuki R, Takizawa T, Utoguchi N, Negishi Y. Drug and gene delivery by ‘‘bubble liposomes’’ and ultrasound. Yakugaku Zasshi 2007;127:781–787. McElroy WD, Strehler BL. Bioluminescence. Bacteriol Rev 1954;18: 177–194. Suslick KS, Grinstaff MW, Kolbeck KJ, Wong M. Characterization of sonochemically prepared proteinaceous microspheres. Ultrason Sonochem 1994;1:S65–S68. Tinkov S, Coester C, Serba S, Geis NA, Katus HA, Winter G, Bekeredjian R. New doxorubicin-loaded phospholipid microbubbles for targeted tumor therapy: In vivo characterization. J. Control. Release 2010a;148(3):368–372.
Volume 38, Number 11, 2012 Tinkov S, Winter G, Coester C, Bekeredjian R. New doxorubicinloaded phospholipid microbubbles for targeted tumor therapy: Part I–Formulation development and in-vitro characterization. J. Control. Release 2010b;143:143–150. Wang YH, Liao AH, Chen JH, Wang CR, Li PC. Photoacoustic/ultrasound dual-modality contrast agent and its application to thermotherapy. J Biomed Opt 2012;17:045001. Yang F, Zhang M, He W, Chen P, Cai X, Yang L, Gu N, Wu J. Controlled release of Fe3O4 nanoparticles in encapsulated microbubbles to tumor cells via sonoporation and associated cellular bioeffects. Small 2011;7:902–910. Yuan B. Ultrasound-modulated fluorescence based on a fluorophorequencher-labeled microbubble system. J Biomed Opt 2009;14:024043.