Molecules of Various Pharmacologically-Relevant Sizes Can Cross the Ultrasound-Induced Blood-Brain Barrier Opening in vivo

Focused ultrasound (FUS) is hereby shown to noninvasively and selectively deliver compounds at pharmacologically relevant molecular weights through the opened blood-brain barrier (BBB). A complete examination on the size of the FUS-induced BBB opening, the spatial distribution of the delivered agents and its dependence on the agent's molecular weight were imaged and quantified using fluorescence microscopy. BBB opening in mice (n=13) was achieved in vivo after systemic administration of microbubbles and subsequent application of pulsed FUS (frequency: 1.525MHz, peak-rarefactional pressure in situ: 570 kPa) to the left murine hippocampus through the intact skin and skull. BBB-impermeant, fluorescent-tagged dextrans at three distinct molecular weights spanning over several orders of magnitude were systemically administered and acted as model therapeutic compounds. First, dextrans of 3 and 70 kDa were delivered trans-BBB while 2000 kDa dextran was not. Second, compared with 70 kDa dextran, a higher concentration of 3 kDa dextran was delivered through the opened BBB. Third, the 3 and 70 kDa dextrans were both diffusely distributed throughout the targeted brain region. However, high concentrations of 70 kDa dextran appeared more punctated throughout the targeted region. In conclusion, FUS combined with microbubbles opened the BBB sufficiently to allow passage of compounds of at least 70 kDa, but not greater than 2000 kDa into the brain parenchyma. This noninvasive and localized BBB opening technique could, thus, provide a unique means for the delivery of compounds of several magnitudes of kDa that include agents with shown therapeutic promise in vitro but whose in vivo translation has been hampered by their associated BBB impermeability. (E-mail: ek2191@columbia.edu)     

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)     

Identifying the Inertial Cavitation Threshold and Skull Effects in a Vessel Phantom Using Focused Ultrasound and Microbubbles

Focused ultrasound (FUS) in combination with microbubbles has been shown capable of delivering large molecules to the brain parenchyma through opening of the blood-brain barrier (BBB). However, the mechanism behind the opening remains unknown. To investigate the pressure threshold for inertial cavitation of preformed microbubbles during sonication, passive cavitation detection in conjunction with B-mode imaging was used. A cerebral vessel was simulated by generating a cylindrical hole of 610 μm in diameter inside a polyacrylamide gel and saturating its volume with microbubbles. Definity microbubbles (Mean diameter range: 1.1-3.3 μm, Lantheus Medical Imaging, N. Billerica, MA, USA) were injected prior to sonication (frequency: 1.525 MHz; pulse length: 100 cycles; PRF: 10 Hz; sonication duration: 2 s) through an excised mouse skull. The acoustic emissions due to the cavitation response were passively detected using a cylindrically focused hydrophone, confocal with the FUS transducer and a linear-array transducer with the field of view perpendicular to the FUS beam. The broadband spectral response acquired at the passive cavitation detector (PCD) and the B-mode images identified the occurrence and location of the inertial cavitation, respectively. Findings indicated that the peak-rarefactional pressure threshold was approximately equal to 0.45 MPa, with or without the skull present. Mouse skulls did not affect the threshold of inertial cavitation but resulted in a lower inertial cavitation dose. The broadband response could be captured through the murine skull, so the same PCD set-up can be used in future in vivo applications. (E-mail: ek2191@columbia.edu)     

Noninvasive, transcranial and localized opening of the blood-brain barrier using focused ultrasound in mice

The feasibility of blood-brain barrier (BBB) opening in the hippocampus of wild-type mice using focused ultrasound (FUS) through the intact skull and skin was investigated. Needle hydrophone measurements through ex vivo skulls revealed minimal attenuation ( approximately 18% of the pressure amplitude), a well-focused beam pattern and minute focus displacement through the parietal bone. In experiments in vivo, the brains of three mice were sonicated transcranially. Pulsed ultrasound sonications at 1.5 MHz and acoustic pressures ranging from 0.8 to 2.7 MPa were used at 20% duty cycle. Before sonication, a bolus of 10 microL of an ultrasound contrast agents (Optison) was injected intravenously. Contrast-enhanced high-resolution magnetic resonance imaging (9.4 T) revealed BBB opening and allowed for the monitoring of the slow permeation of gadolinium in the hippocampus. The region of the brain where BBB opening occurred increased with the pressure amplitude. These findings thus demonstrated the feasibility of locally opening the BBB in mice using FUS through intact skull and skin and serve as the first step in determining and assessing feasibility of drug delivery to specific regions in the mouse brain using FUS.     

Microbubble Type and Distribution Dependence of Focused Ultrasound-Induced Blood–Brain Barrier Opening

Focused ultrasound, in the presence of microbubbles, has been used non-invasively to induce reversible blood–brain barrier (BBB) opening in both rodents and non-human primates. This study was aimed at identifying the dependence of BBB opening properties on polydisperse microbubble (all clinically approved microbubbles are polydisperse) type and distribution by using a clinically approved ultrasound contrast agent (Definity microbubbles) and in-house prepared polydisperse (IHP) microbubbles in mice. A total of 18 C57 BL/6 mice (n = 3) were used in this study, and each mouse was injected with either Definity or IHP microbubbles via the tail vein. The concentration and size distribution of activated Definity and IHP microbubbles were measured, and the microbubbles were diluted to 6 × 10⁸/mL before injection. Immediately after microbubble administration, mice were subjected to focused ultrasound with the following parameters: frequency = 1.5 MHz, pulse repetition frequency = 10 Hz, 1000 cycles, in situ peak rarefactional acoustic pressures = 0.3, 0.45 and 0.6 MPa for a sonication duration of 60 s. Contrast-enhanced magnetic resonance imaging was used to confirm BBB opening and allowed for image-based analysis. Permeability of the treated region and volume of BBB opening did not significantly differ between the two types of microbubbles (p > 0.05) at peak rarefractional acoustic pressures of 0.45 and 0.6 MPa, whereas IHP microbubbles had significantly higher permeability and opening volume (p < 0.05) at the relatively lower pressure of 0.3 MPa. The results from this study indicate that microbubble type and distribution could have significant effects on focused ultrasound-induced BBB opening at lower pressures, but less important effects at higher pressures, possibly because of the stable cavitation that governs the former. This difference may have become less significant at higher pressures, where inertial cavitation typically occurs.     

Influence of Nanobubble Concentration on Blood–Brain Barrier Opening Using Focused Ultrasound Under Real-Time Acoustic Feedback Control

Real-time acoustic feedback control based on harmonic emissions of stimulated microbubbles may serve as a way to achieve reliable blood–brain barrier (BBB) opening with focused ultrasound in the brain. Previously, we demonstrated BBB opening was possible using sub-micron bubbles (aka nanobubbles) and produced comparable results to commercially available microbubbles (Optison, Definity, etc.). The harmonic emissions and acoustic control were observed to be more consistent using nanobubbles, which warrants further study of BBB opening using these agents. This study examined the stimulated acoustic emissions of nanobubbles at different concentrations both in vitro and in vivo and evaluated BBB opening under real-time acoustic feedback control across concentrations. Original nanobubbles (10¹¹ bubbles/mL) have long in vitro persistence (7.3 ± 3.3 min) and circulation time in rats (approximately 10 min) under exposures in this study, and both degraded with dilutions. With all three tested dilutions (1:1, 1:10 and 1:100), successful BBB opening was reliably achieved under real-time feedback control.     

电镜硝酸镧示踪超声微泡造影剂开放血脑屏障

目的 研究超声波破坏微泡造影剂对大鼠血脑屏障通透性的影响。 方法 用电镜硝酸镧示踪法观察超声波破坏微泡造影剂对大鼠血脑屏障通透性的变化。 结果 超声波照射后即刻，即可见镧颗粒通过毛细血管内皮细胞及细胞间的紧密连接进入组织间隙，血脑屏障开放持续至6h，12h时已关闭。电镜下可以见到血管源性脑水肿，细胞器水肿不明显。 结论 超声波破坏微泡造影剂开放血脑屏障，为中枢神经系统疾病的治疗提供了一种无创、具靶向性的药物转运方法。     

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.     

Correlation Between Brain Tissue Damage and Inertial Cavitation Dose Quantified Using Passive Cavitation Imaging

Focused ultrasound (FUS)-induced cavitation-mediated brain therapies have become emerging therapeutic modalities for neurologic diseases. Cavitation monitoring is essential to ensure the safety of all cavitation-mediated therapeutic techniques as inertial cavitation can be associated with tissue damage. The objective of this study was to reveal the correlation between the inertial cavitation dose, quantified by passive cavitation imaging (PCI), and brain tissue histologic-level damage induced by FUS in combination with microbubbles. An ultrasound image-guided FUS system consisting of a single-element FUS transducer (1.5 MHz) and a co-axially aligned 128-element linear ultrasound imaging array was used to perform FUS treatment of mice. Mice were sonicated by FUS with different peak negative pressures (0.5 MPa, 1.1 MPa, 4.0 MPa and 6.5 MPa) in the presence of systemically injected microbubbles. The acoustic emissions from the FUS-activated microbubbles were passively detected by the imaging array. The pre-beamformed channel data were acquired and processed offline using the frequency-domain delay, sum and integration algorithm to generate inertial cavitation maps. All the mice were sacrificed after the FUS treatment, and their brains were harvested and processed for hematoxylin and eosin staining. The obtained inertial cavitation maps revealed the dynamic changes of microbubble behaviors during FUS treatment at different pressure levels. It was found that the inertial cavitation dose quantified based on PCI had a linear correlation with the scale of histologic-level tissue damage. Findings from this study suggested that PCI can be used to predict histologic-level tissue damage associated with the FUS-induced cavitation.     

纳米微泡载药靶向治疗中枢神经系统白血病的研究进展

中枢神经系统白血病（centralnervoussystemleukemia,CNS—L）是一种缓解率低而复发率高、死亡率高的白血病致命合并症。由于血脑屏障（bloodbrainbarrier,BBB）的存在阻碍了药物进入中枢神经系统,因而寻求一种新的给药系统使药物能够高效通过血脑屏障成为亟待解决的问题。检索2012年12月至2014年2月中英文文献发现,针对中枢神经系统白血病已有一种可能有效的解决方案即超声微泡载阿糖胞苷（Ara—C）,其通过空化效应改变细胞膜的通透性并增加细胞间隙而提高阿糖胞苷通过血脑屏障的效率进而达到有效治疗的目的。本文就中枢神经系统白血病的治疗现状及超声微泡载药治疗中枢神经系统白血病方面的进展进行综述。     

Cavitation threshold of microbubbles in gel tunnels by focused ultrasound

The investigation of inertial cavitation in micro-tunnels has significant implications for the development of therapeutic applications of ultrasound such as ultrasound-mediated drug and gene delivery. The threshold for inertial cavitation was investigated using a passive cavitation detector with a center frequency of 1 MHz. Micro-tunnels of various diameters (90 to 800 microm) embedded in gel were fabricated and injected with a solution of Optison(trade mark) contrast agent of concentrations 1.2% and 0.2% diluted in water. An ultrasound pulse of duration 500 ms and center frequency 1.736 MHz was used to insonate the microbubbles. The acoustic pressure was increased at 1-s intervals until broadband noise emission was detected. The pressure threshold at which broadband noise emission was observed was found to be dependent on the diameter of the micro-tunnels, with an average increase of 1.2 to 1.5 between the smallest and the largest tunnels, depending on the microbubble concentration. The evaluation of inertial cavitation in gel tunnels rather than tubes provides a novel opportunity to investigate microbubble collapse in a situation that simulates in vivo blood vessels better than tubes with solid walls do.     

A Comparison of Sonothrombolysis in Aged Clots between Low-Boiling-Point Phase-Change Nanodroplets and Microbubbles of the Same Composition

We present enhanced cavitation erosion of blood clots exposed to low-boiling-point (−2°C) perfluorocarbon phase-change nanodroplets and pulsed ultrasound, as well as microbubbles with the same formulation under the same conditions. Given prior success with microbubbles as a sonothrombolysis agent, we considered that perfluorocarbon phase-change nanodroplets could enhance clot disruption further beyond that achieved with microbubbles. It has been hypothesized that owing to their small size and ability to penetrate into a clot, nanodroplets could enhance cavitation inside a blood clot and increase sonothrombolysis efficacy. The thrombolytic effects of lipid-shell-decafluorobutane nanodroplets were evaluated and compared with those of microbubbles with the same formulation, in an aged bovine blood clot flow model. Seven different pulsing schemes, with an acoustic intensity (I_SPTA) range of 0.021–34.8 W/cm² were applied in three different therapy scenarios: ultrasound only, ultrasound with microbubbles and ultrasound with nanodroplets (n = 5). Data indicated that pulsing schemes with 0.35 W/cm² and 5.22 W/cm² produced a significant difference (p < 0.05) in nanodroplet sonothrombolysis performance compared with compositionally identical microbubbles. With these excitation conditions, nanodroplet-mediated treatment achieved a 140% average thrombolysis rate over the microbubble-mediated case. We observed distinctive internal erosion in the middle of bovine clot samples from nanodroplet-mediated ultrasound, whereas the microbubble-mediated case generated surface erosion. This erosion pattern was supported by ultrasound imaging during sonothrombolysis, which revealed that nanodroplets generated cavitation clouds throughout a clot, whereas microbubble cavitation formed larger cavitation clouds only outside a clot sample.     

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.     

Localized Blood–Brain Barrier Opening in Ovine Model Using Image-Guided Transcranial Focused Ultrasound

Transcranial application of focused ultrasound (FUS) combined with vascular introduction of microbubble contrast agents (MBs) has emerged as a non-invasive technique that can temporarily create a localized opening in the blood–brain barrier (BBB). Under image-guidance, we administered FUS to sheep brain after intravenous injection of microbubbles. BBB opening was confirmed by performing dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) to detect the extravasated gadolinium-based magnetic resonance contrast agents. Through pharmacokinetic analysis as well as independent component analysis of the DCE-MRI data, we observed localized enhancement in BBB permeability at the area that subjected to acoustic pressure of 0.48 MPa (mechanical index = 0.96). On the other hand, application of a higher pressure at 0.58 MPa resulted in localized, minor cerebral hemorrhage. No animals exhibited abnormal behavior during the post-FUS survival periods up to 2 mo. Our data suggest that monitoring for excessive BBB disruption is important for safe translation of the method to humans.     

The Long-Term Fate of the Sonoporated Pancreatic Cancer Cells is Uncorrelated With the Degree of Model Molecular Loading

Studies have determined that ultrasound-activated microbubbles can increase the membrane permeability of tumor cells by triggering membrane perforation (sonoporation) to improve drug loading. However, because of the distinct cavitation events adjacent to each cell, the degree of drug loading appeared to be heterogeneous. The relationship between the long-term fate trend and the degree of drug loading remains unclear. To investigate the time-lapse viability of diversity loading cells, fluorescein isothiocyanate–dextran (FITC-dextrans) was used as a molecular model mixed with 2% v/v SonoVue microbubbles (Bracco, Milan, Italy) and exposed to various peak negative pressures (0.25 MPa, 0.6 MPa, 1.2 MPa), 1 MHz frequency and 300 μs pulse duration. To select a suitable parameter, the cavitation activity was measured, and the cell analysis was performed by flow cytometry under these acoustic pressures. The sonoporated cells were then categorized into 3 sub-groups by flow cytometry according to the various fluorescence intensity distributions to analyze their long-term fate. We observed that the stable cavitation occurred at 0.25 MPa and microbubbles underwent ultra-harmonic emission, and obvious broadband signals were observed at 0.6 MPa and 1.2 MPa, suggesting the occurs of inertial cavitation. The cell analysis further showed the maximum delivery efficiency and cell viability at 0.6 MPa, and it was selected for the following experiment. The categorization displayed that the fluorescence intensity of FITC-dextrans in sub-groups 2 and 3 were approximate 5.62-fold and 19.53-fold higher than that in sub-group 1, respectively. After separation of these sub-groups, the apoptosis and necrosis ratios in all 3 sub-groups of sonoporated cells gradually increased with increasing culture time and displayed no significant difference in either the apoptosis (p > 0.05) or necrosis (p > 0.05) ratio after 6 h and 24 h of culture, respectively. Further analysis using Western blot verified that the long-term fate of sonoporated cells involves the mitochondrial signaling proteins. These results provide better insight into the role of cavitation-enhanced permeability and a critical guide for acoustic cavitation designs.     

Detection of intracerebral hemorrhage and transient blood-supply shortage in focused-ultrasound-induced blood-brain barrier disruption by ultrasound imaging

Focused ultrasound (FUS) in the presence of microbubbles can selectively open the blood-brain barrier (BBB). However, since overexcitation by FUS probably induces intracerebral hemorrhage, it is essential to develop an imaging approach for real-time detection of hemorrhage and blood-flow changes during FUS-induced BBB disruption. Here we investigated the feasibility of using ultrasound imaging to monitor the transient responses of FUS-induced BBB disruption. The BBB was disrupted with in-house-manufactured microbubbles in rats by 1-MHz FUS with a pressure of 1.1 MPa (pulse repetition frequency: 1 Hz, pulse duration: 10 ms, exposure time: 60 s) and imaged for the next 2 h. Ultrasound B-mode imaging was used to detect hyperechoic changes induced by hemorrhage and contrast-enhanced ultrasound (US) imaging was performed to analyze changes in blood flow. Hyperechoic spots appeared in B-mode images at 5 s after FUS sonication and contrast-enhanced US images simultaneously showed a region of transient blood-supply shortage in the sonicated area. Thus, the location of hyperechoic spots correlated with hemorrhagic patterns and the blood-supply-shortage region was consistent with the BBB-disrupted areas. Furthermore, we detected a transient hyperemic response in the unsonicated contralateral hemisphere brain. Our approach has potential as an immediate-feedback control tool for preventing the induction of intracerebral hemorrhage during FUS treatment.     

Bioeffects considerations for diagnostic ultrasound contrast agents.

Diagnostic ultrasound contrast agents have been developed for enhancing the echogenicity of blood and for delineating other structures of the body. Approved agents are suspensions of gas bodies (stabilized microbubbles), which have been designed for persistence in the circulation and strong echo return for imaging. The interaction of ultrasound pulses with these gas bodies is a form of acoustic cavitation, and they also may act as inertial cavitation nuclei. This interaction produces mechanical perturbation and a potential for bioeffects on nearby cells or tissues. In vitro, sonoporation and cell death occur at mechanical index (MI) values less than the inertial cavitation threshold. In vivo, bioeffects reported for MI values greater than 0.4 include microvascular leakage, petechiae, cardiomyocyte death, inflammatory cell infiltration, and premature ventricular contractions and are accompanied by gas body destruction within the capillary bed. Bioeffects for MIs of 1.9 or less have been reported in skeletal muscle, fat, myocardium, kidney, liver, and intestine. Therapeutic applications that rely on these bioeffects include targeted drug delivery to the interstitium and DNA transfer into cells for gene therapy. Bioeffects of contrast-aided diagnostic ultrasound happen on a microscopic scale, and their importance in the clinical setting remains uncertain.     

Functional ultrasound-triggered phase-shift perfluorocarbon nanodroplets for cancer therapy

In recent years, because of their unique properties, the use of perfluorocarbon nanodroplets (PFC NDs) in ultrasound-mediated tumor theranostics has attracted increasing interest. PFC is one of the most stable organic compounds with high hydrophobicity. Phase-shift PFC NDs can be transformed into highly echogenic microbubbles for ultrasound and photoacoustic imaging by ultrasound and laser light. In addition, in the process of acoustic droplet vaporization, PFC NDs with cavitation nuclei can be combined with a variety of ultrasound technologies to produce cavitation effects for tumor ablation, antivascular therapy and release of therapeutic agents loaded in nanodroplets. Moreover, they can also be used to overcome tumor hypoxia by virtue of high oxygen solubility. In this review, first the preparation and stabilization of PFC NDs are summarized and then the issues and outlook are discussed. More importantly, multifunctional platforms based on PFC NDs for cancer diagnostics, therapy and theranostics are reviewed in detail.     

Simulation,Implementation and Measurement of Defined Sound Fields for Blood–Brain Barrier Opening in Rats

The blood–brain barrier (BBB) is the most important obstacle to delivery of therapeutics to the central nervous system. Low-intensity pulsed focused ultrasound (FUS) in combination with microbubbles applied under magnetic resonance imaging (MRI) control provides a non-invasive and safe technique for BBB opening (BBBo). In rodent models, however, settings and application protocols differ significantly. Depending on the strain and size, important variables include ultrasound attenuation and sound field distortion caused by the skull. We examined the ultrasound attenuation of the skull of Wistar rats using a targeted FUS system. By modifying the transducer elements and by varying and simulating the acoustic field of the FUS system, we measured a skull attenuation of about 60%. To evaluate potential application of the targeted FUS system in genetically modified animals with increased sensitivity to brain hemorrhage caused by vascular dysfunction, we assessed safety in healthy animals. Histological and MRI analyses of the central nervous system revealed an increase in the number and severity of hyperacute bleeds with focal pressure. At a pressure of 0.4 MPa, no bleeds were induced, albeit at the cost of a weaker hyperintense MRI signal post BBBo. These results indicate a relationship between pressure and the dimension of permeabilization.     

Encapsulated ultrasound microbubbles: therapeutic application in drug/gene delivery.

Encapsulated gas microbubbles are well known as ultrasound contrast agents for medical ultrasound imaging. Nonetheless, not only do these microbubbles help to image, but they can also be used as drug/gene carriers. The microbubbles as drug/gene carriers have an average size less than that of red blood cells, i.e. they are capable of penetrating even into the small blood capillaries and releasing drug and genes under the action of ultrasound field. The application of ultrasound and microbubbles to targeted drug and gene delivery has been the subject of intense experimental research. Under exposure of sufficiently high-amplitude ultrasound, these targeted microbubbles would rupture, spewing drugs or genes, which are contained in its encapsulating layer, to targeted cells or tissues. Recently, targeting ligands are attached to the surface of the microbubbles (i.e. targeted-microbubbles), which have been widely used in cardiovascular system and tumor diagnosis and therapy. In this paper, the characterization of novel targeted ultrasonic contrast agents or microbubbles and their potential applications in drug delivery or gene therapy are reviewed.