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DOI: 10.1148/radiol.2342030889
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(Radiology 2005;234:431-437.)
© RSNA, 2005

Experimental Studies

Delivery of Systemic Chemotherapeutic Agent to Tumors by Using Focused Ultrasound: Study in a Murine Model1

Esther L. Yuh, MD, PhD, Suzanne G. Shulman, MD, Shilpa A. Mehta, Jianwu Xie, MD, Lili Chen, PhD, Victor Frenkel, PhD, Mark D. Bednarski, MD, PhD and King C. P. Li, MD, MBA

1 From the Lucas Magnetic Resonance Imaging and Sprectroscopy Research Center, Department of Radiology, Stanford University School of Medicine, Stanford, Calif (E.L.Y., L.C., M.D.B.); and Department of Radiology, Warren Grant Magnuson Clinical Center, National Institutes of Health, 10 Center Dr, Bldg 10, Room 1C657, Bethesda, MD 20892 (S.G.S., S.A.M., J.X., V.F., M.D.B., K.C.P.L.). Received June 5, 2003; revision requested August 18; final revision received May 3, 2004; accepted June 17. Supported in part by the Lucas Foundation, the Phil Allen Trust, and the National Institutes of Health. E.L.Y. supported by the National Cancer Institute, Lucas Center for Magnetic Resonance Imaging and Spectroscopy Research Center, Cancer Imaging Fellowship, and Stanford Medical Student Scholars Program. S.G.S. supported by the National Institutes of Health Intramural Research Training Award. Address correspondence to V.F. (e-mail: vfrenkel@cc.nih.gov).


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
PURPOSE: To quantitatively determine the delivery of systemic liposomal doxorubicin to tumors treated with pulsed high-intensity focused ultrasound and to study the mechanism underlying this delivery in a murine model.

MATERIALS AND METHODS: All animal work was performed in compliance with guidelines and approval of institutional animal care committee. C3H mice received subcutaneous injections in the flank of a cell suspension of SCC7, a murine squamous cell carcinoma cell line; mice (n = 32) in drug delivery study received unilateral injections, whereas mice (n = 10) in mechanistic study received bilateral injections. Tumors were treated when they reached 1 cm3 in volume. In the drug delivery study, doxorubicin hydrochloride liposomes were injected into the tail vein: Mice received therapy with doxorubicin injections and high-intensity focused ultrasound, doxorubicin injections alone, or neither form of therapy (controls). Tumors were removed, and the doxorubicin content was assayed with fluorescent spectrophotometry. In the mechanistic study, all mice received an injection of 500-kDa dextran–fluorescein isothyocyanate into the tail vein, and half of them were exposed to high-intensity focused ultrasound prior to injection. Contralateral tumors served as controls for each group. Extravasation of dextran–fluorescein isothyocyanate was observed by using in vivo confocal microscopy.

RESULTS: Mean doxorubicin concentration in tumors treated with pulsed high-intensity focused ultrasound was 9.4 µg · g–1 ± 2.1 (standard deviation), and it was significantly higher (124% [9.4 µg · g–1/4.2 µg · g–1]) than in those that were not treated with high-intensity focused ultrasound (4.2 µg · g–1 ± 0.95) (P < .001, unpaired two-tailed Student t test). Extravasation of dextran–fluorescein isothyocyanate was observed in the vasculature of tumors treated with high-intensity focused ultrasound but not in that of untreated tumors.

CONCLUSION: Pulsed high-intensity focused ultrasound is an effective method of targeting systemic drug delivery to tumor tissue. Potential mechanisms for producing the observed enhancement are discussed.

© RSNA, 2005


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Achievement of uniform drug delivery of chemotherapeutic agents throughout solid tumors is limited by the anomalous vascularization and blood vessel permeability that have been demonstrated to exist in many solid tumors (1–7). In addition, high interstitial pressure at the center of a tumor can prevent diffusion of drug molecules through the interstitium, once they have moved from the intravascular space into the parenchyma (8,9). These impediments to drug delivery create spatial gradients in drug concentrations such that chemotherapeutic agents at clinically acceptable doses often fail to eliminate all cancer cells in a tumor.

Researchers in the past decade have demonstrated that liposomal drugs can increase delivery of the free drug while they decrease systemic toxicity. With incorporation of the drug inside a lipid membrane, liposomal drug formulations allow most of the drug to remain within the vascular system and thus prevent toxic effects (ie, neutropenia, cardiotoxic effects) to other organs. In addition, liposomal drugs release their contents slowly, thereby reducing high peak concentrations that also may cause toxic effects (10). Furthermore, some of the newer liposomal drug formulations have a "stealth" coating (10–13), which prevents uptake of the liposome by the immune system (reticuloendothelial system) and thereby increases the bioavailability of the drug.

Advances have been made in the use of physical methods to enhance drug delivery to solid tumors. One such method is hyperthermia, which can enhance the transportability of drugs from the vasculature to the interstitium in solid tumors by using both nanoparticles and liposomes. In addition, hyperthermia has been used successfully to ameliorate cancer gene therapy (14–16). Hyperthermia, however, also has been found to be directly cytotoxic and must be maintained within a narrow temperature range in order to be effective (17). Another drawback of hyperthermia is that it is accurate only to within centimeters, compared with, for example, high-intensity focused ultrasound, which can be delivered accurately within millimeters (18).

High-intensity focused ultrasound is another clinical method for treatment of cancers. High-intensity focused ultrasound has been clinically used in thermal ablation of prostate cancer in Europe (19–21). This procedure has also been used in clinical trials for treatment of lesions of other organs, such as the kidney and the uterus (22,23). High-intensity focused ultrasound also is used in the research arena at substantially lower energy levels to demonstrate in vitro the ability to release lipid-encapsulated chemotherapeutic drugs or genetic therapies into tumors (24–30). Enhancement of drug delivery to tumors with this method has been demonstrated in vivo in a rat model (31).

High-intensity focused ultrasound, unlike other physical techniques (eg, hyperthermia), can be focused deep into soft tissue. High-intensity focused ultrasound also can be easily combined with other imaging modalities, such as ultrasonography (US) or magnetic resonance (MR) imaging, for more precise localization of the treatment area (32). The purpose of this study was to quantitatively determine the enhancement effects of pulsed high-intensity focused ultrasound exposures on the delivery of systemic liposomal doxorubicin to tumors, as well as to perform a preliminary investigation into how this enhancement occurs.


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

Particle Size Determination
The size of all particles and materials used in the study was measured by using dynamic light scattering with a nanoparticle size analyzer (90 Plus; Brookhaven Instruments, Holtsville, NY): doxorubicin hydrochloride liposome injection (Doxil; Alza, Mountain View, Calif) for the drug delivery study and dextran– fluorescein isothyocyanate (Molecular Probes, Eugene, Ore) for the mechanistic study. This measurement was performed for both studies by one of the authors (M.D.B.).

Experimental Animals, Cell Cultures, and Tumor Implantation
For the drug delivery study, 32 C3H/Km mice were used; for the mechanistic study, 10 C3H/MTV mice were used. At the time of tumor implantation, the mice were 10–12 weeks old for the drug delivery study and 6–12 weeks old for the mechanistic study.

For both studies, SCC7 cells (a murine squamous cell carcinoma line) were grown in RPMI 1640 medium and 25 mmol/L of HEPES, supplemented with 10% fetal bovine serum, 200 mmol/L of 1% glutamine, and 1% pen/strep 100x (BioSource International; Rockville, Md) at 37°C. In the drug delivery study, this procedure was performed by two of the authors (E.L.Y., L.C.). In the mechanistic study, this procedure was performed by two other authors (S.A.M., J.X.).

For the drug delivery study, the mice were anesthetized with an intraperitoneal injection of pentobarbital sodium (Nembutal; Abbott Laboratories, Abbott Park, Ill) (58 mg per kilogram body weight) and the right flanks were shaved. The site was prepared with isopropyl alcohol, and a suspension of 2 x 105 SCC7 cells in 0.5 mL of Hanks solution was injected subcutaneously in each mouse with a 27-gauge needle. This procedure was performed by two of the authors (E.L.Y., L.C.).

For the mechanistic study, the mice were anesthetized by means of inhalation of isoflurane, and both flanks were shaved. These areas were prepared with isopropyl alcohol, and a suspension of 1 x 107 SCC7 cells in 0.1 mL of phosphate-buffered saline was injected subcutaneously in both flanks of each mouse with a 27-gauge needle. In both studies, the growth of the tumors was monitored every 2 days by measuring all three dimensions of the tumors with calipers. Tumor volume (V) was calculated according to previously published information (33) as follows: V = 0.5236(d1 · d2 · d3). Approximately 2 weeks was required for tumors to grow to 1 cm3 in volume for use in the experiments. This procedure was performed by two other authors (S.G.S., J.X.).

Focused Ultrasound Systems
Drug delivery study.—An MR imaging–compatible high-intensity focused ultrasound system, designed for both human and animal use, was used to apply pulsed focused ultrasound to the SCC7 tumors in the mice. A pulse generator (8003A; Hewlett-Packard, Palo Alto, Calif) produced pulses of sinusoidal waves, amplified by a 50-dB radiofrequency power amplifier (2100L; Electronic Navigation Industry, Rochester, NY). The total power available to the system was 500 W. The spherical concave therapeutic transducer possessed a frequency of 1.5 MHz, an aperture of 100 mm, and a focal length of 77 mm. The focal zone (ie, the region at the focal length where the acoustic energy is concentrated) was in the shape of an elongated ellipsoid, with an axial length of 5 mm and a radial diameter of 2 mm. The corresponding focusing factor (ie, transducer surface area divided by focal zone area) was approximately 2.5 x 103.

Mechanistic study.—A modified ultrasound system (Sonoblate; Focus Surgery, Indianapolis, Ind) was used to apply pulsed focused ultrasound to the SCC7 tumors in the mice in this study. The total acoustic power available to the system was 120 W. The probe comprised a spherical concave therapeutic transducer and a collinear imaging transducer. The therapeutic transducer possessed a frequency of 1.0 MHz, an aperture of 50 mm, and a focal length of 40 mm. The focal zone (–3 dB) was in the shape of an elongated ellipsoid, with an axial length of 7.2 mm and a radial diameter of 1.38 mm. The corresponding focusing factor was approximately 1.3 x 103. The imaging transducer possessed a frequency of 10.0 MHz, an aperture of 8 mm, a bandwidth of 80%, and a focal length of 40 mm.

The acoustic output levels for both systems, at the focal point, were determined in water through calibration by using the radiation force technique. Measurements of the focal zone dimensions were performed by using the Schlieren measurement technique (34). This procedure was performed by personnel at the manufacturer of the ultrasound system.

Administration of High-Intensity Focused Ultrasound and Macromolecules
Drug delivery study.—After a single intraperitoneal injection of a mixture of 100 mg/kg ketamine and 5 mg/kg xylazine, individual mice were placed on a specially designed holder and then in a degassed water bath (temperature, approximately 35°C), where the center of the tumor was aligned with the transducer by using a laser-guided system. Each mouse then received an intravenous injection of 9 mg/kg doxorubicin via the tail vein and immediately thereafter was exposed to high-intensity focused ultrasound. All exposures were performed at a single region in the center of the tumor; the axial center of the focal zone was positioned at the center of the tumor with respect to its depth. The focal point of the high-intensity focused ultrasound beam was positioned at the center of the tumor by mounting two laser lights on each side of the transducer, where the intersection of the laser beams occurred at the focal point of the transducer. Duration of all exposures was 5 minutes at a total acoustic power output of 35 W, which corresponded to a spatial average–time average intensity of 1114 W · cm–2 during the pulse. The pulse repetition frequency was 0.5 Hz, and the pulse width was varied, as will be detailed next.

In a preliminary experiment, mice received an injection of doxorubicin and were exposed to high-intensity focused ultrasound with varying pulse widths: 50 msec (n = 2), 70 msec (n = 2), 80 msec (n = 2), 95 msec (n = 3), and 120 msec (n = 3). For the comparative experiment, mice were classified into groups 1 (n = 10), 2 (n = 5), and 3 (n = 5). Mice from group 1 received an injection of doxorubicin and then were exposed to high-intensity focused ultrasound with a pulse width of 80 msec. Mice from group 2 received an injection of doxorubicin but were not exposed to high-intensity focused ultrasound. Mice from group 3 were not exposed to high-intensity focused ultrasound and did not receive doxorubicin injections; these mice served as controls. This procedure was performed by three authors (E.L.Y., M.D.B., K.C.P.L.).

Mechanistic study.—Two groups of five mice each were used. The tumor in the right flank in each mouse was exposed to high-intensity focused ultrasound, but the tumor in the left flank was not and served as a control. Each mouse was anesthetized to a surgical plane of anesthesia (no response to a toe pinch) with the mixture of ketamine and xylazine injected intraperitoneally. The anesthetized mouse was placed in a degassed water bath at 35°C in a specially designed holder. By using the imaging portion of the transducer, the mouse was positioned so that the tumor to be treated was aligned opposite the center of the probe. The tumor was imaged, and the outline of the tumor was demarcated on the display as the region designated for exposure; the axial center of the focal zone was positioned at the center of the tumor with respect to its depth. The exposures were performed at a total acoustic power output of 20.5 W, which corresponded to a spatial average–time average intensity of 1376 W · cm–2 during the pulse. Pulse length was 90 msec at a pulse repetition frequency of 0.5 Hz.

The transducer would raster across the tumor and deliver exposures arranged in a grid at an interval spacing of 2 mm per site, both vertically and laterally. In one group of mice, exposures were delivered at 30 pulses per site; the second group received 150 pulses per site. For tumors with an average surface projection of approximately 1 cm2, the average number of sites per tumor was 20. Average treatment time was 20 and 100 minutes for 30 and 150 pulses per site, respectively. Immediately after treatment with high-intensity focused ultrasound, mice were removed from the water bath, and they received an intravenous injection of 0.1 mL of dextran–fluorescein isothyocyanate (molecular weight, 500 000) at a concentration of 20 mg/mL via the tail vein. The fluorophore conjugate was allowed to circulate for 30 minutes, and then mice were prepared for confocal microscopy. (Because of the relatively shorter half-life of dextran–fluorescein isothyocyanate, it was injected after high-intensity focused ultrasound exposures and not before, as was done with the doxorubicin.) This procedure was performed by three authors (S.G.S., S.A.M., J.X.).

Assay for Intratumoral Doxorubicin Content
Mice from the comparative drug delivery study were sacrificed 2 hours after doxorubicin injection, regardless of whether or not they were exposed to high-intensity focused ultrasound. Mice from group 3 (controls) of the comparative study were sacrificed just prior to the processing of the tumors. The tumors of each of the mice were removed, homogenized in acidic ethanol (3% hydrochloride, 48.5% ethanol, 48.5% double-distilled water) with a micro tissue homogenizer, and stored for 12 hours in the dark at 4°C. The homogenates were then centrifuged at 5000 rpm for 10 minutes at 4°C, and the supernatants were collected. The level of fluorescence for each of the samples was obtained from the supernatants by using fluorescent spectrophotometry and converted to doxorubicin content according to standard published procedures (31). The doxorubicin concentration of each sample was calculated according to the mass of the corresponding tumor. This procedure was performed by one author (E.L.Y.).

Laser Scanning Confocal Microscopy
Mice from the mechanistic study were reassessed for a surgical plane of anesthesia (no response to a toe pinch); additional anesthetic was provided according to need. Next, an incision was made in the skin over the tumor, and the tumor surface was exposed. For confocal microscopy, the mouse was placed on a slide on the stage of an inverted microscope (DM IRB; Leica Microsystems, Bannockburn, Ill), with the exposed tumor facing the objective lens. Blood vessels were localized by using a mercury lamp with a blue excitation/green emission filter set. Representative images of the blood vessels were obtained by using an attached single-photon confocal microscope (TCS SL; Leica Microsystems) with an excitation wavelength of 488 nm. The objective lens used was a 0.3 dry lens with a magnification of x10 (HC PL Fluotar), and scanning was performed at a frequency of 400 Hz. This procedure was performed for both control tumors and tumors treated with high-intensity focused ultrasound by three authors (S.G.S., S.A.M., J.X.).

Statistical Analysis
A two-tailed unpaired Student t test was used to determine whether statistically significant differences existed between mean concentrations of doxorubicin in the tumors in group 1 mice (those treated with doxorubicin and high-intensity focused ultrasound) and those in group 2 mice (those treated with doxorubicin without high-intensity focused ultrasound) in the comparative drug delivery study. The test was performed by using a software package (StatView 4.0; Abacus Concepts, Berkeley, Calif). A P value of .01 or less was designated for determining significant differences. This analysis was performed by two authors (E.L.Y., V.F.).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The effective diameter and polydispersity of doxorubicin were found to be 89.9 nm and 0.072, respectively. The respective values for dextran–fluorescein isothyocyanate (molecular weight, 500 000) were 246.5 nm and 0.379.

In the drug delivery preliminary experiment, a trend over the range of pulse widths evaluated (50–120 msec) indicated that a pulse width of 80 msec produced the highest concentrations of doxorubicin in the tumors. The results appear in Figure 1. According to these results, a pulse width was chosen for the comparative study.



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  		Figure 1. Chart shows doxorubicin concentration in SCC7 tumors for pulse widths ranging from 50 to 120 msec. All other high-intensity focused ultrasound (HIFU) parameters were constant: frequency, 1.5 MHz; pulse repetition frequency, 0.5 Hz; spatial average-time average intensity, 1114 W · cm–2; and time, 5 minutes. A trend over the range of pulse widths evaluated indicated that 80 msec produced the highest concentrations of doxorubicin in tumors.

 
In the drug delivery comparative experiment, doxorubicin administration followed by a high-intensity focused ultrasound exposure (group 1) produced a mean doxorubicin concentration of 9.4 µg · g–1 ± 2.1 (standard deviation) in the treated tumors. In the tumors of mice that received doxorubicin without high-intensity focused ultrasound exposure (group 2), the mean doxorubicin concentration was 4.2 µg · g–1 ± 0.95. There was no detectable doxorubicin in the tumors of mice from group 3 (controls). The enhancement in doxorubicin concentration when high-intensity focused ultrasound was applied was 124% (9.4 µg · g–1/4.2 µg · g–1); this increase was statistically significant (P < .001). The results appear in Figure 2. Figure 3 shows the fluorescence curves for the tumor extracts of doxorubicin; both groups 1 and 2 had emission peaks at 550 nm and 585 nm, which correspond to the major emission peaks of doxorubicin and its metabolites. The extracts of the tumors in mice from group 3 showed no doxorubicin emission peaks.



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  		Figure 2. Graph shows mean doxorubicin content in tumors in mice in group 1 (high-intensity focused ultrasound [HIFU] and doxorubicin [Doxil]), group 2 (doxorubicin without high-intensity focused ultrasound), and group 3 (controls, no doxorubicin or high-intensity focused ultrasound). Analysis of group 3 tumors showed no peaks at the major emission frequencies for doxorubicin and its metabolites. Group 1 tumors showed a 124% (9.4 µg · g–1/4.2 µg · g–1) increase in free doxorubicin over that in group 2 tumors (P < .001).

 


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  		Figure 3. Graph shows fluorescence curves for doxorubicin (Doxil) detected in extracts from tumors in mice in group 1 (doxorubicin and high-intensity focused ultrasound [HIFU]), group 2 (doxorubicin without high-intensity focused ultrasound), and group 3 (control). Both group 1 and group 2 tumors showed fluorescence peaks at 550 nm and 585 nm, which correspond to major emission peaks of doxorubicin and its metabolites. Group 3 tumors, which received no systemic doxorubicin treatment or high-intensity focused ultrasound treatment (controls), showed no peaks at these values.

 
Observations of the confocal micrographs revealed that in the control tumors (Fig 4a, 4c), dextran–fluorescein isothyocyanate was restricted to the vasculature. However, in the tumors treated with high-intensity focused ultrasound (Fig 4b, 4d), extravasation of the fluorophore conjugate into the parenchyma from the vasculature was clearly evident. Moreover, when the number of pulses per site was increased from 30 to 150, an increase in extravasation was observed (Fig 4b, 4d, respectively).



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  		Figure 4a. Representative confocal micrographs of the tumors in the mechanistic study. (a, c) Images of control tumors of mice treated with 30 and 150 pulses per site, respectively. (b, d) Images of tumors treated with 30 and 150 pulses per site, respectively. All other high-intensity focused ultrasound parameters were constant: frequency, 1.0 MHz; pulse repetition frequency, 0.5 Hz; spatial average-time average intensity, 1376 W · cm–2; and time, 2 minutes per site. Increased extravasation is evident in tumors that were exposed to high-intensity focused ultrasound. Relative increase in extravasation is also evident in d, where 150 pulses per site were given, as opposed to b, which received 30 pulses per site. Permeability appears to be isolated to capillary beds and not related to larger vessels. Large arrows = blood vessels, small arrows = postcapillary venules, bar = 80 µm.

 


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  		Figure 4b. Representative confocal micrographs of the tumors in the mechanistic study. (a, c) Images of control tumors of mice treated with 30 and 150 pulses per site, respectively. (b, d) Images of tumors treated with 30 and 150 pulses per site, respectively. All other high-intensity focused ultrasound parameters were constant: frequency, 1.0 MHz; pulse repetition frequency, 0.5 Hz; spatial average-time average intensity, 1376 W · cm–2; and time, 2 minutes per site. Increased extravasation is evident in tumors that were exposed to high-intensity focused ultrasound. Relative increase in extravasation is also evident in d, where 150 pulses per site were given, as opposed to b, which received 30 pulses per site. Permeability appears to be isolated to capillary beds and not related to larger vessels. Large arrows = blood vessels, small arrows = postcapillary venules, bar = 80 µm.

 


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  		Figure 4c. Representative confocal micrographs of the tumors in the mechanistic study. (a, c) Images of control tumors of mice treated with 30 and 150 pulses per site, respectively. (b, d) Images of tumors treated with 30 and 150 pulses per site, respectively. All other high-intensity focused ultrasound parameters were constant: frequency, 1.0 MHz; pulse repetition frequency, 0.5 Hz; spatial average-time average intensity, 1376 W · cm–2; and time, 2 minutes per site. Increased extravasation is evident in tumors that were exposed to high-intensity focused ultrasound. Relative increase in extravasation is also evident in d, where 150 pulses per site were given, as opposed to b, which received 30 pulses per site. Permeability appears to be isolated to capillary beds and not related to larger vessels. Large arrows = blood vessels, small arrows = postcapillary venules, bar = 80 µm.

 


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  		Figure 4d. Representative confocal micrographs of the tumors in the mechanistic study. (a, c) Images of control tumors of mice treated with 30 and 150 pulses per site, respectively. (b, d) Images of tumors treated with 30 and 150 pulses per site, respectively. All other high-intensity focused ultrasound parameters were constant: frequency, 1.0 MHz; pulse repetition frequency, 0.5 Hz; spatial average-time average intensity, 1376 W · cm–2; and time, 2 minutes per site. Increased extravasation is evident in tumors that were exposed to high-intensity focused ultrasound. Relative increase in extravasation is also evident in d, where 150 pulses per site were given, as opposed to b, which received 30 pulses per site. Permeability appears to be isolated to capillary beds and not related to larger vessels. Large arrows = blood vessels, small arrows = postcapillary venules, bar = 80 µm.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
High doses of antineoplastic agents that are needed to achieve a therapeutic response to cancer have severe side effects that limit their use. To circumvent this problem, one solution would be to locally enhance the drug effect, thus decreasing the required systemic drug dose. In this study, we demonstrated how pulsed high-intensity focused ultrasound exposures can significantly enhance the delivery of a systemically administered liposome-encapsulated chemotherapeutic agent locally in a mouse tumor model by increasing vascular permeability in macromolecules as large as 250 nm.

Selectively enhanced delivery of antineoplastic agents is an important approach in the fight against cancer; decreased functional vascularity and increased interstitial pressures in solid tumors (and bulky lymphomas) have repeatedly been demonstrated in both humans and animal models to be limiting factors in such delivery (3,4,35,36). The pharmaceutical industry has tried to ameliorate this problem with biochemical approaches. One approach is the development of sterically stabilized liposomal agents (ie, doxorubicin, which we used in this study) that increase the accumulation of chemotherapeutic agents within tumors by virtue of prolonged circulation time and increased passive transportability across permeable tumor vessels, compared with those characteristics of the free drug (37,38). Another approach to selectively target neoplastic disease is that with the use of monoclonal antibodies. It should be noted that while the targeting antibodies may be high, biodistribution can be problematic. Netti et al (9) showed, for example, that a major difficulty with monoclonal antibody therapy for breast tumors is delivery of the antibodies to the interior of the tumor because of high interstitial pressures therein.

It is thought that pulsed high-intensity focused ultrasound energy disrupts tumor vessel endothelial cell barriers, and this disruption improves vascular permeability. This technique also reduces hydrostatic pressures in tissues, and this reduction enhances diffusion through the interstitium. These effects are summarized in Figure 5. At this time, we can only speculate as to a high-intensity focused ultrasound mechanism for producing effects responsible for these results. One possibility is that increased local and temporal temperatures in the tissues, or hyperthermia, which is thought to increase blood flow to the tumor and to increase microvascular pore size by disassembling the cytoskeleton, a process that allows substances larger than the pore size to extravasate (15), produced the effects. Kong et al (14) showed that extravasation of nanoparticles could be increased with the temperature range of 40°–42°C; at temperatures higher than this range, however, hemorrhage and stasis in tumor vessels occurred.



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  		Figure 5a. Schematic representation of effect of high-intensity focused ultrasound exposures on tumors in conjunction with intravenous injection of liposome-encapsulated doxorubicin (Doxil) as a model. (a) Region of tumor with intact endothelium (E) surrounding vessel (v) and densely packed tumor cells (T). (b) Same region as in a after high-intensity focused ultrasound exposure. It is hypothesized that focused ultrasound energy disrupts endothelial barriers and changes interstitial properties (alters vascular permeability and hydrostatic pressures in the parenchyma), enhancing extravasation and distribution of pharmaceutical agents.

 


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  		Figure 5b. Schematic representation of effect of high-intensity focused ultrasound exposures on tumors in conjunction with intravenous injection of liposome-encapsulated doxorubicin (Doxil) as a model. (a) Region of tumor with intact endothelium (E) surrounding vessel (v) and densely packed tumor cells (T). (b) Same region as in a after high-intensity focused ultrasound exposure. It is hypothesized that focused ultrasound energy disrupts endothelial barriers and changes interstitial properties (alters vascular permeability and hydrostatic pressures in the parenchyma), enhancing extravasation and distribution of pharmaceutical agents.

 
Another possible mechanism that could have contributed to enhanced delivery is acoustic cavitation. Cavitation activity depends on the presence of cavitation nuclei and is difficult to induce in animal tissues, other than in gas-bearing tissues such as the lung and intestine (39). In the parenchyma, the infrastructure of the cells and their relatively high viscosity are severe hindrances for inducing cavitation (40). Intercellular spaces are even more unlikely locations for bubble formation and collapse toward a surface to occur, again because the dimensions of the spaces are orders of magnitude smaller than the bubbles (41). Cavitation within tumors for high-intensity focused ultrasound exposures has been reported by Miller and Song (42) only when cavitation nucleation agents (eg, Optison) were injected directly into the tumors prior to exposures. Cavitation nuclei are also considered to be a limiting factor for producing cavitation in the capillaries. Cavitation was achieved in the blood vessels of tumors only when cavitation nucleation agents were intravenously injected (42). The limiting factor of cavitation nucleation agents (ie, contrast agents) for obtaining cavitation in the blood vessels was clearly demonstrated by Kobayashi et al (43), who showed that endothelial cell injury could be produced in the capillaries and venules even at the low exposure levels of diagnostic contrast material–enhanced US.

While neither thermal nor cavitational mechanisms can be ruled out at this time for production of the enhanced delivery reported here, evidence against either being present during exposures is provided in a previous study that we performed (44). Exposure parameters identical to those in the mechanistic study were used to significantly enhance expression of green fluorescent protein in the SCC7 tumors, when the plasmid encoding for green fluorescent protein was intravenously injected. Histologic sections for the same exposures were stained with both hematoxylin-eosin and terminal transferase-mediated dUTP nick-end labeling (or TUNEL), and showed no significant levels of apoptosis, necrosis, or hemorrhage in the tumors treated with high-intensity focused ultrasound when they were compared with sections in control (untreated) tumors (44). Demonstration that both mechanisms were absent during the exposures will ultimately require further investigations, such as real-time measurements of temperature elevations with thermocouples (thermal mechanisms) and ultrastructural histologic analysis of induced alterations (cavitational mechanisms).

Outside the sphere of thermal and cavitational mechanisms, reports about other mechanisms that may cause ultrasound bioeffects, and especially those for enhancing bioavailability, are more obscure and by far the least well documented. Answers perhaps may be found in recent reports about radiation forces produced by short high-intensity focused ultrasound pulses (similar to those used here), in which the absorption or reflection of acoustic energy causes a transfer of momentum from the ultrasound wave to the medium, with negligible concomitant temperature elevations. Depending on the exposure parameters and the tissue type, displacements in the tissues have been measured at 10 µm without producing damage (45,46). Displacement in the tissues at this scale (eg, comparable to cell dimensions) may produce shear forces between displaced and nondisplaced segments of tissue adjacent to the focal zone boundary, and also within the focal zone, where the intensity is nonuniform along the axial dimension.

The resulting strain from these induced stresses may potentially cause widening of intercellular spaces, an effect that could enhance diffusion. The "detachment of cells from surfaces" (47) and bilayer disorganization (48) are some of the effects that have been attributed to ultrasound exposures. A more specific description of the mechanisms behind these effects was not given. Frenkel et al (49) found that transverse waves produced at a fluid-epithelial interface created shear forces, and this process caused widening of intercellular spaces between cells in the epithelium. This effect was further employed to increase both the depth of penetration and effective rate of diffusion of nanoparticles within the epithelium (50). Determining whether or not this type of mechanism played a part in producing the results presented here will also require further investigation.

Although the results of the work presented here clearly show the beneficial effects of the high-intensity focused ultrasound exposures for drug delivery, both of our experimental exposure devices were limited to a single depth in the axial dimension. This limitation restricted our exposures to relatively small tumors (grown subcutaneously) and did not allow us to determine the additive effects of multiple exposures in the axial dimension (ie, contiguously in this axis). In addition, although our mechanistic study showed the manner in which high-intensity focused ultrasound produced alterations in the tissues for doxorubicin delivery, it would have been preferable to also visualize doxorubicin just as we did for dextran–fluorescein isothyocyanate. In this way, we could have established a correlation between quantitative values of the drug concentration and the visualization of the effects on drug delivery. Unfortunately, the fluorescent signals of doxorubicin are not nearly as luminous, nor accurately distinguished, as are those of dextran–fluorescein isothyocyanate. Ultimately, to gain a greater understanding of the mechanisms of the pulsed high-intensity focused ultrasound exposures and to further optimize delivery and minimize exposure levels, we will need to more accurately determine delivery enhancement, as well as to visualize the manner in which this occurs.

Practical application: Our results show that pulsed high-intensity focused ultrasound with systemic administration of liposomal doxorubicin may selectively increase delivery of the drug to solid tumor tissue. Thus, for the treatment of specific malignancies, pulsed high-intensity focused ultrasound has the potential to serve as an adjuvant therapy in the context of systemic drug administration, whether for primary tumor cure or palliative local care. The authors agree that further studies on the biologic effects of pulsed high-intensity focused ultrasound in different tissues at different exposure parameter levels are needed for clinical optimization to be achieved.


   ACKNOWLEDGMENTS
 
The authors gratefully acknowledge Deloris E. Koziol, PhD, for assisting in the statistical analysis.


   FOOTNOTES
 
Authors stated no financial relationship to disclose.

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


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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