- Email: [email protected]
Ultrasound therapy for stroke and regenerative medicine
International Congress Series 1274 (2004) 153 – 158
www.ics-elsevier.com
Ultrasound therapy for stroke and regenerative medicine Katsuro Tachibana* Department of Anatomy, Fukuoka University School of Medicine, 7-45-1, Nanakuma, Jonan, 814-0180, Fukuoka, Japan
Abstract. Ultrasound has been in use in medicine for half a century as a modality for diagnostic imaging and therapy. Recently, there have been numerous reports on the application of thermal and nonthermal ultrasound energy for treating various diseases in the brain. In addition to thermal ablation of brain tumors, nonthermal ultrasound combined with drugs has led to much excitement especially for vascular diseases in the brain and regenerative medicine. Ultrasound energy can enhance the effects of thrombolytic agents such as urokinase for treatment of stroke. Therapeutic ultrasound catheters are currently being developed. Noninvasive methods such as high-intensity focused ultrasound (HIFU) in conjunction with magnetic resonance image (MRI) and CT are already being applied in the clinical field. Chemical activation of drugs by ultrasound energy for treatment of tumors is another new field recently termed bSonodynamic TherapyQ. Combination of genes and microbubble has induced great hopes for application of gene therapy to the brain. Various examples of ultrasound combined modalities are under investigation which could lead to revolutionary brain therapy. D 2004 Elsevier B.V. All rights reserved.
Keywords: Drug delivery; Brain; Stroke; Gene therapy
1. Introduction The use of ultrasound for diagnostic purposes in medicine has progressed dramatically over the last few decades. Currently, ultrasound is the fastest growing sector of the medical imaging market. Therapeutic ultrasound, on the other hand, has not been widely accepted among clinicians although extensive clinical work was done in the 1950s and 1960s [1]. * Tel.: +81 92 801 1011; fax: +81 92 865 6032. E-mail address: [email protected]. 0531-5131/ D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.ics.2004.07.021
154
K. Tachibana / International Congress Series 1274 (2004) 153–158
The object of this modality was to thermally ablate lesions in very localized area. Today, advances in computers and imaging technologies have initiated a revival or re-evaluation of the application of ultrasound for therapy in various fields [2]. Morocz et al. [3] recently investigated the potential technique for magnetic resonance image (MRI)-guided minimally invasive thermal ablation of brain tumors using focused ultrasound. MRI allowed on-line monitoring of the lesion setting and demonstrated tissue damage after thermal injury. Vaezy [4] reported the use of high-intensity focused ultrasound (HIFU) to arrest the bleeding from incisions made in rabbit livers. An HIFU transducer with a spherically curved aperture was used for the treatment of liver hemorrhage. Accurate targeting with the help of new technologies and localization with phased arrays are capable of producing a rapid rise in temperature within malignant tissues to cytotoxic levels. This minimally invasive technology utilizes a focused ultrasound beam to destroy well-defined tumors without affecting the surrounding tissue. Clinical trials are currently underway to treat prostate cancer and uterine leiomyomas with HIFU. Recently, a completely new concept of using nonthermal ultrasound has broadened the scope of therapeutic ultrasound. Characterized ultrasound thermal bioeffects and their application has been studied previously but relatively few researchers have investigated the possibility of using nonthermal ultrasound as a means to enhance the effectivity of drugs. Nonthermal mechanisms include various forms of energy such as cavitation, acoustic streaming and radiation force which increases drug availability to target tissues and enhances pharmaceutical effectiveness or even chemical activation of certain substances. The level of ultrasound energy alone in this case is minimal but causes bioeffects leading to a beneficial outcome for drug therapy. The most futuristic investigation under progress is the use of ultrasound for gene therapy. Induction of gene transfer by ultrasound to various cells and tissues could open a series of new ways to treat diseases.
2. Stroke therapy Stroke and acute myocardial infarction are the major causes of death in developed countries. Blood clots that occlude major arteries in the brain and the heart are the cause of this life-threatening disease. Drugs that dissolve these clots have proven effective; however in some cases, there is no response to the drug due to unknown reasons. Largescale clinical trials such as the Pro-urokinase in Acute Cerebral Thromboembolism (PROACT) study [5] have been conducted. In general, higher dosages of lytics increase the treatment success rate but have also resulted in higher incidence of side effects such as unwanted bleeding in the brain and the digestive system. Tachibana [6] demonstrated in vitro that relatively low-intensity ultrasound irradiation of clots in the presence of lytic agents can reduce the amount of drug required by one-tenth and shorten the lysis duration to one-fifth of the original time. This phenomenon has been confirmed by many researchers around the world [7,8]. The mechanism, however, has not been clarified other than the fact that nonthermal effects of ultrasound seem to play the major role in accelerating lysis. Although the minimum ultrasound intensity needed to induce acceleration of fibrinolysis is currently under discussion, recent reports have shown that relatively low mechanical index (MI) energy ranging from 0.1 to 1.0 W/cm2 can produce
K. Tachibana / International Congress Series 1274 (2004) 153–158
155
enhanced fibrinolytic effects. Nonthermal effects of ultrasound contribute to the penetration of drugs into the thrombus. Experiments under conditions where cavitation is more easily produced have resulted in further enhancement of thrombolysis. Cavitation, the phenomenon of bubble formation and collapse, can generate violent microstreams, which increase bioavailability of the drugs at the surface of the thrombus. Increased thrombolysis may also be associated to the unidirectional motion of a fluid or drug known as acoustic streaming, which originates within close range of the ultrasound transducers. Researchers have studied the driving force of acoustic streaming of microparticles theoretically and experimentally in various fluid conditions. Another possible explanation for the increased thrombolysis may be the temporary effect of ultrasound on the thrombus itself. It was suggested that bubble formation, growth and collapse causes reversible alteration in the fibrin structure that may result in an increased flow of the drug into the thrombus. Clinical application of this new therapeutic ultrasound method has already started in 2001. Clinical trials in Europe and in the USA have been reported using miniature ultrasound transducers at the tip of catheters that approach the clots via arterial vessels (MicroLysUS infusion catheter, EKOS, USA). The lytic drug, urokinase, is released at the distal end of the catheter during ultrasound irradiation. The major goal of the catheters used in this study was to apply ultrasound at shorter distances with smaller ultrasound probes, and, at the same time minimize damage to surrounding normal tissues. Mahon et al. [9] presented early experience with the MicroLysUS infusion catheter for acute embolic stroke treatment in North America. This study was designed to demonstrate the safety of the device and to determine if ultrasound accelerates thrombolysis and improves clinical outcomes. Fourteen patients aged 40 to 77 years with anterior- or posterior-circulation occlusion presented with cerebral ischemia 3 h after symptom onset. Patients were treated with the catheter and simultaneous intra-arterial thrombolysis. Procedural and clinical information, including time to lysis, degree of recanalization, NIHSS score, and modified Rankin Scale (mRS) score was recorded before treatment and afterward. The numbers in the study were small but the trends demonstrated that the rates of recanalization and neurologic outcomes in patients treated with the new ultrasound catheter were equivalent or slightly better than those in patients treated with standard microinfusion. Ishibashi et al. [10] have challenged the alternative approach of sonicating transcranially to accelerate thrombolysis. A noninvasive method has been tested in an occlusion model of rabbit femoral artery, produced with thrombin after establishment of stenotic flow and endothelial damage. After stable occlusion was confirmed, monteplase (mtPA) was administered intravenously, and ultrasound (490 kHz, 0.13 W/cm2) was applied through a piece of temporal bone (TUS group). The recanalization ratio in the TUS group was higher than that in the tPA group. Alexandrov et al. [11] similarly demonstrated acceleration of thrombolysis in stroke patients during continuous monitoring with 2 MHz transcranial ultrasound doppler flowmeter. The differences between external and internal ultrasound applications are: (1) in external application, relatively high energy is needed at the surface of the body to sufficiently deliver ultrasound to deeply located thrombus, (2) ultrasound must propagate through the skull and prevents sufficient ultrasound energy from reaching the target accurately, (3) multiple transducers and the large size of the ultrasound-generating
156
K. Tachibana / International Congress Series 1274 (2004) 153–158
equipment needed for extracorpreal exposure could elevate medical costs. More clinical studies are needed to evaluate the medical significance of accelerated thrombolysis by ultrasound energy. However, this therapeutic ultrasound application for stroke seems to be the most promising among various ultrasound drug delivery clinical trials known to date.
3. Regenerative medicine Today, the concept of applying ultrasound as a means to alter the pharmacokinetics in various tissues and drug permeability through cell membrane has expanded into a whole new field ranging from transdermal drug delivery to gene therapy. In order to discuss the significance of ultrasound application in conjunction with drugs, one must understand the pharmacological background involved. Although there are thousands of types of drugs available to use today, the number of drugs that never reach the market may be tenfold or even a hundred-fold. These types of drugs either had severe side effects or proved to be ineffective during the preclinical or early clinical trials. One example is the delivery of gene into cells for gene therapy. Certain tissues such as muscle have been reported to take up and express naked plasmid DNA in vivo. In general, however, the level of transfection after direct injection of naked plasmid DNA is variable and low. Different methods have been devised to improve the transfection efficiency. The use of certain viral vectors leads to more efficient transfection compared with naked plasmid DNA; but serious concerns have been voiced about the use of viral vectors, especially when clinical trials are involved. Instead of viral vectors as the carrier of genes to targeted locations, pure plasmid DNA can be attached either to the outside or inside of the microbubble capsule wall. Bubbles can be collapsed by extracorporeal ultrasound or by intravascular ultrasound catheter, permitting the DNA to penetrate directly into the tissue and cells (Fig. 1). Greenleaf et al. [12] demonstrated an increase in the transfection rate of DNA in the presence of albumin microbubbles in vitro. Ultrasound may become a new, effective and safe means for introducing genetic material into the target cells of tissues. Ultrasound can induce cell-membrane porosity [13], and enhance the delivery of naked plasmid DNA into cells in vitro. Moreover, recent studies have shown enhanced permeability of naked plasmid DNA into tumors in vivo [14] and Li et al. [15] recently made a comparison of gene transfection using various microbubbles available in the market. Ogawa et al. [16] also made a comparison with different dissolved gases and found changes in the extent of gene transfection. Although the exact mechanism is still unknown, it is believed that microspheres, upon rupture, create a local increase in membrane fluidity, thereby enhancing cellular uptake of the therapeutic compound. bSonoporationQ as this phenomenon is frequently called is a new means to overcome limitations of other gene transduction methods. Nevertheless, different genes for various purposes are now under intensive investigation for possible use in regenerative medicine. This could be for angiogenesis, anti-angiogenesis, apoptosis, bone generation and other future treatment methods. Antisense oligodeoxynucleotides (AS-ODNs) have been recognized as a new generation of putative therapeutic agents, Miura et al. [17] established a delivery technique that could transfect AS-ODNs, which are designed for endothelin type B receptor (ETB), into cultured human coronary endothelial cells (HCECs) by exposure to ultrasound in the
K. Tachibana / International Congress Series 1274 (2004) 153–158
157
Fig. 1. SEM images of tumor cell treated with sonoporation.
presence of echo contrast microbubbles. Taniyama et al. [18,19] have successfully transfected genes (HGF) for angiogenesis by ultrasound/microbubbles in skeletal muscles. This could lead to a cure for critical limb ischemia. In addition, they transfected an antioncogene (p53) plasmid into carotid artery after balloon injury as a model of gene therapy for restenosis. Bone morphogenetic proteins (BMPs) are morphogens implicated both in embryonic and regenerative odontogenic differentiation. Gene therapy has the potential to improve induction of reparative dentin formation or potent bioactive pulp capping. Nakashima et al. [20] optimized the gene transfer of Growth/differentiation factor 11 (Gdf11)/Bmp11 plasmid DNA into dental pulp stem cells by sonoporation in vivo. Dental pulp tissue treated with plasmid pEGFP or CMV-LacZ in 5–10% Optison and irradiated by ultrasound (1 MHz, 0.5 W/cm2, 30 s) showed significant efficiency of gene transfer and high level of protein production selectively in the insonated region, within 300 Am under the amputated site of the pulp tissue. The Gdf11 cDNA plasmid transferred into dental pulp tissue by sonoporation in vitro, induced the expression of Dentin sialoprotein (Dsp), a differentiation marker for odontoblasts. The transfection of Gdf11 by sonoporation
158
K. Tachibana / International Congress Series 1274 (2004) 153–158
stimulated the large amount of reparative dentin formation on the amputated dental pulp in canine teeth in vivo. These results suggest the possible use of BMPs employing ultrasound-mediated gene therapy for endodontic dental treatment. It is estimated that genes for regeneration tissues could someday become a realistic mode of treatment.
References [1] W.J. Fry, F.J. Fry, Fundamental neurological research and human neurosurgery using intense ultrasound, I.R.E. Transactions on Medical Electronics 17 (1960) 858 – 876. [2] K. Hynynen, et al., MRI-guided noninvasive ultrasound surgery, Medical Physics 20 (1) (1993) 107 – 115. [3] I.A. Morocz, et al., Brain edema development after MRI-guided focused ultrasound treatment, Journal of Magnetic Resonance Imaging 8 (1) (1998) 136 – 142. [4] S. Vaezy, R. Martin, U. Schmiedl, et al., Liver hemostasis using high-intensity focused ultrasound, Ultrasound in Medicine & Biology 23 (9) (1997) 1413 – 1420. [5] G.J. DelZoppo, R.T. Higashida, A.J. Furlan, et al., PROACT: a phase II randomized trial of recombinant pro-urokinase by direct arterial delivery in acute middle cerebral artery stroke, Stroke 29 (1998) 4 – 11. [6] K. Tachibana, Enhancement of fibrinolysis with ultrasound energy, Journal of Vascular and Interventional Radiology 3 (1992) 299 – 303. [7] C.W. Francis, P.T. Onundarson, E.L. Carstensen, Enhancement of fibrinolysis in vitro by ultrasound, Journal of Clinical Investigation 90 (1992) 2063 – 2068. [8] A. Blinc, C.W. Francis, J.L. Trudnowski, Characterization of ultrasound-potentiated fibrinolysis in vitro, Blood 81 (1993) 2636 – 2643. [9] B.R. Mahon, G.M. Nesbit, S.L. Barnwell, et al., North American clinical experience with the EKOS MicroLysUS infusion catheter for the treatment of embolic stroke, Am. J. Neuroradiol. 24 (2003) 535 – 538. [10] T. Ishibashi, et al., Can transcranial ultrasonication increase recanalization flow with tissue plasminogen activator? Stroke 33 (2002) 1399 – 1404. [11] A.V. Alexandrov, A.M. Demchuk, R.A. Felberg, et al., High rate of complete recanalization and dramatic clinical recovery during tPA infusion when continuously monitored with 2-MHz transcranial doppler monitoring, Stroke 31 (2000) 610 – 614. [12] W.J. Greenleaf, et al., Artificial cavitation nuclei significantly enhance acoustically induced cell transfection, Ultrasound in Medicine & Biology 24 (1998) 587 – 595. [13] K. Tachibana, et al., Induction of cell-membrane porosity by ultrasound, Lancet 353 (1999) 1409. [14] S. Bao, B.D. Thrall, D.L. Miller, Transfection of a reporter plasmid into cultured cells by sonoporation in vitro, Ultrasound in Medicine & Biology 23 (1997) 953 – 959. [15] T. Li, et al., Gene transfer with echo-enhanced contrast agents: comparison between albunex, optison, and levovist in mice initial results, Radiology 229 (2003) 423 – 428. [16] R. Ogawa, et al., Effects of dissolved gases and an echo contrast magnet on ultrasound mediated in vitro gene transfection, Ultrasonics Sonochemistry 9 (2003) 197 – 203. [17] S. Miura, et al., In vitro transfer of antisense oligodeoxynucleotides into coronary endothelial cells by ultrasound, Biochemical and Biophysical Research Communication 298 (2002) 587 – 590. [18] Y. Taniyama, K. Tachibana, K. Hiraoka, et al., Development of safe and efficient novel nonviral gene transfer using ultrasound: enhancement of transfection efficiency of naked plasmid DNA in skeletal muscle, Gene Therapy 9 (2002) 372 – 380. [19] Y. Taniyama, K. Tachibana, K. Hiraoka, et al., Local delivery of plasmid DNA into rat carotid artery using ultrasound, Circulation 105 (2002) 1233 – 1239. [20] M. Nakashima, et al., Induction of reparative dentin formation by growth/differentiation factor 11 (Gdf11) employing ultrasound-mediated gene delivery, Human Gene Therapy 14 (2003) 591 – 597.
www.ics-elsevier.com
Ultrasound therapy for stroke and regenerative medicine Katsuro Tachibana* Department of Anatomy, Fukuoka University School of Medicine, 7-45-1, Nanakuma, Jonan, 814-0180, Fukuoka, Japan
Abstract. Ultrasound has been in use in medicine for half a century as a modality for diagnostic imaging and therapy. Recently, there have been numerous reports on the application of thermal and nonthermal ultrasound energy for treating various diseases in the brain. In addition to thermal ablation of brain tumors, nonthermal ultrasound combined with drugs has led to much excitement especially for vascular diseases in the brain and regenerative medicine. Ultrasound energy can enhance the effects of thrombolytic agents such as urokinase for treatment of stroke. Therapeutic ultrasound catheters are currently being developed. Noninvasive methods such as high-intensity focused ultrasound (HIFU) in conjunction with magnetic resonance image (MRI) and CT are already being applied in the clinical field. Chemical activation of drugs by ultrasound energy for treatment of tumors is another new field recently termed bSonodynamic TherapyQ. Combination of genes and microbubble has induced great hopes for application of gene therapy to the brain. Various examples of ultrasound combined modalities are under investigation which could lead to revolutionary brain therapy. D 2004 Elsevier B.V. All rights reserved.
Keywords: Drug delivery; Brain; Stroke; Gene therapy
1. Introduction The use of ultrasound for diagnostic purposes in medicine has progressed dramatically over the last few decades. Currently, ultrasound is the fastest growing sector of the medical imaging market. Therapeutic ultrasound, on the other hand, has not been widely accepted among clinicians although extensive clinical work was done in the 1950s and 1960s [1]. * Tel.: +81 92 801 1011; fax: +81 92 865 6032. E-mail address: [email protected]. 0531-5131/ D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.ics.2004.07.021
154
K. Tachibana / International Congress Series 1274 (2004) 153–158
The object of this modality was to thermally ablate lesions in very localized area. Today, advances in computers and imaging technologies have initiated a revival or re-evaluation of the application of ultrasound for therapy in various fields [2]. Morocz et al. [3] recently investigated the potential technique for magnetic resonance image (MRI)-guided minimally invasive thermal ablation of brain tumors using focused ultrasound. MRI allowed on-line monitoring of the lesion setting and demonstrated tissue damage after thermal injury. Vaezy [4] reported the use of high-intensity focused ultrasound (HIFU) to arrest the bleeding from incisions made in rabbit livers. An HIFU transducer with a spherically curved aperture was used for the treatment of liver hemorrhage. Accurate targeting with the help of new technologies and localization with phased arrays are capable of producing a rapid rise in temperature within malignant tissues to cytotoxic levels. This minimally invasive technology utilizes a focused ultrasound beam to destroy well-defined tumors without affecting the surrounding tissue. Clinical trials are currently underway to treat prostate cancer and uterine leiomyomas with HIFU. Recently, a completely new concept of using nonthermal ultrasound has broadened the scope of therapeutic ultrasound. Characterized ultrasound thermal bioeffects and their application has been studied previously but relatively few researchers have investigated the possibility of using nonthermal ultrasound as a means to enhance the effectivity of drugs. Nonthermal mechanisms include various forms of energy such as cavitation, acoustic streaming and radiation force which increases drug availability to target tissues and enhances pharmaceutical effectiveness or even chemical activation of certain substances. The level of ultrasound energy alone in this case is minimal but causes bioeffects leading to a beneficial outcome for drug therapy. The most futuristic investigation under progress is the use of ultrasound for gene therapy. Induction of gene transfer by ultrasound to various cells and tissues could open a series of new ways to treat diseases.
2. Stroke therapy Stroke and acute myocardial infarction are the major causes of death in developed countries. Blood clots that occlude major arteries in the brain and the heart are the cause of this life-threatening disease. Drugs that dissolve these clots have proven effective; however in some cases, there is no response to the drug due to unknown reasons. Largescale clinical trials such as the Pro-urokinase in Acute Cerebral Thromboembolism (PROACT) study [5] have been conducted. In general, higher dosages of lytics increase the treatment success rate but have also resulted in higher incidence of side effects such as unwanted bleeding in the brain and the digestive system. Tachibana [6] demonstrated in vitro that relatively low-intensity ultrasound irradiation of clots in the presence of lytic agents can reduce the amount of drug required by one-tenth and shorten the lysis duration to one-fifth of the original time. This phenomenon has been confirmed by many researchers around the world [7,8]. The mechanism, however, has not been clarified other than the fact that nonthermal effects of ultrasound seem to play the major role in accelerating lysis. Although the minimum ultrasound intensity needed to induce acceleration of fibrinolysis is currently under discussion, recent reports have shown that relatively low mechanical index (MI) energy ranging from 0.1 to 1.0 W/cm2 can produce
K. Tachibana / International Congress Series 1274 (2004) 153–158
155
enhanced fibrinolytic effects. Nonthermal effects of ultrasound contribute to the penetration of drugs into the thrombus. Experiments under conditions where cavitation is more easily produced have resulted in further enhancement of thrombolysis. Cavitation, the phenomenon of bubble formation and collapse, can generate violent microstreams, which increase bioavailability of the drugs at the surface of the thrombus. Increased thrombolysis may also be associated to the unidirectional motion of a fluid or drug known as acoustic streaming, which originates within close range of the ultrasound transducers. Researchers have studied the driving force of acoustic streaming of microparticles theoretically and experimentally in various fluid conditions. Another possible explanation for the increased thrombolysis may be the temporary effect of ultrasound on the thrombus itself. It was suggested that bubble formation, growth and collapse causes reversible alteration in the fibrin structure that may result in an increased flow of the drug into the thrombus. Clinical application of this new therapeutic ultrasound method has already started in 2001. Clinical trials in Europe and in the USA have been reported using miniature ultrasound transducers at the tip of catheters that approach the clots via arterial vessels (MicroLysUS infusion catheter, EKOS, USA). The lytic drug, urokinase, is released at the distal end of the catheter during ultrasound irradiation. The major goal of the catheters used in this study was to apply ultrasound at shorter distances with smaller ultrasound probes, and, at the same time minimize damage to surrounding normal tissues. Mahon et al. [9] presented early experience with the MicroLysUS infusion catheter for acute embolic stroke treatment in North America. This study was designed to demonstrate the safety of the device and to determine if ultrasound accelerates thrombolysis and improves clinical outcomes. Fourteen patients aged 40 to 77 years with anterior- or posterior-circulation occlusion presented with cerebral ischemia 3 h after symptom onset. Patients were treated with the catheter and simultaneous intra-arterial thrombolysis. Procedural and clinical information, including time to lysis, degree of recanalization, NIHSS score, and modified Rankin Scale (mRS) score was recorded before treatment and afterward. The numbers in the study were small but the trends demonstrated that the rates of recanalization and neurologic outcomes in patients treated with the new ultrasound catheter were equivalent or slightly better than those in patients treated with standard microinfusion. Ishibashi et al. [10] have challenged the alternative approach of sonicating transcranially to accelerate thrombolysis. A noninvasive method has been tested in an occlusion model of rabbit femoral artery, produced with thrombin after establishment of stenotic flow and endothelial damage. After stable occlusion was confirmed, monteplase (mtPA) was administered intravenously, and ultrasound (490 kHz, 0.13 W/cm2) was applied through a piece of temporal bone (TUS group). The recanalization ratio in the TUS group was higher than that in the tPA group. Alexandrov et al. [11] similarly demonstrated acceleration of thrombolysis in stroke patients during continuous monitoring with 2 MHz transcranial ultrasound doppler flowmeter. The differences between external and internal ultrasound applications are: (1) in external application, relatively high energy is needed at the surface of the body to sufficiently deliver ultrasound to deeply located thrombus, (2) ultrasound must propagate through the skull and prevents sufficient ultrasound energy from reaching the target accurately, (3) multiple transducers and the large size of the ultrasound-generating
156
K. Tachibana / International Congress Series 1274 (2004) 153–158
equipment needed for extracorpreal exposure could elevate medical costs. More clinical studies are needed to evaluate the medical significance of accelerated thrombolysis by ultrasound energy. However, this therapeutic ultrasound application for stroke seems to be the most promising among various ultrasound drug delivery clinical trials known to date.
3. Regenerative medicine Today, the concept of applying ultrasound as a means to alter the pharmacokinetics in various tissues and drug permeability through cell membrane has expanded into a whole new field ranging from transdermal drug delivery to gene therapy. In order to discuss the significance of ultrasound application in conjunction with drugs, one must understand the pharmacological background involved. Although there are thousands of types of drugs available to use today, the number of drugs that never reach the market may be tenfold or even a hundred-fold. These types of drugs either had severe side effects or proved to be ineffective during the preclinical or early clinical trials. One example is the delivery of gene into cells for gene therapy. Certain tissues such as muscle have been reported to take up and express naked plasmid DNA in vivo. In general, however, the level of transfection after direct injection of naked plasmid DNA is variable and low. Different methods have been devised to improve the transfection efficiency. The use of certain viral vectors leads to more efficient transfection compared with naked plasmid DNA; but serious concerns have been voiced about the use of viral vectors, especially when clinical trials are involved. Instead of viral vectors as the carrier of genes to targeted locations, pure plasmid DNA can be attached either to the outside or inside of the microbubble capsule wall. Bubbles can be collapsed by extracorporeal ultrasound or by intravascular ultrasound catheter, permitting the DNA to penetrate directly into the tissue and cells (Fig. 1). Greenleaf et al. [12] demonstrated an increase in the transfection rate of DNA in the presence of albumin microbubbles in vitro. Ultrasound may become a new, effective and safe means for introducing genetic material into the target cells of tissues. Ultrasound can induce cell-membrane porosity [13], and enhance the delivery of naked plasmid DNA into cells in vitro. Moreover, recent studies have shown enhanced permeability of naked plasmid DNA into tumors in vivo [14] and Li et al. [15] recently made a comparison of gene transfection using various microbubbles available in the market. Ogawa et al. [16] also made a comparison with different dissolved gases and found changes in the extent of gene transfection. Although the exact mechanism is still unknown, it is believed that microspheres, upon rupture, create a local increase in membrane fluidity, thereby enhancing cellular uptake of the therapeutic compound. bSonoporationQ as this phenomenon is frequently called is a new means to overcome limitations of other gene transduction methods. Nevertheless, different genes for various purposes are now under intensive investigation for possible use in regenerative medicine. This could be for angiogenesis, anti-angiogenesis, apoptosis, bone generation and other future treatment methods. Antisense oligodeoxynucleotides (AS-ODNs) have been recognized as a new generation of putative therapeutic agents, Miura et al. [17] established a delivery technique that could transfect AS-ODNs, which are designed for endothelin type B receptor (ETB), into cultured human coronary endothelial cells (HCECs) by exposure to ultrasound in the
K. Tachibana / International Congress Series 1274 (2004) 153–158
157
Fig. 1. SEM images of tumor cell treated with sonoporation.
presence of echo contrast microbubbles. Taniyama et al. [18,19] have successfully transfected genes (HGF) for angiogenesis by ultrasound/microbubbles in skeletal muscles. This could lead to a cure for critical limb ischemia. In addition, they transfected an antioncogene (p53) plasmid into carotid artery after balloon injury as a model of gene therapy for restenosis. Bone morphogenetic proteins (BMPs) are morphogens implicated both in embryonic and regenerative odontogenic differentiation. Gene therapy has the potential to improve induction of reparative dentin formation or potent bioactive pulp capping. Nakashima et al. [20] optimized the gene transfer of Growth/differentiation factor 11 (Gdf11)/Bmp11 plasmid DNA into dental pulp stem cells by sonoporation in vivo. Dental pulp tissue treated with plasmid pEGFP or CMV-LacZ in 5–10% Optison and irradiated by ultrasound (1 MHz, 0.5 W/cm2, 30 s) showed significant efficiency of gene transfer and high level of protein production selectively in the insonated region, within 300 Am under the amputated site of the pulp tissue. The Gdf11 cDNA plasmid transferred into dental pulp tissue by sonoporation in vitro, induced the expression of Dentin sialoprotein (Dsp), a differentiation marker for odontoblasts. The transfection of Gdf11 by sonoporation
158
K. Tachibana / International Congress Series 1274 (2004) 153–158
stimulated the large amount of reparative dentin formation on the amputated dental pulp in canine teeth in vivo. These results suggest the possible use of BMPs employing ultrasound-mediated gene therapy for endodontic dental treatment. It is estimated that genes for regeneration tissues could someday become a realistic mode of treatment.
References [1] W.J. Fry, F.J. Fry, Fundamental neurological research and human neurosurgery using intense ultrasound, I.R.E. Transactions on Medical Electronics 17 (1960) 858 – 876. [2] K. Hynynen, et al., MRI-guided noninvasive ultrasound surgery, Medical Physics 20 (1) (1993) 107 – 115. [3] I.A. Morocz, et al., Brain edema development after MRI-guided focused ultrasound treatment, Journal of Magnetic Resonance Imaging 8 (1) (1998) 136 – 142. [4] S. Vaezy, R. Martin, U. Schmiedl, et al., Liver hemostasis using high-intensity focused ultrasound, Ultrasound in Medicine & Biology 23 (9) (1997) 1413 – 1420. [5] G.J. DelZoppo, R.T. Higashida, A.J. Furlan, et al., PROACT: a phase II randomized trial of recombinant pro-urokinase by direct arterial delivery in acute middle cerebral artery stroke, Stroke 29 (1998) 4 – 11. [6] K. Tachibana, Enhancement of fibrinolysis with ultrasound energy, Journal of Vascular and Interventional Radiology 3 (1992) 299 – 303. [7] C.W. Francis, P.T. Onundarson, E.L. Carstensen, Enhancement of fibrinolysis in vitro by ultrasound, Journal of Clinical Investigation 90 (1992) 2063 – 2068. [8] A. Blinc, C.W. Francis, J.L. Trudnowski, Characterization of ultrasound-potentiated fibrinolysis in vitro, Blood 81 (1993) 2636 – 2643. [9] B.R. Mahon, G.M. Nesbit, S.L. Barnwell, et al., North American clinical experience with the EKOS MicroLysUS infusion catheter for the treatment of embolic stroke, Am. J. Neuroradiol. 24 (2003) 535 – 538. [10] T. Ishibashi, et al., Can transcranial ultrasonication increase recanalization flow with tissue plasminogen activator? Stroke 33 (2002) 1399 – 1404. [11] A.V. Alexandrov, A.M. Demchuk, R.A. Felberg, et al., High rate of complete recanalization and dramatic clinical recovery during tPA infusion when continuously monitored with 2-MHz transcranial doppler monitoring, Stroke 31 (2000) 610 – 614. [12] W.J. Greenleaf, et al., Artificial cavitation nuclei significantly enhance acoustically induced cell transfection, Ultrasound in Medicine & Biology 24 (1998) 587 – 595. [13] K. Tachibana, et al., Induction of cell-membrane porosity by ultrasound, Lancet 353 (1999) 1409. [14] S. Bao, B.D. Thrall, D.L. Miller, Transfection of a reporter plasmid into cultured cells by sonoporation in vitro, Ultrasound in Medicine & Biology 23 (1997) 953 – 959. [15] T. Li, et al., Gene transfer with echo-enhanced contrast agents: comparison between albunex, optison, and levovist in mice initial results, Radiology 229 (2003) 423 – 428. [16] R. Ogawa, et al., Effects of dissolved gases and an echo contrast magnet on ultrasound mediated in vitro gene transfection, Ultrasonics Sonochemistry 9 (2003) 197 – 203. [17] S. Miura, et al., In vitro transfer of antisense oligodeoxynucleotides into coronary endothelial cells by ultrasound, Biochemical and Biophysical Research Communication 298 (2002) 587 – 590. [18] Y. Taniyama, K. Tachibana, K. Hiraoka, et al., Development of safe and efficient novel nonviral gene transfer using ultrasound: enhancement of transfection efficiency of naked plasmid DNA in skeletal muscle, Gene Therapy 9 (2002) 372 – 380. [19] Y. Taniyama, K. Tachibana, K. Hiraoka, et al., Local delivery of plasmid DNA into rat carotid artery using ultrasound, Circulation 105 (2002) 1233 – 1239. [20] M. Nakashima, et al., Induction of reparative dentin formation by growth/differentiation factor 11 (Gdf11) employing ultrasound-mediated gene delivery, Human Gene Therapy 14 (2003) 591 – 597.