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Sonodynamic therapy
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Ultrasonics 48 (2008) 253–259 www.elsevier.com/locate/ultras
Sonodynamic therapy Katsuro Tachibana *, Loreto B. Feril Jr., Yurika Ikeda-Dantsuji Department of Anatomy, Fukuoka University School of Medicine, 7-45-1 Nanakuma, Jonan, Fukuoka 814-0180, Japan Received 19 July 2007; received in revised form 26 November 2007; accepted 28 February 2008 Available online 7 March 2008
Abstract Recently, there have been numerous reports on the application of non-thermal ultrasound energy for treating various diseases in combination with drugs. Furthermore, the introduction of microbubbles and nanobubbles as carriers/enhancers of drugs has added a whole new dimension to therapeutic ultrasound. Non-thermal mechanisms for effects seen include various forms of energy due to cavitation, acoustic streaming, micro jets and radiation force which increases possibilities for targeting tissue with drugs, enhancing drug effectiveness or even chemically activating certain materials. Examples such as enhancement of thrombolytic agents by ultrasound have proven to be beneficial for acute stroke patients and peripheral arterial occlusions. Non-invasive low intensity focused ultrasound in conjunction with anti-cancer drugs may help to reduce tumor size and lessen recurrence while reducing severe drug side effects. Chemical activation of drugs by ultrasound energy for treatment of atherosclerosis and tumors is another new field recently termed as ‘‘Sonodynamic therapy”. Lastly, advances in molecular imaging have aroused great expectations in applying ultrasound for both diagnosis and therapy simultaneously. Microbubbles or nanobubbles targeted at the molecular level will allow medical doctors to make a final diagnosis of a disease using ultrasound imaging and then immediately proceed to a therapeutic ultrasound treatment. Ó 2008 Elsevier B.V. All rights reserved. Keywords: Ultrasound; Sonodynamic therapy; Sonotransfection
Contents 1. 2.
3.
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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sonodynamic therapy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Ultrasound-induced apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Ultrasound-mediated gene transfection (sonotransfection) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Cavitation and the use of microbubbles. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Synergistic effect of combined ultrasound and antibiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.1. Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.2. Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions and outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Corresponding author. E-mail address: [email protected] (K. Tachibana).
0041-624X/$ - see front matter Ó 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.ultras.2008.02.003
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1. Introduction Although there are thousands of types of drugs available to use today, the number of drugs that have never reached the market may be ten- or even a 100-fold greater. These drugs either had severe side effects or proved to be ineffective for some reason during preclinical or early clinical trials. Examples include various agents for cancer chemotherapy. Some of these agents, when administered systemically, can kill solid tumors, but at the same time severely damage healthy tissues and organs. Researchers have attempted to address these problems using the concept of a ‘‘drug delivery system (DDS)” since the 70s. The two keywords for DDS are ‘‘targeting” and ‘‘controlled release”. The major goal was to avoid severe side effects by reducing the total dosage but at the same time concentrating the drug at the target site. Additionally, controlled drug release permits more control over the amount of drug administered in a certain time frame. Recent progress in the application of ultrasound energy for DDS has demonstrated it to be a promising modality for both ‘‘targeting” and ‘‘controlled release” of drugs. Microbubbles and nanobubbles can carry a certain drug at specific target site and release it at a particular time using ultrasound. Ultrasound-sensitive materials such as hematoporphyrin, a nontoxic agent, can also be activated by ultrasound in a localized volume and can thus result in the killing of cancer cells. A wide variety of applications for drug delivery with ultrasound are currently under investigation in many fields. The most futuristic investigation under progress is the use of ultrasound for gene therapy. DNA can be injected through the cell membrane as if a ‘‘micro syringe” were being used with ultrasound energy and induce transfection. Induction of gene transfer into the cell by ultrasound could result in regeneration of blood vessels, nerves or any other tissue. With the help of microbubbles or nanobubbles, it is possible to change the permeability of drugs at the microscopic cell membrane level. 2. Sonodynamic therapy The term ‘‘Sonodynamic therapy” is often used for all non-thermally related therapeutic ultrasound applications mentioned above ranging from induction of apoptosis, combining with chemotherapy, to ultrasound gene therapy. It was however, originally coined by Yumita and Umemura who first discovered the phenomenon of sonochemical activation of photosensitive materials by ultrasound for cancer therapy. Some researchers tend to prefer this narrower meaning where a sonochemical mechanism is involved. As the exact mechanism is not well determined, further discussion about the definition of this term is needed. Nevertheless, it is well known that Kremkau et al. [1] were the first to study the cytotoxic effects of various anti-cancer drugs, such as nitrogen mustard, to mouse leukemia L1210 cells in combination with ultrasound irradiation. It was discovered from these experiments that the
increase in drug efficiency could be explained by the nonthermal effect of ultrasound interacting with the drugs. Experiments performed by Harrison and Balcer-Kubiczek [2] using low-intensity ultrasound with no temperature increase showed similar results. It was pointed out that low-level ultrasound may have altered the cell membrane, thus changing its permeability to the drug. Marked enhancement by ultrasound of the cytotoxicity of adriamycin and amphotericin B against Chinese hamster ovary cells HA1 at lower energies was demonstrated by Harrison. Compared to radiotherapy and light irradiation for the treatment of leukemia and solid cancers, ultrasonic irradiation is superior in that the applied energy can be focused solely on the tumor volume to be treated, with little effect upon normal tissues, and is better than light irradiation in the degree of penetration. Umemura et al. [3] pioneered the development of non-thermal ultrasound to activate a group of chemicals that were originally used as light activated chemicals for cancer therapy. This new ultrasound therapy falls into the narrow definition of sonodynamic therapy. Ultrasonic waves are known to cause chemical effects if cavitation occurs; for example, irradiation of water can generate hydrogen peroxide. It was found that certain drugs, upon ultrasonic irradiation, create active oxygen species such as superoxide radicals and singlet oxygen, and that the active oxygen thus formed effectively destructs cancer tissues [4]. The agents themselves have no anti-tumor activity and are very low in toxicity, exhibit anti-tumor activity only by the chemical action caused by ultrasonic irradiation. Thus, there is significant less risk of causing any systemic effects. In addition, these drugs act exclusively upon tumor tissues when combined with ultrasonic irradiation, with no adverse effects upon normal tissues. An important factor involved in ultrasound irradiation is the chemical reactions induced during the course of violent microbubble collapse. Short lived free radicals that may alter various compounds leading to cell killing can be created by ultrasound [5]. Sonoluminescence (the production of light by cavitation) may also be related to complex sonochemical or sonodynamic reactions. However, the exact mechanism related to cytotoxicity still remains to be found. Acoustic cavitation can chemically activate photosensitive drugs specifically bound to malignant cell membranes, which could result in cell surface disruption. Recent experiments showed that adult T leukemia cells were specifically killed by low intensity ultrasound of 0.3 W/cm2 in the presence of porfimer sodium [6]. Abe et al. [7] developed a strategy for the selective destruction of cancer cells by ultrasonic irradiation in the presence of an antibody-conjugated photosensitizer. A photoimmunoconjugate (PIC) between ATX-70, a photosensitizer of a gallium–porphyrin analogue, and F11-39, a high affinity monoclonal antibody (MAb) against carcinoembryonic antigen (CEA), which is often overexpressed in various carcinoma cells, was prepared. The conjugate, designated F39/ATX-70, retained immunoreactivity against purified CEA and CEA-expressing cells, as determined by an enzyme-linked immunosorbent assay, flow cytometry
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and immunofluorescence microscopic analysis. The cytotoxicity of F39/ATX-70 against CEA-expressing human gastric carcinoma cells in vitro was found to be greater than that of ATX-70 when applied in combination with ultrasound irradiation. In vivo anti-tumor effects in a mouse xenograft model resulted in a marked growth inhibition of tumor compared with ultrasound alone or ultrasound after administration of ATX-70. 2.1. Ultrasound-induced apoptosis Apoptosis is a process of deliberate life relinquishment by a cell in a multicellular organism such as the human: programmed cell death. In contrast to necrosis, which is a form of cell death that results from acute cellular injury, apoptosis is carried out in an ordered process that generally confers advantages during an organism’s life cycle, particularly in the physiologic balance of tissues in the body. Modalities such as radiation therapy, chemotherapy and hyperthermia have been employed to induce apoptosis of unwanted cells in the body (e.g., cancer cells). It has been shown that low intensity US can induce cell killing even without significant temperature rise and even at very low intensities (less than 0.5 W/cm2). Although lysis is commonly involved in US-induced cell killing in vitro, in vivo it is less unlikely due to structural configurations of cells within the body. Apoptosis induction by US has been observed both in vitro and in vivo. Some factors that enhance these effects and factors that inhibit them have been identified and characterized [8,9]. Here we review the type of apoptosis induced by US, ways of optimizing apoptosis induction, and the possible mechanisms involved. The finding that ultrasound can induce apoptosis in cancer cell lines [10] was a surprise to those who knew that US is a non-ionizing radiation, but exciting to those trying to find therapeutic applications of US. More and more research work is now being undertaken to optimize USmediated apoptosis for possible therapeutic application, while others are trying to investigate the mechanism involved in the apoptosis induction. A role for free radicals in ultrasound-induced apoptosis has been suggested. When active oxygen scavengers were shown to reduce the apoptosis induced by US, it was believed that sonochemical mechanism was involved [8]. Although such inhibition has also been confirmed by other studies, extensive study of the role of free radicals in apoptosis induction has shown that free radicals generated primarily by the sonication did not play a major role in apoptosis induction. However, free radicals generated secondarily by mitochondria are those involved in apoptosis [11]. In these studies, evidence that US-induced apoptosis involves the mitochondria-caspase pathways and that increased intracellular calcium concentration is an important part of the events leading to apoptosis induction was shown. But the question remained as to how US triggers the apoptotic mechanism to set in US as a form of mechan-
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ical wave was considered. However, the radiation forces involved were not enough to trigger a ‘‘stress” sufficient for the cells to commit suicide (induced apoptosis). The important factor of gases trapped within a fluid medium provides a more viable explanation, namely cavitation. Cavitation may be defined as the physical effect of the sound waves on microenvironmental gases within fluid. Both stable cavitations (oscillating bubbles) and inertial cavitation (collapsing bubbles) are capable of producing damage to cell membranes. Using lower intensities of US we have shown that even below the threshold for inertial cavitations, apoptosis can be induced. Since free radicals are produced only at intensities above the threshold for inertial cavitation such a finding confirms that free radicals generated by US do not play an important role in apoptosis induction. Such findings also lead us to believe that membrane damage, or a sonomechanical effect is the trigger for apoptosis. Though apoptosis induction is a result of membrane damage, such membrane damage must be repaired by the cells for the apoptosis process to continue to its final stage of DNA fragmentation. Convinced that mechanical effects are responsible for the bioeffects being described here, we then proposed that membrane damage is pivotal in all the cellular damage, since membranes are the most susceptible structure and are the ones exposed to any gross external mechanical stress—in this case caused by US. The degree of membrane damage and the ability of the cells to repair the damage determines the mode of cell death; it may be instant lysis, necrosis or apoptosis. Although damaged cells which are able to successfully repair the damage may eventually survive, some of these cells will die by apoptosis or necrosis. Apoptosis is a form of cell death that occurs during the natural development of organs and tissues, and in response to specific types of cellular stress in order to delete irreversibly damaged and/or undesirable cells. In contrast, necrosis is always inappropriate or accidental, and usually occurs under extreme adverse environmental conditions. Although apoptotic cells will eventually undergo secondary necrosis, also known as late apoptosis, any disruption of the membrane may abort the process and kill the cell by necrosis or lysis. In such a case, inhibition of the end points of apoptosis such as DNA fragmentation may be observed. Several findings show that in US-induced apoptosis, necrosis is an unavoidable consequence. Such conditions will be likely to have some cells dying in a type of cell death that combines some of the features of apoptosis (requirement for protein synthesis) and necrosis (cytoplasmic vacuolation)—called paraptosis. Electron microscopy of sonicated cells which had been identified as ‘‘apoptotic”, also showed some cytoplasmic vacuolations. In addition, unusually early appearance of secondary necrotic cells and low yield in DNA fragmentation compared to the level of apoptosis detected by flow cytometry and by microscopy, could also be explained by this mechanism. The above findings and the underlying hypothesis of the mechanism of cell killing induced by US led us to consider
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that certain exposure parameters (such as ultrasound frequency, pulse repetition frequency, duty factor and intensity) would optimize killing from a desired mode of cell death, in this case apoptosis. Certain US exposure conditions resulted in optimal apoptosis induction and the intensity needed to induce apoptosis under such optimal conditions was reduced when an echo-contrast agent (ECA), engineered microbubbles designed to enhance echogenicity in sonography, was added to the cell suspension during sonication. Apoptosis induction was observed even at intensities lower than 0.3 W/cm2, which is apparently much lower than that of the maximum limit (intensity considered safe) set by the British Medical Ultrasound Society, (0.72 W/cm2). Apart from optimization of US-induced apoptosis by variation of exposure conditions, US can also be combined with other apoptosis-inducing modalities to attain a higher level of apoptosis. A study shows that hyperthermiainduced apoptosis can be enhanced by US at intensities even below threshold for cell killing with US alone [10]. Use of echo-contrast agents can also result in a higher level of apoptosis [12], even intensities lower than 0.2 W/cm2. Some anti-cancer drugs and other chemical agents with or without therapeutic effects on their own [13,14] have been shown also to augment apoptosis induction by US. Physical modification of cells or tissue may also increase the susceptibility of cells to the bioeffects of US [15] including apoptosis induction. 2.2. Ultrasound-mediated gene transfection (sonotransfection) Most human diseases (e.g., genetic disorders, cancers and metabolic disorders) are somehow linked to a particular gene or genes [16]. This discovery has brought unprecedented progress in the science of therapy. Gene therapy, in particular, is formulated for treating certain ailments and is carried out by introducing recombinant genes into the somatic cells to alter the course of a disease process. Several strategies have been designed to attain transfection and eventual integration into the nucleus of the target cells. Viral-mediated gene transfer is efficient for this task, but cytotoxicity, cytopathy and antigenicity are among the limiting factors of this therapy. The use of US in therapy and also in gene transfection has been investigated both in vitro and in vivo [17]. Greenleaf et al. [18] first used non-viral plasmid DNA in combination with ultrasound. It was postulated that ultrasound would initiate the delivery of DNA through the cellular membrane into the cells and would not at the same time produce irreversible damage to the cells. Bao et al. [19] further added microbubbles to increase the rate of DNA transfer. Ward et al. [20] and Tachibana and Tachibana [21] had earlier suggested that the liquid microjets induced during collapse of microbubbles could be the mechanism by which DNA easily penetrates the cell membrane. Scanning electron microscopy and high-speed video imaging
technologies have recently revealed images of collapsing microbubbles and ruptured cell membrane surfaces to support this theory. Unger et al. [22] introduced the concept of ‘‘tailor made” microbubbles and nanobubbles that target specific tissue lesions to deliver drugs and DNA. Today, the concept of applying ultrasound as a means of altering pharmacokinetics in various tissues and drug permeability through cell membranes has expanded into a whole new field ranging from gene therapy to anti-cancer drugs. A new generation of microbubbles and nano-sized bubbles are under development specifically for the purpose of ‘‘target and deliver” drug treatment with non-thermal ultrasound. So far the mechanisms remain generally unknown, but the leading belief is that US increases DNA uptake by the cells. A study investigated the effects of US on liposome-mediated transfection using three different types of liposomes, which had previously been shown to mediate transfection with different degrees of efficiency, showed that US significantly increased luciferase expression when combined with liposomes [23]. Optimal enhancement was observed when US was given 2 h after incubation of the cells with the liposome–DNA complexes, suggesting that US works to enhance transfection only after cells had enough time to interact with the DNA. More recently, we have shown that improving the survival of cells after induction of membrane damage leads to an improved transfection rate in five cancer cell lines (U937, HeLa, PC-3, Meth A and T-24). It was also shown that optimized US exposure conditions resulted in a better transfection rate than that of electrotransfection and liposome-mediated transfection. Use of engineered microbubbles in sonotransfection has therefore widened the therapeutic potential of this method. 2.3. Cavitation and the use of microbubbles Interaction of diagnostic US with gas bodies or microbubbles produces a useful contrast effect in medical imaging, but the same interaction also represents a mechanism for bioeffects [24]. Because cavitation is primarily responsible in causing cellular membrane damage, the size of the bubbles, the affinity of the bubble to the DNA and the target cells, and the toxicity of the substance and gas that make up the bubbles are important factors in determining the outcome. Microbubble agents can be designed such that they can carry therapeutic genes or agents while being equipped with targeting ligands that would specifically attach to the particular cells being targeted [25]. These types of microbubbles are especially useful in drug or gene delivery by which bubbles that carry the therapeutic agents (e.g., drug or gene) are first localized before sonication, thus focusing much of the desired bioeffects on the target tissues and cells, and therefore also minimizing unwanted effects in the normal, non-target, tissues. Targeted microbubbles and acoustically active perfluorocarbon nanoemulsions with specific ligands can be developed for detecting disease at the molecular level and
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targeted drug and gene delivery. Bioactive compounds can be incorporated into these carriers for site-specific delivery [22]. Use of receptor selective targeted microbubbles has also been shown to improve binding of microbubbles to vascular thrombi in both in vitro and in vivo settings. As a result the use of therapeutic US with intravenous microbubbles has a high success rate in recanalizing deeply located thrombosed arteriovenous grafts when performed under diagnostic US guidance. A study also showed that when a recombinant adenovirus containing beta-galactosidase and driven by a constitutive promoter attached to the surface of albumin-coated perfluoropropane-filled microbubbles was used, sonication resulted to a 10-fold higher beta-galactosidase activity. The above findings show how important engineered and custom-designed microbubbles are in attaining the desired level of bioeffects with lowintensity US in therapy. 2.4. Synergistic effect of combined ultrasound and antibiotics The enhanced effect of chemotherapy against cancer has frequently been investigated using different modalities such as radiation, hyperthermia and ultrasound. In this study, we investigated the effects of ultrasound and antibiotics against chlamydia. These are preliminary results of the first step of research to investigate the in vitro response of Chlamydia trachomatis-infected human epithelial cells to a combination of ultrasound and two types of antibiotics. 2.4.1. Materials and methods Chlamydial strain and cell lines: C. trachomatis serovar E/UW-5/Cx was prepared in McCoy cells and propagated using a previously reported method [26]. The mouse fibroblast cell line McCoy cell (CRL 1696) and human epithelial cell line HeLa 229 cell (CLL 2.1) were maintained in Dulbecco’s modified Eagle medium (DMEM, Invitrogen, Grand Island, NY, USA) supplemented by 10% heat-inactivated fetal calf serum (FCS, Invitrogen) and 100 g/ml streptomycin. Infection of HeLa cells: The HeLa cells were seeded into a 24-well plate with lumoxTM fluorocarbon film base (optically clear, 50 m-thin, gas permeable film, Greiner bio-one, Gottingen, Germany). Stocks of chlamydial strain were diluted with sucrose–phosphate–glutamate (SPG) medium [26]. Chlamydial suspensions of 0.5 104 inclusion-forming units (IFUs) in 0.25 ml SPG medium were inoculated onto the monolayer cultures of HeLa cells (1 104 cells/ well). This is equivalent to a multiplicity of infection of 0.5. Preparation of bubble liposomes: Bubble liposomes were prepared using a previously described method [27]. The sterilized vials containing 2 ml of liposome suspension (lipid concentration: 1 mg/ml) were pressurised with 7.5 ml of perfluoropropane gas. Immunofluorescence staining and fluorescence microscopy: At 48 or 72 h after infection, the infected monolayers were washed with PBS, and the cells were fixed with 20 °C chilled methanol. After the specimens had been
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dried, the inclusion bodies were stained with fluorescein isohtiocyanate (FITC)-labeled monoclonal antibody against C. trachomatis lipopolysaccharides (Progen Biotechnik, Heidelberg, Germany) for 30 min at room temperature. The cells were rinsed with saline, and the films were cut away from the plate, and mounted in a 1:1 solution of PBS–glycerol. The antibody staining resulted in yellowgreen chlamydial proteins, and Evans blue counterstaining yielded red eukaryotic cells. Infectivity of chlamydia was presented as the number of inclusion-forming units (IFUs). Antibiotics and measurements of MIC: Doxycycline (DOX, Sigma Chemicals) and ceftizoxime (CZX, Fujisawa Yakuhin Kogyou, Tokyo, Japan) were obtained in powder form. Both antibiotics were diluted with saline, and were dissolved in maintenance medium at a concentration of 100 g/ml and frozen at 80 °C until used. The minimum inhibitory concentrations (MICs) were determined using a previously described method [28]. To determine the MICs, the cover slips were stained and observed as described above. The lowest concentration of the antimicrobial agent that completely inhibited the formation of visible chlamydial inclusions was determined as the MIC. Ultrasound exposure: An acoustically transparent gel (Pharmaceutical Innovations Inc., Newark, NJ) was placed between the top of ultrasound probe and the film base of the 24-well plate (Fig. 1). Ultrasound irradiation was done using a 1.0 MHz device (SonoPore KTAC-4000, NepaGene, Chiba, Japan) at intensity (ISATA) of 0.15 W/cm2 (duty cycle of 25%) for 20 s immediately after addition of bubble liposomes. Measurement of cell viability: The Trypan blue exclusion test was carried out by mixing 200 ml of the suspension of HeLa cells with an equal amount of 0.3% Trypan blue solution (Sigma Chemicals) in PBS. After 5 min incubation at room temperature, the number of cells excluding Trypan blue was counted using a C-Chip disposable hemocytometer (Digital Bio Technology Co., Gyeonggi, Korea) to estimate the number of viable cells immediately after sonication.
Fig. 1. Ultrasound exposure: an acoustically transparent gel was placed between the ultrasound probe and the film base of 24-well plate. HeLa cells were irradiated using therapeutic ultrasound (1.0 MHz) at an intensity (ISATA) of 0.15 W/cm2 (duty cycle of 25%) for 20 s after addition of bubble liposomes.
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Measurement of infectivity of chlamydiae: The 1.0 ml of chlamydial suspensions in sucrose–phosphate–glutamate medium with application of ultrasound and/or microbubbles were inoculated into triplicate cultures of McCoy cells in order to estimate the infectivity immediately after sonication. 0.25 ml of chlamydial suspensions was inoculated onto the monolayer culture of McCoy cells. After centrifugation at 1000g for 60 min, the inoculum was decanted, and the cells were washed in medium to remove the nonadsorbed chlamydiae, and were then further incubated in 1.0 ml of maintenance medium. 2.4.2. Results Ultrasonic enhancement of antibiotic action on C. trachomatis-infected HeLa cells: the MIC of DOX for C. trachomatis-infected HeLa cells was determined to be 0.03 g/ ml. DOX at 1/2 MIC (0.015 g/ml) was added to the infected cultures, and ultrasound exposures were carried out immediately after addition of bubble liposomes (50 g/ ml). The results of ultrasound experiments showed that ultrasound alone or bubble liposomes alone did not decrease the formation of inclusions administered with DOX (Fig. 2). However, DOX at 1/2 MIC in combination with microbubble-enhanced ultrasound reduced the number of IFUs to 66 ± 39%, at intensity of 0.15 w/cm2, compared with that administered DOX at 1/2 MIC only. The MIC of CZX for C. trachomatis-infected HeLa cells could not be determined because intracellular pathogens were resistant to CZX, we therefore tried to use concentrations of 0.125, 0.25, 0.5 and 1.0 g/ml. These were higher concentrations than expected. The 1.0 g/ml CZX was the most effective of these concentrations (data not shown). Similarly to the administration of DOX, 1.0 g/ml CZX in combination with microbubble-enhanced ultrasound also
Fig. 2. Ultrasonic enhancement of antibiotic action on C. trachomatis infected HeLa cells: the MIC of DOX for C. trachomatis-infected HeLa cells was determined to be 0.03 lg/ml. When DOX at 1/2 MIC (0.015 lg/ ml) was added to the infected cultures, ultrasound irradiation was carried out immediately after addition of bubble liposomes (50 lg/ml). Ultrasound alone (US(0.15) only) or bubble liposomes alone (bubble only) did not decrease the formation of inclusions administered with DOX. Significant decrease in the formation of inclusion bodies was noted when sonication was carried out in the presence of bubble liposomes (bubble + US(0.15)).
reduced the number of IFUs to 53 ± 32% at an intensity of 0.15 w/cm2, compared with that administered with 1.0 g/ml CZX only. However, these reductions were not statistically significant. 3. Conclusions and outlook Research on the bioeffects of ultrasound alone and in the presence of various drugs in patients has only just begun. Most investigations are still highly experimental and far from being applicable in the clinical situation. However, such techniques as HIFU therapy for cancer are already beginning to be used widely for patients as alternative non-invasive modalities. Additionally, there definitely exists an interesting biological phenomenon that cannot be ignored when non-thermal ultrasound mechanisms are involved. The interaction between ultrasound and drugs can range from a change in permeability of biological membrane to the manipulation of DNA into the cells. Understanding the mechanism of micro/nano bubble collapse or chemical activation of drugs will eventually result in optimization of the acoustics and the design of ultrasound devices for sonodynamic therapy. Acknowledgements This review was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas (15300187, 16500328 and 18800075) from the Ministry of Education, Culture, Sports, Science and Technology, Japan and from Fukuoka University Central Research Institute. References [1] F.W. Kremkau, J.S. Kaufmann, M.M. Walker, P.G. Burch, C.L. Spurr, Ultrasonic enhancement of nitrogen mustard cytotoxicity in mouse leukemia, Cancer 37 (4) (1976) 1643–1647. [2] G.H. Harrison, E.K. Balcer-Kubiczek, Pulsed ultrasound and neoplastic transformation in vitro, Ultrasound in Medicine and Biology 17 (6) (1991) 627–632. [3] S. Umemura, N. Yumita, R. Nishigaki, K. Umemura, Mechanism of cell damage by ultrasound in combination with hematoporphyrin, Japanese Journal of Cancer Research 81 (9) (1990) 962–966. [4] N. Miyoshi, V. Misik, P. Riesz, Sonodynamic toxicity of gallium– porphyrin analogue ATX-70 in human leukemia cells, Radiation Research 148 (1) (1997) 43–47. [5] N. Yumita, I. Sakata, S. Nakajima, S. Umemura, Ultrasonically induced cell damage and active oxygen generation by 4-formyloximeetylidene-3-hydroxyl-2-vinyl-deuterio-porphynyl(IX)-6-7-diaspartic acid: on the mechanism of sonodynamic activation, Biochimica et Biophysica Acta 1620 (1–3) (2003) 179–184. [6] K. Tachibana, T. Uchida, S. Hisano, E. Morioka, Eliminating adult T-cell leukaemia cells with ultrasound, Lancet 349 (9048) (1997) 325. [7] H. Abe, M. Kuroki, K. Tachibana, T. Li, A. Awasthi, A. Ueno, et al., Targeted sonodynamic therapy of cancer using a photosensitizer conjugated with antibody against carcinoembryonic antigen, Anticancer Research 22 (3) (2002) 1575–1580. [8] L.B. Feril Jr., T. Kondo, Biological effects of low intensity ultrasound: the mechanism involved, and its implications on therapy and on biosafety of ultrasound, Journal of Radiation Research 45 (4) (2004) 479–489.
K. Tachibana et al. / Ultrasonics 48 (2008) 253–259 [9] L.B. Feril Jr., T. Kondo, Major factors involved in the inhibition of ultrasound-induced free radical production and cell killing by presonication incubation or by high cell density, Ultrasonics Sonochemistry 12 (5) (2005) 353–357. [10] L.B. Feril Jr., T. Kondo, Z.G. Cui, Y. Tabuchi, Q.L. Zhao, H. Ando, et al., Apoptosis induced by the sonomechanical effects of low intensity pulsed ultrasound in a human leukemia cell line, Cancer Letters 221 (2) (2005) 145–152. [11] H. Honda, T. Kondo, Q.L. Zhao, L.B. Feril Jr., H. Kitagawa, Role of intracellular calcium ions and reactive oxygen species in apoptosis induced by ultrasound, Ultrasound in Medicine and Biology 30 (5) (2004) 683–692. [12] H. Ando, L.B. Feril Jr., T. Kondo, Y. Tabuchi, R. Ogawa, Q.-L. Zhao, Z.-G. Cui, S. Umemura, H. Yoshikawa, T. Misaki, An echo-contrast agent, Levovist, lowers the ultrasound intensity required to induce apoptosis of human leukemia cells, Cancer Letters 242 (2006) 37–45. [13] L.B. Feril Jr., T. Kondo, S. Umemura, T. Tachibana, A.H. Manalo, P. Riesz, Sound waves and antineoplastic drugs: the possibility of an enhanced combined anticancer therapy, Journal of Medical Ultrasonics 29 (Winter) (2002) 173–187. [14] L.B. Feril Jr., Y. Tsuda, T. Kondo, Q.L. Zhao, R. Ogawa, Z.G. Cui, et al., Ultrasound-induced killing of monocytic U937 cells enhanced by 2,20 -azobis(2-amidinopropane) dihydrochloride, Cancer Science 95 (2) (2004) 181–185. [15] L.B. Feril Jr., T. Kondo, K. Takaya, P. Riesz, Enhanced ultrasoundinduced apoptosis and cell lysis by a hypotonic medium, International Journal of Radiation Biology 80 (2) (2004) 165–175. [16] K.T. Zondervan, L.R. Cardon, The complex interplay among factors that influence allelic association, Nature Reviews Genetics 5 (2) (2004) 89–100. [17] C.M. Newman, A. Lawrie, A.F. Brisken, D.C. Cumberland, Ultrasound gene therapy: on the road from concept to reality, Echocardiography 18 (4) (2001) 339–347. [18] W.J. Greenleaf, M.E. Bolander, G. Sarkar, M.B. Goldring, J.F. Greenleaf, Artificial cavitation nuclei significantly enhance acousti-
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Ultrasonics 48 (2008) 253–259 www.elsevier.com/locate/ultras
Sonodynamic therapy Katsuro Tachibana *, Loreto B. Feril Jr., Yurika Ikeda-Dantsuji Department of Anatomy, Fukuoka University School of Medicine, 7-45-1 Nanakuma, Jonan, Fukuoka 814-0180, Japan Received 19 July 2007; received in revised form 26 November 2007; accepted 28 February 2008 Available online 7 March 2008
Abstract Recently, there have been numerous reports on the application of non-thermal ultrasound energy for treating various diseases in combination with drugs. Furthermore, the introduction of microbubbles and nanobubbles as carriers/enhancers of drugs has added a whole new dimension to therapeutic ultrasound. Non-thermal mechanisms for effects seen include various forms of energy due to cavitation, acoustic streaming, micro jets and radiation force which increases possibilities for targeting tissue with drugs, enhancing drug effectiveness or even chemically activating certain materials. Examples such as enhancement of thrombolytic agents by ultrasound have proven to be beneficial for acute stroke patients and peripheral arterial occlusions. Non-invasive low intensity focused ultrasound in conjunction with anti-cancer drugs may help to reduce tumor size and lessen recurrence while reducing severe drug side effects. Chemical activation of drugs by ultrasound energy for treatment of atherosclerosis and tumors is another new field recently termed as ‘‘Sonodynamic therapy”. Lastly, advances in molecular imaging have aroused great expectations in applying ultrasound for both diagnosis and therapy simultaneously. Microbubbles or nanobubbles targeted at the molecular level will allow medical doctors to make a final diagnosis of a disease using ultrasound imaging and then immediately proceed to a therapeutic ultrasound treatment. Ó 2008 Elsevier B.V. All rights reserved. Keywords: Ultrasound; Sonodynamic therapy; Sonotransfection
Contents 1. 2.
3.
*
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sonodynamic therapy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Ultrasound-induced apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Ultrasound-mediated gene transfection (sonotransfection) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Cavitation and the use of microbubbles. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Synergistic effect of combined ultrasound and antibiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.1. Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.2. Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions and outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Corresponding author. E-mail address: [email protected] (K. Tachibana).
0041-624X/$ - see front matter Ó 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.ultras.2008.02.003
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1. Introduction Although there are thousands of types of drugs available to use today, the number of drugs that have never reached the market may be ten- or even a 100-fold greater. These drugs either had severe side effects or proved to be ineffective for some reason during preclinical or early clinical trials. Examples include various agents for cancer chemotherapy. Some of these agents, when administered systemically, can kill solid tumors, but at the same time severely damage healthy tissues and organs. Researchers have attempted to address these problems using the concept of a ‘‘drug delivery system (DDS)” since the 70s. The two keywords for DDS are ‘‘targeting” and ‘‘controlled release”. The major goal was to avoid severe side effects by reducing the total dosage but at the same time concentrating the drug at the target site. Additionally, controlled drug release permits more control over the amount of drug administered in a certain time frame. Recent progress in the application of ultrasound energy for DDS has demonstrated it to be a promising modality for both ‘‘targeting” and ‘‘controlled release” of drugs. Microbubbles and nanobubbles can carry a certain drug at specific target site and release it at a particular time using ultrasound. Ultrasound-sensitive materials such as hematoporphyrin, a nontoxic agent, can also be activated by ultrasound in a localized volume and can thus result in the killing of cancer cells. A wide variety of applications for drug delivery with ultrasound are currently under investigation in many fields. The most futuristic investigation under progress is the use of ultrasound for gene therapy. DNA can be injected through the cell membrane as if a ‘‘micro syringe” were being used with ultrasound energy and induce transfection. Induction of gene transfer into the cell by ultrasound could result in regeneration of blood vessels, nerves or any other tissue. With the help of microbubbles or nanobubbles, it is possible to change the permeability of drugs at the microscopic cell membrane level. 2. Sonodynamic therapy The term ‘‘Sonodynamic therapy” is often used for all non-thermally related therapeutic ultrasound applications mentioned above ranging from induction of apoptosis, combining with chemotherapy, to ultrasound gene therapy. It was however, originally coined by Yumita and Umemura who first discovered the phenomenon of sonochemical activation of photosensitive materials by ultrasound for cancer therapy. Some researchers tend to prefer this narrower meaning where a sonochemical mechanism is involved. As the exact mechanism is not well determined, further discussion about the definition of this term is needed. Nevertheless, it is well known that Kremkau et al. [1] were the first to study the cytotoxic effects of various anti-cancer drugs, such as nitrogen mustard, to mouse leukemia L1210 cells in combination with ultrasound irradiation. It was discovered from these experiments that the
increase in drug efficiency could be explained by the nonthermal effect of ultrasound interacting with the drugs. Experiments performed by Harrison and Balcer-Kubiczek [2] using low-intensity ultrasound with no temperature increase showed similar results. It was pointed out that low-level ultrasound may have altered the cell membrane, thus changing its permeability to the drug. Marked enhancement by ultrasound of the cytotoxicity of adriamycin and amphotericin B against Chinese hamster ovary cells HA1 at lower energies was demonstrated by Harrison. Compared to radiotherapy and light irradiation for the treatment of leukemia and solid cancers, ultrasonic irradiation is superior in that the applied energy can be focused solely on the tumor volume to be treated, with little effect upon normal tissues, and is better than light irradiation in the degree of penetration. Umemura et al. [3] pioneered the development of non-thermal ultrasound to activate a group of chemicals that were originally used as light activated chemicals for cancer therapy. This new ultrasound therapy falls into the narrow definition of sonodynamic therapy. Ultrasonic waves are known to cause chemical effects if cavitation occurs; for example, irradiation of water can generate hydrogen peroxide. It was found that certain drugs, upon ultrasonic irradiation, create active oxygen species such as superoxide radicals and singlet oxygen, and that the active oxygen thus formed effectively destructs cancer tissues [4]. The agents themselves have no anti-tumor activity and are very low in toxicity, exhibit anti-tumor activity only by the chemical action caused by ultrasonic irradiation. Thus, there is significant less risk of causing any systemic effects. In addition, these drugs act exclusively upon tumor tissues when combined with ultrasonic irradiation, with no adverse effects upon normal tissues. An important factor involved in ultrasound irradiation is the chemical reactions induced during the course of violent microbubble collapse. Short lived free radicals that may alter various compounds leading to cell killing can be created by ultrasound [5]. Sonoluminescence (the production of light by cavitation) may also be related to complex sonochemical or sonodynamic reactions. However, the exact mechanism related to cytotoxicity still remains to be found. Acoustic cavitation can chemically activate photosensitive drugs specifically bound to malignant cell membranes, which could result in cell surface disruption. Recent experiments showed that adult T leukemia cells were specifically killed by low intensity ultrasound of 0.3 W/cm2 in the presence of porfimer sodium [6]. Abe et al. [7] developed a strategy for the selective destruction of cancer cells by ultrasonic irradiation in the presence of an antibody-conjugated photosensitizer. A photoimmunoconjugate (PIC) between ATX-70, a photosensitizer of a gallium–porphyrin analogue, and F11-39, a high affinity monoclonal antibody (MAb) against carcinoembryonic antigen (CEA), which is often overexpressed in various carcinoma cells, was prepared. The conjugate, designated F39/ATX-70, retained immunoreactivity against purified CEA and CEA-expressing cells, as determined by an enzyme-linked immunosorbent assay, flow cytometry
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and immunofluorescence microscopic analysis. The cytotoxicity of F39/ATX-70 against CEA-expressing human gastric carcinoma cells in vitro was found to be greater than that of ATX-70 when applied in combination with ultrasound irradiation. In vivo anti-tumor effects in a mouse xenograft model resulted in a marked growth inhibition of tumor compared with ultrasound alone or ultrasound after administration of ATX-70. 2.1. Ultrasound-induced apoptosis Apoptosis is a process of deliberate life relinquishment by a cell in a multicellular organism such as the human: programmed cell death. In contrast to necrosis, which is a form of cell death that results from acute cellular injury, apoptosis is carried out in an ordered process that generally confers advantages during an organism’s life cycle, particularly in the physiologic balance of tissues in the body. Modalities such as radiation therapy, chemotherapy and hyperthermia have been employed to induce apoptosis of unwanted cells in the body (e.g., cancer cells). It has been shown that low intensity US can induce cell killing even without significant temperature rise and even at very low intensities (less than 0.5 W/cm2). Although lysis is commonly involved in US-induced cell killing in vitro, in vivo it is less unlikely due to structural configurations of cells within the body. Apoptosis induction by US has been observed both in vitro and in vivo. Some factors that enhance these effects and factors that inhibit them have been identified and characterized [8,9]. Here we review the type of apoptosis induced by US, ways of optimizing apoptosis induction, and the possible mechanisms involved. The finding that ultrasound can induce apoptosis in cancer cell lines [10] was a surprise to those who knew that US is a non-ionizing radiation, but exciting to those trying to find therapeutic applications of US. More and more research work is now being undertaken to optimize USmediated apoptosis for possible therapeutic application, while others are trying to investigate the mechanism involved in the apoptosis induction. A role for free radicals in ultrasound-induced apoptosis has been suggested. When active oxygen scavengers were shown to reduce the apoptosis induced by US, it was believed that sonochemical mechanism was involved [8]. Although such inhibition has also been confirmed by other studies, extensive study of the role of free radicals in apoptosis induction has shown that free radicals generated primarily by the sonication did not play a major role in apoptosis induction. However, free radicals generated secondarily by mitochondria are those involved in apoptosis [11]. In these studies, evidence that US-induced apoptosis involves the mitochondria-caspase pathways and that increased intracellular calcium concentration is an important part of the events leading to apoptosis induction was shown. But the question remained as to how US triggers the apoptotic mechanism to set in US as a form of mechan-
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ical wave was considered. However, the radiation forces involved were not enough to trigger a ‘‘stress” sufficient for the cells to commit suicide (induced apoptosis). The important factor of gases trapped within a fluid medium provides a more viable explanation, namely cavitation. Cavitation may be defined as the physical effect of the sound waves on microenvironmental gases within fluid. Both stable cavitations (oscillating bubbles) and inertial cavitation (collapsing bubbles) are capable of producing damage to cell membranes. Using lower intensities of US we have shown that even below the threshold for inertial cavitations, apoptosis can be induced. Since free radicals are produced only at intensities above the threshold for inertial cavitation such a finding confirms that free radicals generated by US do not play an important role in apoptosis induction. Such findings also lead us to believe that membrane damage, or a sonomechanical effect is the trigger for apoptosis. Though apoptosis induction is a result of membrane damage, such membrane damage must be repaired by the cells for the apoptosis process to continue to its final stage of DNA fragmentation. Convinced that mechanical effects are responsible for the bioeffects being described here, we then proposed that membrane damage is pivotal in all the cellular damage, since membranes are the most susceptible structure and are the ones exposed to any gross external mechanical stress—in this case caused by US. The degree of membrane damage and the ability of the cells to repair the damage determines the mode of cell death; it may be instant lysis, necrosis or apoptosis. Although damaged cells which are able to successfully repair the damage may eventually survive, some of these cells will die by apoptosis or necrosis. Apoptosis is a form of cell death that occurs during the natural development of organs and tissues, and in response to specific types of cellular stress in order to delete irreversibly damaged and/or undesirable cells. In contrast, necrosis is always inappropriate or accidental, and usually occurs under extreme adverse environmental conditions. Although apoptotic cells will eventually undergo secondary necrosis, also known as late apoptosis, any disruption of the membrane may abort the process and kill the cell by necrosis or lysis. In such a case, inhibition of the end points of apoptosis such as DNA fragmentation may be observed. Several findings show that in US-induced apoptosis, necrosis is an unavoidable consequence. Such conditions will be likely to have some cells dying in a type of cell death that combines some of the features of apoptosis (requirement for protein synthesis) and necrosis (cytoplasmic vacuolation)—called paraptosis. Electron microscopy of sonicated cells which had been identified as ‘‘apoptotic”, also showed some cytoplasmic vacuolations. In addition, unusually early appearance of secondary necrotic cells and low yield in DNA fragmentation compared to the level of apoptosis detected by flow cytometry and by microscopy, could also be explained by this mechanism. The above findings and the underlying hypothesis of the mechanism of cell killing induced by US led us to consider
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that certain exposure parameters (such as ultrasound frequency, pulse repetition frequency, duty factor and intensity) would optimize killing from a desired mode of cell death, in this case apoptosis. Certain US exposure conditions resulted in optimal apoptosis induction and the intensity needed to induce apoptosis under such optimal conditions was reduced when an echo-contrast agent (ECA), engineered microbubbles designed to enhance echogenicity in sonography, was added to the cell suspension during sonication. Apoptosis induction was observed even at intensities lower than 0.3 W/cm2, which is apparently much lower than that of the maximum limit (intensity considered safe) set by the British Medical Ultrasound Society, (0.72 W/cm2). Apart from optimization of US-induced apoptosis by variation of exposure conditions, US can also be combined with other apoptosis-inducing modalities to attain a higher level of apoptosis. A study shows that hyperthermiainduced apoptosis can be enhanced by US at intensities even below threshold for cell killing with US alone [10]. Use of echo-contrast agents can also result in a higher level of apoptosis [12], even intensities lower than 0.2 W/cm2. Some anti-cancer drugs and other chemical agents with or without therapeutic effects on their own [13,14] have been shown also to augment apoptosis induction by US. Physical modification of cells or tissue may also increase the susceptibility of cells to the bioeffects of US [15] including apoptosis induction. 2.2. Ultrasound-mediated gene transfection (sonotransfection) Most human diseases (e.g., genetic disorders, cancers and metabolic disorders) are somehow linked to a particular gene or genes [16]. This discovery has brought unprecedented progress in the science of therapy. Gene therapy, in particular, is formulated for treating certain ailments and is carried out by introducing recombinant genes into the somatic cells to alter the course of a disease process. Several strategies have been designed to attain transfection and eventual integration into the nucleus of the target cells. Viral-mediated gene transfer is efficient for this task, but cytotoxicity, cytopathy and antigenicity are among the limiting factors of this therapy. The use of US in therapy and also in gene transfection has been investigated both in vitro and in vivo [17]. Greenleaf et al. [18] first used non-viral plasmid DNA in combination with ultrasound. It was postulated that ultrasound would initiate the delivery of DNA through the cellular membrane into the cells and would not at the same time produce irreversible damage to the cells. Bao et al. [19] further added microbubbles to increase the rate of DNA transfer. Ward et al. [20] and Tachibana and Tachibana [21] had earlier suggested that the liquid microjets induced during collapse of microbubbles could be the mechanism by which DNA easily penetrates the cell membrane. Scanning electron microscopy and high-speed video imaging
technologies have recently revealed images of collapsing microbubbles and ruptured cell membrane surfaces to support this theory. Unger et al. [22] introduced the concept of ‘‘tailor made” microbubbles and nanobubbles that target specific tissue lesions to deliver drugs and DNA. Today, the concept of applying ultrasound as a means of altering pharmacokinetics in various tissues and drug permeability through cell membranes has expanded into a whole new field ranging from gene therapy to anti-cancer drugs. A new generation of microbubbles and nano-sized bubbles are under development specifically for the purpose of ‘‘target and deliver” drug treatment with non-thermal ultrasound. So far the mechanisms remain generally unknown, but the leading belief is that US increases DNA uptake by the cells. A study investigated the effects of US on liposome-mediated transfection using three different types of liposomes, which had previously been shown to mediate transfection with different degrees of efficiency, showed that US significantly increased luciferase expression when combined with liposomes [23]. Optimal enhancement was observed when US was given 2 h after incubation of the cells with the liposome–DNA complexes, suggesting that US works to enhance transfection only after cells had enough time to interact with the DNA. More recently, we have shown that improving the survival of cells after induction of membrane damage leads to an improved transfection rate in five cancer cell lines (U937, HeLa, PC-3, Meth A and T-24). It was also shown that optimized US exposure conditions resulted in a better transfection rate than that of electrotransfection and liposome-mediated transfection. Use of engineered microbubbles in sonotransfection has therefore widened the therapeutic potential of this method. 2.3. Cavitation and the use of microbubbles Interaction of diagnostic US with gas bodies or microbubbles produces a useful contrast effect in medical imaging, but the same interaction also represents a mechanism for bioeffects [24]. Because cavitation is primarily responsible in causing cellular membrane damage, the size of the bubbles, the affinity of the bubble to the DNA and the target cells, and the toxicity of the substance and gas that make up the bubbles are important factors in determining the outcome. Microbubble agents can be designed such that they can carry therapeutic genes or agents while being equipped with targeting ligands that would specifically attach to the particular cells being targeted [25]. These types of microbubbles are especially useful in drug or gene delivery by which bubbles that carry the therapeutic agents (e.g., drug or gene) are first localized before sonication, thus focusing much of the desired bioeffects on the target tissues and cells, and therefore also minimizing unwanted effects in the normal, non-target, tissues. Targeted microbubbles and acoustically active perfluorocarbon nanoemulsions with specific ligands can be developed for detecting disease at the molecular level and
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targeted drug and gene delivery. Bioactive compounds can be incorporated into these carriers for site-specific delivery [22]. Use of receptor selective targeted microbubbles has also been shown to improve binding of microbubbles to vascular thrombi in both in vitro and in vivo settings. As a result the use of therapeutic US with intravenous microbubbles has a high success rate in recanalizing deeply located thrombosed arteriovenous grafts when performed under diagnostic US guidance. A study also showed that when a recombinant adenovirus containing beta-galactosidase and driven by a constitutive promoter attached to the surface of albumin-coated perfluoropropane-filled microbubbles was used, sonication resulted to a 10-fold higher beta-galactosidase activity. The above findings show how important engineered and custom-designed microbubbles are in attaining the desired level of bioeffects with lowintensity US in therapy. 2.4. Synergistic effect of combined ultrasound and antibiotics The enhanced effect of chemotherapy against cancer has frequently been investigated using different modalities such as radiation, hyperthermia and ultrasound. In this study, we investigated the effects of ultrasound and antibiotics against chlamydia. These are preliminary results of the first step of research to investigate the in vitro response of Chlamydia trachomatis-infected human epithelial cells to a combination of ultrasound and two types of antibiotics. 2.4.1. Materials and methods Chlamydial strain and cell lines: C. trachomatis serovar E/UW-5/Cx was prepared in McCoy cells and propagated using a previously reported method [26]. The mouse fibroblast cell line McCoy cell (CRL 1696) and human epithelial cell line HeLa 229 cell (CLL 2.1) were maintained in Dulbecco’s modified Eagle medium (DMEM, Invitrogen, Grand Island, NY, USA) supplemented by 10% heat-inactivated fetal calf serum (FCS, Invitrogen) and 100 g/ml streptomycin. Infection of HeLa cells: The HeLa cells were seeded into a 24-well plate with lumoxTM fluorocarbon film base (optically clear, 50 m-thin, gas permeable film, Greiner bio-one, Gottingen, Germany). Stocks of chlamydial strain were diluted with sucrose–phosphate–glutamate (SPG) medium [26]. Chlamydial suspensions of 0.5 104 inclusion-forming units (IFUs) in 0.25 ml SPG medium were inoculated onto the monolayer cultures of HeLa cells (1 104 cells/ well). This is equivalent to a multiplicity of infection of 0.5. Preparation of bubble liposomes: Bubble liposomes were prepared using a previously described method [27]. The sterilized vials containing 2 ml of liposome suspension (lipid concentration: 1 mg/ml) were pressurised with 7.5 ml of perfluoropropane gas. Immunofluorescence staining and fluorescence microscopy: At 48 or 72 h after infection, the infected monolayers were washed with PBS, and the cells were fixed with 20 °C chilled methanol. After the specimens had been
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dried, the inclusion bodies were stained with fluorescein isohtiocyanate (FITC)-labeled monoclonal antibody against C. trachomatis lipopolysaccharides (Progen Biotechnik, Heidelberg, Germany) for 30 min at room temperature. The cells were rinsed with saline, and the films were cut away from the plate, and mounted in a 1:1 solution of PBS–glycerol. The antibody staining resulted in yellowgreen chlamydial proteins, and Evans blue counterstaining yielded red eukaryotic cells. Infectivity of chlamydia was presented as the number of inclusion-forming units (IFUs). Antibiotics and measurements of MIC: Doxycycline (DOX, Sigma Chemicals) and ceftizoxime (CZX, Fujisawa Yakuhin Kogyou, Tokyo, Japan) were obtained in powder form. Both antibiotics were diluted with saline, and were dissolved in maintenance medium at a concentration of 100 g/ml and frozen at 80 °C until used. The minimum inhibitory concentrations (MICs) were determined using a previously described method [28]. To determine the MICs, the cover slips were stained and observed as described above. The lowest concentration of the antimicrobial agent that completely inhibited the formation of visible chlamydial inclusions was determined as the MIC. Ultrasound exposure: An acoustically transparent gel (Pharmaceutical Innovations Inc., Newark, NJ) was placed between the top of ultrasound probe and the film base of the 24-well plate (Fig. 1). Ultrasound irradiation was done using a 1.0 MHz device (SonoPore KTAC-4000, NepaGene, Chiba, Japan) at intensity (ISATA) of 0.15 W/cm2 (duty cycle of 25%) for 20 s immediately after addition of bubble liposomes. Measurement of cell viability: The Trypan blue exclusion test was carried out by mixing 200 ml of the suspension of HeLa cells with an equal amount of 0.3% Trypan blue solution (Sigma Chemicals) in PBS. After 5 min incubation at room temperature, the number of cells excluding Trypan blue was counted using a C-Chip disposable hemocytometer (Digital Bio Technology Co., Gyeonggi, Korea) to estimate the number of viable cells immediately after sonication.
Fig. 1. Ultrasound exposure: an acoustically transparent gel was placed between the ultrasound probe and the film base of 24-well plate. HeLa cells were irradiated using therapeutic ultrasound (1.0 MHz) at an intensity (ISATA) of 0.15 W/cm2 (duty cycle of 25%) for 20 s after addition of bubble liposomes.
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Measurement of infectivity of chlamydiae: The 1.0 ml of chlamydial suspensions in sucrose–phosphate–glutamate medium with application of ultrasound and/or microbubbles were inoculated into triplicate cultures of McCoy cells in order to estimate the infectivity immediately after sonication. 0.25 ml of chlamydial suspensions was inoculated onto the monolayer culture of McCoy cells. After centrifugation at 1000g for 60 min, the inoculum was decanted, and the cells were washed in medium to remove the nonadsorbed chlamydiae, and were then further incubated in 1.0 ml of maintenance medium. 2.4.2. Results Ultrasonic enhancement of antibiotic action on C. trachomatis-infected HeLa cells: the MIC of DOX for C. trachomatis-infected HeLa cells was determined to be 0.03 g/ ml. DOX at 1/2 MIC (0.015 g/ml) was added to the infected cultures, and ultrasound exposures were carried out immediately after addition of bubble liposomes (50 g/ ml). The results of ultrasound experiments showed that ultrasound alone or bubble liposomes alone did not decrease the formation of inclusions administered with DOX (Fig. 2). However, DOX at 1/2 MIC in combination with microbubble-enhanced ultrasound reduced the number of IFUs to 66 ± 39%, at intensity of 0.15 w/cm2, compared with that administered DOX at 1/2 MIC only. The MIC of CZX for C. trachomatis-infected HeLa cells could not be determined because intracellular pathogens were resistant to CZX, we therefore tried to use concentrations of 0.125, 0.25, 0.5 and 1.0 g/ml. These were higher concentrations than expected. The 1.0 g/ml CZX was the most effective of these concentrations (data not shown). Similarly to the administration of DOX, 1.0 g/ml CZX in combination with microbubble-enhanced ultrasound also
Fig. 2. Ultrasonic enhancement of antibiotic action on C. trachomatis infected HeLa cells: the MIC of DOX for C. trachomatis-infected HeLa cells was determined to be 0.03 lg/ml. When DOX at 1/2 MIC (0.015 lg/ ml) was added to the infected cultures, ultrasound irradiation was carried out immediately after addition of bubble liposomes (50 lg/ml). Ultrasound alone (US(0.15) only) or bubble liposomes alone (bubble only) did not decrease the formation of inclusions administered with DOX. Significant decrease in the formation of inclusion bodies was noted when sonication was carried out in the presence of bubble liposomes (bubble + US(0.15)).
reduced the number of IFUs to 53 ± 32% at an intensity of 0.15 w/cm2, compared with that administered with 1.0 g/ml CZX only. However, these reductions were not statistically significant. 3. Conclusions and outlook Research on the bioeffects of ultrasound alone and in the presence of various drugs in patients has only just begun. Most investigations are still highly experimental and far from being applicable in the clinical situation. However, such techniques as HIFU therapy for cancer are already beginning to be used widely for patients as alternative non-invasive modalities. Additionally, there definitely exists an interesting biological phenomenon that cannot be ignored when non-thermal ultrasound mechanisms are involved. The interaction between ultrasound and drugs can range from a change in permeability of biological membrane to the manipulation of DNA into the cells. Understanding the mechanism of micro/nano bubble collapse or chemical activation of drugs will eventually result in optimization of the acoustics and the design of ultrasound devices for sonodynamic therapy. Acknowledgements This review was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas (15300187, 16500328 and 18800075) from the Ministry of Education, Culture, Sports, Science and Technology, Japan and from Fukuoka University Central Research Institute. References [1] F.W. Kremkau, J.S. Kaufmann, M.M. Walker, P.G. Burch, C.L. Spurr, Ultrasonic enhancement of nitrogen mustard cytotoxicity in mouse leukemia, Cancer 37 (4) (1976) 1643–1647. [2] G.H. Harrison, E.K. Balcer-Kubiczek, Pulsed ultrasound and neoplastic transformation in vitro, Ultrasound in Medicine and Biology 17 (6) (1991) 627–632. [3] S. Umemura, N. Yumita, R. Nishigaki, K. Umemura, Mechanism of cell damage by ultrasound in combination with hematoporphyrin, Japanese Journal of Cancer Research 81 (9) (1990) 962–966. [4] N. Miyoshi, V. Misik, P. Riesz, Sonodynamic toxicity of gallium– porphyrin analogue ATX-70 in human leukemia cells, Radiation Research 148 (1) (1997) 43–47. [5] N. Yumita, I. Sakata, S. Nakajima, S. Umemura, Ultrasonically induced cell damage and active oxygen generation by 4-formyloximeetylidene-3-hydroxyl-2-vinyl-deuterio-porphynyl(IX)-6-7-diaspartic acid: on the mechanism of sonodynamic activation, Biochimica et Biophysica Acta 1620 (1–3) (2003) 179–184. [6] K. Tachibana, T. Uchida, S. Hisano, E. Morioka, Eliminating adult T-cell leukaemia cells with ultrasound, Lancet 349 (9048) (1997) 325. [7] H. Abe, M. Kuroki, K. Tachibana, T. Li, A. Awasthi, A. Ueno, et al., Targeted sonodynamic therapy of cancer using a photosensitizer conjugated with antibody against carcinoembryonic antigen, Anticancer Research 22 (3) (2002) 1575–1580. [8] L.B. Feril Jr., T. Kondo, Biological effects of low intensity ultrasound: the mechanism involved, and its implications on therapy and on biosafety of ultrasound, Journal of Radiation Research 45 (4) (2004) 479–489.
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