Katsuro Tachibana

Development of safe and efficient novel nonviral gene transfer using ultrasound: enhancement of transfection efficiency of naked plasmid DNA in skeletal muscle

Although clinical trials of stimulation of angiogenesis by transfection of angiogenic growth factors using naked plasmid DNA or adenoviral vector have been successful, there are still unresolved problems for human gene therapy such as low transfection efficiency and safety. From this viewpoint, it is necessary to develop safe and efficient novel nonviral gene transfer methods. As therapeutic ultrasound induces cell membrane permeabilization, ultrasound irradiation might increase the transfection efficiency of naked plasmid DNA into skeletal muscle. Thus, we examined the transfection efficiency of naked plasmid DNA using ultrasound irradiation with echo contrast microbubble (Optison) in vitro and in vivo experiments. First, we examined the feasibility of ultrasound-mediated transfection of naked plasmid DNA into skeletal muscle cells. Luciferase plasmid mixed with or without Optison was transfected into cultured human skeletal muscle cells using ultrasound (1 MHz; 0.4 W2) for 30 s. Interestingly, luciferase activity was markedly increased in cells treated with Optison, while little luciferase activity could be detected without Optison (P < 0.01). Electron microscopy demonstrated the transient formation of holes (less than 5 μM) in the cell surface, which could possibly explain the rapid migration of the transgene into the cells. Next, we studied the in vivo transfection efficiency of naked plasmid DNA using ultrasound with Optison into skeletal muscle. Two days after transfection, luciferase activity in skeletal muscle transfected with Optison using ultrasound was significantly increased about 10-fold as compared with plasmid alone. Successful transfection was also confirmed by β-galactosidase staining. Finally, we examined the feasibility of therapeutic angiogenesis using naked hepatocyte growth factor (HGF) plasmid in a rabbit ischemia model using the ultrasound–Optison method. Five weeks after transfection, the angiographic score and the number of capillary density in rabbits transfected with Optison using ultrasound was significantly increased as compared with HGF plasmid alone (P < 0.01), accompanied by a significant increase in blood flow and blood pressure ratio (P < 0.01). Overall, the ultrasound transfection method with Optison enhanced the transfection efficiency of naked plasmid DNA in vivo as well as in vitro. Transfection of HGF plasmid by the ultrasound-Optison method could be useful for safe clinical gene therapy to treat peripheral arterial disease without a viral vector system.

Induction of cell-membrane porosity by ultrasound.

We previously reported that leukaemia cells can be selectively eliminated by low-intensity ultrasound in the presence of photosensitive drugs. 1 Moreover, recent studies have shown enhanced permeability of genes 2,3 and other structures 4,5 within cells with acoustic energy. The cell-killing mechanism of these treatments has not been clearly determined. To identify the site and degree of damage to tumour cells by sonication, we studied the structure of the cell surface with scanning electron microscopy. An ultrasound emitting transducer (660·62 mm) was inserted directly into the HL-60 cell line suspension (3·010 6 cells/mL). Cells were sonicated with continuous wave ultrasound (255 kHz) in the presence of the photosensitive drug merocyanine 540 (MC 540: 15 g/mL) at an intensity of 0·4 W/cm 2 for a duration of 30 s. Cells were observed with a scanning electron microscope (Hitachi, S450) operating at 10 kV. Cells exposed to ultrasound in the presence of MC 540 showed multiple surface pores (figure, A‐D). Dimple-like craters were seen as well as destroyed cells where the cytoplasm seemed to have extruded through the surface boundary (figure, D). In contrast, cells exposed to identical ultrasound conditions in the absence of MC 540 showed none of the above features. Minor disruptions of the surface were observed in almost all of the cells (figure, F). The microvilli on the cell surface disappeared and several flaplike wrinkles were seen. This remarkable difference in cell surface morphology suggests a completely different ultrasound cell damaging phenomenon induced only in the presence of MC 540. No structural changes compared with intact cells were observed in cells added with MC 540 alone without ultrasound irradiation (not shown). Ultrasound alone had no cytotoxic effects (cell survival: mean [SE], 95·1 [3·4]%), whereas ultrasound with MC 540 showed significant reduction in the number of colonies (47·2 [5·3]%, p<0·001, ANOVA). MC 540 alone at this concentration showed no cytotoxicity (96·5 [5·8]%). These results suggest that cell killing was induced by a very rapid process of cell membrane porosity during sonication in the presence of MC 540. Although more evaluation is needed to control the number and sizes of holes in the cell membrane, this modality could also be used to selectively deliver genes and various agents into tumour cells.

Albumin microbubble echo-contrast material as an enhancer for ultrasound accelerated thrombolysis

BACKGROUND Recent findings suggest that acoustic cavitation is responsible for acceleration of thrombolysis by ultrasound (US) energy. It is known that albumin microbubbles lower the threshold of acoustic cavitation production. METHODS AND RESULTS The present study was designed to determine whether the presence of albumin microbubbles used for echo-contrast material (Albunex) can further accelerate fibrinolysis of US. Artificial thrombus was produced by Chandlers loop method with blood extracted from a healthy subject. Urokinase (UK, 1200 IU/mL) was added to the artificial thrombi placed in test tubes. Each thrombus was exposed to US (170 kHz) at a distance of 1.2 cm for a total of 60 seconds at an intensity of 0.5 W/cm2 at intervals of 2 seconds on and 4 seconds off. Echo-contrast material (0.6 x 10(6) microspheres per mL) or 5% albumin (for control) was circulated near the thrombus at a rate of 1 mL/min during the US exposure. Fibrinolysis was later determined by percentage of weight loss of thrombus after 60 minutes of incubation (n = 15). Fibrinolysis with UK alone was 26.6 +/- 4.8%. Fibrinolysis with UK+US treatment was 33.3 +/- 5.8%. Further increase of fibrinolysis to 51.3 +/- 7.7% occurred in the presence of Albunex (UK+US+Albunex). Statistical differences were obtained between all these groups (ANOVA). CONCLUSIONS The presence of the echo-contrast agent induced further acceleration of thrombolysis by US energy. It is suggested that this diagnostic echo-contrast material can be used as an alternative therapeutic US drug enhancer for thrombolysis.

Enhancement of ultrasound-induced apoptosis and cell lysis by echo-contrast agents.

To determine the effects of echo-contrast agents (ECAs) on ultrasound (US)-induced apoptosis and cell lysis, human myelomonocytic lymphoma U937 cells in suspension were exposed to 1 MHz continuous waves US for 1 min at an intensity of 0.5, 1.0, 2.0 or 4.0 W/cm(2) with or without non-shell type ECA, Levovist (2 mg/ml), and shell type, Optison (1 microl/ml) or YM454 (1 microl/ml). Levovist minimally enhanced the US-induced apoptosis at 1.0 W/cm(2) while Optison and YM454 did at 2.0 and 4.0 W/cm(2), as detected by flow cytometry. Cell lysis was also augmented when Levovist was combined with US at 2.0 W/cm(2), and when Optison was combined with US at 2.0 and 4.0 W/cm(2). YM454 showed the highest rate of enhanced cell lysis at 1.0, 2.0 and 4.0 W/cm(2). Therefore, this study shows that Optison and YM454 are effective in augmenting the US-induced cell killing, but not Levovist. Another result indicates that cavitation plays a role in the augmented effects and that inertial cavitation appears necessary for Optison and YM454 to effect their actions. In addition, results show that the rate of apoptosis is lower in the presence of ECAs with higher free radical scavenging activity, suggesting a possible role for free radicals in apoptosis. These findings suggest that some ECAs have potential to be adjuncts in cases wherein augmented US-induced cell killing is needed, such as in cancer therapy with US.

Transdermal Delivery of Insulin to Alloxan-Diabetic Rabbits by Ultrasound Exposure

4,029,084 6/1977 Soldner. 4,309,989 1/1982 Fahim. 4,372,296 2/1983 Fahim. 4,542,751 9/1985 Webster et al. ......................... 128,760 4,657,022 4/1987 Holscher .......... ... 128/635 4,657,543 4/1987 Langer et al. ... 604/89 4,706,676 11/1987 Peck ................. ... 128/632 4,756,314 7/1988 Eckenhoff et al. ... 128,760 4,767,402 8/1988 Kost et al. ....... ... 604/290 4,780,212 10/1988 Kost et al. ... 210,646 4,787,888 11/1988 Fox ........................................... 604/20

The Use of Ultrasound for Drug Delivery

Ultrasound has been in use for the last three decades as a modality for diagnostic imaging in medicine. Recent studies have shown that nonthermal ultrasound energy could be applied for targeting or controlling drug release. This new concept of therapeutic ultrasound combined with drugs has induced interest in various medical fields. Enhanced effects of thrombolytic agents such as urokinase and TPA with acoustic energy have been demonstrated. Ultrasound transducer‐tipped catheters are being developed for treatment of cardiovascular diseases. Other devices with ultrasound transducers implanted in transdermal drug patches are also being evaluated for possible delivery of insulin through the skin. Echo contrast microbubbles could also be used to carry and release genes to various tissues and lesions. Chemical activation of drugs by ultrasound energy for treatment of cancers is another new field recently termed “sonodynamic therapy.” Various examples of ultrasound application are under investigation that could lead to revolutionary drug delivery systems of the future.

An efficient gene transfer method mediated by ultrasound and microbubbles into the kidney.

Safety issues are of paramount importance in clinical human gene therapy. From this point of view, it would be better to develop a novel non‐viral efficient gene transfer method. Recently, it was reported that ultrasound exposure could induce cell membrane permeabilization and enhance gene expression.

Enhancement of Fibrinolysis with Ultrasound Energy

The effect of ultrasound energy on fibrinolysis of artificial thrombus in vitro was investigated. Thrombi produced by the Chandler loop method were exposed to low-energy ultrasound (5,000-6,000 Pa) in an ultrasound bath (48 kHz) for 60 seconds. Fibrinolysis with urokinase was enhanced from 40.6% +/- 1.8% to 59.2% +/- 2.6% (mean +/- standard deviation) with ultrasound exposure after a 60-minute incubation. Ultrasound alone without urokinase resulted in no fibrinolysis. In a second experiment, a newly developed miniature ultrasound-emitting ceramic element (2 x 1 x 5 mm) was attached to the tip of a catheter. Ultrasound exposure (225 kHz) from this device markedly enhanced fibrinolysis with urokinase from 8.9% +/- 1.5% to 37.3% +/- 0.8% (total ultrasound exposure 60 seconds, intensity 30 mW/cm2) after a 30-minute incubation. After a 120-minute incubation, fibrinolysis with ultrasound exposure was 61.1% +/- 1.8% versus 46.7% +/- 0.5% for the unexposed group. Ultrasound enhancement of fibrinolysis was less pronounced with longer incubation time (60 or 120 minutes). Ultrasound energy enhanced fibrinolysis with urokinase, especially in the early phase of lysis. This new device may shorten the time needed to complete fibrinolysis and reduce total drug dosage needed for treatment of thromboembolic diseases.

Induction of Reparative Dentin Formation by Ultrasound-Mediated Gene Delivery of Growth/Differentiation Factor 11

Bone morphogenetic proteins (BMPs) are morphogens implicated in embryonic and regenerative odontogenic differentiation. Gene therapy has the potential to induce reparative dentin formation for potential pulp capping. We have 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 (Molecular Biosystems Inc., San Diego, CA) and stimulated by ultrasound (1 MHz, 0.5 W/cm(2), 30 sec) showed significant efficiency of gene transfer and high level of protein production selectively in the local region, within 500 microm of 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 stimulated a large amount of reparative dentin formation on the amputated dental pulp in canine teeth in vivo. These results suggest the possible use of BMPs using ultrasound-mediated gene therapy for endodontic dental treatment.

Transdermal delivery of insulin by ultrasonic vibration

Abstract— Ultrasonic vibration has been used to deliver insulin through the skin of hairless mice fasted overnight and partially immersed in an aqueous solution of insulin (20 units mL−1). The skin surface was exposed to ultrasonic vibration in two ultrasonic energy ranges (3000–5000 Pa and 5000–8000 Pa) at 48 kHz for 5 min. Blood glucose concentration was measured before and after exposure to insulin and ultrasonic vibration. In the group subjected to the lower energy vibrations, blood glucose fell rapidly to reach 34 ± 11.9% of control values in 120 min, while when the animals were exposed to higher energy vibrations, the fall in blood glucose was 22.4 ± 3.9% of control values at 120 min. The values remained low for the length of the experiment (240 min). Those exposed to insulin alone or ultrasonic vibration alone revealed no significant change in blood glucose concentration. It is postulated that ultrasonic vibration may alter skin permeability resulting in the absorption of insulin. That the blood glucose decrease was greater at the higher of the two energy ranges, suggests this factor could control insulin delivery.