Ultrasound can selectively and specifically visualize upregulated vascular receptors through the detection of bound microbubbles. However, most current ultrasound molecular imaging methods incur delays that result in longer acquisition times and reduced frame rates. These delays occur for two main reasons: 1) multi-pulse imaging techniques are used to differentiate microbubbles from tissue and 2) acquisition occurs after free bubble clearance (>6 minutes) in order to differentiate bound from freely circulating microbubbles. In this paper, we validate tumor imaging with a broadband single pulse molecular imaging method that is faster than the multi-pulse methods typically implemented on commercial scanners. We also combine the single pulse method with interframe filtering to selectively image targeted microbubbles without waiting for unbound bubble clearance, thereby reducing acquisition time from 10 to 2 minutes. The single pulse imaging method leverages non-linear bubble behavior by transmitting at low and receiving at high frequencies (TLRH). We implemented TLRH imaging and visualized the accumulation of intravenously administrated integrin-targeted microbubbles in a phantom and a Met-1 mouse tumor model. We found that the TLRH contrast imaging has a ~2-fold resolution improvement over standard contrast pulse sequencing (CPS) imaging. By using interframe filtering, the tumor contrast was 24.8±1.6 dB higher after the injection of integrin-targeted microbubbles than non-targeted control MBs, while echoes from regions lacking the target integrin were suppressed by 26.2±2.1 dB as compared with tumor echoes. Since real-time three-dimensional (3D) molecular imaging provides a more comprehensive view of receptor distribution, we generated 3D images of tumors to estimate their volume, and these measurements correlated well with expected tumor sizes. We conclude that TLRH combined with interframe filtering is a feasible method for 3D targeted ultrasound imaging that is faster than current multi-pulse strategies.
In ultrasound molecular imaging, a sequence of high-pressure ultrasound pulses is frequently applied to destroy bound targeted microbubbles, to quantify accumulated microbubbles or to prepare for successive microbubble injections; however, the potential for biological effects from such a strategy has not been fully investigated. Here, we investigate the effect of high-pressure insonation of bound microbubbles and the potential for thrombogenic effects.
Acquisition of the epithelial-mesenchymal transition (EMT) tumor phenotype is associated with impaired chemotherapeutic delivery and a poor prognosis. In this study, we investigated the application of therapeutic ultrasound methods available in the clinic to increase nanotherapeutic particle accumulation in epithelial and EMT tumors by labeling particles with a positron emission tomography tracer. Epithelial tumors were highly vascularized with tight cell-cell junctions, compared with EMT tumors where cells displayed an irregular, elongated shape with loosened cell-cell adhesions and a reduction in E-cadherin and cytokeratins 8/18 and 19. Without ultrasound, the accumulation of liposomal nanoparticles administered to tumors in vivo was approximately 1.5 times greater in epithelial tumors than EMT tumors. When ultrasound was applied, both nanoaccumulation and apparent tumor permeability were increased in both settings. Notably, ultrasound effects differed with thermal and mechanical indices, such that increasing the thermal ultrasound dose increased nanoaccumulation in EMT tumors. Taken together, our results illustrate how ultrasound can be used to enhance nanoparticle accumulation in tumors by reducing their intratumoral pressure and increasing their vascular permeability.
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