Sonodynamic therapy (SDT) is a noninvasive treatment, often used for tumor irradiation, that utilizes a sonosensitizer and the deep penetration of ultrasound to treat lesions of varying depths by reducing target cell number and preventing future tumor growth. Many existing cancer treatment strategies cause systemic toxicity or cannot penetrate tissue deep enough to reach the entire tumor; however, emerging ultrasound stimulated therapies could offer an alternative to these treatments with their increased efficiency, greater penetration depth, and reduced side effects. Sonodynamic therapy could be used to treat cancers and other diseases, such as atherosclerosis, and diminish the risk associated with other treatment strategies since it induces cytotoxic effects only when externally stimulated by ultrasound and only at the cancerous region, as opposed to the systemic administration of chemotherapy drugs.
Reactive oxygen species (ROS) are an essential component of SDT as they provide the cytotoxicity of sonodynamic therapy; they are produced when ultrasound is coupled with a sensitizing drug and molecular oxygen. Without ultrasound, the drug is not toxic. However, once the drug is exposed to ultrasound and molecular oxygen, it becomes toxic. Photodynamic therapy, from which sonodynamic therapy was derived, uses a similar mechanism. Instead of ultrasound, light is used to activate the drug. SDT allows the ultrasound to reach deeper into the tissue (to about 30 centimeters) compared to photodynamic therapy (PDT) since it can be highly focused. This increased penetration depth ultimately means that SDT can be utilized to treat deeper, less accessible tumors and is more cost-effective than PDT. Photodynamic therapy can be used in combination with sonodynamic therapy and is expanded upon in the Applications section of this article. Sonodynamic therapy can be used synergistically with other therapeutic methods such as drug-loaded microbubbles, nanoparticles, exosomes, liposomes, and genes for improved efficacy. Currently, SDT does not have any clinical products and acts as an adjuvant for the aforementioned therapeutic methods, but it has been explored for use in atherosclerosis and cancer treatment to reduce tumor size in breast, pancreas, liver, and spinal sarcomas.
Mechanism of Action
The mechanism of action for sonodynamic therapy is the use of low-intensity ultrasound through the use of focused mechanical waves to create a cytotoxic effect. However, SDT itself is non-thermal, non-toxic, and is able to non-invasively penetrate deep into tissue compared to other delivery methods such as photodynamic therapy. SDT is often performed alongside the use of a sonosensitizer such as porphyrin, phthalocyanines, xanthenes, and antitumor drugs. Ultrasound waves are also classified as acoustic waves, and the effect they have on the tissue of application can be described by a process called cavitation. Cavitation occurs as a specific interaction between ultrasound and aqueous surroundings and causes gas bubbles to break upon exposure to particular ultrasonic parameters, thus promoting penetration of the therapeutic into the biological tissues by generating cavities near the edge of the membrane. Cavitation can be broken down into stable and inertial cavitation. In stable cavitation, the oscillation of gas bubbles causes the environmental media to intermix. In inertial cavitation, gas bubbles increase in volume and almost reach their resonance volume, swelling before aggressively collapsing. The implosion of vesicles results in a drastic temperature and pressure change, thereby increasing the cell membrane's permeability to various drugs. Microbubbles are created by the acoustic waves from the ultrasound that expand and collapse, releasing energy, bringing the sonosensitizer into an excited state, and generating a ROS. The cavitation of this gas bubble can form the ROS with different methodologies such as sonoluminescence and pyrolysis. Apoptosis results from the formation of ROS and mechanical forces of SDT through membrane disruption in a process called lipid peroxidation. Necrosis is also a potential result of SDT.
The influence of sonoluminescence on SDT and ROS has not been fully elaborated within literature. Currently, it is understood that sonoluminescence allows the emission of light upon bubble collapse which can activate sensitizers. A study by Hachimine et al. highlights the use of SDT as a method to activate a low photosensitive sonosensitizer, DCPH-P-Na(I), for cancer that is too deep within the tissue to combat utilizing PDT without skin irritation. Pyrolysis raises the surrounding temperature, enhances the cavitation process, breaks down the sensitizer, generating free radicals, and the free radicals interact within their environment to generate ROS. For both methods, the importance of the singlet oxygen compared to the hydroxyl radical to induce cytotoxicity has been highlighted. While other studies have found the singlet oxygen to not have a substantial effect. Overall, both of these methodologies lack significant breadth in literature to fully explain their role in ROS formation. However, literature has shown success in their analysis and application.
Sonoluminescence
Two primary mechanisms of ROS generation exist in sonodynamic therapy: sonoluminescence and pyrolysis. Sonoluminescence occurs when ultrasound produces light after irradiating an aqueous solution The exact mechanism with which light is produced remains unclear. However, it is suggested that inertial cavitation is a key element for this process. Other studies also indicate the potential role of stable cavitation
Pyrolysis Pyrolysis is believed to occur when inertial cavitation induces an extreme temperature increase, degrades the sonosensitizers, thus producing free radicals that can react and ultimately produce ROS necessary for SDT. The localized temperature increase assists in the inertial cavitation and breakdown of the sonosensitizer in order to create ROS. The pyrolysis within the cavitation bubbles will produce H+ and OH- via weak bonding within the solute molecule.
Lipid Peroxidation
In addition to chemical methods, mechanical properties of the acoustic wave generated from the ultrasound can assist in initiating cytotoxic effects. This occurs through disruption of the membrane with a hydrophobic sonosensitizer. The mechanical disruption of the membrane causes a process called lipid peroxidation and adjustments to the cell membrane can change cell drug permeability. Both sonochemical and sonomechanical methodologies are used to generate ROS and release cargo from vesicles for applications such as tumor targeting.
Apoptosis Low intensity ultrasound has been shown within past literature to induce apoptotic effects within surrounding cells. It has been found that it is not the initial ROS that causes apoptosis within the cells, but the free radicals within the mitochondria. In a study by Honda et al., it was determined that the mitochondria-caspase pathway is responsible for apoptosis through the increase of intracellular calcium. Outside of ROS induced apoptosis, cavitation is another factor involved within apoptosis of surrounding cells. Both cavitation types are able to induce apoptosis through damage to the membrane. Conditions such as frequency, duty cycle, pulse, and intensity can be manipulated to optimize cell death conditions such as necrosis, lysis, or apoptosis.
Autophagy This method of cell death can occur by cell organelles becoming entrapped into autophagosomes that combine with lysosomes. Continuation of this process will lead to cell death and autophagy inhibitors or promoters can be controlled to encourage or discourage cell death and uptake of chemotherapeutics.
Sonosensitizers Sonosensitizers, or sonosensitizing therapeutics, are the primary element of SDT and can be tailored to treat various cancers and generate different effects. These therapeutics, often involving the use of porphyrin or xanthene, will initiate a toxic effect via the ROS upon exposure to ultrasound.
Porphyrin-based sensitizers
Porphyrin-based sensitizers, initially used as a photosensitizer in PDT, are fairly hydrophobic molecules derived from hematoporphyrin. Single oxygen atoms or hydroxyl radicals are produced by porphyrin-based sensitizers upon exposure to ultrasound or light, providing the cytotoxic effects desired with sonodynamic and photodynamic therapies. However, the result of porphyrin-based sensitizers is not as local as desired for sonodynamic therapy since they are also located in non-targeted tissue between the tumor and the ultrasound emitter.
Xanthene-based sensitizers
Xanthene-based sensitizers, on the other hand, have shown successful cytotoxicity in vitro by producing reactive oxygen species after being triggered by ultrasound. More research is necessary to improve its potential in vivo performance since it is quickly processed by the liver and cleared from the body. Rose Bengal is a commonly used xanthene-based sonosensitizer.
Additional sensitizers Other sensitizers that have been investigated for their potential in sonodynamic therapy (and have also been used previously in PDT) include acridine orange, methylene blue, curcumin, and indocyanine green. A study by Suzuki et al. used acridine orange, a fluorescent cationic dye that can insert itself into nucleic acids, for treating sarcoma 180 cells with ultrasound and demonstrated that reactive oxygen species are a critical element of SDT considering that their absence decreased the efficacy of SDT. Similar to the previous study, a recent study by Komori et al. utilized ultrasound coupled with methylene blue (a phenothiazine dye commonly used in PDT that exhibits low toxicity) to irradiate sarcoma 180 cells and found that methylene blue was an effective sonosensitizer in decreasing cell viability. Interestingly, curcumin is a spice that also can act as a sensitizer for PDT and SDT. In a study by Waksman et al., curcumin was able to impact macrophages, which are important for development of plaques found in atherosclerosis patients, thus reducing the amount of plaque in an animal model. These findings along with other research indicate that curcumin sensitizers could be used in SDT cancer treatments. Indocyanine green is a dye that absorbs near infrared wavelengths and is another sensitizer that has been shown to reduce cell viability when coupled with ultrasound and/or light. An in vivo study demonstrated that treating a mouse tumor model with indocyanine green coupled with ultrasound and light resulted in a 98% reduction in tumor volume by 27 days after treatment.
+ Name and structure of additional sensitizers Name Structure Phthalocyanine Indocyanine Green Phenothiazine Curcumin-keto Curcumin-enol
Carriers As aforementioned, sonosensitizers are often used in conjunction with different drug carriers such as microbubbles, nanobubbles, liposomes, and exosomes to improve therapeutic agent concentration and penetration.
Liposomes