study by cheng et al. determined that thp-1 macrophage apoptosis is induced by an increase in ppix concentration, leading to the production of large amounts of ros. the use of sdt for as treatment has also shown success in promoting the repopulation of vascular smooth muscle cells (vmscs) through inducing further expression and autophagy to prevent vmsc evolution into plaque-holding macrophages. a study performed by dan et al. showed the increase in smooth muscle a-actin, smooth muscle 22a, p38 mitogen-activated protein kinase phosphorylation. while a study by geng et al. showed improved vmsc autophagy. each of these factors contributed to the improved differentiation and development of vmscs. in vitro in vitro experimentation provides great insight and knowledge to characterize the potential of sonosensitizer behavior in vivo. in addition, sdt has shown success through its low intensity allowing increased plasma membrane permeability without cell death. sonosensitizers have also been used in vitro in applications with different cell lines and to further understand the mechanism of action for cell death. it is currently understood that pdt and sdt have similar mechanisms for free radical generation for inducing apoptosis and necrosis. however, each cell line is unique and can cause cell death with different efficacy. some examples of in vitro work include initial studies that were performed by yumita et al., 1989 who used haematoprophyrin and sdt for mouse sarcoma 180 and rat ascites hepatoma (ah) that showed a relationship between dosage and ultrasound, and microbubbles causing cavitation leading to cell damage without the use of drugs. this study also emphasized the difference in efficacy between cell lines through sdt 180 having less lysis compared to ah-130 cells. another study by hachimine et al. emphasized efficacy between cell lines by examining seven different cancers with 17 cell lines total under the use of dcph-p-na(i). this study revealed that the stomach and lung cancer lines of mkn-28 and lu65a respectively had the highest survival rate, but the stomach and lung cancer lines of rerflc-kj and mkn-45 respectively had the lowest survival rates. another study by honda et al., with u937 and k562 showed that sonication increases the intracellular calcium ion levels and decreases gsh concentration respectively. this increased concentration of calcium plays a significant role in cell death through dna fragmentation and mitochondrial membrane disruption. while a decreased concentration of gsh plays a significant role in allowing the formation of more free radicals. a study by umemura et al., found that atx-70 versus hematoporphyrin has increased cytotoxic activity. current research typically focuses on using tumor xenograft models to determine the effect of sdt on target cells and delivery efficacy. in vivo building upon the study by umemura et al. and atx-70, it was found that 24h after administration of the sonosensitizer had improved efficacy when ultrasound was applied compared to immediate administration. it was also determined that most ultrasound frequencies range between 1-3 mhz and 0.5-4w/cm^2. higher frequencies at values such as 20w/cm^2 and 25w/cm^2 resulted in large necrotic lesions. this established a relationship between sonosensitizer formulation and ultrasound intensity to necrosis. other studies have continued to innovate upon this by controlling drug ultrasound interval (dui) for different sonosensitizers in order to determine the optimal time period to apply the ultrasound for improved efficacy. in addition, it has been shown that sdt can disturb surrounding vasculature in tumors. this has been shown in studies by gao et al. with 5-ala in mice and human umbilical vein endothelial cell lines through inhibition of microvessel density and cell proliferation, migration, and invasion. challenges and development ultrasound imaging vs. ultrasound therapy one of the many advantages of sdt compared to pdt is the ability of sdt to penetrate deeply placed solid tumors allowing a wider treatment range. despite this fact, there are limitations to sdt that must be overcome or have optimized components in order to expand the effect and application of sdt. sdt does allow for precise activation of the therapeutic, but is limited in the delivery and accumulation of the delivery modality to penetrate deeply into the desired tumor site. this is often accommodated for through delivery vessels such as nanoparticles or liposomes. however, nanomedicine is limited by the enhanced permeability and retention effect and struggles to deliver in targeted abundance depending on the delivery vesicle. this can be seen in nanoparticles struggling with non-specific delivery. future research has been focused on developing high targeting and penetrating nanoparticles for improved delivery and pharmacokinetics. due to the complex nature of tumors and their microenvironments, they are difficult to treat with only one therapy. in order to enhance the oftentimes low production of reactive oxygen species to address the hypoxic tumor environment, sdt can be combined with other therapies, such as pdt, chemotherapy, and immunotherapy to improve patient outcomes. sdt alone does not respond well in hypoxic environments. however, bioreductive therapy could be used to reduce the impact of sdt's limitations regarding hypoxia in the tumor while leaving healthy/normal tissue alone. sonosensitizers also require continuous high levels of oxygen to create ros, which is not readily available within a hypoxic tumor microenvironment. however, strategies such as oxygen supplementation and production to supply the required oxygen and enhance cavitation, and glutathione depletion to avoid the reduction of the free radicals produced have been implemented alongside sonosensitizers to supply the required oxygen or reduce the combative function. in addition to its relatively low generation of reactive oxygen species, sdt also can cause permanent destruction of normal tissues. this lack of selectivity is caused by ultrasound divergence, resulting in heat and shear that impacts off-target tissues. although advantages of organic sonosensitizers exist, such as high reproducibility, biocompatibility, production of reactive oxygen species, they also have limitations. factors that limit the translation of organic sensitizers to clinical applications include low water solubility, sonotoxicity, and targetability as well as high phototoxiticty. other properties could promote rapid clearance of the drug, which is why various nano and microparticles are used to transport the drug to the desired location. in addition, sonosensitizers in sdt often require increased dosage, and the relationship between therapeutic dosage and toxicity of sonosensitizers has not been properly characterized alongside other variables such as tissue type and acoustic pressure. inorganic sensitizers produce reactive oxygen species, but in lower concentrations than desirable for sdt, limiting their ability to be used in a clinical setting. another challenge is reflected in vitro and in vivo work. an example of this can be seen in a study using rose bengal, a xanthene dye. it was found to be successful in vitro, but in vivo showed significantly less efficacy due to liver squestation and clearance. lastly, there are no current standardized computer simulations to predict the characteristics of different sonosenistizers within tissue, which would provide further insight into how sonosensitizers may behave. current clinical use photofrin sdt has been researched most commonly to combat cancers and atherosclerosis such as breast cancer, pancreatic cancer, liver, and spinal sarcomas. currently, there are no fda approved clinical applications of sdt. however, for pdt, photofrin is an fda approved hematoporphyrin (photofrin®). however, sdt has been used in a clinical trial in combination with pdt to assess for reduction in tumor size in patients with breast cancer. however, it was difficult to determine if sdt pdt or the drug dosage was the primary mechanism of treatment. another case study expanded on this by using sdt as a standalone treatment with a gc protein hormone therapy with the use of 5-ala or chlorin e6 as a sonosensitizer. it was shown that tumor markers significantly decreased during treatment. future directions enhanced drug uptake using acoustic targeted drug delivery (atdd). the effectiveness of sonodynamic therapy as a cancer treatment is supported by many in vitro and in vivo studies. however, large-scale clinical trials are necessary for translation into the clinical setting. in order to mitigate the limitations aforementioned, new sonosensitizers are being developed and sdt is being combined with other therapies in novel ways. particularly, organic sonosensitizers with high solubility in water, high sonotoxocity, increased ability to target tumors, and low phototoxicity need to be developed in order to improve the therapeutic efficacy of sdt and allow it to be used for treating cancers. in addition, the mechanisms by which ros are produced by sonosensitizers upon exposure to ultrasound is yet to be determined, reducing the ability to control its function and outcomes. ultimately, the synergistic effects of combining sdt with other therapies would allow each to compensate for the limitations of the other, improving their therapeutic efficacy and increasing their ability to destroy tumors.