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Published online before print March 29, 2005, 10.1148/radiol.2352040254
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(Radiology 2005;235:541-546.)
© RSNA, 2005

Molecular Imaging

Pulsed High-Intensity Focused Ultrasound Enhances Systemic Administration of Naked DNA in Squamous Cell Carcinoma Model: Initial Experience1

Kristin M. Dittmar, MD, Jianwu Xie, MD, Finie Hunter, BS, Cameron Trimble, BS, Monica Bur, BS, Victor Frenkel, PhD and King C. P. Li, MD, MBA

1 From the Department of Radiology, Warren Grant Magnuson Clinical Center, National Institutes of Health, Bethesda, Md. Received February 9, 2004; revision requested April 20; revision received June 6; accepted July 8. K.M.D. supported by NIH Clinical Research Training Program. F.H., C.T. supported by NIH Pre–Intramural Research Training Award Fellowship Program. Address correspondence to V.F., Department of Diagnostic Radiology, Clinical Center, NIH, 10 Center Dr, Bldg 10, Room 1C657, Bethesda, MD 20892 (e-mail: vfrenkel@cc.nih.gov).


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
PURPOSE: To determine whether exposures to pulsed high-intensity focused ultrasound can enhance local delivery and expression of a reporter gene, administered with systemic injection of naked DNA, in tumors in mice.

MATERIALS AND METHODS: The study was performed according to an approved animal protocol and in compliance with guidelines of the institutional animal care and use committee. Squamous cell carcinoma (SCC7) tumors were induced subcutaneously in both flanks of female C3H mice (n = 3) and allowed to grow to average size of 0.4 cm3. In each mouse, one tumor was exposed to pulsed high-intensity focused ultrasound while a second tumor served as a control. Immediately after ultrasound exposure, a solution containing a cytomegalovirus–green fluorescent protein (GFP) reporter gene construct was injected intravenously via the tail vein. The mouse was sacrificed 24 hours later. Tissue specimens were viewed with fluorescence microscopy to determine the presence of GFP expression, and Western blot analysis was performed, at which signal intensities of expressed GFP were quantitated. A paired Student t test was used to compare mean values in controls with those in treated tumors. Histologic analyses were performed with specific techniques (hematoxylin-eosin staining, terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling) to determine whether tumor cells had been damaged by ultrasound exposure.

RESULTS: GFP expression was present in all sections of tumors that received ultrasound exposure but not in control tumors. Results of signal intensity measurement at Western blot analysis showed expressed GFP to be nine times greater in ultrasound-exposed tumors (160.2 ± 24.5 [standard deviation]) than in controls (17.4 ± 11.8) (P = .004, paired Student t test). Comparison of histologic sections from treated tumors with those from controls revealed no destructive effects from ultrasound exposure.

CONCLUSION: Local exposure to pulsed high-intensity focused ultrasound in tumors can enhance the delivery and expression of systemically injected naked DNA.

© RSNA, 2005


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Gene therapy research has emerged and holds great hopes of translating knowledge about genetics into medical treatments. Clinical trials of potential new treatments for a range of diseases, including cardiovascular disorders, multiple sclerosis, cystic fibrosis, acquired immunodeficiency syndrome, and a variety of cancers, are presently under way. Moreover, genetic vaccination, which involves the administration of a plasmid vector that mediates the expression of a pathogen-derived gene or genes in the vaccinated individual, is considered a promising alternative to traditional antigen-based vaccines (1).

As the number of diseases that may potentially be treated with gene therapy increases, a variety of methods to enhance gene delivery are being investigated. Ultimately, a specific gene therapy protocol will be dependent on the nature of the target disease, as well as on the biology of the target cell population (2). In recent years, there has been a great deal of attention paid to the development of different gene carriers to more effectively deliver DNA (3). In addition to naked DNA, both viral (eg, adenovirus) and nonviral (eg, liposome) vectors have been used (1). DNA may also be incorporated into nanospheres with biopolymers such as chitosan and gelatin (4).

A wide range of methods are currently being investigated for enhancing local DNA delivery. The gene gun has been used successfully to ballistically propel DNA into target tissues such as the skin (5), a liver graft before transplantation (6), and even a beating heart (7). DNA can also be injected into a target tissue such as the skin (8), skeletal muscle (9), or tumors (10). Gene delivery, and subsequent expression, with direct DNA administration may be enhanced by a number of methods. Locally applied in vivo electroporation, for example, was found to significantly enhance gene delivery and expression in the skin (8), muscle (9), and lung (11). The use of infrared lasers has also been demonstrated for enhancing transfection and expression of naked DNA injected directly into muscle (12), as have moderate-field-strength square-wave electric pulses (13).

Delivery of DNA may also be achieved systemically. Uptake and gene expression, however, are often limited because of nuclease degradation in the serum and clearance by mononuclear phagocytes (14). Tail vein injections will result in expression predominantly in the liver (15), whereas local intravascular injections can produce significantly higher expression in the muscle (1,15). Today, gene carriers are being designed to deliver DNA to specific cell types (14). Local gene delivery and expression of systemically injected DNA can also be enhanced by using physical methods such as electroporation (16,17).

Recently, interest has been shown in using high-intensity focused ultrasound to enhance local gene delivery. Ultrasonic waves, because of their very short wavelengths, may be focused in very small and defined tissue volumes. Focusing of ultrasonic waves greatly increases their intensity. Most of the current interest in high-intensity focused ultrasound has been for its use in ablation of living tissue, such as tumors, an application in which relatively long and continuous exposures are used. The focused beam passes through the skin over a wide area, at intensities that cause no damage, while the tissue at the focal point is selectively destroyed. The advantages of using high-intensity focused ultrasound include the elimination of scar formation, the limitation of blood loss and infections, and the reduction of risk for other complications. Shortened recovery time, a significant reduction in cost, and the ability to perform many procedures on an outpatient basis are additional advantages (18).

Unlike the continuous high-intensity focused ultrasound exposures used to ablate tissue, pulsed exposures involve noncontinuous deposition of mechanical energy, rather than the accumulation of thermal energy; duty cycles are short enough to allow for heat dissipation between the pulses (19). In previous studies (20,21), we showed how pulsed high-intensity focused ultrasound exposures can be used to locally enhance the delivery of systemically injected macromolecules to specific targets. Thus, the purpose of this study was to determine whether pulsed high-intensity focused ultrasound exposures can enhance local delivery and expression of a reporter gene, administered with systemic injection of naked DNA, in tumors in mice.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
All animal experiments were performed in compliance with institutional animal care committee guidelines and with the approval of the animal care committee. All experiments were designed and supervised by one of the authors (K.C.P.L.).

Cell Line and Animal Model
Cells from a murine squamous cell carcinoma line (SCC7) were cultured in medium (RPMI 1640; Biofluids, Gaithersburg, Md) containing 10% fetal calf serum, 2 mmol/L glutamine, and a mixture of 50 IU/mL penicillin and 50 µg/mL streptomycin (BioSource International; Rockville, Md) at 37°C with 5% CO2. At pretesting (Research Animal Diagnostic Laboratory, University of Missouri, Columbia, Mo), the cells were mycoplasma-negative.

For the establishment of the squamous cell carcinoma model, female C3H mice (age range, 6–9 weeks) were obtained from a commercial vendor (Charles River Laboratories; Gaithersburg, Md). The animal experiments and all handling of animals were performed according to an approved animal protocol and in compliance with guidelines of the animal care and use committee at the Clinical Center, National Institutes of Health. Prior to the implantation of the tumor cells, the mice were anesthetized with inhalation of isoflurane, and both the right and the left hind flanks of each mouse were shaved. The skin at the implantation site on both flanks was prepared with isopropyl alcohol, and a suspension of SCC7 cells (1 x 106 cells in 100 µL of phosphate-buffered saline) was injected subcutaneously in each flank of each mouse with a 27-gauge needle. The mice (n = 3) then were monitored for tumor growth, and the tumor volume was measured every second day by using calipers. On average, tumors required 7–10 days to grow to a volume of approximately 0.4 cm3 before they underwent treatment with pulsed high-intensity focused ultrasound. Tumor induction was completed by three of the authors (K.M.D., J.X., M.B.).

High-Intensity Focused Ultrasound System
A commercially available high-intensity focused ultrasound system (Sonoblate 500; Focus Surgery, Indianapolis, Ind) was customized for use in this study. The probe comprised a therapeutic 1-MHz transducer and a collinear 10-MHz imaging transducer, both with a focal length of 4 cm. The therapeutic transducer was concave and spherical, with a diameter of 5 cm; the aperture of the imaging transducer was 0.8 cm. A maximum power output of 120 W was available with the therapeutic transducer. Exposures were performed with the mouse placed vertically in a tank of degassed water by using a specially designed holder that was positioned directly opposite the transducer.

The focal zone of the therapeutic transducer was ellipsoid, with a radial diameter of 1.38 mm and an axial length of 7.2 mm. These dimensions were determined with the schlieren measurement technique (22). The radiation force technique was used (in water) to calibrate total acoustic power levels. The overall focusing factor of the therapeutic transducer was approximately 1.3 x 103.

Plasmid Administration and Ultrasound Exposures
A green fluorescent protein (GFP) gene expression vector driven by a cytomegalovirus promoter (Invitrogen, Carlsbad, Calif) was selected as a marker for tracking gene delivery and expression. The endotoxin-free plasmids were amplified and purified at an independent laboratory (GenoQuest; Ijamsville, Md). Three mice with two tumors each (one in each flank) were used in the study.

Pulsed high-intensity focused ultrasound exposures were performed when the tumors reached the size of approximately 0.4 cm3. Individual mice were placed in the holder opposite the transducer, and the imaging transducer was used to localize the tumor that was to undergo exposure. A three-dimensional stage was used to position the mouse so that the tumor was located in the center of the focal zone of the therapeutic transducer. One tumor in each mouse underwent pulsed high-intensity focused ultrasound exposure. Settings for the exposures were as follows: total acoustic power of 20.5 W, pulse frequency of 0.5 Hz, and duty cycle of 4.5% (duration of exposure, 90 msec; interval between exposures, 2000 msec). The raster used for exposure was a grid pattern with a lateral and vertical spacing of 2 mm per site and with each site receiving a 2-minute exposure. Immediately after exposure, each mouse was given an injection of the GFP plasmids (5 µg/g) in the tail vein. These steps were performed by three of the authors (K.M.D., J.X., M.B.).

Animal Sacrifice and Tissue Use
The animals were then returned to the animal care facility. Twenty-four hours after ultrasound exposure and systemic administration of the GFP plasmids, the mice were sacrificed, and both tumors were harvested from each mouse. Each of the six tumors was sliced longitudinally into three approximately equal sections. One slice was used for histopathologic analysis, the second was used for fluorescence microscopy, and the third was used for Western blot analysis.

Standard hematoxylin-eosin staining was performed to evaluate histologic changes that may have occurred as a result of the ultrasound exposures. The tissue samples were fixed with 10% formalin, embedded in paraffin, sectioned, deparaffinized in xylene, rehydrated in graded alcohol, placed on slides, and stained with hematoxylin and eosin. In situ detection of apoptotic cells was performed by using terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay. A separate set of slides not previously stained with hematoxylin and eosin were pretreated with proteinase K before undergoing the TUNEL assay. The TUNEL assay was performed by using a commercially available kit (In Situ Cell Death Detection Kit; Roche Molecular Biochemicals, Indianapolis, Ind) in accordance with the manufacturer’s instructions, and then the slides were counterstained with hematoxylin and eosin. These steps were completed by three of the authors (J.X., F.H., C.T.).

Observation of GFP Expression in Tumor Tissue
Twenty-four hours after pulsed high-intensity focused ultrasound exposures and the injection of GFP plasmids, one-third of each of the tumors (treated and untreated) was sectioned with a cryostat. The samples then were counterstained with a mounting medium containing 4',6-diamidine-2-phenylindole (DAPI) stain (VECTASHIELD; Vector Laboratories, Burlingame, Calif) and were mounted on glass slides. Observations were performed by using a fluorescence microscope (Axioplan 2; Carl Zeiss MicroImaging, Thornwood, NY) with an objective of x10, and representative digital images were captured. These steps were performed by two of the authors (K.M.D., J.X).

Western Immunoblotting and Expression Quantitation
Western blot analysis was performed to quantify the expression of GFP in tissue samples. One-third of each excised tumor was weighed, and the tissue was homogenized in a lysis buffer solution that contained a combination of protease inhibitors (10 mmol/L Tris-HCl with pH of 7.6, 5 mmol/L ethylenediaminetetraacetic acid, 50 mmol/L NaCl, 30 mmol/L sodium pyrophosphate, 50 mmol/L sodium fluoride, 1 mmol/L sodium orthovanadate, 1% Triton X-100 surfactant, 1 mmol/L phenylmethylsulfonyl fluoride, 5 µg/mL aprotinin, 1 µg/mL pepstatin A, and 2 µg/mL leupeptin) and then rotated for 60 minutes at 4°C. The insoluble components of the tumor were pelleted by using a centrifuge at 12 000 rpm for 30 minutes at 4°C. The protein concentration in the clarified tissue lysates was measured with a commercially available kit (Micro BCA Protein Assay Kit; Pierce, Rockford, Ill). Anti-GFP antiserum (Invitrogen) and an {alpha}-actin antibody (Santa Cruz Biotechnology, Santa Cruz, Calif) were used. A total amount of 50 µg whole cell lysate from each tissue sample was analyzed with sodium dodecylsulfate–polyacrylamide gel electrophoresis and with immunoblotting by using polyvinylidene difluoride membranes (Millipore, Bedford, MA) and horseradish peroxidase–conjugated secondary antibodies in conjunction with an enhanced-chemiluminescence substrate mixture (Amersham Biosciences, Piscataway, NJ). Radiographic film was then exposed to the blots, and signal intensities were determined from the resultant film images by using densitometry software (NIH Image; National Institute of Mental Health, National Institutes of Health, Bethesda, Md). These steps were completed by three of the authors (J.X., F.H., C.T.)

Statistical Analysis
The signal intensities at Western blot analysis of control and treated tumors were compared by using a paired Student t test performed with statistical software (JMP; SAS Institute, Cary, NC) by two of the authors (K.C.P.L., V.F.).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of GFP was observed in sections from all three tumors that received ultrasound exposures. Fluorescence, indicating GFP expression, was visible in the SCC7 epithelial cells, being predominantly in regions surrounding blood vessels. Fluorescence was not observed in control (nonexposed) tumors (Fig 1).



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  		Figure 1. Representative histologic sections from, A, control tumor and, B, tumor treated with pulsed high-intensity focused ultrasound, viewed at fluorescence microscopy. Expression of GFP reporter gene (green) is visible in tumor that underwent ultrasound exposure but not in control. DAPI staining (blue) indicates nuclei of tumor cells. Bar = 50 µm. (Original magnification, x100.)

 
Quantitation of signal intensities with the Western blot analysis (Fig 2) showed that, at 24 hours posttreatment, GFP expression levels were significantly greater in tumors that received ultrasound exposures (160.2 au ± 24.5) than in control tumors (17.4 au ± 11.8) (P = .004, n = 3).



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  		Figure 2. Left: Western blot analysis shows expression of GFP gene (top row) against that of {alpha}-actin (bottom row) in tissue samples from control tumors (a, c, e) and treated tumors (b, d, f). Right: Graph of results of densitometric analysis shows significant increase in GFP expression in tumors treated with pulsed high-intensity focused ultrasound (160.2 arbitrary units [au] ± 24.5) compared with that in control tumors (17.4 au ± 11. 8) (paired Student t test, P = .004). Values were normalized to those of the housekeeping gene, {alpha}-actin.

 
Observations at light microscopy in tumor sections excised at 24 hours posttreatment and stained with hematoxylin and eosin indicated that pulsed high-intensity focused ultrasound exposures did not produce any acute destruction of the tissues (Fig 3). There was also an absence of any thermal lesions, and no evidence of hemorrhage or coagulation necrosis was observed. Moreover, none of the cells appeared to be dead; no nuclear pyknosis or cellular debris or dissolution was seen. The results of the TUNEL assay did not show a noticeable increase in the number of apoptotic cells in the treated tumors compared with the control tumors, and this result indicated that the ultrasound exposures did not cause cell death in the tumors.



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  		Figure 3. Comparison of histologic sections, A, C, from control tumors and, B, D, tumors treated with pulsed high-intensity focused ultrasound indicates no difference between the two tumor groups with regard to the absence of hemorrhage or necrosis and, thus, no destructive effects from ultrasound exposures. (A, B, Hematoxylin-eosin stain; C, D, TUNEL assay; original magnification, x100.) Bar = 50 µm.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The results presented here demonstrate that pulsed high-intensity focused ultrasound can be used to locally enhance the delivery and expression of systemically injected plasmid DNA. Ultrasound exposure has been investigated as a means for enhancing gene transfection. By far, the majority of studies have used the process of sonoporation, whereby inertial cavitation produces "small, reparable tears or holes, which permeate a cell membrane and allow the insertion" of a substance into a cell without compromising its viability (23). These studies have primarily been performed in vitro (24), and in most of them, contrast agents, serving as cavitation nucleation agents, were added to improve transfection rates (25–28). Studies, however, have also been performed in vivo, and in these studies, DNA was injected either systemically (29,30) or locally (31), and ultrasound exposures were administered to enhance transfection and expression. As in the in vitro studies, contrast agents were added to locally delivered DNA to improve transfection, with delivery achieved via the carotid artery in rabbits (2) and rats (31), as well as directly in tumors (32). In all these studies, the mechanism of transfection is assumed to be predominantly or perhaps even exclusively sonoporation, since ultrasound exposures followed administration of DNA, and relatively little or no transfection was obtained without the addition of the contrast agents.

Evidence of longer-lasting effects of ultrasound exposures for enhancing gene delivery does, however, exist. Schratzberger et al (33) showed that the expression of ß-galactosidase after injections of naked DNA directly into muscle in rabbits could be increased as much as 20 times by a transcutaneous ultrasound exposure. Gene expression was greater when injections were given prior to exposures, as opposed to after them, and this result indicated the presence of an immediate and very transient sonoporation that enhanced gene transfection. The fact that enhanced expression was obtained in animals exposed to ultrasound (compared with controls not exposed to ultrasound), even when exposures were administered prior to injections, points to a more long-term mechanism than sonoporation, because the permeability-enhancing effects of sonoporation last only seconds (34). This hypothesis is borne out by the results from our study, in which systemic injections of naked DNA were administered after the application of pulsed high-intensity focused ultrasound exposures.

Enhanced gene delivery and subsequent gene expression may be the result solely of improved bioavailability of the DNA in the target tissues, and not necessarily because effects were produced that facilitated its transfection into cells (as is the case with sonoporation). DNA introduced intravascularly can be taken up and expressed in cells of certain tissues, without the presence of additional factors that assist uptake (14,15). In previous work, we showed that pulsed high-intensity focused ultrasound exposures can enhance local delivery of a systemically administered liposome-encapsulated magnetic resonance contrast agent in rabbit muscle (20). We then demonstrated the use of this procedure to locally enhance the delivery of a liposome-encapsulated chemotherapeutic agent in SCC7 tumors in mice (21). In the latter study, we also conducted preliminary investigations into the mechanism of enhancement. At in vivo confocal microscopy, we observed the extravasation of a nonpermeating fluorophore (fluorescein isothiocyanate–dextran) in regions of tumors that received pulsed high-intensity focused ultrasound exposures.

Hyperthermia and cavitation have been the most widely described ultrasound-related mechanisms for producing effects in biologic tissues, including effects that contribute to enhanced bioavailability of an administered substance (35). Sections of tissue exposed to pulsed high-intensity focused ultrasound and viewed with light microscopy did not show any of the destructive effects that are often associated with exposures in which significant heat transfer or cavitation occurs. Ultimately, ultrastructural investigations (eg, with electron microscopy) will have to be performed to confirm this. Additional credence, however, is provided for the absence of these destructive effects by the very fact that the exposed cells expressed the DNA that was administered. The authors believe that enhanced delivery resulting from pulsed high-intensity focused ultrasound exposures was more likely the result of radiation forces occurring from transient energy absorption, which can produce displacements in the tissues on the order of whole cells (19,36). In focal-zone boundary regions between displaced and nondisplaced tissue, widening of intercellular spaces could produce enhanced diffusion. Ultrasound exposures have been shown to induce gaps between endothelial cells (37,38) and widen epithelial intercellular spaces (39). The nondestructive opening of intercellular spaces was shown to increase diffusion of nanoparticles through the epithelium (40).

Although direct evidence is not presently available of how enhanced transfection may occur as a result of pulsed high-intensity focused ultrasound exposures, the possibility of this occurrence cannot be discounted out of hand. Stress induced by ultrasound exposure is known to affect the state of cell membranes in ways that may alter their permeability (41). The literature is replete with accounts of the successful application of nondestructive (and noncavitational) physical stimuli to increase cell membrane permeability to a variety of substances. Stretching myotubes increased cell membrane permeability to fluorescein isothiocyanate–dextran (42), and fluid shear stress increased the permeability of endothelial cell membranes to merocyanine 540 (43) and bovine serum albumin (44). In these studies, however, increased membrane permeability occurred only in the presence of the stimuli. Long-term effects were produced in mouse breast cancer cell membranes by laser-induced stress waves that increased membrane permeability to the fluorophore calcein-acetomethylester, effects that were observed until 80 seconds after cessation of induction (45). Effects detected in bovine aortic endothelial cells exposed to physiologic levels of fluid shear stress lasted considerably longer: Permeability to fluorescein isothiocyanate–conjugated bovine serum albumin increased 10-fold (compared to no shear) and returned to pre-shear values only after 60 minutes (46).

The results presented here are from a pilot study, of which the specific objective was to determine whether pulsed high-intensity focused ultrasound exposures, which have been used for enhanced targeted delivery of other compounds, could be used in a similar manner for gene delivery. We acknowledge that small sample sizes were used, as required by our animal care and use committee. These results are preliminary, and plans are under way to further evaluate this procedure by using different genes and tumor types, as well as by optimizing the pulsed high-intensity focused ultrasound exposures for gene delivery.

Practical applications: In this study, we have shown how a local exposure of pulsed high-intensity focused ultrasound can enhance the delivery and expression of systemically injected plasmid DNA. Potential applications for such a procedure vary widely and continually increase with advancements in gene therapy research. These applications include possible treatments of pathologic conditions such as ischemia (through the delivery of genes for angiogenic factors) and cancer (through increased levels of tumor necrosis factor {alpha}). Researchers are avidly pursuing the latter application as a way to improve the eradication of tumors.


   ACKNOWLEDGMENTS
 
The authors gratefully acknowledge Deloris E. Koziol, PhD, for assistance with the statistical analysis.


   FOOTNOTES
 
Abbreviations: DAPI = 4',6-diamidine-2-phenylindole, GFP = green fluorescent protein, TUNEL = terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling

Authors stated no financial relationship to disclose.

Author contributions: Guarantors of integrity of entire study, K.M.D., K.C.P.L.; study concepts and design, K.M.D., J.X., K.C.P.L.; literature research, K.M.D., F.H., C.T., V.F.; experimental studies, K.M.D., J.X., F.H., C.T., M.B.; data acquisition, K.M.D., J.X.; data analysis/interpretation, K.M.D., J.X., V.F., K.C.P.L.; statistical analysis, V.F., K.C.P.L.; manuscript preparation, K.M.D., J.X., V.F.; manuscript definition of intellectual content, K.M.D.; manuscript editing, V.F., K.C.P.L.; manuscript revision/review and final version approval, K.M.D., F.H., C.T., M.B., V.F., K.C.P.L.


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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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