Patent Application
20230277831
Not applicable.
The present disclosure generally relates to focused ultrasound devices for use in microbubble-mediated blood-brain barrier (BBB) opening (FUS-BBBO).
Focused ultrasound combined with microbubble-mediated blood-brain barrier opening (FUS-BBBO) has been established as a promising technique for the noninvasive and localized delivery of various therapeutic agents to the brain. Its feasibility and safety have been demonstrated in patients with various brain diseases, including brain tumors, Parkinson's disease, amyotrophic lateral sclerosis, and Alzheimer's disease. However, FUS-BBBO is not only a promising technique for clinical applications but also a powerful preclinical research tool that has the potential to be adopted by a broad research community, including but not limited to neuroscience, neuro-oncology, and neurology. For example, FUS-BBBO can facilitate the delivery of gene vectors encoding channelrhodopsin to the mouse brains for optogenetic neuromodulation and engineered G-protein-coupled receptors for chemogenetic neuromodulation. It can also modulate brain function by delivering neurotransmitters (e.g., GABA) to a targeted brain location. Besides these applications in neuroscience, FUS-BBBO has been used in neuro-oncology research to evaluate the delivery efficiency and therapeutic efficacy of various agents in murine models of brain tumors, such as chemotherapeutic agents (e.g., BCNU), monoclonal antibodies (e.g., Herceptin and bevacizumab), and nanoparticles (e.g., brain-penetrating nanoparticles and radiolabeled copper nanoclusters). FUS-BBBO without any drugs was found to reduce the amyloid plaque and improve cognitive performance in Alzheimer's disease mouse models. FUS-BBBO can also be used to deliver therapeutic agents (e.g., GSK-3 inhibitor) to further reduce plaque deposition in mouse models. Despite the great promise, the adoption of FUS-BBBO by the broad research community is limited by the lack of affordable, easy-to-use, and high-precision FUS devices for mouse studies.
Existing FUS devices for FUS-BBBO in mice are expensive, bulky, and with a high technical barrier. There are only a few commercially available FUS devices for preclinical FUS-BBBO research, for example, magnetic resonance imaging (MRI)-guided FUS devices provided by Image Guided Therapy (IGT, Pessac, France), MRI- and stereotactic-guided FUS devices from FUS Instruments Inc. (Toronto, Ontario, Canada), and VIFU 2000 from Alpinion US Inc. (Bothell, Wash., USA). These commercial devices are expensive (˜$50,000 to ˜$250,000). There are also custom-made FUS devices, but the cost of those systems is within the same range. Single-element FUS transducers have been the most widely used devices because they are relatively more affordable than phased arrays. However, even single-element FUS transducers are often bulky with large apertures (˜50 mm). These bulky transducers require heavy 3D motors to control their positioning for brain targeting. Moreover, MRI or ultrasound imaging is often needed to guide the spatial targeting of the FUS transducer at a specific brain location, limiting the usage of FUS-BBBO to mainly groups with expertise in ultrasound and/or MRI. A stereotactic-guided FUS system was introduced that uses a stereotactic frame to stabilize the mouse head and use the brain atlas to guide the positioning of the FUS transducer, which avoids the need for MRI or ultrasound imaging guidance. However, same as all other FUS devices, the FUS transducer is bulky. The cost of the device is still high (˜$50,000).
The broad application of FUS-BBBO in small animal research is also limited by the low spatial precision of existing FUS devices. The commonly used FUS transducers have low frequencies 1.5 MHz) with a focal region at the scale of 1×1×10 mm3, which essentially covers the entire depth of the mouse brain (˜6 mm). Low-frequency transducers are needed to minimize skull-induced attenuation and beam aberration, which is critical in clinical applications. However, the mouse skull is much thinner than the human skull. Successful BBBO was reported using a diagnostic ultrasound imaging probe with a center frequency of 8 MHz in combination with microbubbles. However, the focal region size of the ultrasound imaging probe was large, which was not suitable for spatially precise BBBO in mice. FUS transducers with optimized design are needed to precisely target individual structures in the mouse brain.
Precise control of BBBO volume and drug delivery efficiency is also needed to ensure the robust application of FUS-BBBO. Both mechanical index (defined by the ratio between acoustic pressure and the squared root of frequency, MI=P/√{square root over (f)}) and cavitation index (defined by the ratio between acoustic pressure and the squared root of frequency, MI=P/√{square root over (f)}) have been proposed to evaluate the likelihood of FUS-BBBO as well as the drug delivery efficiency. McDannold et al. found that MI was correlated with the threshold of FUS-induced BBB opening. Chu et al. found both MI and CI were highly correlated with the delivery efficiency of FUS-BBBO, and the correlation with MI was slightly higher than that of CI.
Among the various aspects of the present disclosure is the provision of a focused ultrasound device.
Briefly, therefore, the present disclosure is directed toward a focused ultrasound device and uses thereof.
The present teachings include a description of a focused ultrasound (FUS) device. In one aspect, the device is configured to deliver FUS to a targeted tissue. The FUS device includes an adapter configured to an actuated mounting shaft of a stereotaxic system. The adapter includes an adapter platform. The adapter platform defines a mounting bore at one end, where the mounting bore is configured to receive the actuated mounting shaft. The adapter further includes a transducer housing bore configured to receive a transducer housing. The FUS device further includes a focused ultrasound (FUS) transducer. The FUS device further includes the transducer housing. The transducer housing includes a transducer bore connecting a transducer receptacle and a coupling cone positioned on opposite faces of the transducer housing. The transducer housing further includes the transducer receptacle configured to receive and house the focused ultrasound (FUS) transducer. The transducer housing further includes a coupling cone projecting downward from the transducer bore that is configured to insert through the transducer housing bore. In some aspects, the adapter further includes a first pair of magnets attached to the adapter within a corresponding pair of magnet insets formed within the adapter on either side of the transducer housing bore. In some aspects, the transducer housing further includes a second pair of magnets attached to the transducer housing within a corresponding second pair of magnet insets formed within the transducer housing on either side of the transducer bore. In some aspects, the first and second sets of magnets are configured to lock the transducer housing to the adapter when the coupling cone is inserted through the transducer housing bore. In some aspects, the coupling cone is further configured to contain an impedance-matched ultrasound coupling material, where the impedance-matched ultrasound coupling material is configured to provide a low-impedance acoustic path for delivery of FUS to the targeted tissue. In some aspects, the FUS device further includes a pointer insert configured to insert into the transducer mounting bore and to provide a visual indication of the position and orientation of the FUS produced by the device. In some aspects, the pointer insert includes a flanged insert configured to insert into the transducer mounting bore and a shaft extending downward and ending in a pointer tip, wherein the pointer tip is positioned at the focus point of the focused ultrasound produced by the device.
The present teachings include a description of a FUS system. In one aspect, the FUS system includes an adapter configured to couple to an actuated mounting shaft of a stereotaxic system. The adapter includes an adapter platform. The adapter platform includes a mounting bore at one end, where the mounting bore is configured to receive the actuated mounting shaft. The adapter platform further includes a transducer housing bore configured to receive a transducer housing. The FUS system further includes a focused ultrasound (FUS) transducer and at least one transducer housing. Each transducer housing includes a transducer bore connecting a transducer receptacle and a coupling cone positioned on opposite faces of the transducer housing, as well as a transducer receptacle configured to receive and house the focused ultrasound (FUS) transducer. The transducer house further includes a coupling cone projecting downward from the transducer bore, where the coupling cone is configured to insert through the transducer housing bore. The FUS system further includes a transducer driving system operatively coupled to focused ultrasound (FUS) transducer. The transducer driving system includes a function generator configured to control the operation of the FUS transducer to produce FUS, and a power amplifier configured to control the operation of the FUS transducer to produce FUS.
The present teachings also include a description of a stereotaxic-guided focused ultrasound-blood brain barrier (FUS-BBB) system. The FUS-BBB system includes a FUS system that includes an adapter configured to couple to an actuated mounting shaft of a stereotaxic system, a FUS transducer, and at least one transducer housing. In another aspect, the FUS-BBB system can include a stereotaxic system that can include an actuated mounting shaft, wherein the actuated mounting shaft is inserted into a mounting bore defined within the platform of the FUS device to couple the FUS device to the stereotaxic system. The adapter includes an adapter platform defining a mounting bore at one end, where the mounting bore is configured to receive the actuated mounting shaft as well as a transducer housing bore configured to receive at least one transducer housing. Each transducer housing includes a transducer bore connecting a transducer receptacle and a coupling cone positioned on opposite faces of the transducer housing, where the transducer receptacle is configured to receive and house the focused ultrasound (FUS) transducer. The transducer housing further includes a coupling cone projecting downward from the transducer bore, where the coupling cone is configured to insert through the transducer housing bore. The stereotaxic system include the actuated mounting shaft, wherein the actuated mounting shaft is inserted into the mounting bore defined within the platform of the FUS device to couple the FUS device to the stereotaxic system.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
Those of skill in the art will understand that the drawings, described below, are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.
In various aspects, an affordable and easy-to-use FUS device for spatially accurate and precise FUS-BBBO is disclosed.
Referring again to
In various aspects, the FUS device further includes a transducer housing configured to receive and house a focused ultrasound (FUS) transducer and to reversibly couple to the adapter. As illustrated in
Referring again to
In various aspects, at least one magnet inset configured to receive and house at least one magnet is defined within the transducer housing, as illustrated in
In various additional aspects, the FUS device further includes a pointer insert for positioning the adapter platform in an initial position prior to mounting the FUS transducer and transducer housing. The pointer is configured to fit within the transducer housing bore of the adapter (
In some aspects, the landmark to which the pointer tip is aligned includes, but is not limited to, an external feature of the FUS-BBBO subject. In other aspects, the external feature used as a landmark marks a desired location for a FUS-BBO treatment on a subject. In other aspects, the external feature provides an origin or reference point. In these other aspects, the platform may be repositioned relative to the origin or reference point according to a surgical map or atlas describing the position of the desired treatment location relative to the origin or reference point. By way of non-limiting example, the bregma of a mouse, which is visible through the mouse's shaved scalp, may be selected as an origin or reference point to which the pointer tip is aligned, after which specific brain structure of the mouse may be located by moving the platform and attached transducer relative to the bregma as specified by a mouse brain atlas.
Referring to
By way of non-limiting example, a stereotactic-guided FUS system (
The FUS transducers of this example were miniature FUS transducers that had an aperture of 13 mm and a focal length of 10 mm. The FUS transducers included lead zirconate titanate (PZT) ceramic piezo material (DL-47, Del Piezo Specialties LLC, West Palm Beach, Fla., USA). These miniaturized transducers provided sufficient output pressure because PZT is a material commonly used material for high-power ultrasound transmission. As described in the Examples herein, FUS transducers producing three different frequencies (1.5 MHz, 3 MHz, and 6 MHz) were included to investigate the relationship between frequency and BBBO outcome. The manufacturing of these transducers was straightforward as it only required gluing two wires to the positive and negative electrodes of the piezoelectric element.
In this non-limiting example, the transducer element was encapsulated in the 3D-printed transducer housing using epoxy (Devcon Epoxy Adhesive, Devcon Corp., Danvers, Mass.). The back of the transducer directly contacted the air to form air backing. The transducers were then connected through these wires to a power amplifier coupled with a function generator. No electrical impedance matching was needed because the real part of the transducer impedance at the resonance frequency as measured by an E5061A ENA Network Analyzer with the 85070E Dielectric Probe Kit (Agilent Technologies, Santa Clara, Calif., USA) was in the range of 31 to 59 ohms, which was close to 50 ohms needed for a perfect impedance match.
The transducer housing, shown in
In order to achieve precise targeting of the FUS transducer at a specific brain location, a pointer was manufactured by 3D printing (
As illustrated in the Examples herein, the disclosed FUS device achieved FUS-BBBO with sub-millimeter accuracy as measured by the offset between the desired target location and the BBBO centroid. Overall, the present disclosure describes an affordable and easy-to-use FUS device for spatially accurate, precise, and tunable drug delivery to the mouse brain.
The FUS device can have the following features: (1) The FUS transducer elements are widely available at a low cost (˜$80 per element). Transducer manufacturing only required connecting wires to the electrodes on the elements. All other components can be 3D printed. (2) The integration of the FUS transducers with a stereotactic frame for targeting desired brain location using established stereotactic procedures decreased the barrier to the adoption of the FUS technique. The device achieved sub-millimeter targeting accuracy. (3) The use of higher frequency FUS transducers (3 MHz and 6 MHz) decreased the BBBO volume and improved the spatial precision of FUS-BBBO in targeting individual structures in the mouse brain. (4) The drug delivery outcome was tunable by adjusting the CI or MI. In some embodiments, the device may be manufactured by research groups without an ultrasound background and used in various applications with minimal training needed.
In an exemplary embodiment, the targeting accuracy of the stereotactic-guided FUS system was high, reaching 0.63±0.19 mm (
Multiple strategies have been proposed to improve the spatial precision of FUS in achieving BBBO in individual brain structures. One strategy is to replace a single-element FUS transducer with a large-aperture phased array, but the high cost and complexity of the phased array limit its broad adoption. Other strategies include the use of two transducers with frequency-modulated crossed beams and the use of chirp and random frequency-modulated ultrasound waveforms. The focal region size of a single-element FUS transducer can be approximately estimated using λF # in the lateral direction and 7λ(F #)2 in the axial direction, where λ is the wavelength and F #=focal length/aperture. It is well-known that increasing the transducer frequency can decrease the focal region size. It is demonstrated herein that under the same pressure level, a higher frequency FUS transducer achieved a small drug delivery volume (
The FUS-BBBO drug delivery outcome can be tuned by the CI and MI. Strong linear correlations were found between Gadolinium delivery volume and CI or MI (
The methods and algorithms of the invention may be enclosed in a controller or processor. Furthermore, methods and algorithms of the present invention, can be embodied as a computer-implemented method or methods for performing such computer-implemented method or methods, and can also be embodied in the form of a tangible or non-transitory computer-readable storage medium containing a computer program or other machine-readable instructions (herein “computer program”), wherein when the computer program is loaded into a computer or other processor (herein “computer”) and/or is executed by the computer, the computer becomes an apparatus for practicing the method or methods. Storage media for containing such computer programs include, for example, floppy disks and diskettes, compact disk (CD)-ROMs (whether or not writeable), DVD digital disks, RAM and ROM memories, computer hard drives and backup drives, external hard drives, “thumb” drives, and any other storage medium readable by a computer. The method or methods can also be embodied in the form of a computer program, for example, whether stored in a storage medium or transmitted over a transmission medium such as electrical conductors, fiber optics or other light conductors, or by electromagnetic radiation, wherein when the computer program is loaded into a computer and/or is executed by the computer, the computer becomes an apparatus for practicing the method or methods. The method or methods may be implemented on a general-purpose microprocessor or on a digital processor specifically configured to practice the process or processes. When a general-purpose microprocessor is employed, the computer program code configures the circuitry of the microprocessor to create specific logic circuit arrangements. Storage medium readable by a computer includes medium being readable by a computer per se or by another machine that reads the computer instructions for providing those instructions to a computer for controlling its operation. Such machines may include, for example, machines for reading the storage media mentioned above.
Definitions and methods described herein are provided to better define the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art.
In some embodiments, numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, used to describe and claim certain embodiments of the present disclosure are to be understood as being modified in some instances by the term “about.” In some embodiments, the term “about” is used to indicate that a value includes the standard deviation of the mean for the device or method being employed to determine the value. In some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the present disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the present disclosure may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. The recitation of discrete values is understood to include ranges between each value.
In some embodiments, the terms “a” and “an” and “the” and similar references used in the context of describing a particular embodiment (especially in the context of certain of the following claims) can be construed to cover both the singular and the plural, unless specifically noted otherwise. In some embodiments, the term “or” as used herein, including the claims, is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive.
The terms “comprise,” “have” and “include” are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as “comprises,” “comprising,” “has,” “having,” “includes” and “including,” are also open-ended. For example, any method that “comprises,” “has” or “includes” one or more steps is not limited to possessing only those one or more steps and can also cover other unlisted steps. Similarly, any composition or device that “comprises,” “has” or “includes” one or more features is not limited to possessing only those one or more features and can cover other unlisted features.
All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the present disclosure and does not pose a limitation on the scope of the present disclosure otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the present disclosure.
Groupings of alternative elements or embodiments of the present disclosure disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.
All publications, patents, patent applications, and other references cited in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other reference was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Citation of a reference herein shall not be construed as an admission that such is prior art to the present disclosure.
Having described the present disclosure in detail, it will be apparent that modifications, variations, and equivalent embodiments are possible without departing from the scope of the present disclosure defined in the appended claims. Furthermore, it should be appreciated that all examples in the present disclosure are provided as non-limiting examples.
The following non-limiting examples are provided to further illustrate the present disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent approaches the inventors have found function well in the practice of the present disclosure, and thus can be considered to constitute examples of modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.
The objective of this study was to design and fabricate an affordable and easy-to-use FUS device for spatially accurate and precise FUS-BBBO. Mini-FUS transducers ($80 each in material cost) with different frequencies (1.5, 3.0, and 6.0 MHz) were manufactured in-house and integrated with a commercially available stereotactic frame using 3D-printed parts. It was found that this device achieved FUS-BBBO with sub-millimeter accuracy as measured by the offset between the desired target location and the BBBO centroid. It was also shown that FUS-BBBO volume could be decreased to perform spatially precise BBBO. The drug delivery volume and signal intensity were tunable by the CI and MI.
The stereotactic-guided FUS device (
The key hardware component was the FUS transducer. The miniature FUS transducers had an aperture of 13 mm and a focal length of 10 mm. They were made by lead zirconate titanate (PZT) ceramic piezo material (DL-47), which was purchased from Del Piezo Specialties LLC. (West Palm Beach, Fla., USA) at a cost of ˜$80 per element. These miniaturized transducers could provide sufficient output pressure because PZT is the most commonly used material for high-power ultrasound transmission. Elements with three different frequencies were chosen (
The element was then encapsulated in a 3D-printed housing using epoxy (Devcon Epoxy Adhesive, Devcon Corp., Danvers, Mass.). The back of the transducer directly contacted the air to form air backing. The transducers were then connected through these wires to a power amplifier coupled with a function generator. No electrical impedance matching was needed because the real part of the transducer impedance at the resonance frequency as measured by an E5061A ENA Network Analyzer with the 85070E Dielectric Probe Kit (Agilent Technologies, Santa Clara, Calif., USA) was in the range of 31 to 59 ohms, which was close to 50 ohms needed for a perfect impedance match.
The design of the transducer housing is shown in
In order to achieve precise targeting of the FUS transducer at a specific brain location, a pointer was manufactured by 3D printing (
To assess the performance of the disclosed FUS device, the following experiments were conducted. The acoustic pressure fields generated by the FUS transducers in a mouse head were simulated using a k-space pseudospectral method-based solver (Matlab toolbox, k-Wave). Experimental calibration of each FUS transducer was performed in degassed water without and with three degassed ex vivo mouse skulls.
The acoustic pressure fields generated by the FUS transducers in a mouse head were simulated using a k-space pseudospectral method-based solver (Matlab toolbox, k-Wave). A mouse head was placed in a μCT scanner (Rigaku, Tokyo, Japan). The acquired CT images consisted of 512×512×679 voxels with a spatial resolution of 0.08 mm. Linear interpolation was performed to adjust the voxel spacing of the images to 0.12 mm for 1.5 MHz, 0.06 mm for 3 MHz, and 0.03 mm for 6 MHz, to ensure the voxel spacing was less than ⅛ of the corresponding FUS transducer's wavelength. The density and sound speed of the skull and brain tissue were converted from the Hounsfield units of the CT images using the function ‘hounsfield2density’ in the k-Wave toolbox. This function uses a piecewise linear fit to the data reported by Schneider and Mast. The sound speed of the coupling gel was set to be the same as that of the water (1484 m/s). A Courant-Friedrichs-Lewy (CFL) stability factor of 0.17 was used in all the simulations. Experimental calibration of each FUS transducer was performed in degassed water without and with three degassed ex vivo mouse skulls. The skulls were positioned in front of the transducer. The acoustic pressure fields were measured using a hydrophone (HGL-200, ONDA Corporation, Sunnyvale, Calif.), which was moved in 3D using a computer-controlled 3D stage (PK245-01AA, Velmex Inc., NY, USA). The transmission efficiency of each FUS transducer was measured by the ratio between the maximum peak negative pressures measured with and without the skull.
To assess the in vivo targeting accuracy of FUS-BBBO performed using the FUS device disclosed herein, the following experiments were conducted. Mice were administered intravenously with a mixture of 1 mL/kg of Gadolinium (Dotarem, Guerbet, Aulnay sous Bois, France), 60 μL of 2% Evans Blue, and 10 μL/kg of Definity (Lantheus Medical Imaging, Billerica, Mass., USA) were subjected to FUS-BBBO using the disclosed device. FUS-BBB opening was measured by detecting gadolinium hyperenhancement within T1-weighted images obtained post-treatment, indicating the leakage of gadolinium associated with BBB disruption.
Adult female mice (IACUC protocol number: 21-0187, C57BL/6, 8 weeks old, female, Charles River Laboratory, Wilmington, Mass., USA) were used in this study. A total of 36 mice were randomly assigned to 9 groups to evaluate FUS-BBBO using FUS transducers with different frequencies (1.5, 3.0, 6.0 MHz) and different pressures (0.20, 0.40, 0.57 MPa) at each frequency. Prior to FUS sonication, animals were placed in the stereotaxic frame under isoflurane anesthesia (1.5-2% v/v isoflurane in oxygen). The fur on the mouse head was shaved using a hair removal cream while the skull and the scalp remained intact. Ultrasound gel was applied to the exposed skin above the skull and inside the housing of the small transducer. Mice were placed on a heating pad throughout the experiment. A catheter was placed in the mouse tail vein for intravenous injection.
The desired brain target was selected to be a point in the left thalamus using the following coordinates: −1.94 mm in the anterior-posterior (AP) direction, −1.50 mm in the medial-lateral (ML) direction, and −3.30 mm in the dorsal-ventral (DV) direction relative to the bregma according to the mouse brain atlas. The desired target location is indicated by the yellow dot in the transverse view (
The targeting accuracy of the stereotactic-guided FUS device was quantified by the offset between the desired target location and the centroid of the BBBO detected by CE-MRI. Representative post-treatment CE-MRI images of the mouse brain are shown in
For each CE-MRI brain image, a region of interest was manually drawn to cover the non-treated right side of the brain. Three times the standard deviation above the mean pixel intensity within the non-treated region was calculated to represent the background intensity. Then, a region of interest was manually drawn to cover the FUS-treated left side of the brain. Pixels with intensities above the background intensity were identified for each CE-MRI image. Gadolinium delivery volume was calculated by the total number of the identified pixels in the whole brain.
MRI images in transverse and coronal views at different frequencies and pressures are shown in
To assess the efficacy of drug delivery via FUS-BBBO performed using the FUS device disclosed herein, the following experiments were conducted. Following the FUS-BBBO treatment as described in Example 3, the brains of the mice were harvested and harvested sections were fluorescence imaged using an LI-COR imaging system (Pearl Trilogy; 700 Channel laser source (Ex 785 nm/Em 820 nm); resolution 85 μm). Enhanced fluorescence signals associated with the delivery of Evans blue through the FUS-disrupted BBB were determined by comparing sections from FUS-BBBO and corresponding untreated brain halves.
Evans Blue has been commonly used as a model drug for evaluating the FUS-BBBO drug delivery outcome. The harvested mouse brains were cut into 1-mm thick coronal sections using the mouse brain matrix (World Precision Instrument, Sarasota, Fla., USA). Fluorescence images of brain sections were taken using a LI-COR imaging system (Pearl Trilogy; 700 Channel laser source (Ex 785 nm/Em 820 nm); resolution 85 μm) and analyzed using Matlab (Mathworks, Natick, Mass., USA). Same to gadolinium delivery quantification, a region of interest was manually drawn to cover the non-treated right side of the brain for each brain section. Three times the standard deviation above the mean pixel intensity within the non-treated region was calculated to represent the background autofluorescence intensity. Then, a region of interest was manually drawn to cover the FUS-treated left side of the brain. Pixels with intensities above the background autofluorescence intensity were identified for each brain section. Evans blue delivery volume was calculated by the total number of pixels with enhanced fluorescence signal in all the brain sections. The mean fluorescence intensity of pixels with enhanced fluorescence signal in all the brain sections was calculated to represent the signal intensity of the delivered Evans blue.
Bright-field images of coronal sections of the mouse brains and corresponding fluorescence images are presented in
The correlation between Evans blue delivery volume and signal intensity with CI is shown in
To assess the safety of FUS-BBBO performed using the FUS device disclosed herein, the following experiments were conducted. Following fluorescent microscope examination, a portion of the brain sections described in Example 3 containing the highest Evans blue fluorescence signal was subjected to histologic examination using hematoxylin and eosin (H&E) staining to detect microhemorrhage resulting from FUS-BBBO.
Histologic examination was performed for all mice using hematoxylin and eosin (H&E) staining. After fluorescence imaging, brain slices containing the highest Evans blue fluorescence signal were cryoprotected in 30% sucrose and embedded at −20° C. Brain slices were then sectioned into 5 μm coronal sections and stained with H&E. Bright-field images of stained sections were obtained using an all-in-one microscope (BZ-X810, Keyence, Osaka, Japan) coupled with 2× and 20× objectives.
Linear curve fitting was performed using OriginLab software (Origin, Mass., USA) for the following four groups: (a) Evans blue volume v.s. CI; (b) Evans blue volume v.s. MI; (c) Evans blue intensity v.s. CI; and (d) Evans blue intensity v.s. MI. The coefficient of determination (R2) was calculated. All data are presented in the format of mean±standard deviation. Statistical significance was evaluated by the unpair two-tail student t-test using GraphPad Prism (Version x, La Jolla, Calif., USA), and a p-value<0.05 was defined as statistically significant.
Of all groups tested, we only detected microhemorrhage in 2 out of 4 mice treated with FUS delivered at 1.5 MHz at 0.57 MPa. Representative slides with microhemorrhage observed are shown in
This application claims priority from U.S. Provisional Application Ser. No. 63/317,387 filed on Mar. 7, 2022, which is incorporated herein by reference in its entirety.
This invention was made with government support under EB027223, EB030102, and MH116981 awarded by the National Institutes of Health and under N00014-19-1-2335 awarded by the Office of Naval Research. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 63317387 | Mar 2022 | US |
