[Show abstract][Hide abstract] ABSTRACT: Previously, we demonstrated the feasibility to monitor ultrasound-mediated uptake of a cell-impermeable model drug in real time with fibered confocal fluorescence microscopy. Here, we present a complete post-processing methodology, which corrects for cell displacements, to improve the accuracy of pharmacokinetic parameter estimation.
Nucleus detection was performed based on the radial symmetry transform algorithm. Cell tracking used an iterative closest point approach. Pharmacokinetic parameters were calculated by fitting a two-compartment model to the time-intensity curves of individual cells.
Cells were tracked successfully, improving time-intensity curve accuracy and pharmacokinetic parameter estimation. With tracking, 93 % of the 370 nuclei showed a fluorescence signal variation that was well-described by a two-compartment model. In addition, parameter distributions were narrower, thus increasing precision.
Dedicated image analysis was implemented and enabled studying ultrasound-mediated model drug uptake kinetics in hundreds of cells per experiment, using fiber-based confocal fluorescence microscopy.
Molecular imaging and biology: MIB: the official publication of the Academy of Molecular Imaging 02/2014; · 2.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Imaging has become a cornerstone for medical diagnosis and the guidance of patient management. A new field called image-guided drug delivery (IGDD) now combines the vast potential of the radiological sciences with the delivery of treatment and promises to fulfill the vision of personalized medicine. Whether imaging is used to deliver focused energy to drug-laden particles for enhanced, local drug release around tumors, or it is invoked in the context of nanoparticle-based agents to quantify distinctive biomarkers that could risk stratify patients for improved targeted drug delivery efficiency, the overarching goal of IGDD is to use imaging to maximize effective therapy in diseased tissues and to minimize systemic drug exposure in order to reduce toxicities. Over the last several years, innumerable reports and reviews covering the gamut of IGDD technologies have been published, but inadequate attention has been directed toward identifying and addressing the barriers limiting clinical translation. In this consensus opinion, the opportunities and challenges impacting the clinical realization of IGDD-based personalized medicine were discussed as a panel and recommendations were proffered to accelerate the field forward. WIREs Nanomed Nanobiotechnol 2014, 6:1-14. doi: 10.1002/wnan.1247 Conflict of interest: The authors have declared no conflicts of interest for this article. For further resources related to this article, please visit the WIREs website.
[Show abstract][Hide abstract] ABSTRACT: 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.
Advanced drug delivery reviews 11/2013; · 11.96 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Magnetic Resonance Imaging-guided High-Intensity Focused Ultrasound (MR-HIFU) is a promising technique for non-invasive breast tumor ablation. The purpose of this study was to investigate the effects of HIFU ablation and thermal exposure on ex vivo human breast tissue. HIFU ablations were performed in three unembalmed cadaveric breast specimens using a clinical MR-HIFU system. Sonications were performed in fibroglandular and adipose tissue. During HIFU ablation, time-resolved anatomical MR images were acquired to monitor macroscopic tissue changes. Furthermore, the breast tissue temperature was measured using a thermocouple to investigate heating and cooling under HIFU exposure. After HIFU ablation, breast tissue samples were excised and prepared for histopathological analysis. In addition, thermal exposure experiments were performed to distinguish between different levels of thermal damage using immunohistochemical staining. Irreversible macroscopic deformations up to 3.7mm were observed upon HIFU ablation both in fibroglandular and in adipose tissue. No relationship was found between the sonication power or the maximum tissue temperature and the size of the deformations. Temperature measurements after HIFU ablation showed a slow decline in breast tissue temperature. Histopathological analysis of sonicated regions demonstrated ablated tissue and morphologically complete cell death. After thermal exposure, samples exposed to three different temperatures could readily be distinguished. In conclusion, the irreversible macroscopic tissue deformations in ex vivo human breast tissue observed during HIFU ablation suggest that it might be relevant to monitor tissue deformations during MR-HIFU treatments. Furthermore, the slow decrease in breast tissue temperature after HIFU ablation increases the risk of heat accumulation between successive sonications. Since cell death was inflicted after already 5minutes at 75°C, MR-HIFU may find a place in non-invasive treatment of breast tumors.
European journal of pharmacology 04/2013; · 2.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: PURPOSE: This study aims to quantitatively analyze cellular uptake following local ultrasound (US)-mediated cell permeabilization. PROCEDURES: A 2 μM cell-impermeable dye Sytox Green was co-injected with 3 × 107 microbubbles in the presence of C6 rat glioblastoma cell monolayer in total volume of 10 ml. A 5.8-mm diameter mono-element US transducer was positioned at a distance of 8 mm to the Opticell® membrane. Acoustical pressure of pulsed US was varied from 0.62 MPa peak-to-peak (p-p) to 1.25 MPa p-p. Large field of view (FOV = 15 × 15 mm) 22 × 22 mosaic acquisitions were done under epifluorescence Leica DMR microscope and analyzed in Metamorph software to evaluate cell density as well as model drug uptake percentage. RESULTS: The size of acoustical field of the transducer closely matches the spatial pattern of the model drug internalized into the cells by US. Maximum of uptake percentage (42 ± 15 %) was found at 0.88 MPa p-p. CONCLUSIONS: Spatial aspect of US-mediated model drug uptake has been quantitatively evaluated on adherent cells using robust 2D-mapping approach.
Molecular imaging and biology: MIB: the official publication of the Academy of Molecular Imaging 03/2013; · 2.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: OBJECTIVES: Volumetric magnetic resonance (MR)-guided high-intensity focused ultrasound (HIFU) is a completely noninvasive image-guided thermal ablation technique. Recently, there has been growing interest in the use of MR-HIFU for noninvasive ablation of malignant tumors. Of particular interest for noninvasive ablation of malignant tumors is reliable treatment monitoring and evaluation of response. At this point, there is limited evidence on the evolution of the ablation region after MR-HIFU treatment. The purpose of the present study was to comprehensively characterize the evolution of the ablation region after volumetric MR-HIFU ablation in a Vx2 tumor model using MR imaging, MR temperature data, and histological data. MATERIALS AND METHODS: Vx2 tumors in the hind limb muscle of New Zealand White rabbits (n = 30) were ablated using a clinical MR-HIFU system. Twenty-four animals were available for analyses. Magnetic resonance imaging was performed before and immediately after ablation; MR temperature mapping was performed during the ablation. The animals were distributed over 7 groups with different follow-up lengths. Depending on the group, animals were reimaged and then killed on day 0, 1, 3, 7, 14, 21, or 28 after ablation. For all time points, the size of nonperfused areas (NPAs) on contrast-enhanced T1-weighted (CE-T1-w) images was compared with lethal thermal dose areas (ie, the tissue area that received a thermal dose of 240 equivalent minutes or greater [EM] at 43°C) and with the necrotic tissue areas on histology sections. RESULTS: The NPA on CE-T1-w imaging showed an increase in median size from 266 ± 148 to 392 ± 178 mm during the first day and to 343 ± 170 mm on day 3, followed by a gradual decrease to 113 ± 103 mm on day 28. Immediately after ablation, the NPA was 1.6 ± 1.4 times larger than the area that received a thermal dose of 240 EM or greater in all animals. The median size of the necrotic area on histology was 1.7 ± 0.4 times larger than the NPA immediately after ablation. After 7 days, the size of the NPA was in agreement with the necrotic tissue area on histology (ratio, 1.0 ± 0.2). CONCLUSIONS: During the first 3 days after MR-HIFU ablation, the ablation region increases in size, after which it gradually decreases in size. The NPA on CE-T1-w imaging underestimates the extent of tissue necrosis on histology in the initial few days, but after 1 week, the NPA is reliable in delineating the necrotic tissue area. The 240-EM thermal dose limit underestimates the necrotic tissue area immediately after MR-HIFU ablation. Reliable treatment evaluation techniques are particularly important for noninvasive, image-guided tumor ablation. Our results indicate that CE-T1-w imaging is reliable for MR-HIFU treatment evaluation after 1 week.
[Show abstract][Hide abstract] ABSTRACT: Ultrasound (US) and microbubbles can be used to facilitate cellular uptake of drugs through a cavitationinduced enhancement of cell membrane permeability. The mechanism is, however, still incompletely understood. A direct contact between microbubbles and cell membrane is thought to be essential to create membrane perturbations lasting from seconds to minutes after US exposure of the cells. A recent study showed that the effect may even last up to 8 h after cavitation (with residual permeability up to 24 h after cavitation). In view of possible membrane damage, the purpose of this study was to further investigate the evolution of cell viability in the range of the 24-h temporal window. Furthermore, a description of the functional changes in tumor cells after US exposure was initiated to obtain a better understanding of the mechanism of membrane perturbation after sonication with microbubbles. Our results suggest that US does not reduce cell viability up to 24 h post-exposure. However, a perturbation of the entire cell population exposed to US was observed in terms of enzymatic activity and characteristics of the mitochondrial membrane. Furthermore, we demonstrated that US cavitation induces a transient loss of cell membrane asymmetry, resulting in phosphatidylserine exposure in the outer leaflet of the cell membrane.
IEEE transactions on ultrasonics, ferroelectrics, and frequency control 01/2013; 60(1):34-45. · 1.80 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Modern Magnetic Resonance Imaging (MRI) methods now allow the rapid acquisition of images with an excellent tissue contrast and high spatial resolution. Complex organ deformations can thus be estimated using image registration techniques applied to anatomical information. This opens great perspectives for the use of MRI to retroactively target an interventional procedure in mobile organs in real-time. For this purpose, both the update time and the latency of the motion information are two key points. In the current paper, the organ deformation is estimated on a voxel-by-voxel basis and a Kalman predictor is used to compensate for the residual latency. The implementation benefitted from the parallel architecture of Graphical Processing Units (GPU) for accelerating computation times. The efficiency and the potential of the method to anticipate organ displacements in real-time was evaluated on the abdomen of twelve free-breathing volunteers. The deformation of both kidney and liver could be updated with a rate of 10 Hz over sustained periods of several minutes, and the employed Kalman predictor reduced the tracking error in average by 30%.
Biomedical Imaging (ISBI), 2013 IEEE 10th International Symposium on; 01/2013
[Show abstract][Hide abstract] ABSTRACT: Real time magnetic resonance (MR) thermometry has evolved into the method of choice for the guidance of highintensity focused ultrasound (HIFU) interventions. For this role, MR-thermometry should preferably have a high temporal and spatial resolution and allow observing the temperature over the entire targeted area and its vicinity with a high accuracy. In addition, the precision of real time MR-thermometry for therapy guidance is generally limited by the available Signal to Noise ratio (SNR) and the influence of physiological noise. MR-guided HIFU would benefit of the large coverage volumetric temperature maps, including characterization of volumetric heating trajectories as well as near- and far-field heating. In this paper, continuous volumetric MR-temperature monitoring was obtained as follows: The targeted area was continuously scanned during the heating process by a multi-slice sequence. Measured data and a priori knowledge of 3D data derived from a forecast based on a physical model were combined using an Extended Kalman Filter (EKF). The proposed reconstruction improved the temperature measurement resolution and precision while maintaining guaranteed output accuracy. The method was evaluated experimentally ex-vivo on a phantom, and in-vivo on a porcine kidney, using HIFU heating. On the in-vivo experiment, it allowed the reconstruction from a spatio-temporally under-sampled data set (with an update rate for each voxel of 1.143 s) to a 3D dataset covering a field of view of 142.528554 mm3 with a voxel size of 336 mm3 and a temporal resolution of 0.127 s. The method also provided noise reduction, while having a minimal impact on accuracy and latency.
[Show abstract][Hide abstract] ABSTRACT: Optimizing the treatment of breast cancer remains a major topic of interest. In current clinical practice, breast-conserving therapy is the standard of care for patients with localized breast cancer. Technological developments have fueled interest in less invasive breast cancer treatment. Magnetic resonance-guided high-intensity focused ultrasound (MR-HIFU) is a completely noninvasive ablation technique. Focused beams of ultrasound are used for ablation of the target lesion without disrupting the skin and subcutaneous tissues in the beam path. MRI is an excellent imaging method for tumor targeting, treatment monitoring, and evaluation of treatment results. The combination of HIFU and MR imaging offers an opportunity for image-guided ablation of breast cancer. Previous studies of MR-HIFU in breast cancer patients reported a limited efficacy, which hampered the clinical translation of this technique. These prior studies were performed without an MR-HIFU system specifically developed for breast cancer treatment. In this article, a novel and dedicated MR-HIFU breast platform is presented. This system has been designed for safe and effective MR-HIFU ablation of breast cancer. Furthermore, both clinical and technical challenges are discussed, which have to be solved before MR-HIFU ablation of breast cancer can be implemented in routine clinical practice.
CardioVascular and Interventional Radiology 12/2012; · 2.09 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The objective of this study is to evaluate the feasibility to integrate
ultrasound echography as an additional imaging modality for continuous
target tracking, while performing simultaneously real-time MR-
thermometry to guide a High Intensity Focused Ultrasound (HIFU)
ablation. Experiments on a moving phantom were performed with MRI-guided
HIFU during continuous ultrasound echography. Real-time US
echography-based target tracking during MR-guided HIFU heating was
performed with heated area dimensions similar to those obtained for a
static target. The combination of both imaging modalities shows great
potential for real-time beam steering and MR-thermometry.
[Show abstract][Hide abstract] ABSTRACT: A novel large aperture transducer design is proposed for MR-HIFU
treatment of breast tumors. The phased array is composed of 384 elements
placed on a circular structure with a lateral beam direction. This beam
path orientation considerably reduces the risk of damaging nearby vital
organs. In addition, this transducer shape induces a well delineated
sharp focal point with low energy density in the near and far field for
safe, accurate and efficient treatment. The performance of the
transducer is demonstrated with acoustic field measurements and
temperature maps of a phantom sonication.
[Show abstract][Hide abstract] ABSTRACT: PURPOSE: Transport across the plasma membrane is a critical step of drug delivery for weakly permeable compounds with intracellular mode of action. The purpose of this study is to demonstrate real-time monitoring of ultrasound (US)-mediated cell-impermeable model drug uptake with fibered confocal fluorescence microscopy (FCFM). PROCEDURES: An in vitro setup was designed to combine a mono-element US transducer, a cell chamber with a monolayer of tumor cells together with SonoVue microbubbles, and a FCFM system. The cell-impermeable intercalating dye, SYTOX Green, was used to monitor US-mediated uptake. RESULTS: The majority of the cell population showed fluorescence signal enhancement 10 s after US onset. The mean rate constant k of signal enhancement was calculated to be 0.23 ± 0.04 min(-1). CONCLUSIONS: Feasibility of real-time monitoring of US-mediated intracellular delivery by FCFM has been demonstrated. The method allowed quantitative assessment of model drug uptake, holding great promise for further local drug delivery studies.
Molecular imaging and biology: MIB: the official publication of the Academy of Molecular Imaging 06/2012; · 2.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Tight regulation of gene expression in the region where therapy is necessary and for the duration required to achieve a therapeutic effect and to minimise systemic toxicity is very important for clinical applications of gene therapy. Hyperthermia in combination with a temperature sensitive heat shock protein (Hsp70) promoter presents a unique approach allowing non-invasive spatio-temporal control of transgene expression. In this study we investigated the in vivo and ex vivo relationship between temperature and duration of thermal stress with respect to the resulting gene expression using an Arrhenius analysis.
A transgenic mouse expressing the luciferase reporter gene under the transcriptional control of a thermosensitive promoter was used to assure identical genotype for in vivo (mouse leg) and ex vivo (bone marrow mononuclear and embryonic fibroblast cells) studies. The mouse leg and cells were heated at different temperatures and different exposure times. Bioluminescence imaging and in vitro enzymatic assay were used to measure the resulting transgene expression.
We showed that temperature-induced Hsp70 promoter activation was modulated by both temperature as well as duration of hyperthermia. The relationship between temperature and duration of hyperthermia and the resulting reporter gene expression can be modelled by an Arrhenius analysis for both in vivo as well as ex vivo.
However, the increase in reporter gene expression after elevating the temperature of the thermal stress with 1°C is not comparable for in vivo and ex vivo situations. This information may be valuable for optimising clinical gene therapy protocols.
International Journal of Hyperthermia 06/2012; 28(5):441-50. · 2.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Many chemotherapeutic drugs are characterized by high systemic toxicity and/or suffer from limited bioavailability. Thermosensitive liposomes (TSLs) encapsulating drugs in their aqueous lumen are promising activatable nanocarriers for ultrasound (US)-mediated drug delivery in response to mild hyperthermia. On the other hand, US is known to locally break biological barriers and as a consequence enable internalization of molecules. In this work, a two-step protocol for intracellular delivery of cell-impermeable molecules comprising of US-induced permeabilization followed by temperature-controlled release of the model drug from thermosensitive liposomes has been developed. TSLs containing TO-PRO-3, a cell-impermeable molecule that displays a significant increase in fluorescence upon binding to nucleic acids thus serving as a 'sensor' for internalization have been prepared and characterized in detail. US-mediated permeabilization followed by temperature-controlled release was applied to tumor bearing mice following i.v. injection of TSLs and microbubbles. The efficacy of this approach was evaluated by in vivo fluorescence imaging followed by histological analysis. A 2.4-fold increase of fluorescence signal was observed and intracellular delivery of TO-PRO-3 was confirmed by a characteristic nuclear staining. These results demonstrate the feasibility of novel drug delivery system to tumors comprising of local cell permeabilization by US followed by in situ release of the payload from thermosensitive liposomes. Possible applications include local and controlled intracellular delivery of molecules with otherwise limited bioavailability.
Journal of Controlled Release 04/2012; 161(1):90-7. · 7.63 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Online MR temperature monitoring during radiofrequency (RF) ablation of cardiac arrhythmias may improve the efficacy and safety of the treatment. MR thermometry at 1.5 T using the proton resonance frequency (PRF) method was assessed in 10 healthy volunteers under normal breathing conditions, using a multi-slice, ECG-gated, echo planar imaging (EPI) sequence in combination with slice tracking. Temperature images were post-processed to remove residual motion-related artifacts. Using an MR-compatible steerable catheter and electromagnetic noise filter, RF ablation was performed in the ventricles of two sheep in vivo. The standard deviation of the temperature evolution in time (TSD) was computed. Temperature mapping of the left ventricle was achieved at an update rate of approximately 1 Hz with a mean TSD of 3.6 ± 0.9 °C. TSD measurements at the septum showed a higher precision (2.8 ± 0.9 °C) than at the myocardial regions at the heart-lung and heart-liver interfaces (4.1 ± 0.9 °C). Temperature rose maximally by 9 °C and 16 °C during 5 W and 10 W RF applications, respectively, for 60 s each. Tissue temperature can be monitored at an update rate of approximately 1 Hz in five slices. Typical temperature changes observed during clinical RF application can be monitored with an acceptable level of precision.
NMR in Biomedicine 04/2012; 25(4):556-62. · 3.45 Impact Factor