Dynamic Fluorescence Microscopy of Cellular Uptake of Intercalating Model Drugs by Ultrasound-Activated Microbubbles.

PURPOSE: The combination of ultrasound and microbubbles can facilitate cellular uptake of (model) drugs via transient permeabilization of the cell membrane. By using fluorescent molecules, this process can be studied conveniently with confocal fluorescence microscopy. This study aimed to investigate the relation between cellular uptake and fluorescence intensity increase of intercalating model drugs. PROCEDURES: SYTOX Green, an intercalating fluorescent dye that displays >500-fold fluorescence enhancement upon binding to nucleic acids, was used as a model drug for ultrasound-induced cellular uptake. SYTOX Green uptake was monitored in high spatiotemporal resolution to qualitatively assess the relation between uptake and fluorescence intensity in individual cells. In addition, the kinetics of fluorescence enhancement were studied as a function of experimental parameters, in particular, laser duty cycle (DC), SYTOX Green concentration and cell line. RESULTS: Ultrasound-induced intracellular SYTOX Green uptake resulted in local fluorescence enhancement, spreading throughout the cell and ultimately accumulating in the nucleus during the 9-min acquisition. The temporal evolution of SYTOX Green fluorescence was substantially influenced by laser duty cycle: continuous laser (100 % DC) induced a 6.4-fold higher photobleaching compared to pulsed laser (3.3 % DC), thus overestimating the fluorescence kinetics. A positive correlation of fluorescence kinetics and SYTOX Green concentration was found, increasing from 0.6 × 10-3 to 2.2 × 10-3 s-1 for 1 and 20 µM, respectively. Finally, C6 cells displayed a 2.4-fold higher fluorescence rate constant than FaDu cells. CONCLUSIONS: These data show that the temporal behavior of intracellular SYTOX Green fluorescence enhancement depends substantially on nuclear accumulation and not just on cellular uptake. In addition, it is strongly influenced by the experimental conditions, such as the laser duty cycle, SYTOX Green concentration, and cell line.

Performance analysis of a dedicated breast MR-HIFU system for tumor ablation in breast cancer patients.

MR-guided HIFU ablation is a promising technique for the non-invasive treatment of breast cancer. A phase I study was performed to assess the safety and treatment accuracy and precision of MR-HIFU ablation in breast cancer patients (n=10) using a newly developed MR-HIFU platform dedicated to applications in the breast. In this paper a technical analysis of the performance of the dedicated breast MR-HIFU system during breast tumors ablation is described. The main points of investigation were the spatial targeting accuracy and precision of the system and the performance of real-time respiration-corrected MR thermometry.The mean targeting accuracy was in the range of 2.4-2.6 mm, whereas the mean targeting precision was in the range of 1.5-1.8 mm. To correct for respiration-induced magnetic field fluctuations during MR temperature mapping a look-up-table (LUT)-based correction method was used. An optimized procedural sedation protocol in combination with the LUT-based correction method allowed for precise MR thermometry during the ablation procedure (temperature standard deviation <3 °C). No unwanted heating in the near field (i.e. skin) nor in the far field (pectoral muscle) was detected.The newly developed dedicated breast MR-HIFU system allows for safe, accurate and precise ablation of breast tumors.

Understanding ultrasound induced sonoporation: definitions and underlying mechanisms.

In the past two decades, research has underlined the potential of ultrasound and microbubbles to enhance drug delivery. However, there is less consensus on the biophysical and biological mechanisms leading to this enhanced delivery. Sonoporation, i.e. the formation of temporary pores in the cell membrane, as well as enhanced endocytosis is reported. Because of the variety of ultrasound settings used and corresponding microbubble behavior, a clear overview is missing. Therefore, in this review, the mechanisms contributing to sonoporation are categorized according to three ultrasound settings: i) low intensity ultrasound leading to stable cavitation of microbubbles, ii) high intensity ultrasound leading to inertial cavitation with microbubble collapse, and iii) ultrasound application in the absence of microbubbles. Using low intensity ultrasound, the endocytotic uptake of several drugs could be stimulated, while short but intense ultrasound pulses can be applied to induce pore formation and the direct cytoplasmic uptake of drugs. Ultrasound intensities may be adapted to create pore sizes correlating with drug size. Small molecules are able to diffuse passively through small pores created by low intensity ultrasound treatment. However, delivery of larger drugs such as nanoparticles and gene complexes, will require higher ultrasound intensities in order to allow direct cytoplasmic entry.

Magnetic resonance-guided high-intensity focused ultrasound (MR-HIFU) ablation of liver tumours.

Recent decades have seen a paradigm shift in the treatment of liver tumours from invasive surgical procedures to minimally invasive image-guided ablation techniques. Magnetic resonance-guided high-intensity focused ultrasound (MR-HIFU) is a novel, completely non-invasive ablation technique that has the potential to change the field of liver tumour ablation. The image guidance, using MR imaging and MR temperature mapping, provides excellent planning images and real-time temperature information during the ablation procedure. However, before clinical implementation of MR-HIFU for liver tumour ablation is feasible, several organ-specific challenges have to be addressed. In this review we discuss the MR-HIFU ablation technique, the liver-specific challenges for MR-HIFU tumour ablation, and the proposed solutions for clinical translation.

Volumetric feedback ablation of uterine fibroids using magnetic resonance-guided high intensity focused ultrasound therapy.

OBJECTIVE: The purpose of this prospective multicenter study was to assess the safety and technical feasibility of volumetric Magnetic Resonance-guided High Intensity Focused Ultrasound (MR-HIFU) ablation for treatment of patients with symptomatic uterine fibroids. METHODS: Thirty-three patients with 36 fibroids were treated with volumetric MR-HIFU ablation. Treatment capability and technical feasibility were assessed by comparison of the Non-Perfused Volumes (NPVs) with MR thermal dose predicted treatment volumes. Safety was determined by evaluation of complications or adverse events and unintended lesions. Secondary endpoints were pain and discomfort scores, recovery time and length of hospital stay. RESULTS: The mean NPV calculated as a percentage of the total fibroid volume was 21.7%. Correlation between the predicted treatment volumes and NPVs was found to be very strong, with a correlation coefficient r of 0.87. All patients tolerated the treatment well and were treated on an outpatient basis. No serious adverse events were reported and recovery time to normal activities was 2.3 ± 1.8 days. CONCLUSION: This prospective multicenter study proved that volumetric MR-HIFU is safe and technically feasible for the treatment of symptomatic uterine fibroids. KEY POINTS: • Magnetic-resonance-guided high intensity focused ultrasound allows non-invasive treatment of uterine fibroids. • Volumetric feedback ablation is a novel technology that allows larger treatment volumes • MR-guided ultrasound ablation of uterine fibroids appears safe using volumetric feedback.