Venous thromboembolism has been ranked as the third most common cause of disability and cardiovascular death in the world, encompassing deep vein thrombosis (DVT) and pulmonary embolism (PE), which frequently occur due to trauma, stress, pregnancy, and postoperative complications (Khan et al., 2021; Jackson et al., 2022). Due to the intensive study of thrombosis, venous thrombosis has been revealed be closely associated with the inflammatory response of the venous wall. Slowed blood flow, hypoxia, inflammatory cell activation, and the release of various active substances correlated with thrombosis (Poredos and Poredos, 2022). Studies have revealed that TNF-α is closely linked to the interaction between leukocytes and endothelial cells (Aguilar et al., 2021). In the initial stage of thrombosis, TNF-α induces the expression of adhesion molecules on the surface of endothelial cells. Therefore, the inflammatory response can be neutralized by inhibiting TNF-α. The use of antibody to TNF-α is one of the most effective methods to reduce neutrophil infiltration (Presicce et al., 2020). Aue et al. (2011) found that the upregulation of TNF-α was significantly related to thrombosis, and the upregulation degree was more predictive than the medical history of non-drug-related (DVT).

The above descriptions indicate that TNF-α can be used as a target for thrombosis therapy. Anti-inflammatory and targeted therapy of thrombosis can be achieved by using bioactive materials (Liu et al., 2022a) or ultrasound microbubble contrast agents labeled with targeting guides. The combination of a microbubble contrast agent and ultrasound targeted microbubble destruction (UTMD) technology can deliver bioactive substances to various targets non-invasively and specifically (Liu et al., 2022b). At present, it is mainly used in the treatment of heart disease, tumors, and atherosclerotic plaque (Zhang et al., 2021; He et al., 2022). UTMD technology breaks up targeted microbubbles and releases large amounts of therapeutic substances in the target area, which optimizes the local treatment effect while reducing systemic side effects (Yang et al., 2019; Xu et al., 2022).

There is no doubt that ultrasound can accelerate thrombolysis. Both high-frequency ultrasound and low-power ultrasound can significantly promote thrombolysis (Notten et al., 2020; Avgerinos et al., 2021). Microbubbles combined with thrombolytic medication, such as urokinase (UK) or tissue plasminogen activator, can further enhance the success rate of thrombolysis (Wang et al., 2019; Wang et al., 2020). Studies have demonstrated that UTMD can mechanically remove dialysis grafts with thrombosis in experimental animals even without the delivery of any substance (Liang et al., 2021). Ultrasound with a frequency greater than 0.5 MHz can enhance the dissolution of thrombus in vitro (Ebben et al., 2015), and the study by Zhong et al. (2019) has confirmed that ultrasound can accelerate thrombolysis in vivo. In recent years, microbubbles carrying medication to treat thrombosis have also become a research hotspot.

Microbubbles carrying the antibody of TNF-α played multiple roles in thrombolytic therapy. Firstly, targeted microbubbles were an ultrasound contrast agent, which could effectively identify thrombus through ultrasound contrast imaging and evaluate the thrombolytic effect. Secondly, the monoclonal antibody to TNF-α carried by targeted microbubbles bound the local TNF-α produced by thrombus and alleviated the inflammatory response. Finally, the cavitation effect and thermal effect generated by breaking the microbubble through UTMD technology stimulated the regeneration of blood vessels, loosened the internal structure of thrombus, and promoted the effect of thrombolytic drugs.

In this study, an animal model of DVT was established and treated by low-power ultrasound combined with targeted microbubbles (MBt) bearing TNF-α antibody and UK. UTMD technology promoted monoclonal antibodies to TNF-α release from MBt and alleviated inflammation and subsequent chain reaction. At the same time, the cavitation effect produced by ultrasonic crushing microbubbles also promoted thrombus dissolution, in the hope of achieving the effect of complete dissolution of thrombus, recanalization, and providing new ideas for thrombus treatment.

The application of ultrasound in thrombus therapy was first noted in 1976 (Trübestein et al., 1976). The pivotal mechanism is that the cavitation effect produced by ultrasonic irradiation promotes thrombolysis. Sonic flow generated by low-frequency ultrasound causes mechanical displacement of the thrombus and exposes the fiber structure inside the thrombus to thrombolytic medicine. The micro jet caused by the cavitation effect generates a shear force on the surface of the thrombus and then dissolves the thrombus (Pan et al., 2023).

In recent years, research on the application of ultrasound targeted contrast agents has improved rapidly (Liu et al., 2022c). CEUS is a highly secure inspection technology developed in recent years. The principle of this method is that ultrasound contrast agents reflect ultrasound and create different echo signals for the diagnosis of related diseases. MBt contrast agents are not only used for the diagnosis of diseases, but also carry genes or medicine to treat various diseases (Han et al., 2022). MBt swiftly expands and ruptures under the action of low-power focused ultrasound, then releases the genes or medication, so as to increase the local medicine concentration and enhance the efficacy without systemic side effects (Yu et al., 2020; Liu et al., 2023).

Many studies have verified that there is a close connection between inflammation and venous thrombosis (Weitz and Chan, 2020; Stark and Massberg, 2021). TNF-α is one of the critical factors in many diseases complicated with venous thrombosis (Zheng et al., 2021). It plays a vital role in mediating inflammatory events related to venous thrombosis, including early inflammatory response and neutrophil infiltration into the venous wall. Previous studies confirmed that the TNF-α content continued to rise after thrombosis. And the MBt contrast agent carrying monoclonal antibodies against IL-6, IL-8, and TNF-α was successfully prepared. Therefore, this research followed the preceding preparation method of MBt contrast agent, prepared MBt carrying TNF-α antibody, and selected TNF-α as the target to study the treatment of DVT.

This study concluded that there was no significant difference in PT, APTT, and TT of rabbits in each group before modeling and treatment, but the contents of FIB, D-dimer, and TNF-α increased significantly before treatment. Although relevant literature (Heestermans et al., 2021) reported that Pt and APTT would be shortened after thrombosis, the four indexes of coagulation were affected by numerous factors. Pt and APTT were not the most sensitive indicators to predict thrombosis. The relationship between thrombosis and the fibrinolytic system was complicated. In addition, this experimental model was an acute phase thrombus model. Therefore, these two indicators did not change significantly in this research after thrombosis. As the most sensitive index for clinical prediction of thrombosis, the contents of D-dimer and FIB increased markedly after thrombosis.

After treatment with different schemes, the four coagulation indexes and D-dimer content in Group I did not change significantly compared with that before treatment. But the content of TNF-α increased compared with those before treatment, indicating that ultrasonic irradiation did not increase the risk of bleeding or activate the fibrinolytic system in vivo, and had no effect on inflammatory reactions. The PT, APTT, and TT in Group II-IV were longer than those before and after modeling, while the contents of FIB and D-dimer were significantly lower than those before modeling and before treatment, indicating that the use of UK to treat thrombosis could also increase the risk of bleeding. Among the four groups, only the TNF-α content in Group IV decreased significantly after treatment, indicating that TNF-α content increased with the extension of thrombosis time, and MBt effectively reduced the level of the inflammatory factors.

Meanwhile, there was no significant difference in the four indexes of coagulation and the contents of D-dimer and TNF-α among the rabbits in Group I-IV before modeling and before treatment. After treatment, the PT, APTT, and TT of rabbits in Group II-IV were longer than those in Group I, and the contents of FIB and D-dimer of rabbits in Group II-IV were lower than those in Group I; in addition, there was no significant difference in the four indexes of coagulation and the content of D-dimer among Group II-IV. It showed that both MBc and MBt would not cause changes in the coagulation system. As for the content of TNF-α, Group IV was higher than Group I-III, but there was no significant difference among Group I-III. It showed that in thrombotic diseases, the increase of inflammatory factor level was not only related to thrombosis, but may also be linked to reperfusion injury after thrombolysis.

Through the analysis of CDFI, CEUS, and pathological results, the study found that the thrombolysis of focused ultrasound alone was limited. When focused ultrasound was combined with UK, the thrombus was partially dissolved. With the addition of a microbubble contrast agent, the thrombus almost disappeared, which indicated that the treatment method significantly accelerated the thrombolysis and promoted the recanalization of blood vessels. Furthermore, the combination of UK, focused ultrasound, and MBt produced the best therapeutic effect. More MBt accumulates locally in the thrombus through an antigen-antibody reaction, facilitating the therapeutic effect of UK on the thrombus, so as to completely dissolve the thrombus while alleviating the inflammatory response.

Combined with previous studies, we found that CEUS can be used not only to diagnose thrombosis but also to treat it. The thrombolytic effect of ultrasound was one of its clinical applications, which mainly depended on the cavitation effect to enhance the efficacy of thrombolytic medicine (Porter et al., 2017; Ma et al., 2020). Relevant studies showed that a microbubble contrast agent, as an artificially added cavitation core, could enhance the induced ultrasonic cavitation effect and improve the thrombolytic effect of ultrasound (Yang and Zhou, 2017).

In summary, this study had some innovations for thrombosis treatment. At first, the combination of targeted microbubbles and UK was innovatively used in the study of acute DVT. Thrombus was diagnosed by ultrasound imaging and treated at the same time. This treatment has been rarely reported. Secondly, TNF-α was used as the target of thrombus to attract a large number of targeted microbubbles to enrich locally in the thrombus and neutralize the inflammatory response, which was another innovative way to treat thrombus. This experiment opens up new ways to study the effect of UTMD technology targeting inflammatory factors on thrombosis and provides new ideas for the diagnosis and treatment of acute DVT.

This study has a few limitations in experimental design. This experiment only treated acute thrombosis, ignoring the long-term damage of thrombus. By extending the experimental time, we will prepare animal models of thrombosis in the subacute and chronic phases and conduct therapeutic studies on them. Secondly, using low-power focused ultrasound to thrombi could affect the perfusion of microcirculation of the surrounding tissues. The main concern in the experiment was the recanalization of vascular lumen. We will study the damage to the vessel wall of the thrombus segment and use more refined ultrasound irradiation to avoid damage to the tissue surrounding the thrombus.