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Published online before print September 25, 2003, 10.1148/radiol.2292020500
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(Radiology 2003;229:423-428.)
© RSNA, 2003

Experimental Studies

Gene Transfer with Echo-enhanced Contrast Agents: Comparison between Albunex, Optison, and Levovist in Mice—Initial Results1

Tieli Li, MD, PhD, Katsuro Tachibana, MD, PhD, Motomu Kuroki, PhD and Masahide Kuroki, MD, PhD

1 From the Molecular Oncology Center (T.L., Motumu Kuroki, Masahide Kuroki) and Department of Anatomy (K.T.), Fukuoka University School of Medicine, Nanakuma 7–45-1, Jonan-ku, Fukuoka 814-0180, Japan. Received April 29, 2002; revision requested July 10; final revision received January 31, 2003; accepted March 10. Supported in part by the Central Research Institute of Fukuoka University and Grant-in-Aid for High-Technology Research and Scientific Research [C] from the Japanese Ministry of Education, Science, Sports and Culture. Address correspondence to K.T.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
 REFERENCES
 
PURPOSE: To determine if commercially available echo-enhanced microbubble contrast agents could be used to increase gene transfection efficiency by means of relatively low-intensity ultrasound-mediated microbubble destruction in skeletal muscles.

MATERIALS AND METHODS: Three types of ultrasound microbubble contrast agents (0.01 mL of albumin [Albunex] and human albumin [Optison] and 10 mg/mL of SH U 508A [Levovist]) were each separately mixed with the reporter plasmid DNA (25 µg) encoding green fluorescent protein (GFP) prior to intramuscular injection into the quadriceps muscle of a mouse thigh bilaterally (seven mice per contrast agent). One of the muscle sites that was injected with plasmid DNA was irradiated with low-intensity therapeutic ultrasound (1 MHz) at an intensity of 2.0 W/cm2 for 2 minutes. Mice were sacrificed 7 days after ultrasound treatment for gene expression assay. The number of GFP-expressing muscle fibers was counted. Statistical significance was determined with a two-tailed Student t test. P < .05 was considered to indicate statistically significant difference.

RESULTS: Muscle tissue exposed to ultrasound with air-filled Albunex or Levovist microbubbles revealed no difference in the number of GFP-expressing muscle fibers compared with the control non–ultrasound-exposed muscle. Albumin-coated octafluoropropane gas-filled Optison microbubbles showed a 10-fold increase in the number of GFP-expressing fibers (P < .05).

CONCLUSION: Low-intensity ultrasound with echo-enhanced Optison induced efficient gene transfer unlike that with Albunex or Levovist.

© RSNA, 2003

Index terms: Animals • Experimental study • Genes and genetics • Microbubbles • Ultrasound (US), contrast media


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
 REFERENCES
 
Arterial occlusive diseases cause serious ischemic conditions in various areas, such as the heart, brain, and legs. Therapeutic angiogenesis is believed to be beneficial for such conditions. Intramuscular injection of naked plasmid DNA encoding angiogenic growth factors offers a promising new approach for such purposes; however, only a small amount will pass through the cell membrane, which leads to low gene transfer efficiency. Findings of recent studies have shown that therapeutic ultrasound can induce or increase cell membrane permeabilization of various agents, including genes. It is currently suggested that the mechanism of this phenomenon is closely related to acoustic cavitation. High ultrasound intensities are required to create cavitation within tissues such as the skeletal muscles and myocardium. Ultrasound alone can lead to temperature increase and mechanical damage to the tissue itself. Thus, the purpose of our study was to determine if commercially available echo-enhanced microbubble contrast agents could be used to increase gene transfection efficiency by means of relatively low-intensity ultrasound-mediated microbubble destruction in skeletal muscles.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
 REFERENCES
 
Plasmid Preparations
A commercial reporter plasmid pQBI25 (Takara Biomedicals, Otsu, Japan) encoding green fluorescent protein (GFP) and rhodamine-labeled plasmid (pGeneGrip; GTS, San Diego, Calif) with human cytomegalovirus immediate early promoter and/or enhancer driving the GFP gene were used for insertion and subsequent transient expression within the cells. The plasmid DNA obtained from Escherichia coli DH5 cultures was prepared with a kit (Qiagen Maxi; Qiagen, Chatsworth, Calif) according to the company protocol. Agarose gel electrophoresis was performed before and after restriction endonucleases digestion to verify the identity and purity of the plasmid DNA.

In Vitro Studies
Chinese hamster ovary cells were maintained in alpha minimal essential medium (Life Technologies, Gaithersburg, Md) that was supplemented with 10% heat-inactivated fetal bovine serum (Bio-Whittaker, Walkersville, Md), 2 mM glutamine, 100 units/mL penicillin, and 100 µg/mL streptomycin. The cell cultures were maintained at 37°C in a humidified atmosphere of 5% CO2 in air. Exponentially growing cells were used in all experiments. Cell viability was determined by means of exclusion with trypan blue dye.

Echo-enhanced Contrast Agents
Culture cells were harvested and washed once in phosphate-buffered saline and resuspended at 2 x 106 cells/mL of plain phosphate-buffered saline per well of a 48-well plate (Corning, New York, NY). Three types of ultrasound microbubble contrast agents were evaluated: Albunex (Molecular Biosystems, San Diego, Calif, distributed by Mallinckrodt, St Louis, Mo), an albumin-shelled ultrasound contrast agent composed of air-filled microbubbles with a median diameter of 3–4 µm; Optison (human albumin; Nycomed-Amersham, Oslo, Norway), a second-generation perfluorocarbon-filled contrast agent with similar microbubble diameter and concentration (5–8 x108 bubbles per milliliter); and Levovist (SH U 508A; Schering, Berlin, Germany), a galactose-based air-filled microbubble contrast agent, 99% of which is smaller than 7 µm. Each contrast agent was added to the cell medium and diluted to various concentrations (Levovist: 10, 20, and 40 mg/mL; Albunex and Optison: 5%, 10%, and 20%). Plasmid-encoding GFPs were then added immediately to the cell supernatant to a final DNA concentration of 20 µg/mL at a volume of 1 mL.

Cell Ultrasound Exposure
The ultrasound probe and well plate were firmly fixed to a stand to avoid dislocation during ultrasound administration. Immediately after addition of plasmid DNA and microbubbles into the well, the cells were exposed with 1.0-MHz ultrasound (Sonitron 1000; Rich Mar, Inola, Okla) for 20 seconds at an intensity of 0.5 or 1.0 W/cm2 and duty cycle of 20%. The ultrasound probe was inserted directly into the cell suspension (Figs 1, 2). A miniature stirrer was placed within the well and centrifuged at a speed of 300 rpm by means of a magnetic rotator placed under the container (PC-220; Corning, Boston, Mass). The cells were placed at least a row apart from each other to prevent overlap or interaction by transmission of ultrasound along the plastic well container. All tests were conducted in triplets and repeated five times on different days by the author (T.L.). Immediately after exposure, the cell viability was tested by counting the cells stained with trypan blue. To verify that ultrasound exposure did not produce thermal effects, the temperature in the sample was measured before and immediately after ultrasound exposure with a needle-type digital thermometer (PTC-201; Unique Medical, Tokyo, Japan). The increase in temperature within the samples was found to be less than 1°C. Thus, any ultrasound bioeffects observed in this study were considered to be nonthermal.



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  		Figure 1. Diagram of the experimental set up for in vitro ultrasound irradiation to the cells.

 


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  		Figure 2. Ultrasound generator and probe.

 
Quantification of GFP Expression
After the cell viability analysis, the cell suspensions were harvested from the wells, separated by centrifugation, and resuspended in alpha minimal essential medium. Viable cells (2 x 105) were plated onto 6-cm dishes. After 48-hour culture, the GFP-positive cells were detected with a digital camera (FUJIX HC-300; Fujifilm, Tokyo, Japan), image processor (ARGUS-20; Hamamatsu Photonics K.K., Hamamatsu, Japan), and a chilled charge-coupled device camera (Hamamatsu Photonics K.K.) mounted on a fluorescence microscope (Eclipse E 600; Nikon, Tokyo, Japan) equipped with plain apochromat lenses and a fluorescein isothiocyanate-HVQ filter (EX, 450–490 nm; DM, 505 nm; BA, 520 nm). The images were printed on hard copy (FUJIX Pictrography 3000; Fujifilm).

In Vivo Studies
Six-week-old female BALB/C mice (Charles River Breeding Laboratories, Osaka, Japan) were anesthetized with a interperitoneal injection (T.L.) of 60 mg per kilogram of body weight sodium pentobarbital (Nembutal; Abbott Laboratories, North Chicago, Ill). Approval of the Institutional Animal Care and Use Committee was obtained prior to initiation of these studies.

Immediately prior to intramuscular injection, three types of ultrasound microbubble contrast agents (0.01 mL of Albunex and Optison and 10 mg/mL of Levovist) were each separately mixed with the commercial reporter plasmid DNA (25 µg; pGeneGrip) encoding GFP.

Mice Ultrasound Exposure
After the thigh quadriceps muscles of seven mice were surgically exposed bilaterally, the plasmid-ultrasound contrast agent was carefully injected into the muscles with a 27-gauge needle (Terumo, Atsugi, Japan), with direct visualization by two of the authors (T.L., K.T.). An acoustically transparent gel (Aqusonic; Parker Laboratories, Orange, NJ) was placed between the ultrasound probe tip and the muscle surface (Fig 3). Therapeutic ultrasound (1 MHz) was irradiated directly from the surface of the muscle at an intensity of 2.0 W/cm2 (duty cycle of 50%) for 2 minutes. The alternative leg was unexposed to ultrasound. The procedure was repeated for each contrast agent. The control mice (n = 7) were exposed to ultrasound in exactly the same way, except that the plasmid contained saline instead of the ultrasound contrast agents. The temperature increase at the surface of the ultrasound exposed to the lesion was monitored with a digital thermister probe. The temperature difference before and immediately after ultrasound treatment was less than 2°C in all cases.



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  		Figure 3. Schematic of the procedure of ultrasound exposure to the legs of mice. Ultrasound was given at the site where the plasmid DNA was intramuscularly injected. The ultrasound probe was in direct contact with the tissue.

 
Histologic Analysis
Mice were sacrificed for gene expression assay 7 days after ultrasound treatment with an overdose of pentobarbital sodium (0.2 mL/kg). The muscle specimens were immediately excised and snap frozen. Cross sections (7-µm-thick slices) were cut with a cryostat and affixed to glass slides. Fluorescence distribution patterns were observed with confocal laser microscopy (LSM 410UV; Zeiss, Jena, Germany). A dual-wavelength laser (480 and 530 nm) and digital image subtraction method were devised to eliminate autofluorescence of normal muscle tissue. The total number of GFP-positive muscles fibers within the entire cross section was counted by one observer (K.T.) who had no knowledge of the treatment performed on the samples. Hematoxylin-eosin staining was performed on separate tissue specimens for histopathologic evaluation of hemorrhage, necrosis, and inflammation.

Statistical Analysis
The statistical significance of the data was determined with a Student t test. P < .05 was considered to indicate a statistically significant difference. Differences between groups were assessed with a two-way repeated-measures analysis of variance (ANOVA). All statistics were computed by using a statistical package (Statview; Abacus Concepts, Berkeley, Calif).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
 REFERENCES
 
In Vitro Experiments
Preliminary measurement of cell viability after ultrasound administration alone at various intensities (0.5, 1.0 W/cm2) and duration (10, 20, 40 seconds) was performed. Ultrasound alone had minimal cell-killing effects at the conditions examined (Table 1), whereas addition of echo-enhanced contrast agents resulted in a significant reduction of cell viability. Addition of 10 mg/mL Levovist resulted in a cell viability reduction at baseline from 90.6% ± 2.2 (SD) to 60.7% ± 5.3 (P < .01, ANOVA). Similar percentages of cell viability were observed with the addition of Levovist concentrations of 20 and 40 mg/mL (Fig 4). Addition of Albunex and Optison also induced significant cell killing compared with that after ultrasound administration alone (Fig 5). Cell viability with Optison was significantly different from that with Albunex at the same 5% microbubble concentration (73.1 ± 2.7 vs 33.3 ± 1.0; P < .01, ANOVA). The cell-killing rate with Optison remained at approximately the same level at bubble concentrations of 10% and 20%. On the other hand, cell viability tended to decrease at higher bubble concentrations (62.0% ± 4.3 vs 54.8% ± 1.4) with use of Albunex. On the basis of these results, the concentration of each echo-enhanced contrast agent was adjusted to induce similar cell-killing rates at identical ultrasound administration conditions in the following gene transfection experiments. Optison was diluted to a concentration of 2%, whereas Albunex was adjusted to that of 10%. Levovist microbubble concentration was adjusted to 10 mg/mL. Cell viability under these conditions after ultrasound administration was approximately 60% for each of the agents tested (Fig 6a). However, the mean number of GFP-transfected cells in the presence of Optison was approximately eightfold greater than that in groups that contained Albunex or Levovist (Fig 6b).


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  		Cell Viability after Ultrasound Irradiation without Echo-enhanced Contrast Agents

 


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  		Figure 4. Graph depicts cell viability after ultrasound irradiation with Levovist at various concentrations. Error bars represent the SD of the mean (n = 5). Ultrasound was given for 20 seconds at an intensity of 0.5 W/cm2 (20% duty cycle).

 


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  		Figure 5. Graph depicts cell viability after ultrasound administration in the presence of Albunex (white bars) and Optison (black bars). Values are expressed as mean ± SD (n = 5). Ultrasound was given for 20 seconds at an intensity of 0.5 W/cm2 (20% duty cycle).

 


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  		Figure 6a. (a) Bar graph shows cell viability (mean ± SD, n = 5) after 1 MHz, 20% duty cycle, and 20-second duration. Concentrations of each echo-enhanced contrast agent were 10 mg/mL of Levovist, 10% of Albunex, and 2% of Optison. (b) Bar graph shows the percentage of GFP-positive cells at identical conditions. * = significant difference from the other echo-enhanced contrast agents tested (P < .05, ANOVA).

 


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  		Figure 6b. (a) Bar graph shows cell viability (mean ± SD, n = 5) after 1 MHz, 20% duty cycle, and 20-second duration. Concentrations of each echo-enhanced contrast agent were 10 mg/mL of Levovist, 10% of Albunex, and 2% of Optison. (b) Bar graph shows the percentage of GFP-positive cells at identical conditions. * = significant difference from the other echo-enhanced contrast agents tested (P < .05, ANOVA).

 
In Vivo Experiments
Muscle tissue exposed to ultrasound with air-filled Albunex and Levovist microbubbles revealed no significant difference (n = 7, two-tailed Student t test) in the number of GFP-expressing muscle fibers compared with the nonexposed muscles (Albunex and DNA vs Albunex, DNA, and US; Levovist and DNA vs Levovist, DNA, and US). In contrast, albumin-coated octafluoropropane gas–filled Optison microbubbles exposed to ultrasound showed a 10-fold increase in the number of GFP-expressing fibers (Figs 7, 8). Control group comparison between DNA injection alone versus DNA and ultrasound resulted in no significant difference in the numbers of GFP-expressing muscle fibers (n = 7, two-tailed Student t test). No significant differences in the distribution of rhodamine-labeled DNA were observed in all intramuscularly injected groups. Tissue damage was not observed with naked eye at the surface of the muscles where ultrasound was irradiated. Histologic examination with hematoxylin-eosin staining of the tissue specimen of ultrasound-irradiated muscle revealed no evidence of inflammation or necrosis.



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  		Figure 7. Bar graph shows the number of GFP-positive skeletal muscle fibers within the cross-sectional view of the muscles in the presence of each echo-enhanced contrast agent. Error bars represent the SD of the mean (n = 7). * = significant difference (P < .05, two-tailed Student t test. NS = not significant, (-) and (+) indicate absence and presence, respectively, of ultrasound

 


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  		Figure 8. Confocal laser microscopic image of GFP in muscle tissue (cross-sectional view) treated with plasmid DNA and ultrasound irradiation in the presence of Optison depicts rhodamine-labeled DNA (arrows).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
 
It is well known that microbodies or microglobules of air or a gas (eg, microspheres such as microbubbles suspended in a liquid) are exceptionally efficient ultrasound reflectors for echography. There has been a considerable degree of excitement in diagnostic US imaging with the introduction of these echo-enhanced contrast agents. Second-generation microbubbles contain perfluorocarbon gas rather than air, which results in an even longer life span of contrast agents within the circulatory system. This permits a longer time for the echographers to observe patients. Recently, findings of various in vitro and in vivo experiments have demonstrated that echo-enhanced microbubble contrast agents can be intentionally ruptured with use of diagnostic and therapeutic ultrasound. This acoustically induced destruction and collapse of the microbubbles produces a high-amplitude response. Violent microstreaming can be produced during microbubble collapse. Researchers have hypothesized that these microjets or microstreaming could be applied to promote diffusion of drugs into various tissues and lesions (1–3). Scanning electron microscopy has actually demonstrated micropores and crater-like disruption on the cell surface caused by ultrasound (4). Researchers have recently termed this phenomenon sonoporation (5,6).

In the present study, the commercially available echo-enhanced contrast agents Optison, Albunex, and Levovist were evaluated in vitro and in vivo to see if there are differences in the induction of gene transfer. Perfluorocarbon-filled second-generation Optison proved to be more effective compared with the other agents in increasing the rate of gene transfection.

It has been previously reported that certain microbubbles significantly increase drug uptake into cells and gene transfection (7–22). Although the exact mechanisms that cause this phenomenon are still unclear, the rapid collapse of microbubbles is thought to play a major role. It is also speculated that the presence of microbubbles can significantly reduce the acoustic pressure amplitude threshold for cavitation. Miller et al (22) have supported this hypothesis with the use of a 20-MHz passive cavitation detector system. Greenleaf et al (9) demonstrated an increase in the transfection rate of DNA in vitro in the presence of albumin microbubbles, Albunex. Unger et al (5) described similar results with microbubble liposomes. Guzman et al (23) succeeded in quantification of molecular uptake of drugs in DU145 prostate cancer and aortic smooth muscle cells. High acoustic pressure resulted in greater uptake of drugs per cell in the presence of Optison. Furthermore, several groups have recently performed a comparison between different types of microbubbles such as Albunex and Optison. It was reported by Miller et al (22) that Optison had greater sonolytic potential than Albunex in destruction of human erythrocytes in vitro. Others have similarly shown more lysis of cervical cancer cells (HeLaS3) in the presence of Optison rather than of Albunex (24). Although drug uptake per cell generally increased with increasing bubble concentration and acoustic pressure, it has been pointed out that significant cell killing does occur, thus altering the absolute number of living cells that actually receive therapeutic dosages of drugs. In addition, detailed measurement of the amount of calcein fluorescence within the cells after ultrasound administration has demonstrated heterogeneous drug uptake among the cells, which would be classified as low-, nominal-, and high-uptake cell population. Other researchers have suggested bubble-cell spacing as an important factor in accurate prediction of sonoporation (25).

From the aforementioned information, it has been generally believed that lethal sonoporation and reparable sonoporation, which results in drug and/or gene uptake, can be theoretically discussed in a single dimension. That is, lethal sonoporation is merely an extension of the severity of damage induced when microbubbles burst at various distances from the cell surface, and cells with drug uptake are at a prelethal stage, or reparable sonoporation. From a gene therapy point of view, a high gene transfection rate is often required to produce sufficient therapeutic outcome. However, too great an effect by microbubble and ultrasound to achieve this objective will cause cell death. There are circumstances in which genes are required to be injected into the cells with minimal cell damage. Furthermore, the ideal microbubbles would be those that deliver drugs and/or genes to cells with maximum efficiency but without lethal effects. Researchers have previously questioned if such bubbles would ever exist or be produced. To our surprise, the present in vitro study demonstrated that Optison was by far the most effective enhancer for gene transfer, despite that Albunex and Levovist induced almost identical degree of mechanical cell death. This suggests that cell killing and gene delivery by microbubble collapse do not necessarily respond in a side-by-side effect. It is suggested that certain bubbles are more appropriate or efficient than others to achieve the delivery of drugs and/or genes into cells. The in vivo experiments in this study resulted in marked increase of gene transfection with Optison, whereas Albunex and Levovist showed no difference compared with the controls. These results were consistent with the in vitro data; however, no damages were detected with histologic evaluation in skeletal muscles. It is postulated that suspended naked cells in vitro are less tolerable to mechanical stress in the event of microbubble collapse and ultrasound. From this aspect, low-intensity ultrasound used in the present study might not be a major safety problem in the clinical situation.

Results of this study demonstrated that low-intensity ultrasound increased the transfection of naked plasmid DNA with Optison microbubbles but not with air-filled Albunex or Levovist. It is believed that microbubbles, upon rupture, create a local increase in membrane fluidity, thereby enhancing cellular uptake of the therapeutic compound. Furthermore, because Optison has a longer life span than does a bubble, gene transfection may be attributed to repeated or slower bubble destruction during ultrasound exposure, which results in a greater number of cell membrane poration.

The major limitations of this study were as follows: (a) only commercially available microbubbles were studied, (b) a limited number of ultrasound frequencies and intensities were evaluated, and (c) the plasmid DNA was limited to marker genes in Chinese hamster ovary cell lines and mice skeletal muscles. Next-generation echo-enhanced contrast agents could be developed as a carrier of genes to targeted locations, and pure plasmid DNA can be attached either to the outside or inside of the microbubble capsule wall (26). Bubbles can be collapsed by using an extracorporeal or intravascular ultrasound catheter (27), which permits the DNA to penetrate directly into the tissue and cells. Nevertheless, more experiments are anticipated to determine the optimal echo-enhanced contrast agent and ultrasound conditions for drug and/or gene delivery.

Practical applications:: Recently, a therapeutic strategy in which angiogenic growth factors were used to expedite or augment collateral artery development has entered the realm of treatment of ischemic diseases. The clinical utility of gene therapy with use of the vascular endothelial growth factor gene has been reported for the treatment of limb ischemia and myocardial ischemia (28–31). Most clinical trials have used intramuscular injection of naked plasmid DNA for therapeutic angiogenesis. However, the low transfection efficiency still remains a major obstacle to adequate angiogenesis without side effects. In clinical trials, researchers have used adenoviral gene transfer instead of naked plasmid DNA. This method, however, has potential toxicity of adenovirus, such as strong immunogenicity. Therefore, it is critical to develop a safe gene transfer method with high efficiency. From this viewpoint, we focused on enhanced plasmid DNA-based gene transfer by means of echo-enhanced contrast agents and therapeutic ultrasound. Given the high transfection efficiency of gene transfection with use of Optison in this study, this modality has potential as a nonviral, safe, and efficient therapeutic strategy for angiogenesis or treatment of various diseases in the future.


   FOOTNOTES
 
Abbreviations: ANOVA = analysis of variance, GFP = green fluorescent protein

Author contributions: Guarantors of integrity of entire study, all authors; study concepts, all authors; study design, T.L., K.T., Motomu Kuroki; literature research, K.T.; experimental studies, T.L., K.T.; data acquisition, T.L., K.T.; data analysis/interpretation, T.L., K.T., Motomu Kuroki; statistical analysis, T.L.; manuscript preparation and editing, T.L., K.T.; manuscript definition of intellectual content, revision/review, and final version approval, all authors

See also Science to Practice in this issue.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
 REFERENCES
 

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