Evaluation of Dose Distribution of Molecular Delivery After Blood-Brain Barrier Disruption by Focused Ultrasound with Treatment Planning

The permeability of the blood-brain barrier (BBB) can be enhanced by focused ultrasound (FUS) in localized regions with applications of ultrasound contrast agent (UCA). The purpose of this study was to evaluate the dose distribution of Evans blue (EB) in the targeted brain by sonication with treatment strategy. FUS exposure was applied with an ultrasound frequency of 1 MHz, a 5% duty cycle and a repetition frequency of 1 Hz. Single sonication with two doses of UCA and two sonications at the same location or an interval of 3 mm to induce BBB disruption for assessing dose distribution. The permeability of the BBB was measured quantitatively based on EB extravasation. Gadolinium deposition was monitored by contrast enhanced MR imaging for dose distribution of the focal plane. Hematoxylin and eosin staining was performed for histologic observation. No significant difference was found for EB in the focal regions between the single sonication with UCA at a dose of 300 μL/kg and repeated sonication with UCA at a lower dose of 150 μL/kg. There was a sharper dose distribution in the brain with repeated sonication at the same location, compared with the brain receiving two sonications at an interval of 3 mm. Compared with a single sonication with UCA at a dose of 150 μL/kg, the histologic evaluation of the sonicated regions indicated that more erythrocytes were seen in the brain treated with single sonication at a higher dose of 300 μL/kg or repeated sonication at a dose of 150 μL/kg. This study demonstrated that the dose distribution of molecular delivery could be regulated by sonication with treatment planning.     

Transcranial Photoacoustic Detection of Blood-Brain Barrier Disruption Following Focused Ultrasound-Mediated Nanoparticle Delivery

Molecular Imaging and Biology - Blood-brain barrier disruption (BBBD) is of interest for treating neurodegenerative diseases and tumors by enhancing drug delivery. Focused ultrasound (FUS) is a...     

脑超声造影中超声强度对血脑屏障通透性的影响

目的探讨不同机械指数的诊断性超声在超声造影中对血脑屏障通透性的影响,以了解脑超声造影检查中超声强度的安全应用范围。方法50只清洁级SD大鼠,经尾静脉注射剂量为1ml/kg的“脂氟显”超声造影剂,辅以不同机械指数的超声进行辐照,观察超声照射后血脑屏障通透性的变化。结果在MI等于0.4时,血脑屏障通透性与对照组相比无统计学差异,当MI≥0.8时,血脑屏障通透性增加,且随着超声能量的进一步提高血脑屏障的通透性增加。结论高机械指数的体表超声在超声造影中可导致血脑屏障通透性增加,但应用适当强度的超声在进行大脑超声造影时仍是安全的。     

Ultrasound Molecular Imaging and Drug Delivery

Ultrasound is a rapidly advancing field with many emerging diagnostic and therapeutic applications. For diagnostics, new vascular targets are routinely identified and mature technologies are being translated to humans, while other recent innovations may bring about the creation of acoustic reporter genes and micron-scale resolution with ultrasound. As a cancer therapy, ultrasound is being explored as an adjuvant to immune therapies and to deliver acoustically or thermally active drugs to tumor regions. Ultrasound-enhanced delivery across the blood brain barrier (BBB) could potentially be very impactful for brain cancers and neurodegenerative diseases where the BBB often impedes the delivery of therapeutic molecules. In this minireview, we provide an overview of these topics in the field of ultrasound that are especially relevant to the interests of World Molecular Imaging Society.     

Multi-Modality Safety Assessment of Blood-Brain Barrier Opening Using Focused Ultrasound and Definity Microbubbles: A Short-Term Study

As a potentially viable method of brain drug delivery, the safety profile of blood-brain barrier (BBB) opening using focused ultrasound (FUS) and ultrasound contrast agents (UCA) needs to be established. In this study, we provide a short-term (30-min or 5-h survival) histological assessment of murine brains undergoing FUS-induced BBB opening. Forty-nine mice were intravenously injected with Definity microbubbles (0.05 μL/kg) and sonicated under the following parameters: frequency of 1.525 MHz, pulse length of 20 ms, pulse repetition frequency of 10 Hz, peak rarefactional acoustic pressures of 0.15–0.98 MPa and two 30-s sonication intervals with an intermittent 30-s delay. The BBB opening threshold was found to be 0.15–0.3 MPa based on fluorescence and magnetic resonance imaging of systemically injected tracers. Analysis of three histological measures in hematoxylin and eosin–stained sections revealed the safest acoustic pressure to be within the range of 0.3–0.46 MPa in all examined time periods post sonication. Across different pressure amplitudes, only the samples 30 min post opening showed significant difference (p < 0.05) in the average number of distinct damaged sites, microvacuolated sites, dark neurons and sites with extravasated erythrocytes. Enhanced fluorescence around severed microvessels was also noted and found to be associated with the largest tissue effects, whereas mildly diffuse BBB opening with uniform fluorescence in the parenchyma was associated with no or mild tissue injury. Region-specific areas of the sonicated brain (thalamus, hippocampal fissure, dentate gyrus and CA3 area of hippocampus) exhibited variation in fluorescence intensity based on the position, orientation and size of affected vessels. The results of this short-term histological analysis demonstrated the feasibility of a safe FUS-UCA–induced BBB opening under a specific set of sonication parameters and provided new insights on the mechanism of BBB opening. (E-mail: ek2191@columbia.edu)     

Improved Anti-Tumor Effect of Liposomal Doxorubicin After Targeted Blood-Brain Barrier Disruption by MRI-Guided Focused Ultrasound in Rat Glioma

The blood-brain barrier (BBB) inhibits the entry of the majority of chemotherapeutic agents into the brain. Previous studies have illustrated the feasibility of drug delivery across the BBB using focused ultrasound (FUS) and microbubbles. Here, we investigated the effect of FUS-enhanced delivery of doxorubicin on survival in rats with and 9L gliosarcoma cells inoculated in the brain. Each rat received either: (1) no treatment (control; N = 11), (2) FUS only (N = 9), (3) IV liposomal doxorubicin (DOX only; N = 17), or (4) FUS with concurrent IV injections of liposomal doxorubicin (FUS+DOX; N = 20). Post-treatment by magnetic resonance imaging (MRI) showed that FUS+DOX reduced tumor growth compared with DOX only. Further, we observed a modest but significant increase in median survival time after a single treatment FUS+DOX treatment (p = 0.0007), whereas neither DOX nor FUS had any significant impact on survival on its own. These results suggest that combined ultrasound-mediated BBB disruption may significantly increase the antineoplastic efficacy of liposomal doxorubicin in the brain.     

脑超声造影中超声造影剂剂量对血脑屏障通透性的影响

目的探讨不同剂量超声造影剂在超声造影中对血脑屏障通透性的影响。方法60只清洁级SD大鼠,给予相同机械指数的超声进行辐照,并经尾静脉注射不同剂量的“脂氟显”超声造影剂,观察超声照射后血脑屏障通透性的变化。结果在造影剂剂量为50μl/kg时,血脑屏障通透性即与对照组相比产生显著性统计学差异,且随着超声造影剂剂量的增加其引起血脑屏障通透性也随之增加。结论在相同能量强度超声场中,超声造影剂微泡数目是引起不同生物学效应的重要因素。     

Ultrafast Imaging of Ultrasound Contrast Agents

The disappearance of ultrasound contrast agents after disruption can provide useful information on their environment. However, in vivo acoustical imaging of this transient phenomenon, which has a duration on the order of milliseconds, requires high frame rates that are unattainable by conventional ultrasound scanners. In this article, ultrafast imaging is applied to microbubble tracking using a 128-element linear array and an elastography scanner. Contrast agents flowing in a wall-less tissue phantom are insonified with a high-intensity disruption pulse followed by a series of plane waves emitted at a 5 kHz PRF. A collection of compounded images depicting the evolution of microbubbles is obtained after the echoes are beamformed in silico. The backscattering of the microbubbles appears to increase in the first image after disruption (4 ms) and decrease following an exponential decay in the next hundred milliseconds. This microbubble dynamic depends on the length and amplitude of the high-intensity pulse. Furthermore, confined microbubbles are found to differ significantly from their free-flowing counterparts in their dissolution curves. The high temporal resolution provided by ultrafast imaging could help distinguish targeted microbubbles during molecular imaging. (E-mail: olicou@gmail.com)     

Focused Ultrasound Platform for Investigating Therapeutic Neuromodulation Across the Human Hippocampus

Pulsed low-intensity focused ultrasound (PLIFUS) has shown promise in inducing neuromodulation in several animal and human studies. Therefore, it is of clinical interest to develop experimental platforms to test repetitive PLIFUS as a therapeutic modality in humans with neurologic disorders. In the study described here, our aim was to develop a laboratory-built experimental device platform intended to deliver repetitive PLIFUS across the hippocampus in seizure onset zones of patients with drug-resistant temporal lobe epilepsy. The system uses neuronavigation targeting over multiple therapeutic sessions. PLIFUS (548 kHz) was emitted across multiple hippocampal targets in a human subject with temporal lobe epilepsy using a mechanically steered piezoelectric transducer. Stimulation was delivered up to 2.25 W/cm² spatial peak temporal average intensity (free-field equivalent), with 36%–50% duty cycle, 500-ms sonications and 7-s inter-stimulation intervals lasting 140 s per target and repeated for multiple sessions. A first-in-human PLIFUS course of treatment was successfully delivered using the device platform with no adverse events.     

包裹Gd-DTPA的高分子材料超声造影剂显影效果兔实验研究

目的 观察自制包裹Gd-DTPA的高分子材料超声造影剂(Gd-PLGA)的显影效果、特点及其安全性.方法 质量浓度为10%的Gd-PLGA溶液,按0.5 ml/kg剂量经兔耳缘静脉团注,分别对肝血管、肝实质进行超声及磁共振成像,并对肝实质信号强度进行动态定量分析;所有兔在Gd-PLGA注射之前以及注射之后7、14、30 d静脉取血进行肝功能和肾功能检测.结果 Gd-PLGA在超声显像中能明显增强肝血管、肝实质回声强度,而在磁共振成像中肝实质信号强度亦较造影前增强,作用时间均持续约30 min;所有兔在实验过程中以及实验后观察1个月均未发现明显异常,且各组肝、肾功能指标差异无统计学意义.结论 Gd-PLGA是一安全、显影效果好、持续时间长、能实现实时超声及磁共振双重造影的新型多功能高分子材料超声造影剂.     

Critical Appraisal of Targeted Ultrasound Contrast Agents for Molecular Imaging in Large Arteries

Molecular imaging may provide new insights into the early detection and development of atherosclerosis before first symptoms occur. One of the techniques in use employs noninvasive ultrasound. In the past decade, experimental and clinical validation studies showed that for the microcirculation targeted ultrasound contrast agents, such as echogenic liposomes, microbubbles and perfluorocarbon emulsions, do improve visualization of specific structures. For large arteries, however, successful application is less obvious. In this review, we will address the challenges for molecular imaging of large arteries. We will discuss the problems encountered in the use of targeted ultrasound contrast agents presently available, mainly based on data obtained in flow chambers and animal studies because clinical studies are lacking. We conclude that molecular imaging of activated endothelium in large- and middle-sized arteries by site-specific accumulation of contrast material is still difficult to achieve due to wall shear stress conditions in these vessels. (A.Hoeks@bf.unimaas.nl)     

Non-invasive Magnetic Resonance Imaging Follow-up of Sono-sensitive Liposome Tumor Delivery and Controlled Release After High-Intensity Focused Ultrasound

This work examines the use of lanthanide-based contrast agents and magnetic resonance imaging in monitoring liposomal behavior in vivo. Dysprosium (Dy) and gadolinium (Gd) chelates, Dy-diethylenetriaminepentaacetic acid bismethylamide (Dy-DTPA-BMA) and Gd-DTPA-BMA, were encapsulated in pegylated distearoylphosphatidylethanolamine-based (saturated) liposomes, and then intravenously injected into Copenhagen rats with subcutaneous Dunning AT2 xenografts. Liposome-encapsulated Dy chelate shortens transverse relaxation times (T₂ and T₂*) of tissue; thus, liposomal accumulation in the tumor can be monitored by observing the decrease in T₂* relaxation time over time. The tumor was treated at the time of maximum liposomal accumulation (48 h) with confocal, cavitating high-intensity focused ultrasound to induce liposomal payload release. Using liposome-encapsulated Gd chelate at high enough concentrations and saturated liposomal phospholipids induces an exchange-limited longitudinal (T₁) relaxation when the liposomes are intact; when the liposomes are released, exchange limitation is relieved, thus allowing in vivo observation of payload release as a decrease in tumor T₁.     

Distribution and Diffusion of Macromolecule Delivery to the Brain via Focused Ultrasound using Magnetic Resonance and Multispectral Fluorescence Imaging

Focused ultrasound (FUS), in combination with microbubble contrast agents, can be used to transiently open the blood–brain barrier (BBB) to allow intravascular agents to cross into the brain. Often, FUS is carried out in conjunction with magnetic resonance imaging (MRI) to evaluate BBB opening to gadolinium-based MRI contrast agents. Although MRI allows direct visualization of the distribution of gadolinium-based contrast agents in the brain parenchyma, it does not allow measurements of the distribution of other molecules crossing the BBB. Therapeutic molecules (e.g., monoclonal antibodies) are much different in size than MRI contrast agents and have been found to have different distributions in the brain after FUS-mediated BBB opening. In the work described here, we combined in vivo MRI and ex vivo multispectral fluorescence imaging to compare the distributions of MRI contrast and dextran molecules of different molecular weights (3, 70 and 500 kDa) after FUS-mediated BBB opening through a range of ultrasound pressures (0.18–0.46 MPa) in laboratory mice. The volume of brain exposed was calculated from the MRI and fluorescence images and was significantly dependent on both molecular weight and ultrasound pressure. Diffusion coefficients of the different-molecular-weight dextran molecules in the brain parenchyma were also calculated from the fluorescence images and were negatively correlated with the molecular weight of the dextran molecules. The results of this work build on a body of knowledge that is critically important for the FUS technique to be used in clinical delivery of therapeutics to the brain.     

Calibration of Ultrasound Backscatter Temperature Imaging for High-Intensity Focused Ultrasound Treatment Planning

High-intensity focused ultrasound (HIFU) is rapidly gaining acceptance as a non-invasive method for soft tissue tumor ablation, but improvements in the methods of treatment delivery, planning and monitoring are still required. Backscatter temperature imaging (BTI) uses ultrasound to visualize heating-induced echo strain and may be used to indicate the position of the HIFU focal region using low-power “sub-lesioning” exposure. The technique may also provide a quantitative tool for assessing the efficacy of treatment delivery if apparent strain measurements can be related to the underlying temperature rise. To obtain temperature estimates from strain measurements, the relationship between these variables has to be either measured or otherwise assumed from previous calibrations in similar tissues. This article describes experimental measurements aimed at deriving the relationship between temperature rise and apparent strain in the laboratory environment using both ex vivo bovine liver tissue samples and normothermically perfused porcine livers. A BTI algorithm was applied to radiofrequency ultrasound echo data acquired from a clinical ultrasound scanner (Z.One, Zonare Medical Systems, Mountain View, CA, USA) where the imaging probe was aligned with the focal region of a HIFU transducer. Temperature measurements were obtained using needle thermocouples implanted in the liver tissue. A series of “non-ablative” HIFU exposures giving peak temperatures below 10°C were made in three separate ex vivo bovine livers, yielding an average strain/temperature coefficient of 0.126 ± 0.088 percentage strain per degree Celsius. In the perfused porcine livers at a starting temperature of 38°C (normal body temperature) the strain/temperature coefficients were found to be 0.040 ± 0.029 percentage strain per degree Celsius. The uncertainty in these results is directly linked to the precision of the strain measurement, as well as the naturally occurring variance between different tissue samples, indicating that BTI may lack the accuracy required to be implemented successfully in practice as a quantitative treatment planning technique at a sub-lesioning exposure level. This is because, to be of use in treatment planning, temperature-rise estimates may require an accuracy greater (<10%) than that offered by BTI measurement. BTI may, however, still play a role in ensuring the correct positioning of the focal region and as a treatment monitoring modality capable of detecting an increased rate of heating in tissue after HIFU ablation.     

A Comparison of Focused and Unfocused Ultrasound for Microbubble-Mediated Gene Delivery

We compared focused and unfocused ultrasound-targeted microbubble destruction (UTMD) for delivery of reporter plasmids to the liver and heart in mice. Optimal hepatic expression was seen with double-depth targeting at 5 and 13 mm in vivo, incorporating a low pulse repetition frequency and short pulse duration. Reporter expression was similar, but the transfection patterns were distinct, with intense foci of transfection using focused UTMD (F-UTMD). We then compared both approaches for cardiac delivery and found 10-fold stronger levels of reporter expression for F-UTMD and observed small areas of intense luciferase expression in the left ventricle. Non-linear contrast imaging of the liver before and after insonation also showed a substantially greater change in signal intensity for F-UTMD, suggesting distinct cavitation mechanisms for both approaches. Overall, similar levels of hepatic transgene expression were observed, but cardiac-directed F-UTMD was substantially more effective. Focused ultrasound presents a new frontier in UTMD-directed gene therapy.     

Ultrasound Molecular Imaging of Cardiovascular Disease

Molecular imaging with ultrasound contrast agents relies on the detection of microbubbles within diseased tissue. Microbubbles produce an acoustic signal owing to their resonant properties in an ultrasound field. Microbubble targeting is accomplished by either manipulating the microbubble shell for attachment of microbubbles to activated leukocytes, or by conjugation of disease-specific ligands to the microbubble surface. Inflammation, angiogenesis, and thrombus formation are central pathophysiologic processes in many cardiovascular diseases and produce phenotypic changes in the vascular compartment that can be imaged with targeted ultrasound contrast agents. In the future, targeted contrast ultrasound could aid in the diagnosis of atherosclerosis, myocardial ischemia, transplant rejection, and thrombosis syndromes and could be used for assessing angiogenesis.     

Focused Ultrasound and Microbubble Treatment Increases Delivery of Transferrin Receptor-Targeting Liposomes to the Brain

The blood-brain barrier (BBB) is a major obstacle to treating several brain disorders. Focused ultrasound (FUS) in combination with intravascular microbubbles increases BBB permeability by opening tight junctions, creating endothelial cell openings, improving endocytosis and increasing transcytosis. Here we investigated whether combining FUS and microbubbles with transferrin receptor-targeting liposomes would result in enhanced delivery to the brain of post-natal rats compared with liposomes lacking the BBB-targeting moiety. For all animals, increased BBB permeability was observed after FUS treatment. A 40% increase in accumulation of transferrin receptor-targeting liposomes was observed in the FUS-treated hemisphere, whereas the isotype immunoglobulin G liposomes showed no increased accumulation. Confocal laser scanning microscopy of brain sections revealed that both types of liposomes were mainly observed in endothelial cells in the FUS-treated hemisphere. The results demonstrate that FUS and microbubble treatment combined with BBB-targeting liposomes could be a promising approach to enhance drug delivery to the brain.