CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Nos. 61/244,311 entitled "Improved Opening of Tissue Barrier," filed on September 21,

2009, 61/353,611 entitled "Systems and Methods for Opening a Tissue Utilizing Certain Sonication Pulse Values," filed on June 10, 2010, and 61/353,631 entitled "Brain Drug Delivery Using Focused Ultrasound and Microbubbles," filed June 10,

2010, each of which is incorporated by reference in its entirety herein and from which priority is claimed. This application is also related to U.S. Patent Application No.

12/077,612, filed March 19, 2008, and International Patent Application No.

PCT/US09/056565, filed September 10, 2009, each of which is incorporated by reference in its entirety herein. STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH

This invention was made with government support under R01 EB009041 and R21 EYO 18505 awarded by the National Institutes of Health and CAREER 0644713 awarded by the National Science Foundation. The government has certain rights in the invention.

TECHNICAL FIELD

The present application relates to systems and methods for opening a tissue utilizing acoustic parameters in conjunction with microbubbles. BACKGROUND

Recent advances in molecular engineering and neuro science have led to an increasing number of biomarkers and therapeutic agents for the monitoring and treatment of neurological disorders. Many of these agents have proven in vitro specificity or neurological potency, but their in vivo efficacy remains limited by their inability to reach their target due to the blood-brain barrier. This interface regulates the exchange of molecules across the cerebral capillaries through passive, transport, and metabolic barriers, resulting in the exclusion of nearly all agents larger than 400 Da from the brain's extracellular space. Biomarkers and therapeutic agents, such as inhibitors to enzymes (~1 kDa) and antibodies (30 to 300 kDa), are thus rendered ineffective because they do not reach their intended targets.

SUMMARY

Systems and methods for opening a tissue to a target value are disclosed herein.

In an embodiment of a method for opening a tissue to a target value using microbubbles, a region of the tissue is targeted for opening, an acoustic parameter corresponding to the target value is determined and an ultrasound beam is applied to the target region at the acoustic parameter such that the tissue at the target region is opened to the target value with the microbubbles. The method can further include positioning microbubbles in proximity to the targeted region and, in some embodiments, positioning the microbubbles can include performing an injection of the microbubbles such that the microbubbles are positioned proximate to the targeted region. The method can further include determining a number of injections and/or a duration of an injection corresponding to the target value. In some embodiments, the injection can be a systemic injection, a bolus injection and/or a slow diffusion injection. The acoustic parameter can be selected to control an acoustic cavitation event and, in some embodiments, controlling an acoustic cavitation event can include controlling a location, number and/or magnitude of acoustic cavitation events. The acoustic parameter can be a pulse length, a pulse repetition frequency, a burst length, a burst repetition frequency, an ultrasound frequency, a pressure range, and/or a duration corresponding to the target value. In some embodiments, the pressure range can correspond to the resonance frequency of the microbubbles proximate to the targeted region.

The method can include determining a concentration range of microbubbles corresponding to the target value and applying an ultrasound beam to move the microbubbles into vessels of the tissue. In some embodiments, the microbubbles can have a size range of 1 to 10 microns, and in other embodiments can have a size range of 1 to 2 microns, 4 to 5 microns, or 6 to 8 microns. The microbubbles can be acoustically activated and/or molecule-carrying. The molecule- carrying microbubbles can carry or be coated with medicinal molecules and/or a contrast agent and/or a biomarker and/or a liposome. Medicinal molecules and/or contrast agents can also be separately positioned in proximity to the targeted region.

The method can further include imaging the targeted region, to form an image of the opened tissue. In some embodiments imaging the targeted region includes applying an ultrasound beam to the targeted region, while in other embodiments imaging the targeted region includes utilizing a magnetic resonance imaging device and/or a fluorescence imaging device to image the targeted region.

An embodiment of a system for opening a tissue to a target value using a solution of microbubbles having a size range corresponding to the target value includes a targeting assembly for targeting a region of the tissue, an introducer for delivering the solution to a location proximate to the targeted region and a transducer, coupled to the targeting assembly, for applying an ultrasound beam to the targeted region at an acoustic parameter corresponding to the target value thereby opening the tissue with the microbubbles to the target value. The acoustic parameter can be selected to control an acoustic cavitation event.

The system can further include an imaging device for capturing image data of the opened tissue of the targeted region, and a processor, operatively coupled to the imaging device, for processing the image data to form an image therefrom. In some embodiments the imaging device includes a transducer for applying an ultrasound beam to the targeted region, while in other embodiments the imaging device includes a magnetic resonance imaging device and/or a fluorescence imaging device to image the targeted region. BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated and constitute part of this disclosure, illustrate some embodiments of the disclosed subject matter.

FIG. 1 illustrates a method for opening a blood-brain barrier in a brain of a subject to a target value in accordance with an exemplary embodiment of the disclosed subject matter.

FIG. 2 illustrates a method for imaging the opening of a blood-brain barrier in a brain of a subject to a target value in accordance with an exemplary embodiment of the disclosed subject matter. FIG. 3(a) illustrates a system for opening and/or imaging the opening of a blood-brain barrier in a brain of a subject to a target value in accordance with an exemplary embodiment of the disclosed subject matter.

FIG. 3(b) illustrates another system for opening and/or imaging the opening of a blood-brain barrier in a brain of a subject to a target value in accordance with an exemplary embodiment of the disclosed subject matter.

FIGS. 4(a)-(e) illustrate a targeting system for locating a target region of the brain of a subject in accordance with an exemplary embodiment of the disclosed subject matter.

FIG. 5 illustrates a system for opening the blood-brain barrier used in connection with an experiment on mice in accordance with an exemplary

embodiment of the disclosed subject matter.

FIG. 6(a) illustrates a fluorescence image at ten times magnification of a mouse brain after application of tissue opening techniques in accordance with an exemplary embodiment of the disclosed subject matter.

FIGS. 6(b)-(c) illustrate fluorescence images at four times magnification of a mouse brain after application of tissue opening techniques in accordance with an exemplary embodiment of the disclosed subject matter.

FIG. 7 is a graph illustrating the effects of varying the microbubble concentration in accordance with an exemplary embodiment of the disclosed subject matter.

FIG. 8(a) is a graph illustrating the effects of varying the pulse repetition frequency in accordance with an exemplary embodiment of the disclosed subject matter.

FIG. 8(b) is a graph illustrating the probability of blood-brain barrier opening as a function of varying the pulse repetition frequency in accordance with an exemplary embodiment of the disclosed subject matter.

FIG. 9(a) is a graph illustrating the effects of varying the pulse length in accordance with an exemplary embodiment of the disclosed subject matter.

FIGS. 9(b)-(c) are graphs illustrating the probability of blood-brain barrier opening as a function of varying the pulse length in accordance with an exemplary embodiment of the disclosed subject matter. FIGS. 10(a)-(h) illustrate histological images of a mouse brain after focused ultrasound sonication with a varying pulse length in accordance with an exemplary embodiment of the disclosed subject matter.

FIG. 11 illustrates an exemplary pulse and burst sequence in accordance with an exemplary embodiment of the disclosed subject matter.

FIGS. 12(a)-(c) are graphs illustrating the effects of varying the burst repetition frequency for three different pulse repetition frequencies in accordance with an exemplary embodiment of the disclosed subject matter.

FIG. 12(d) is a graph illustrating the effects of varying the burst length for a certain pulse repetition frequency and burst length in accordance with an exemplary embodiment of the disclosed subject matter.

FIGS. 13(a)-(d) illustrate images of a mouse brain subjected to sonication using pulse length of three cycles in accordance with an exemplary embodiment of the disclosed subject matter.

Throughout the figures and specification the same reference numerals are used to indicate similar features and/or structures.

DETAILED DESCRIPTION

The systems and methods described herein are useful for opening a tissue utilizing microbubbles and focused ultrasound at certain acoustic parameters. Although the description provides as an the example opening the blood-brain barrier, the systems and methods herein are useful for opening other tissues, such as muscular tissue, liver tissue or tumorous tissue, among others.

The subjected matter disclosed herein are methods and systems for determining the acoustic parameters for opening a tissue with the assistance of microbubbles to allow for the passage of certain molecules over selected areas.

Accordingly, the techniques described herein make use of selected acoustic parameters chosen to produce a desired opening effect in a tissue when subjected to focus ultrasound utilizing microbubbles of selected sizes and in selected

concentrations. The techniques described herein for determining the acoustic parameters for opening a tissue can also be employed in conjunction with other ultrasound techniques, e.g., diagnostic techniques, where opening of a tissue should be avoided. The techniques described herein can be used to determine the acoustic parameters that can be avoided in order to prevent unwanted tissue opening when utilizing such other techniques that, for example, use microbubbles.

In focused ultrasound (FUS), acoustic waves propagate several centimeters through water or tissue and converge onto a focal region while its surroundings remain relatively unaffected. Noninvasive and localized drug delivery systems have emerged from advances in FUS and microbubble technologies. For example, techniques such as blood-brain barrier (BBB) disruption for the treatment of neurological diseases, delivery of nanoparticles to tumors, gene therapy for treating heart conditions, and enhancement of renal ultrafiltration have all shown promise due to their ability to increase uptake of luminal molecules into the interstitial space.

The mechanistic event underlying the tissue opening in such examples is the reaction of microbubbles to ultrasonic pulses, which can result in an array of behaviors known as acoustic cavitation. In stable cavitation, the microbubble expands and contracts with the acoustic pressure rarefaction and compression over several cycles, and such action can result in the displacement of the vessel diameter through dilation and contraction. In inertial cavitation, the bubble can expand to several factors greater than its equilibrium radius and subsequently collapse due to the inertia of the surrounding media, thus also inducing an alteration of the vascular physiology. The type and magnitude of each cavitation activity can be dictated by, among other things, the microbubble composition and distribution, the ultrasonic pulse shape and sequence, and the in vivo environment in which the bubbles circulate. Control of molecular delivery using FUS can therefore be facilitated by selecting microbubbles and the acoustic environment conditions that they interact with.

Generally, changing ultrasonic parameters has been associated with a tradeoff between efficacy (e.g., high dose, homogeneous distribution, and

consistency) and safety (e.g., erythrocyte extravasations and neuronal damage). The acoustic pressure can have a large influence on the type and magnitude of acoustic cavitation activity. Thus, increasing the pressure increases the likelihood and extent of BBB opening, but also is associated with neurovascular and neuronal damage. At acoustic pressures near the threshold of BBB disruption, histological assessments reveal no detectable damage (e.g., erythrocyte extravasations and/or neuronal damage) but at such low pressures there is also a reduction in molecular delivery. Lowering the transmitted center frequency of the ultrasound can result in a decrease of the acoustic pressure threshold.

The pulse repetition frequency (PRF) can effect the ability of microbubbles to reperfuse the vasculature since each pulse can destroy microbubbles. Further, it has been thought that a long pulse length (PL) was a necessary

characteristic of an ultrasonic pulse for inducing BBB disruption, for example PLs of 10 or 20 ms (1 200 and 30400 cycles at 1.5 MHz) and such long PLs have been associated with inhomogeneity of drug delivery. However, in accordance with an embodiment herein, it is shown that BBB disruption is feasible at a low pressure (less than 1 MPa) using a PL of 33 μ3 (50 cycles at 1.5 MHz). Utilizing such a low PL and pressure, an improved distribution of the delivered agent was observed, but an associated decrease in concentration of molecules delivered was also observed. As further shown in an embodiment herein, pulse sequences based on the use of a PL of 2.3 (3.5 cycles at .5 MHz) can enhance the dose and distribution of delivery without compromising safety.

An acoustic parameter design can be formed on the basis that BBB disruption is dependent on the number, magnitude and/or location of cavitation events occurring throughout the cerebral microvasculature. Further, acoustic cavitation activity within the microvasculature can be modified by taking into account concepts of microbubble persistence, fragmentation, and microvascular replenishment. In one embodiment, a series of pulses can be grouped into a burst, with a sufficient duration between bursts to allow for microbubble replenishment in the microvasculature before arrival of the subsequent acoustic pulses. Grouping pluses into bursts can increase the persistence and mobility of the microbubbles, which can result in a single bubble generating cavitation activity at multiple sites along the cerebral

microvasculature. Thus, in some embodiments, short PLs used in a burst sequence can enhance the dose and distribution of molecular delivery without attendant damage to the microvasculature. In the same or other embodiments, the number injections and duration of each injection of bubbles can be altered to enhance the microbubble persistence, for example.

Figure 1 illustrates a method 100 for opening a tissue to a target value, e.g., a measure of increased ability of the tissue to pass molecules through. The target value can be expressed in terms of an increase in the size of vessels in the tissue, as an area of the tissue that has been opened, or in terms of a rate at which molecules pass through, e.g., a permeability, or as a combination of any of these measures. The method 100 involves targeting 110 a region of the tissue for opening, determining 120 at least one acoustic parameter corresponding to the target value, positioning 140 microbubbles in proximity to the targeted region, and applying 170 an ultrasound beam at the acoustic parameter to the targeted region such that the tissue is opened with the assistance of the microbubbles to the target value. In some embodiments, positioning 140 the microbubbles can include performing an injection of the microbubbles such that the microbubbles are positioned proximate to the targeted region. The method 100 can further include determining a number of injections and/or a duration of an injection corresponding to the target value. In some embodiments, the injection 140 can be a systemic injection, a bolus injection and/or a slow diffusion injection.

As illustrated in Figure 1 , in one exemplary embodiment, the method 100 can further include of determining 130 a concentration range of microbubbles corresponding to the target value and the positioning 140 of the microbubbles can also include positioning the microbubbles of the concentration range that corresponds to the target value. The method 100 can also include positioning 150 a contrast agent and/or medicinal molecule (e.g., a drug) in proximity to the target region.

In one exemplary embodiment, method 100 can include applying 160 an ultrasound beam to move the microbubbles into vessels of the tissue. This application 160 of the ultrasound beam can be the same, or a different, than the application 170 that is used to open the tissue. Further, the application 160 of the ultrasound beam can be at the same, or at a different, acoustic parameter than that determined 120 for the purposes of opening the tissue.

The acoustic parameter to be determined 120 as corresponding to the target value can be selected to control one or more acoustic cavitation events. The acoustic parameter (s) can be selected such that the location, number and/or magnitude of acoustic cavitation events can be controlled in the targeted tissue. In some embodiments, the acoustic parameter can be at least one of the pulse length, the pulse repetition frequency, the burst length, or the burst repetition frequency, or a combination thereof. In other embodiments, the acoustic parameters that are determined 120 can be the pressure range, the frequency, and the duration of the application 170 of ultrasound.

In some embodiments, the target value of the tissue can be selected based on the size of the molecule that is to pass through the tissue, e.g., the BBB, or based on the size, e.g. , area, of the region that is to be exposed to the molecule, or a combination of the two. Thus, in some embodiments, the acoustic parameters can be determined 120 such that the tissue is subject to a certain number of acoustic cavitations at selected locations and of selected magnitudes, which can result in a selected number of molecules of a given size passing through the tissue at selected locations. In one exemplary embodiment, the target value can be such that molecules up to the megaDalton size range are able to pass through the BBB, e.g., 2 MDa molecules.

In one embodiment, the target value can be measured in terms of the normalized optical density (NOD) of a contrast agent such as a dextran, e.g., Texas- Red ^® fluorescent dye and a molecular weight of 3 kiloDaltons (kDa), using the equation NOD = F _L^RO1 - F _R _ _R0} , where FL-ROI is the sum of the pixel values in left region of interest (ROI) of the brain and RL-ROJ is the sum of the pixel values in the right ROI of the brain, and where the ultrasound was applied 170 to the left ROI.

In some embodiments, the acoustic parameter can be determined 120 by finding the lowest acoustic parameter value for which the tissue will open to the target value, where the target value is considered to be the minimum amount of opening. In one example involving murine brains, the acoustic parameters to be determined 120 were the PRF and the PL. In accordance with an exemplary experiment detailed below, a PRF value of 1 Hz was experimentally determined 120 to be the lowest PRF for which the BBB of a mouse subject was observed to open based on an observed NOD of approximately 2 x 10 ⁷ . A PL value of 0.033 ms was experimentally determined 120 to be the lowest PL for which the BBB was observed to open based on an observed NOD of approximately 0.5 x 10 ⁷ . In other

embodiments, a PL of 3 cycles (less than 2.5 με) was experimentally determined 120 to open the BBB in mice, in an experiment conducted in accordance with an exemplary embodiment described below.

In some embodiments, the acoustic parameter can be determined 120 by finding the lowest reliable acoustic parameter value which will reliably open the tissue to the target value. In an example involving murine brains where the target value was considered the minimum NOD for which BBB opening was observed, the acoustic parameters to be determined 120 were the PRF and the PL. In accordance with an exemplary experiment detailed below, a PRF value of 5 Hz was

experimentally determined 120 to be the lowest PRF for which the BBB of a mouse subject was observed to reliably open based on an observed NOD of approximately 2 x 10 ⁷ . A PL value of 0.2 ms was experimentally determined 120 to be the lowest PL for which the BBB was observed to reliably open based on an observed NOD of approximately 0.5 x 10 ⁷ .

In yet other embodiments, the acoustic parameter can be determined

120 by finding the acoustic parameter value above which no further significant increase in opening of the tissue is achieved. In an example involving murine brains where the target value was considered to be the NOD above which no further increase in BBB opening was observed, the acoustic parameters to be determined 120 were the PRF and the PL. In accordance with an exemplary experiment detailed below, a PRF value of 5 Hz was experimentally determined 120 to be the PRF for which no further significant increase in the opening of the BBB of a mouse subject was observed and such opening corresponded to an NOD of approximately 2,5 x 10 ⁷ . A PL value of 10 ms was experimentally determined 120 to be the lowest PL for which no further significant increase in the opening of the BBB of a mouse subject was observed and such opening corresponded to an observed NOD of approximately 3 x 10 ⁷ .

In the same or other embodiment involving murine brains, the acoustic parameters to be determined 120 can be the burst repetition frequency (BRF) and burst length (BL), where each burst can represent a cluster of pulses. A BRF of 10 Hz was experimentally determined 120 to open the BBB of a mouse subject, where the PRF was set at 100 kHz, and a BRF of 5 Hz was experimentally determined 120 to produce the maximal BBB opening at the same frequency. A BL of 100 pulses was experimentally determined 120 to open the BBB where the PRF was set at 100 kHz and the BRF was set at 5 Hz.

In some embodiments determining 130 a concentration range of microbubbles corresponding to the target value can include determining the minimum microbubble concentration range that will open the tissue to the target value. In an example involving murine brains, a microbubble concentration of 0.01 μΐ/g was experimentally determined 130 to be the minimum concentration needed to open the BBB. In same or another embodiment, the appropriate concentration of microbubbies can be determined 130 based on the nature of the subject, e.g., a human or a mouse, based on the size of the target region, e.g., the surface area of the BBB that one wishes to open, and based on the vessel size in the target region, e.g., 4-8 μιη, or a combination of these factors. In the example of opening a BBB area on the order of millimeters, a concentration range of 10 to 10 bubbles/mL can be appropriate. In one exemplary embodiment, the total concentration for both size ranges of bubbles, e.g., 1-2 and 4-5 μηι, was kept constant at approximately 8.5 x 10 ⁸ number of bubbles per mL. In order to ensure accuracy of concentration, the bubbles were generated at an initial yield larger than the desired concentration and then diluted in PBS one minute before intravenous injection into the mouse.

In one exemplary embodiment, the bubble concentration can be chosen to be the same across different size distributions as opposed to the volume fraction, because it can be assumed that BBB opening occurs discretely, e.g. , the sites of molecular leakage highly correlated with the instantaneous locations of the bubbles at the time of sonication. This implies that BBB opening sites are punctuated along the length of the capillaries. In the case where the volume fraction was kept the same for both sets of bubbles, it is deemed that the imaging protocol used would have the required sensitivity to detect minute increases in fluorescence.

As detailed in commonly assigned International Patent Pub. WO 2010/030819, which is incorporated by reference in its entirety herein, in some embodiments the appropriate size range of microbubbies can be determined by comparing the bubble size to the cerebral vasculature size and selecting a bubble size that is small enough to perfuse the vessels while at the same time large enough to induce sufficient mechanical stress on the vessel walls, such that the vessels are opened to the target value.

Figure 2 illustrates a method 200 in accordance with the disclosed subject matter for imaging the opening of a tissue. The method 200 includes the same basic techniques for opening the tissue to a target value: targeting 110 a region of the tissue for opening, determining 120 at least one acoustic parameter corresponding to the target value, positioning 140 microbubbies of a known size range in proximity to the targeted region, and applying 170 an ultrasound beam at the acoustic parameter to the targeted region such that the tissue is opened with the assistance .of the

microbubbles to the target value. The method 200 further includes imaging 21 the opened tissue. In some embodiments, imaging 210 the opened tissue can be the same as the application 170 of an ultrasound beam to open the tissue. In another embodiment, imaging 210 can include utilizing an MRI device to image the opening of the tissue.

Figure 3(a) illustrates a system 300 for opening a tissue to a target value. System 300 has many of the same features as the system described in U.S. Patent Pub. No. 2009/000571 1 and International Patent Pub. No. WO 2010/030819, commonly assigned patent applications, each of which is incorporated by reference in its entirety herein. Ultrasound waves are generated by a focused ultrasound transducer (FUS) 302, which can be a single-element circular-aperture FUS transducer. In one exemplary embodiment the FUS transducer 302 can be a single- element, spherical segment FUS transducer with center frequency of 1.525 MHz, a focal depth of 90 mm, an outer radius of 30 mm, and an inner radius of 1 1.2 mm (Riverside Research Institute, New York, NY, USA). The FUS transducer can be provided with hole in its center for receipt of an imaging transducer 304, which can be a single-element diagnostic transducer having a center frequency of 7.5 MHz with a focal length of 60 mm (Riverside Research Institute, New York, NY, USA). The FUS transducer 302 and the diagnostic transducer 304 can be positioned so that the foci of the two transducers are properly aligned, e.g., overlap.

Further illustrated in Figure 3(a), an exemplary system 300 can include a cone 306 filled with degassed and distilled water and mounted on system 300. The cone 306 can, for example, be manufactured from a clear plastic, such as

polyurethane. The water is contained in the cone 306 by capping it with a material considered substantially "transparent" to the ultrasound beam, such as an ultrathin polyurethane membrane 308 (Trojan; Church & Dwight Co., Princeton, N.J., USA).

The transducer assembly, which can include the FUS transducer 302 and the diagnostic transducer 304, can be mounted to a computer-controlled 3-D positioning system 310 (Velmex Inc., Lachine, QC, Canada), including motors VXM- 1 and VXM-2 used in the exemplary embodiment. It is understood that other positioning systems can be incorporated for positioning the transducer assembly with respect to the targeted tissue. In the same or another sxemplary embodiment, the FUS transducer 302 can be driven by a function generator 320, e.g. , function generator HP33150A, manufactured by Agilent Technologies, Palo Alto, Calif, USA, through an amplifier 322, such as a 50-dB power amplifier 3100L (ENI, Inc., Rochester, N.Y., USA). The diagnostic transducer 304 can be driven by a pulser-receiver system 342, for example a pulser-receiver 5052PR (Panametrics, Waltham, Mass., USA), connected to a digitizer 326, e.g., digitizer CS 14200 (Gage Applied Technologies, Inc., Lachine, QC, Canada). It is understood that the above-described components can be modified or replaced with other components, as is known in the art, for producing the ultrasound beams described herein. Computer 328 typically includes a processor, such as a CPU (not shown), and can be any appropriate personal computer or distributed computer system including a server and a client. For example, a computer useful for this system is a Dell Precision 380 personal computer. It is understood that any personal computer, laptop, or other processor that can load software and communicate with the various components discussed herein can be used. A memory unit (not shown), such as a disk drive, flash memory, volatile memory, etc., can be used to store software for positioning and operating the transducer assembly, image data, a user interface software, and any other software which can be loaded onto the CPU.

In another exemplary embodiment illustrated in Figure 3(b), system

300' can include a transducer assembly having an array of a plurality of single- element FUS transducers 304 and 305 which can be targeted to different regions of the tissue of the subject. Each FUS transducer 304, 305 in the array can be fired individually, thereby permitting opening of the BBB in several locations without repositioning the transducer assembly.

Prior to sonication and in order to verify undistorted propagation through the skull, a scan, such as a 3-D raster-scan (lateral step size: 0.2 mm; axial step size: 1.0 mm), of the beam of the FUS transducer 302, can optionally be performed in a large water tank containing degassed water with a needle hydrophone having a needle diameter on the order of about 0.2 mm (Precision Acoustics Ltd., Dorchester, Dorset, UK). In this manner the pressure amplitudes and three- dimensional beam dimensions of the FUS transducer 302 can be measured. The pressure amplitudes can be measured by calculating the peak-rarefactional pressure values and accounting for an pressure attenuation due to transcranial propagation, e.g., arrl8% pressure attenuation. The dimensions of the beam provided by the PUS transmitter 302 can have a lateral and axial full-width at half-maximum (FWHM) intensity of approximately 1.32 and 13.0 mm, respectively, and in some embodiments can be approximately equal to the dimensions of the beam after propagation through the skull.

System 300 also includes a liquid container 334 containing an appropriate liquid 336, e.g., degassed and distilled water, which is sealed at the bottom with a membrane 338, which can be a polyurethane membrane that is acoustically and transparent, e.g., plastic wrap. The system 300 can also include an optical imaging device 340, such as a digital camera, for imaging the skull of the subject 332 and a MRI device 350 for imaging the brain of the subject 332.

System 300 also includes a platform 330 for the subject. In one exemplary embodiment, the platform 330 for the subject can be a polyurethane bed for a smaller subject 332, such as a mouse. In this configuration, the membrane 338 can be placed over the subject 332. In other embodiments, the platform 330 can be a hospital bed or surgical table, in which a larger subject 332 (such as a human subject) can be laid prone or supine and the transducer assembly positioned on top of the region of the skull targeted.

Additional components of the system 300 include a targeting system

400, coupled to the FUS transducer 302, for locating the focus of the FUS transducer 302 in the brain of the subject 332. The targeting system 400 can be coupled by any known method that permits the targeting system 400 to aid in properly targeting the FUS transducer 302 to the region of interest for opening of the target tissue, e.g., acoustic and/or optical coupling. Figures 4(a)-(d) illustrate a targeting system 400 for use with an embodiment where the subject 332 is a mouse. Figure 4(a) illustrates mouse skull 401, where the skull's sutures can be seen through the skin and used as anatomic landmarks for targeting purposes. As illustrated in Figure 4(a), the landmarks of mouse skull 401 include the sagittal suture 402, the frontal bone 404, the interparietal bone 406, the left parietal bone 408, and the right parietal bone 410.

Figure 4(b) illustrates the placement of targeting system 400 on skull 401 in accordance with an exemplary embodiment. The targeting system 400 can include a plurality of members 420, 422, 424, such as thin metal bars, e.g., 0.3 mm thin metal bars, fabricated from an acoustically reflective material, e.g. , paper clips. The metal bars 420, 422, 424 can be placed on several landmarks of the skull of the subject to create a layout, or grid. As illustrated in Figure 4(b), a grid consisting of three equally spaced 0.3 -mm thin F2 metal bars 420, 422, 424 were placed in the water bath 334 on top of the skull 401 and in alignment with these landmarks, e.g., bone sutures. The first bar 420 was aligned parallel and along the sagittal suture 402, and the second bar 424 was attached perpendicularly to the first bar and in alignment with the suture between the parietal 408 and interparietal bone 406. The third bar 422 was placed 4 mm away from and parallel to the second bar 424.

Figure 4(c) illustrates the location of a brain structure 440 to be targeted, here the hippocampus, relative to the landmarks noted above. The location of the hippocampi are assumed relative to the sutures based on the mouse brain and known skull anatomy. In this exemplary embodiment, using the grid positioning system 400, the location of one of the hippocampi (indicated by circle 440) was reproducibly targeted when assumed to be at mid-distance (arrow 442) between the parallel bars 422, 424 and 2 mm away from the center bar 420 (arrow 444).

To locate the desired brain structure 440 an image, such as a lateral 2- D raster scan, of the grid configuration can be made using the diagnostic transducer 304. The focus of the FUS transducer 302 can then be positioned to precisely target the desired brain structure 440. In another exemplary embodiment, the targeting system can include other imaging devices, such as a digital camera 340. For example, a digital camera 340 can be used to photograph the head of the subject 332. The relevant landmarks can be identified in the photograph, and the focus of the FUS transducer 302 targeted to a location relative to the landmarks. In addition, other MRI targeting equipment, as is known in the art, can be used for targeting the desired brain structure 440 or other targeted tissue structure.

Figure 4(d) illustrates the actual location of the hippocampus 446 as indicated in the histology slice. Figure 4(e) illustrates a lateral 2-D raster-scan 490 of the grid 400 using the diagnostic transducer 304. The location of the hippocampus can be identified relative to this grid. The focus of the FUS transducer 302 was placed 3 mm beneath the top of the skull by measuring distance with the diagnostic transducer 304. Using the grid positioning system 400 and depth calculations, precise, accurate and reproducible targeting of the hippocampus or other brain structures can be performed. In one exemplary embodiment, the grid positioning-, system 400 ^' allowed for sonication of the same location with good accuracy across different mice. This allowed for not only good reproducibility across different mice, but also a good comparison of BBB opening effects in different regions 440 within the sonicated area.

An exemplary method 100 for opening the BBB will be described in connection with the above-referenced figures. The subject 332 is positioned on a platform 330. Subject 332 can be positioned in a prone position, and can be anesthetized for the sonication procedure. The degassed and distilled water bath 334 is suspended over the subject's 332 head. Ultrasound gel can be used to reduce any remaining impedance mismatches between the thin plastic layer 338 and the subject's 332 skin. The transducer assembly can be placed in the water bath 334 with its beam axis perpendicular to the surface of the skull 401.

The focus of the transducer is positioned inside the subject's 332 brain. The focus can be targeted 110 to a region of the brain 440, such as the desired brain tissue, e.g., the hippocampus 446, or to the vasculature of the brain, e.g. , arteries, ventricles, arterioles, and capillaries of the brain, or to other target tissue regions at different locations in the subject 332. The targeted region 440 of the brain can be located 110 utilizing the targeting system as discussed above.

Example

Figure 5 illustrates a system 300 used in an experiment, approved by the Columbia University Institutional Animal Care and Use Committee, on seventy- nine wild-type mice (strain: C57BL/6, mass: 28.0 ± 4.5 g, sex: male; Harlan,

Indianapolis, IN, USA) which were studied in accordance with the techniques described herein. As illustrated in Figure 7, the system 300 can include a FUS transducer 302, a pulse-echo diagnostic transducer 304, a cone 306, a latex membrane 308, a 3-D positioning system 310 all operatively connected to a function generator 320, a power amplifier 322, a pulse-receiver system 324, a digitizer 326 and a computer 327. The cone 306 can be inserted into a water container 334 which is sealed at the bottom by a polyurethane membrane 338 and placed on the shaved skull 502 of the mouse subject 332. The mouse subject 332 is held in place using a stereotaxic apparatus 504. In the experiment, the mice. were anesthetized using 1.25-2.50% isoflurane (SurgiVet, Smiths Medical PM, Inc., Wisconsin, USA) throughout both the BBB opening and transcardial perfusion procedures. After being anesthetized, each mouse 332 was placed prone with its head immobilized by the stereotaxic apparatus 504 (David opf Instruments, Tujunga, CA, USA). The hair on the skull was removed using an electric trimmer and a depiatory cream. A degassed water-filled container 334 sealed at the bottom with thin, acoustically and optically transparent, Saran™ Wrap 338 (Saran™; SC Johnson, Racine, WI, USA) was placed on top of the mouse head 602 while ultrasound coupling gel was used to eliminate any remaining impedance mismatch between the two surfaces. The FUS transducer 302 was then submerged in the water of the container 334 with its beam axis perpendicular to the surface of the skull 332.

The focus of the transducer was positioned inside the mouse brain using a grid-positioning method that utilized the pulse-echo diagnostic transducer 304, as discussed above. The grid was constructed from three 0.30 mm thin metal bars (i.e., paper clips) with two of the bars parallel to one another and separated by 4.00 mm. At the center of the parallel bars, and perpendicular to the two, was soldered the third bar. The grid was placed in the water bath 334, on top of the skull, and in alignment with sutures visible through the skin. The center bar was aligned along the sagittal suture and one of the parallel bars with the lambdoid suture. A lateral two-dimensional raster-scan of the grid using the diagnostic transducer was made and the transducer's beam axis was positioned 2.25 and 2.00 mm away from the sagittal and lambdoid suture, respectively. Finally, the focal point was placed 3.00 mm beneath the top of the skull so that the acoustic wave propagated through the left parietal bone and overlapped with the left hippocampus and a small portion of the lateral region of the thalamus. The right hippocampus was not targeted and was used as the control. The grid positioning method was sufficiently precise to have the FUS beam consistently overlap the hippocampus of the murine brain.

The tissue opening procedure 100 involved injection 140, 150 a 25 μΐ bolus of Definity ^® microbubbles (1-10 μηι) and a dextran contrast agent (Texas-Red ^® fluorescent dye with a molecular weight of 3 kDa) into the tail vein 1 minute after the start of sonication 170, with the injection taking place over a 30 second period. Sonication. was performed for 1 1 minutes total using pulsed FUS at a set pressure of ,

0.51 MPa peak rarefactional at a single location (e.g., the hippocampus).

After the 1 1 minutes of sonication 170, the dextran was allowed to circulate and accumulate in the mouse brain for 10 minutes, after which a transcardial perfusion with phosphate buffer saline (138 mM sodium chloride, 10 mM phosphate, pH 7.4) and 60 ml of 4% paraformaldehyde was performed. The brain was extracted from the skull and then post-fixed in the paraformaldehyde overnight. Following the aforementioned procedures, the brain was prepared for frozen sections. The frozen sectioning protocol provided an efficient means of analyzing fluorescence in order to determine the threshold for BBB opening. In preparation of frozen sectioning, the brain was cryoprotected by soaking it in 30% sucrose overnight. The brain was then embedded in a cutting temperature compound (Sakura Tissue-Tek O.C.T. Compound; Torrance, CA, USA), frozen in a square mold, and then sectioned using a cryostat into nine sections of 100 μηι slices in the horizontal orientation.

Bright field and fluorescent images of the frozen sections were acquired using an inverted light and fluorescence microscope (IX- 81 ; Olympus, Melville, NY, USA) at 4X magnification and with a motorized stage-scanner. Images of the paraffin sections were acquired using an upright light and fluorescence microscope (BX61 ; Olympus, Melville, NY, USA) at 4X and 10X magnification. The Texas Red-tagged dextrans were excited at 568 ± 24 nm while emissions were filtered for 610 ± 40 nm.

As noted above, the nine horizontal sections were chosen at defined cross-sections of the hippocampus. Figure 6(a) illustrates a horizontal section at l Ox magnification, with the left and right ROIs shown in the left and right boxes. Figure 6(b) shows the left ROI, which was subjected to sonication procedures, as detailed above, and Figure 6(c) shows the right ROI, which was the control. In order to process the image, the regions of interest (ROIs) for each of the nine sections were outlined using Adobe® Photoshop® CS2 (San Jose, CA, USA), as illustrated Figures 6(b)-(c). The outlines were then loaded into MATLAB® (Natick, MA, USA) and used to isolate the hippocampus in the fluorescent images. The images were normalized by dividing both the left and right images by the spatially averaged right (control) image of the hippocampus, thus calculating FL-HIP- The threshold for an image was were the pixel value was greater than 2 standard deviations of F _L- HIP; images exceeding the threshold were excluded from the calculations. The normalized optical density (NOD) was then calculated for each section, using the equation NOD = F _L _ _RO1 - F _R _ _R01 , where F _L-R O _! is the sum of the pixel values in left ROI of the brain and R _L -ROI is the sum of the pixel values in the right ROI of the brain. The NOD for the brain was calculated by averaging the NOD of across all nine sections. The resulting averaged NOD was then used to determine whether, to what extent, the BBB had opened.

Figure 7 is a graph illustrating the effects of varying the microbubble concentration in an exemplary embodiment where the FUS pressure was 0.46 MPa, the microbubbles used where Defmity® bubbles, the PRF was 10 Hz and the PL was 20 ms. The asterisks (*) indicates a significant difference in NOD from the control. As can be seen in Figure 7, there was a significant NOD increase for all

concentrations test and further there was not a significant difference between the tested concentrations, indicating that with Definity microbubbles a concentration of 0.01 μΐ/g of body mass is both sufficient to open the BBB in mice and also reliable for doing the same.

Figure 8(a) is a graph illustrating the effects of varying the PRF in an exemplary embodiment where the FUS pressure was 0.46 MPa, the microbubbles used were Definity® bubbles, the microbubble concentration was set at 0.05 μΐ/g of body mass, and the PL was 20 ms. The asterisks (*) indicates a significant difference in NOD from the control. As illustrated in Figure 8(a), at least one pulse is needed to open the BBB in a mouse brain prepared in accordance with the procedures set forth above. Further, the lowest PRF that was observed to open the BBB was 1 Hz, while the lowest PRF that was observed to reliably open the BBB was 5 Hz. Figure 8(b) further illustrates these findings, showing the PRF as a function of the probability of BBB opening. As illustrated in Figure 8(b), at 5 Hz and above there is a 100% probability of the BBB opening in the mice subjects prepared in accordance with the exemplary procedures set forth above. Figure 8(a) also illustrates that no additional benefits are gained from using a PRF greater than 5 Hz.

Figure 9(a) is a graph illustrating the effects of varying the PL in an exemplary embodiment where the FUS pressure was 0.46 MPa, the microbubbles used were Definity® bubbles, the microbubble concentration was set at 0.05 μί/g of body mass, and the PRF was 10 Hz. The asterisks (*) indicates a significant difference.jn NOD from the control. As illustrated in Figure 9(a), lowest PL which was resulted in BBB opening was 0.033 ms, while 0.2 ms was the lowest PL which produced reliable BBB opening in mice prepared in accordance with the above- detailed exemplary procedures. The results illustrated in Figure 9(a) demonstrate that, contrary to prior understanding, pulse lengths shorter than 10 ms can reliably open the BBB for FUS applications utilizing pressures less than 0.5 MPa. Figure 9(a) also illustrates that no additional benefits are gained from using a PL greater than 10 ms. Figures 9(b) and 9(c) illustrate the PL as a function of the probability of BBB opening, with Figure 9(b) showing the data across all PLs tested in the exemplary experiment discussed herein and Figure 9(c) illustrating the shortest PLs tested. As illustrated in Figure 9(c), a PL of as short as 0.1 ms has been shown to reliably open the BBB of mice prepared in accordance with the exemplary procedures described herein, which is contrary to prior understanding of the necessary PL for FUS applications employing pressures less than 0.5 MPa.

Figures 10(a)-(h) are images of murine brains prepared in accordance with the above-detailed exemplary procedures and illustrate the results of varying the PL from 0.033 ms (50 cycles) to 30 ms (45600 cycles). Figures 10(a) and 10(b) illustrate the left sonicated ROI and right control ROI, respectively, at a PL of 0.033 ms (50 cycles). Figures 10(c) and 10(d) illustrate the left sonicated ROI and right control ROI, respectively, at a PL of 0.1 ms (152 cycles). Figures 10(e) and 10(f) illustrate the left sonicated ROI and right control ROI, respectively, at a PL of 20 ms (30400 cycles). Figures 10(g) and 10(h) illustrate the left sonicated ROI and right control ROI, respectively, at a PL of 30 ms (45600 cycles).

Figure 1 1 illustrates an exemplary pulse and burst sequence, where each burst is composed set of pulses operating at a certain pulse rate frequency (PRF). For example and as illustrated in Figure 11 , one pulse can have a PL of 2.5 μ5 (approximately 3.5 cycles) at a pressure of 0.6 MPa. As illustrated, the pulses can be repeated at a PRF of, for example, 100 kHz. The pulsing can be continued for a period of time, e.g. , 10 ms, which comprises a single burst of a certain length (BL). Finally, the bursts can be repeated at a certain rate, for example 50 Hz, which is the BRF.

Figures 12(a)-(c) illustrate the NOD as a function of the BRF for three different PRFs, for an exemplary experiment involving mice preformed in accordance with the above-detailed procedures. Figure 12(a). is a. graph illustrating the effects of varying the BRF, in an exemplary embodiment where the FUS pressure was 0.46 MPa, the microbubbles used were Definity® bubbles, the microbubble concentration was set at 0.05 μΐ/g of body mass, the sonication duration was 11 minutes, the BL was 1000 pulses, and the PRF was set at 100 kHz. Figure 12(b) is a graph illustrating the effects of varying the BRF, in an exemplary embodiment having the same parameters as noted for Figure 12(a), except at a PRF of 25 kHz. Figure 12(b) is a graph illustrating the effects of varying the BRF, in an exemplary embodiment having the same parameters as noted for Figure 12(a), except at a PRF of 6.25 kHz.

Figure 12(d) is a graph illustrating the effects of varying the BL, in an exemplary embodiment where the FUS pressure was 0.46 MPa, the microbubbles used were Definity® bubbles, the microbubble concentration was set at 0.05 μΐ/g of body mass, the sonication duration was 11 minutes, the BRF was 5 Hz, and the PRF was 100 kHz.

Figures 13(a)-(b) illustrate images of a mouse brain, prepared in accordance with the exemplary methods described herein, showing BBB opening where the FUS pressure was 0.6 MPa, the microbubbles used were Definity® bubbles, the microbubble concentration was set at 0.05 μΐ/g of body mass, the sonication duration was 11 minutes, the PL was 3 cycles (less than 2.5 μ3), the PRF was 6.25 kHz, the BL was 1000 pulses, and the BRF was 5 kHz. Figure 13(b) is an enlarged image of the area of interest illustrated by the box in Figure 13(a), and it illustrates that the BBB was opened at a PL of 3 cycles.

Figures 13(c)-(d) illustrate images of a mouse brain, prepared in accordance with the exemplary methods described herein, showing BBB opening where the FUS pressure was 0.6 MPa, the microbubbles used were Definity® bubbles, the microbubble concentration was set at 0.05 μΐ/g of body mass, the sonication duration was 1 1 minutes, the PL was 3 cycles (less than 2.5 μΞ), the PRF was 100 kHz, the BL was 1000 pulses, and the BRF was 2 Hz. Figure 13(d) is an enlarged image of the area of interest illustrated by the box in Figure 13(c), and the boxed area in Figure 13(d) illustrates that the neuronal uptake occurred at a PL of 3 cycles.

Table 1 below illustrates the results of another experiment preformed on ninety-nine male mice (strain: C57B16; 24.71 ± 1.77 g) in accordance with the exemplary methods described herein and the procedures of the Columbia University Institutional Animal Care and Use Committee. Table 1 illustrates eighteen different experimental conditions, varying the microbubble concentration ^L/g of body mass), the PRF (Hz), and the PL (ms). The results are shown in terms of the NOD (mean ± s.d. x 10 ⁹ ) and the number of mice with delivered dextran. The first entry

corresponds to a sham mouse, where no ultrasound was applied; in the second entry the microbubbles and dextran were admimstered 1 minute before a 30-second sonication; and in the final entry the microbubbles were injected over a 180 second period. In the remaining entries the mice were intravenously injected with a solution of dextran and microbubble 1 minute after the start of an 11 -minute sonication. All sonications were performed with an 1.525 MHz acoustic beam and at a peak- rarefactional pressure of 0.46 MPa.

Table 1

aSham mouse. No ultrasound was applied.

bMicrobubble and dextran were administered 1 minute before a 30-second sonication. cMicrobubbles were injected over 180 seconds. Table 2 below illustrates the results of another experiment preformed on ninety-five C57B16 male mice in accordance with the exemplary methods described herein and the procedures of the Columbia University Institutional Animal Care and Use Committee. Table 2 illustrates eighteen different experimental conditions, varying the PRF (kHz), BRF (Hz), the BL (# of pulses) and the peak- rarefactional pressure. The results are shown in terms of the NOD (mean ± s.d. x 10 ⁹ ) and the number of mice with an incidence of NOD increase, calculated as detailed above. Those entries marked with an asterisk (*) correspond to sham mice, where no ultrasound was applied and those entries marked with a double asterisk (**) correspond to parameters where pulses were emitted continuously and without bursts. For all entries the sonication was for 11 minutes at a center frequency of 1.5 MHz, in the presence of systemically administered Definity® microbubbles (0.05 μΐ/g of body mass) and fluorescently-tagged dextran (molecular weight: 3 kDa, fluorescent tag: Texas Red®) and the PL was 3.5 cycles (2.3 μβ).

Table 2

Sham mice where no ultrasound was applied.

**Parameters where pulses were emitted continuously and without bursts.

As illustrated in Table 2, emission of a continuous train of pulses at 0.51 MPa and a PRF of 6.25, 25, and 100 kHz produced no significant increase in NOD, which is a measure of the relative increase in fluorescence in the left (target) ROI relative to the right (control) ROI. Under each condition, only 1 out of 3 mice had an increase in NOD, and in these instances, the fluorescence was faint and distributed along or near large vessels. Select intervals were evaluated by grouping 1000 pulses into bursts and emitting them at a BRF of 0.1, 1, 2, 5, or 10 Hz, which corresponded to a burst repetition period (BRP) of 10, 1, 0.5, 0.2, and 0.1 s, respectively. At a 100-kHz PRF, significant increases in NOD were observed at 1 and 5 Hz, while no increase was observed at 0.1, 2, and 10 Hz. At a 25-kHz PRF, significant increases were observed at 1, 2, and 5 Hz, while no increase was observed at 0.1 and 10 Hz. At a 6.25-kHz PRF no significant increase was observed at any of the BRFs evaluated although some mice had observable increases in fluorescence. In general and as illustrated in Table 2, the NOD increased with the interval between bursts and then decreased beyond a particular duration. Also, both the level and incidence of NOD decreased with the PRF. The maximum average NOD increase was observed with a 100-kHz PRF and a 5-Hz BRF.

Table 2 further illustrates, the dependence of acoustic peak- rarefactional pressure on BBB disruption was evaluated in a sham (0 MPa) and pressures of 0.13, 0.25, 0.37, and 0.51 MPa. A significant increase in NOD was only observed at 0.51 MPa. Although 0.37 MPa had no significant increase in NOD, 2 out of 3 mice had detectable levels of fluorescence. Therefore, the pressure threshold for BBB disruption for a 3.5-cycle pulse was between 0.25 and 0.51 MPa. The effect of BL was evaluated from 1 to 1000 pulses. A single pulse was insufficient in disrupting the BBB. The lowest pressure show feasible in disrupting the BBB was at 5 pulses and was observed in 1 out of 3 mice. A significant increase in NOD was observed from 50 pulses and higher. In general, increasing the number of pulses increased the likelihood and magnitude of NOD increase.

In an experiment using a 100-kHz PRF, a 5-Hz BRF, a 0.51 MPa pressure and a 1000 pulse BL, the relevance of the pulse-sequence to

pharmacologically-sized agents was evaluated with dextrans at molecular weights of 3-, 10-, and 70-kDa. Significant increases in NOD were observed using 3 and 70 kDa dextrans. The 10-kDa dextran was successfully delivered in all 3 mice, but the increase was not as significant (P = 0.06). The 3-kDa agent was distributed most homogeneously and across a larger area than the other two molecular weights. The distribution of 10-kDa was diffuse as well, but did not extend spatially as far as the 3- kDa dextran. The 70-kDa dextran had heterogeneous spots of high levels of fluorescence on top of diffusely distributed fluorescence. In certain instances, when high concentrations of 3-kDa dextran were diffusely delivered to the target ROI, the morphology of neurons and/or glial cells can be seen. For example and as illustrated in Figures 13(c) and 13(d), at a PRF of 100 kHz, a BL of 1000 pulses, and a BRF of 2 Hz, a neuronal axon with an approximately 1 μιη diameter can be observed extending from its cellular body and attached to a capillary, which had a diameter for approximately 4.5 μηι.

It will be understood that the foregoing is only illustrative of the principles described herein, and that various modifications can be made by those skilled in the art without departing from the scope and spirit of the disclosed subject matter. For example, the system and methods described herein are used for opening the blood-brain barrier of a subject. It is understood that that techniques described herein are useful for opemng of other tissues. Further, the techniques described have been performed on mice but it is understood the techniques are applicable to other subject, such as humans. Moreover, features of embodiments described herein can be combined and/or rearranged to create new embodiments.