Methods and uses for enhancing the ultrasound responsiveness of target neurons by combined sonodynamic-sonogene therapy

Abstract

The present disclosure provides a method for enhancing the ultrasound responsiveness of target neurons of a target brain region of a subject, a method for treating, preventing or alleviating a brain disease or a disease related to the behavior of a subject or controlling the behavior of a subject, and the use of a combination of a nucleic acid vector encoding an exogenous mechanically sensitive ion channel and nanobubbles (and optionally an ultrasound generator) in the preparation of a related composition / kits. The technology of the present disclosure utilizes nanobubbles and exogenous mechanical ion channels, and uses low-frequency and low-intensity ultrasound waves to achieve effective and specific regulation of target neurons, which has a wide application prospect and great potential in the field of neural regulation of humans and other mammals.

Description

Technical Field

[0001] This disclosure pertains to the field of biomedical engineering. It relates to methods and uses for enhancing the ultrasound responsiveness of target neurons through combined acoustic-sonogenetics. Specifically, this disclosure relates to methods for enhancing the ultrasound responsiveness of target neurons in a target brain region of a subject using nanobubbles (NB) and exogenous mechanosensitive ion channels; methods for treating, preventing, or alleviating brain diseases or behavioral disorders of a subject, or for controlling the behavior of a subject; and related compositions / kits and uses. Background Technology

[0002] Ultrasound technology offers non-invasive access to deep brain regions and boasts high spatiotemporal resolution, thus showing great potential in treating a variety of brain diseases. However, traditional transcranial ultrasound technology lacks sufficient precision in selectively modulating specific neuron types.

[0003] Sonogenetics uses gene manipulation techniques to express mechanosensitizers on neurons to activate specific cells. This method utilizes ultrasound to achieve precise and specific modulation of neurons. However, due to the complexity of brain cell types and varying levels of sonosensitivity, targeting specific neural circuits and achieving effective neuromodulation in complex brain regions presents challenges. Furthermore, high-intensity ultrasound may induce off-target stimulation, and concerns about biosafety and viral loads carrying mechanosensitizers further limit the effective application of sonogenetics. Therefore, achieving precise, comprehensive, and efficient neuromodulation using sonogenetics faces numerous challenges.

[0004] Compared to optogenetic opsins in optogenetics, existing mechanosensitive ion channels (such as TRAAK, TRPA1, TRPC6, Piezo1, and MScL) have limitations in terms of distribution uniformity and sensitivity optimization. Simply increasing the expression of channel proteins may not be sufficient to achieve precise regulation of neural circuits.

[0005] Therefore, given the shortcomings of existing technologies, there is an urgent need to develop a safe and effective method to optimize the precision and efficiency of neural modulation and minimize undesirable side effects. Summary of the Invention

[0006] To address the problems in the prior art, this disclosure introduces the "acoustic effect-acoustic genetics" technique. Acoustic effect-acoustic genetics is a novel approach that combines nanobubble technology with acoustic genetics. It activates target neurons by aggregating nanoparticles or nanobubbles in designated brain regions and enhancing the ultrasound effect, thereby activating specific neural circuits and achieving effective specific neuromodulation as well as the treatment and prevention of related diseases.

[0007] A first aspect of this disclosure provides a method for enhancing the ultrasound responsiveness of target neurons in a target brain region of a subject, comprising: causing target neurons in the target brain region of the subject to express exogenous mechanosensitive ion channels; and applying ultrasound to the target brain region in the presence of nanobubbles.

[0008] A second aspect of this disclosure provides a method for treating, preventing, or alleviating a subject's brain disease or a behavior-related disease, or controlling the subject's behavior, by enhancing the ultrasound responsiveness of target neurons in a target brain region of the subject, comprising: causing target neurons in the target brain region of the subject to express exogenous mechanosensitive ion channels; and applying ultrasound to the target brain region in the presence of nanobubbles.

[0009] In some embodiments across various aspects, the exogenous mechanosensitive ion channel includes MscL, TRAAK, TRPA (e.g., TRPA1 and hsTRPA1), TRPC (e.g., TRPC6), Piezo (e.g., Piezo1 and Piezo2), or any combination thereof. In some embodiments, the exogenous mechanosensitive ion channel includes or is MscL. In some embodiments, the MscL is MscL-G22S.

[0010] In some embodiments of various aspects, the exogenous mechanosensitive ion channel (e.g., through a promoter connection specific to the target neuron) is specifically targeted and expressed in the target neuron.

[0011] In some embodiments, the method includes delivering the exogenous mechanosensitive ion channel to the target neuron via a nucleic acid vector encoding the exogenous mechanosensitive ion channel. In some embodiments, the method includes delivering the nucleic acid vector to a subject, thereby causing the exogenous mechanosensitive ion channel to be expressed in the target neuron. In some embodiments, the nucleic acid vector comprises a polynucleotide encoding the exogenous mechanosensitive ion channel, or the nucleic acid vector is a nucleic acid vector containing a polynucleotide encoding the exogenous mechanosensitive ion channel. In some embodiments, the nucleic acid vector further comprises a target neuron-specific promoter to specifically target the neuron, thereby specifically expressing the exogenous mechanosensitive ion channel in the target neuron. In some embodiments, the nucleic acid vector comprises a polynucleotide encoding the exogenous mechanosensitive ion channel and a target neuron-specific promoter.

[0012] In some embodiments across various aspects, the nucleic acid vector is a viral vector. In some embodiments, the viral vector comprises a polynucleotide encoding the exogenous mechanosensitive ion channel. In some embodiments, the viral vector comprises a polynucleotide encoding the exogenous mechanosensitive ion channel and a target neuron-specific promoter. In some embodiments, the viral vector includes, but is not limited to, adeno-associated virus vectors, adenovirus vectors, lentiviral vectors, or any combination thereof. In some embodiments, the viral vector includes a recombinant viral vector. In some embodiments, the viral vector is an adeno-associated virus (AAV) vector, including a recombinant adeno-associated virus (rAAV) vector.

[0013] In some implementations of various aspects, the method includes delivering nanobubbles to a target brain region of a subject (e.g., by injection).

[0014] In some embodiments across various aspects, the method includes delivering nanobubbles and a nucleic acid carrier encoding an exogenous mechanosensitive ion channel, respectively, to a subject. For example, the method includes delivering nanobubbles first, followed by the delivery of the nucleic acid carrier; or the method includes delivering the nucleic acid carrier first, followed by the delivery of the nanobubbles. In some embodiments, the method includes simultaneously delivering nanobubbles and the nucleic acid carrier to a subject.

[0015] In some embodiments across various aspects, the nucleic acid carrier and nanobubbles are present in solution form. In some embodiments, the nucleic acid carrier and nanobubbles are present in separate solutions (e.g., a first solution and a second solution) or in the same solution (e.g., a single solution). In some embodiments, the solution also contains a pharmaceutically acceptable carrier or excipient.

[0016] In some embodiments across various aspects, the solution (e.g., a first solution, a second solution, or a single solution) is administered by injection, such as local injection, for example, intracerebral local injection. In some embodiments, the solution (e.g., a first solution, a second solution, or a single solution) is administered by stereotactic injection.

[0017] In some embodiments, the solution (or the first solution) contains 5 × 10 11 -5×10 13 vg / mL (e.g. 6×10) 11 -4×10 12 vg / mL, 8×10 11 -3×10 12 vg / mL, 9×10 11 -2×10 12 vg / mL, 1×10 12 The viral vector is present at a concentration of vg / mL or any value or range thereof. In some embodiments, the solution (or the second solution) contains 4-6 nM / L (e.g., 4.5-6 nM / L, 5-5.5 nM / L, 5-6 nM / L, or any value or range thereof).

[0018] In some implementations of various aspects, the method includes delivering approximately 2 × 10⁻⁶ ppm to the target brain region of the subject. 10 -2.5×10 10 vg / kg or approximately 2.2×10 10 -2.4×10 10 Vg / kg of nucleic acid or viral vector, and / or, approximately 1.6 × 10⁻⁶. -4 -2.0×10 -4 nM / kg or approximately 1.8 × 10 -4 nanobubbles or nanobags with a density of nM / kg.

[0019] In some embodiments across various aspects, the nanobubbles have a particle size of approximately 30-600 nm.

[0020] In some embodiments across various aspects, the nanobubbles are modified to increase biocompatibility. In some embodiments, PEG is grafted onto the surface of the nanobubbles. In some embodiments, the nanobubbles are nano-airbags. In some embodiments, the surface of the nano-airbags is modified with PEG, for example, by grafting PEG onto its surface. In some embodiments, the width (diameter) of the nano-airbag is approximately 60-90 nm or approximately 65-85 nm; the length is approximately 300-600 nm or approximately 350-550 nm; and / or the aspect ratio is approximately 4.5-10 or 5-8.

[0021] In some embodiments, the nanobubbles do not contain molecules or portions connected to the mechanosensitive ion channel. In some embodiments, the nanobubbles do not contain antibodies. In some embodiments, the mechanosensitive ion channel in the target neuron and the nanobubbles are not connected. In some embodiments, the mechanosensitive ion channel or the polynucleotide or nucleic acid carrier encoding the mechanosensitive ion channel does not contain molecules or portions capable of connecting to the nanobubbles.

[0022] In some embodiments, the method includes applying the ultrasonic waves at a frequency of 0.5-5 MHz. In some embodiments, the method includes applying the ultrasonic waves at a sound pressure level of 0.1-0.6 MPa.

[0023] In some embodiments across various aspects, the ultrasound waves are applied via an ultrasound generator. In some embodiments, the ultrasound generator includes a benchtop ultrasound generator, a portable ultrasound generator, a wearable ultrasound generator, an implantable ultrasound generator, or any combination thereof.

[0024] In some implementations of various aspects, the target brain region includes, but is not limited to, all brain regions containing target neurons (e.g., the hypothalamus, including the lateral hypothalamus).

[0025] In some embodiments across various aspects, the target neurons include, but are not limited to, glutamatergic neurons, GABAergic neurons, dopaminergic neurons, cholinergic neurons, serotonergic neurons, adrenergic neurons, or any combination thereof.

[0026] In some embodiments of various aspects, the target neurons include, but are not limited to, excitatory neurons, inhibitory neurons, interneurons, or any combination thereof.

[0027] In some embodiments of various aspects, the target neuron comprises one neuron or a combination of two or more (e.g., two, three, four, five or more) neurons.

[0028] In some embodiments across various aspects, the method is performed in vivo or is an in vivo method. In some embodiments, the method is used to enhance the ultrasound responsiveness of target neurons in a target brain region within a subject.

[0029] In some implementations of various aspects, the subject is an animal, such as a mammal, such as a rodent like a mouse or rat, or a primate like a rabbit, bovine, equine, sheep, canine, feline, human, monkey, chimpanzee, or gorilla.

[0030] In some embodiments, the brain diseases include, but are not limited to, Alzheimer's disease, Parkinson's disease, multiple sclerosis, epilepsy, stroke, traumatic brain injury, sleep disorders, depression, schizophrenia, pain, migraine, compulsive bulimia, anorexia, or any combination thereof. In some embodiments, diseases associated with the subject's behavior include, but are not limited to, obesity, diabetes, cardiovascular disease, or any combination thereof. In some embodiments, the subject's behavior includes, but is not limited to, foraging, escape, reward, punishment, reproduction, socialization, aggression, memory, learning, or any combination thereof.

[0031] A third aspect of this disclosure provides the use of a combination of a nucleic acid carrier encoding an exogenous mechanosensitive ion channel and nanobubbles in the preparation of a composition, wherein the composition is used in a method according to any embodiment of the first and / or second aspects of this disclosure.

[0032] The fourth aspect of this disclosure provides the use of a combination of an ultrasound generator, a nucleic acid carrier encoding an exogenous mechanosensitive ion channel, and nanobubbles in the preparation of a kit, wherein the kit is used in the methods of any embodiment of the first and / or second aspects of this disclosure.

[0033] The fifth aspect of this disclosure provides the use of a combination of a nucleic acid carrier encoding an exogenous mechanosensitive ion channel and nanobubbles in the preparation of a composition for enhancing the ultrasound responsiveness of target neurons in a target brain region of a subject, wherein the nucleic acid carrier targets the target neurons in the target brain region and expresses the exogenous mechanosensitive ion channel therein.

[0034] The sixth aspect of this disclosure provides the use of a combination of a nucleic acid carrier encoding an exogenous mechanosensitive ion channel and nanobubbles in the preparation of a composition for treating, preventing or alleviating a brain disease or a disease related to the behavior of a subject, or for controlling the behavior of the subject, wherein the nucleic acid carrier targets target neurons in the target brain region and expresses the exogenous mechanosensitive ion channel therein.

[0035] The seventh aspect of this disclosure provides the use of a combination of an ultrasound generator, a nucleic acid carrier encoding an exogenous mechanosensitive ion channel, and nanobubbles in the preparation of a kit for enhancing the ultrasound responsiveness of target neurons in a target brain region of a subject, wherein the nucleic acid carrier targets the target neurons in the target brain region and expresses the exogenous mechanosensitive ion channel therein.

[0036] The eighth aspect of this disclosure provides the use of a combination of an ultrasound generator, a nucleic acid carrier encoding an exogenous mechanosensitive ion channel, and nanobubbles in the preparation of a kit for treating, preventing, or alleviating a brain disease or a disease related to the behavior of a subject, or for controlling the behavior of the subject, wherein the nucleic acid carrier targets target neurons in the target brain region and expresses the exogenous mechanosensitive ion channel therein.

[0037] In some embodiments of various aspects, the composition or kit is formulated to deliver the nucleic acid carrier and nanobubbles to the target brain region prior to ultrasound treatment of the subject.

[0038] In some embodiments across various aspects, the composition or kit contains approximately 5 × 10 11 -5×10 12 vg / mL or approximately 9×10 11 -2×10 12 vg / mL, 1×10 12 The nucleic acid carrier is present at a concentration of vg / mL, and / or nanobubbles at a concentration of approximately 4-6 nM / L or approximately 4-5.5 nM / L. In some embodiments, the nucleic acid carrier and nanobubbles are present in the same or different solutions.

[0039] In some embodiments across various aspects, the composition or kit is used to treat, prevent, or alleviate a subject's brain disease or a disease related to the subject's behavior, or to control the subject's behavior, by enhancing the ultrasound responsiveness of target neurons in a target brain region of the subject.

[0040] In some embodiments across various aspects, the composition or kit is administered to the subject prior to ultrasound treatment to deliver the nucleic acid carrier with nanobubbles to the target brain region.

[0041] In some embodiments, the composition or kit is formulated to deliver the nucleic acid carrier and nanobubbles separately (e.g., delivering the nucleic acid carrier first, then the nanobubbles; or delivering the nanobubbles first, then the nucleic acid carrier). In some embodiments, the composition or kit is formulated to deliver the nucleic acid carrier and nanobubbles simultaneously.

[0042] In some embodiments, the nucleic acid vector comprises a polynucleotide encoding the exogenous mechanosensitive ion channel, or the nucleic acid vector is a nucleic acid vector containing a polynucleotide encoding the exogenous mechanosensitive ion channel. In some embodiments, the nucleic acid vector further comprises a target neuron-specific promoter to specifically target the neuron, thereby specifically expressing the exogenous mechanosensitive ion channel in the target neuron. In some embodiments, the nucleic acid vector comprises a polynucleotide encoding the exogenous mechanosensitive ion channel and a target neuron-specific promoter.

[0043] In some embodiments across various aspects, the nucleic acid vector is a viral vector. In some embodiments, the viral vector includes, but is not limited to, adeno-associated virus vectors, adenovirus vectors, lentiviral vectors, or any combination thereof. In some embodiments, the viral vector includes recombinant viral vectors. In some embodiments, the viral vector is an adeno-associated virus (AAV) vector, such as a recombinant adeno-associated virus (rAAV) vector.

[0044] In some embodiments across various aspects, the exogenous mechanosensitive ion channel includes MscL, TRAAK, TRPA (e.g., TRPA1 and hsTRPA1), TRPC (e.g., TRPC6), Piezo (e.g., Piezo1 and Piezo2), or any combination thereof. In some embodiments, the exogenous mechanosensitive ion channel includes or is MscL. In some embodiments, the MscL is MscL-G22S.

[0045] In some embodiments of various aspects, the kit comprises compositions defined in various aspects of this disclosure, such as compositions comprising nucleic acid carriers and nanobubbles.

[0046] In some embodiments across various aspects, the composition is a pharmaceutical composition. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier or excipient.

[0047] In some embodiments across various aspects, the composition is a solution, such as an injection. In some embodiments, the composition comprises: a first solution containing a nucleic acid carrier (e.g., a first injection) and a second solution containing nanobubbles (e.g., a second injection). In some embodiments, the first and second solutions are present as separate injections. In some embodiments, the composition is a single solution (e.g., a single injection) containing nanobubbles and a nucleic acid carrier. In some embodiments, the solution or injection (e.g., a first / second injection or a single injection) further contains a pharmaceutically acceptable carrier or excipient.

[0048] In some embodiments, the composition is administered by injection, such as local injection, for example, intracerebral injection. In some embodiments, the composition is administered by stereotactic injection.

[0049] In some embodiments, the composition (or the first solution) contains 5 × 10 11 -5×10 12 vg / mL (e.g. 6×10) 11 -4×10 12 vg / mL, 8×10 11 -3×10 12 vg / mL, 9×10 11 -2×10 12 vg / mL, 1×10 12 Viral vector (vg / mL or any value or range thereof). In some embodiments, the composition (or the second solution) contains 4-6 nM / L (e.g., 4.5-6 nM / L, 5-6 nM / L, 4.5-5.5 nM / L, or any value or range thereof).

[0050] In some embodiments, the composition or kit is configured to deliver approximately 2 × 10⁻⁶ units to a target brain region of a subject. 10 -2.5×10 10 vg / kg or approximately 2.2×10 10 -2.4×10 10 Vg / kg of nucleic acid or viral vector, and / or, approximately 1.6 × 10⁻⁶. -4 -2.0×10 -4 nM / kg or approximately 1.8 × 10 -4 nanobubbles or nanobags with a density of nM / kg.

[0051] In some embodiments, the target brain region of the subject is subjected to ultrasound waves at a frequency of 0.5-5 MHz. In some embodiments, the target brain region of the subject is subjected to ultrasound waves at a sound pressure level of 0.1-0.6 MPa.

[0052] In some embodiments across various aspects, the nanobubbles have a particle size of approximately 30-600 nm.

[0053] In some embodiments, the nanobubbles do not contain molecules or portions connected to a nucleic acid carrier or an exogenous mechanosensitive ion channel. In some embodiments, the nanobubbles do not contain antibodies. In some embodiments, the exogenous mechanosensitive ion channel in the target neuron is not connected to the nanobubbles. In some embodiments, the nucleic acid carrier or polynucleotide encoding the exogenous mechanosensitive ion channel does not contain molecules or portions capable of connecting to the nanobubbles.

[0054] In some embodiments across various aspects, the nanobubbles are modified to increase biocompatibility. In some embodiments, PEG is grafted onto the surface of the nanobubbles. In some embodiments, the nanobubbles are nano-airbags. In some embodiments, the surface of the nano-airbags is modified with PEG, for example, by grafting PEG onto its surface. In some embodiments, the nano-airbags have a width of approximately 60-90 nm or approximately 65-85 nm; a length of approximately 300-600 nm or approximately 350-550 nm; and / or an aspect ratio of approximately 4.5-10 or 5-8.

[0055] In some embodiments across various aspects, the ultrasound waves are applied via an ultrasound generator. In some embodiments, the ultrasound generator includes a benchtop ultrasound generator, a portable ultrasound generator, a wearable ultrasound generator, an implantable ultrasound generator, or any combination thereof.

[0056] In some implementations of various aspects, the target brain region includes, but is not limited to, all brain regions containing target neurons (e.g., the hypothalamus, including the lateral hypothalamus).

[0057] In some embodiments of various aspects, the target neurons include, but are not limited to, glutamatergic neurons, GABAergic neurons, dopaminergic neurons, cholinergic neurons, serotonergic neurons, adrenergic neurons, or any combination thereof.

[0058] In some embodiments of various aspects, the target neuron includes, but is not limited to, excitatory neurons, inhibitory neurons, interneurons, or any combination thereof.

[0059] In some embodiments of various aspects, the target neuron comprises one neuron or a combination of two or more (e.g., two, three, four, five or more) neurons.

[0060] In some implementations of various aspects, the subject is an animal, such as a mammal, such as a rodent like a mouse or rat, or a primate like a rabbit, bovine, equine, sheep, canine, feline, human, monkey, chimpanzee, or gorilla.

[0061] In some embodiments, the brain diseases include, but are not limited to, Alzheimer's disease, Parkinson's disease, multiple sclerosis, epilepsy, stroke, traumatic brain injury, sleep disorders, depression, schizophrenia, pain, migraine, obsessive-compulsive eating disorder, anorexia, or any combination thereof. In some embodiments, diseases associated with the subject's behavior include, but are not limited to, obesity, diabetes, cardiovascular disease, or any combination thereof. In some embodiments, the subject's behavior includes, but is not limited to, foraging, escape, reward, punishment, reproduction, socialization, aggression, memory, learning, or any combination thereof.

[0062] The methods or uses disclosed herein combine sonogenetic techniques with nanobubbles capable of localizing ultrasound waves and amplifying their energy to neighboring cells. By enhancing the ultrasound effect through nanobubbles (e.g., nanobladders), and leveraging the advantages of sonogenetics, the methods or uses disclosed herein can lower the ultrasound activation threshold, thereby achieving precise, targeted neuromodulation of specific neural circuits within complex brain regions with lower acoustic energy, without adversely affecting non-target brain regions. The methods or uses disclosed herein can specifically activate target neurons and modulate specific neural circuits, thereby effectively controlling animal behavior (e.g., foraging behavior), and therefore have significant application value in the treatment and prevention of brain diseases or diseases related to animal behavior. Attached Figure Description

[0063] It should be understood that the accompanying drawings described herein are for illustrative purposes only. The drawings are not necessarily drawn to scale and are intended to illustrate the principles taught in this disclosure. The drawings are not intended to limit the scope of this disclosure in any way.

[0064] Figure 1 This is a schematic diagram illustrating how the exemplary method according to this disclosure controls the feeding behavior of mice by enhancing the ultrasonic responsiveness of target neurons in a target brain region of the mouse.

[0065] Figure 2 This is a transmission electron micrograph (TEM) of a nanobag used in an exemplary embodiment of this disclosure.

[0066] Figure 3This is a spatial distribution map of sound pressure used in an exemplary embodiment of this disclosure.

[0067] Figure 4 This demonstrates that in the presence of nanobags, MScL before and after ultrasound stimulation... + Calcium response in cells and MScL-cells. Figure 4 The small image in the lower left corner of the left figure is a red fluorescence image, which shows MScL. + Cells (red), the larger image is a calcium signal map. Figure 4 The image on the right is a calcium signal map, where the cells indicated by the arrows are MScL cells. + Cells without arrows are MScL-cells. The rightmost color bar in the figure indicates the intensity of the calcium signal.

[0068] Figure 5 The photograph shows the miniature ultrasonic transducer worn by mice in the experiment of Example 2, wherein the ultrasonic adapter has a diameter of 9 mm.

[0069] Figure 6 illustrates the results of regulating feeding behavior in mice using the exemplary method of this disclosure. a) Mouse movement paths before (top) and during (bottom) ultrasound stimulation. b) Amount of food consumed by mice in each treatment group before and after ultrasound stimulation. c) Time spent in the food zone by mice in each treatment group before and after ultrasound stimulation. N = 6 mice per group, ns indicates no significant difference, **p < 0.01. Two-tailed ANOVA analysis was performed on Figures b) and c), and error bars represent mean ± SD.

[0070] Figure 7 This study assesses the in vivo biosafety of mice during a 10-day evaluation period following injection of nanobags and a viral vector containing a polynucleotide encoding an exogenous mechanosensitive ion channel into the lateral hypothalamus (LH). a) Body weight of mice measured during the 10-day evaluation period (N=6 mice in all groups). b) Histological staining of brain tissue sections from the LH region using H&E staining 10 days later (N=6). Detailed Implementation Plan

[0071] This disclosure employs sonogenetics to induce the expression of exogenous mechanosensitive ion channels in target neurons of a target brain region, while simultaneously delivering nanobubbles to the target brain region. Nanobubbles (NBs) play a crucial role in improving the precision of ultrasound neuromodulation by lowering the neural stimulation threshold of the target brain region. Under the influence of ultrasound, the nanobubbles generate significant mechanical effects, leading to the opening of mechanosensitive ion channels in the target neurons. The opening of these channels allows ions (such as calcium ions) to flow into the neurons, altering their membrane potential and thereby activating the neurons and related neural circuits.

[0072] Figure 1The working process of an exemplary method of this disclosure is illustrated. For example... Figure 1 As shown, nanobubbles (NBs) and a viral vector containing a polynucleotide encoding exogenous mechanosensitive ion channels were injected into the lateral hypothalamus (LH) region of mice. Ultrasonic stimulation (US ON) of the LH region, in the presence of NBs, opened the exogenous mechanosensitive ion channels expressed on the target neurons. The opening of these channels facilitated the release of calcium ions (Ca). 2+ (The black dots in the image indicate that) glutamate flow activates target neurons, leading to glutamate release. This glutamate release activates the LH glutamatergic neural circuit, preventing the mouse from exhibiting feeding behavior. When ultrasound stimulation is turned off (US OFF), the LH glutamatergic neural circuit is inactivated and cannot prevent the mouse from feeding.

[0073] This disclosure utilizes nanobubbles to amplify ultrasonic energy, while the introduction of exogenous mechanosensitive ion channels selectively enhances the ultrasonic responsiveness of target neurons. Therefore, the method of this disclosure allows for specific activation of target neurons with lower acoustic energy, making it safer and more effective in application. Notably, the combined use of nanobubbles and exogenous mechanosensitive ion channels produces a synergistic effect under the influence of ultrasound. Compared to using ultrasound + nanobubbles or ultrasound + exogenous mechanosensitive ion channels (e.g., MScL) alone, the combination of ultrasound + nanobubbles + exogenous mechanosensitive ion channels (e.g., MScL) significantly enhances the ultrasonic responsiveness of target neurons. This synergistic effect is particularly pronounced in brain regions such as the hypothalamus. As demonstrated in Figure 6, applying the combination of ultrasound + nanobubbles + MScL to the LH region of mice produced a statistically significant difference in foraging / feeding behavior compared to a combination of just two of these three methods.

[0074] As demonstrated in the embodiments, the exemplary method of this disclosure allows for the modulation of neural circuits controlling feeding behavior by activating mouse LH-glutamatergic neurons with low acoustic energy specificity, while minimizing the impact on adjacent brain regions. Unbound by any particular theory, the inventors believe that the reduction in feeding behavior is attributed to LH-glutamatergic input to the ventral tegmental area of ​​the midbrain.

[0075] The state of neurons is crucial for maintaining normal physiological functions. Effective activation of target neurons can reshape neural pathways, regulate the release and transmission of neurotransmitters, and thus have a positive therapeutic effect on a variety of diseases. For example, activation of specific neurons can: (1) improve bradykinesia and tremor in patients, thereby helping to treat diseases such as Parkinson's disease; (2) enhance signal transmission between neural synapses and slow cognitive decline, thereby helping to treat diseases such as Alzheimer's disease; (3) regulate the levels of neurotransmitters such as serotonin and alleviate depressive symptoms; (4) block the transmission of pain signals, thereby treating or alleviating pain such as chronic pain symptoms; (5) (e.g., activating related neurons in the hypothalamus, brainstem, etc.) regulate the secretion of sleep-related hormones such as melatonin, helping to treat or alleviate sleep disorders and improve symptoms of excessive sleepiness; (6) promote the normal secretion of insulin, thereby helping to stabilize blood sugar levels, treat diabetes or alleviate its symptoms; (7) (e.g., activating related neurons in the hypothalamus) suppress appetite and reduce energy intake, thereby controlling or treating obesity and helping patients control their weight.

[0076] In some embodiments of various aspects, the methods or compositions / kits of this disclosure are used to selectively modulate neural circuits in a subject by enhancing the ultrasound responsiveness of target neurons. Neural circuits include, but are not limited to, for example, neural circuits controlling subject behavior (e.g., foraging, escape, reward, punishment, reproduction, socialization, aggression, memory, learning, or any combination thereof), or neural circuits associated with brain diseases (Alzheimer's disease, Parkinson's disease, multiple sclerosis, epilepsy, stroke, traumatic brain injury, sleep disorders, depression, schizophrenia, pain, migraine, obsessive-compulsive eating disorder, anorexia). In some embodiments, the neural circuits include glutamatergic neural circuits, GABAergic neural circuits, dopaminergic neural circuits, norepinephrine neural circuits, serotonergic neural circuits, acetylcholinestergic neural circuits, or any combination thereof. In some embodiments, the neural circuits include glutamatergic neural circuits, such as LH glutamatergic neural circuits.

[0077] In some embodiments across various aspects, the ultrasound generator includes a benchtop ultrasound generator, a portable ultrasound generator, a wearable ultrasound generator, an implantable ultrasound generator, or any combination thereof. In some embodiments, the ultrasound generator includes, but is not limited to, an ultrasonic transducer or an ultrasonic exciter. In some embodiments, the ultrasound generator is a wearable or implantable ultrasound generator, such as a wearable or implantable ultrasonic transducer or ultrasonic exciter, a head-mounted miniature ultrasonic transducer, etc.

[0078] In some embodiments across various aspects, the exogenous mechanosensitive ion channel includes MscL, TRAAK, TRPA (e.g., TRPA1 and hsTRPA1), TRPC (e.g., TRPC6), Piezo (e.g., Piezo1 and Piezo2), or any combination thereof. In some embodiments, the exogenous mechanosensitive ion channel includes or is MscL. In some embodiments, MscL is MscL-G22S.

[0079] In some embodiments, the nucleic acid vector contains a polynucleotide encoding an exogenous mechanosensitive ion channel. In some embodiments, the nucleic acid vector also contains a target neuron-specific promoter. In some embodiments, the nucleic acid vector contains both a polynucleotide encoding an exogenous mechanosensitive ion channel and a target neuron-specific promoter.

[0080] In various embodiments, target neuron-specific promoters include, but are not limited to, CaMKIIα, NSE, GAD67, VGAT, TH, VGLUT1, ChAT, DBH, or any combination thereof. In some embodiments, the target neuron-specific promoter includes or is CaMKIIα.

[0081] In some embodiments, the surface of the nanobubbles is modified with a biocompatible material. In some embodiments, the biocompatible material may be selected from one or more of the following: PEG, chitosan, polyurethane, polylactic acid, polyolefin, polysulfone, polycarbonate, polyacrylonitrile, and any combination thereof. In some embodiments, the nanobubbles are modified by grafting PEG onto their surface.

[0082] In some embodiments across various aspects, the nanobubbles have a particle size of approximately 30-600 nm, such as approximately 40-580 nm, approximately 50-550 nm, approximately 60-540 nm, approximately 70-530 nm, approximately 75-520 nm, approximately 80-510 nm, approximately 90-500 nm, approximately 100-500 nm, approximately 150-480 nm, approximately 200-450 nm, approximately 250-420 nm, approximately 280-400 nm, approximately 300-400 nm, approximately 350-550 nm, or any subrange or value thereof. In some embodiments, the nanobubbles have a particle size of approximately 275-575 nm, approximately 300-550 nm, approximately 325-252 nm, approximately 350-500 nm, approximately 375-500 nm, approximately 400-475 nm, approximately 425-450 nm, or any subrange or value thereof.

[0083] In some embodiments across various aspects, the nanobubbles are nano-airbags. In some embodiments, the surface of the nano-airbag is modified with PEG. In some embodiments, PEG is grafted onto the surface of the nano-airbag. In some embodiments, the nano-airbag is a nanoscale airbag isolated from nature (e.g., aquatic photosynthetic organisms, algae, such as *Anabaena*). In some embodiments, the nano-airbag is a nanoscale airbag isolated from nature (e.g., aquatic photosynthetic organisms, algae, such as *Anabaena*) and modified (e.g., surface grafted with PEG). In some embodiments, the nano-airbag is a nanoscale airbag isolated from *Anabaena flos-aquae* and surface grafted with PEG.

[0084] In some implementations across various aspects, the length of the nanobubbles is approximately 200-600 nm, 250-550 nm, 275-575 nm, approximately 300-550 nm, approximately 325-252 nm, approximately 350-500 nm, approximately 375-500 nm, approximately 400-475 nm, approximately 425-450 nm, or any subrange or value thereof.

[0085] In some implementations of various aspects, the diameter (width) of the nanobag is approximately 30-100 nm, approximately 40-90 nm, approximately 45-95 nm, approximately 50-90 nm, approximately 55-85 nm, approximately 60-90 nm, approximately 65-75 nm, approximately 70-90 nm, or any subrange or value between these values.

[0086] In some embodiments across various aspects, the aspect ratio (length to diameter) of the nanobubbles is approximately 3-20, approximately 4-18, approximately 5-15, approximately 8-12, approximately 10-13, or any subrange or value therebetween. In some embodiments, the aspect ratio of the nanobubbles is approximately 4.5-10, 4.5-8, approximately 5-9, approximately 5-8, approximately 6-8, or any subrange or value therebetween.

[0087] In some embodiments across various aspects, the diameter (equivalent to the width) of the nanobag is approximately 60-90 nm or approximately 65-85 nm; the length is approximately 300-600 nm or approximately 350-550 nm; and / or the aspect ratio is approximately 4.5-10 or 5-8.

[0088] In some implementations across various aspects, nanobubbles and / or nucleic acid carriers (e.g., viral carriers) are delivered to target brain regions via injection (e.g., stereotactic injection).

[0089] In some implementation schemes across various aspects, with 1×10 12Nucleic acid or viral vectors are delivered (e.g., injected) into the target brain region of the subject at a concentration of approximately 20-25 μL / kg (e.g., approximately 20-24 μL / kg, approximately 21.5-23.5 μL / kg, approximately 22-23 μL / kg, approximately 21.5-22.5 μL / kg, approximately 23 μL / kg, approximately 22 μL / kg, approximately 24 μL / kg, or any subrange or value between these values).

[0090] In some embodiments across various aspects, nanobubbles or nanocapsules are delivered (e.g., injected) into the target brain region of a subject at a concentration of 4 nM / L, in amounts of approximately 40-50 μL / kg (e.g., approximately 42-48 μL / kg, approximately 43-47 μL / kg, approximately 44-46 μL / kg, approximately 45 μL / kg, or any subrange or value thereof).

[0091] In some implementation schemes across various aspects, nucleic acid vectors or viral vectors (at a concentration of approximately 1 × 10⁻⁶) are used. 12 The nanobubbles or nanocapsules (at a concentration of about 4 nM / L) are delivered (e.g., injected) to the target brain region of the subject at a dose of about 20-25 μL / kg or about 22-24 μL / kg; and / or nanobubbles or nanocapsules (at a concentration of about 4 nM / L) are delivered (e.g., injected) to the target brain region of the subject at a dose of about 40-50 μL / kg or about 45 μL / kg.

[0092] In some embodiments, the frequency of the ultrasound applied is approximately 0.5-5 MHz, such as approximately 0.8-4.5 MHz, approximately 1.0-4.0 MHz, approximately 1.0-3.0 MHz, approximately 1.0-2.0 MHz, approximately 1.5-3.5 MHz, approximately 2-2.5 MHz, or any subrange or value between these values. In some embodiments, the frequency of the ultrasound applied is approximately 0.8-1.5 MHz, approximately 1.0-2.5 MHz, approximately 1.0-2.0 MHz, approximately 1.0-1.5 MHz, or any subrange or value between these values.

[0093] In some embodiments, the sound pressure applied by the ultrasound is approximately 0.1-0.6 MPa, approximately 0.1-0.5 MPa, approximately 0.1-0.4 MPa, approximately 0.1-0.35 MPa, approximately 0.2-0.45 MPa, approximately 0.2-0.35 MPa, approximately 0.25-0.35 MPa, approximately 0.3-0.4 MPa, approximately 0.3-0.35 MPa, or any subrange or value between these values. In some embodiments, the sound pressure applied by the ultrasound is approximately 0.2-0.4 MPa, approximately 0.25-0.35 MPa, approximately 0.3 MPa, approximately 0.31 MPa, approximately 0.32 MPa, or any subrange or value between these values.

[0094] In some embodiments across various aspects, the frequency of the ultrasonic waves applied is approximately 0.8-2.0 MHz or approximately 1.0-1.5 MHz, and / or the sound pressure applied by the ultrasonic waves is approximately 0.25-0.35 MPa or approximately 0.3-0.35 MPa.

[0095] In some implementations of various aspects, brain diseases that can be treated or prevented by the methods or uses disclosed herein include Alzheimer's disease, Parkinson's disease, multiple sclerosis, epilepsy, stroke, traumatic brain injury, sleep disorders, depression, schizophrenia, pain, migraine, bulimia, anorexia, or any combination thereof.

[0096] In some embodiments of the present disclosure, subject behaviors that can be treated or controlled by the methods or uses of this disclosure include foraging (including feeding), escape, reward, punishment, reproduction, socialization, aggression, memory, learning, or any combination thereof. In some embodiments, subject behaviors include foraging behaviors, such as feeding behaviors.

[0097] In some implementation schemes, diseases that can be treated or prevented by controlling the behavior of the subject include, but are not limited to, obesity, diabetes, cardiovascular disease, or any combination thereof.

[0098] In some embodiments of the present disclosure, the methods or uses include delivering nanobubbles and / or nucleic acid carriers to a target brain region before applying ultrasound, or applying ultrasound simultaneously with the delivery of nanobubbles and / or nucleic acid carriers. In some embodiments, the methods or uses of the present disclosure include delivering nanobubbles and nucleic acid carriers to a target brain region before applying ultrasound. In some embodiments, the methods or uses of the present disclosure include delivering nanobubbles to a target brain region, then delivering a nucleic acid carrier to the target brain region, and then applying ultrasound. In some embodiments, the methods or uses of the present disclosure include delivering a nucleic acid carrier to a target brain region, then delivering nanobubbles to the target brain region, and then applying ultrasound.

[0099] In some implementations across various aspects, the kit includes instructions for use.

[0100] This disclosure successfully triggered specific neural circuits in awake, freely moving mice through exemplary ultrasound neuromodulation in the LH region, thereby influencing feeding behavior by activating glutamatergic neurons. Notably, this behavioral change was absent in mice receiving NB+ultrasound or ultrasound neuromodulation alone, indicating a beneficial synergistic effect between NB+ultrasound and exogenous mechanosensitive ion channels. Furthermore, no significant tissue damage was observed in the treated brain regions after the implementation of the method of this disclosure. Therefore, the methods or uses of this disclosure (and the compositions / kits) provide neuron-specific, spatially targeted, temporally precise, and reproducible activation of deep brain circuits for neuromodulation. This has significant application value for precise and effective neuromodulation in non-human primates and humans.

[0101] definition

[0102] As used herein, “nanobubble” or “NB” are used interchangeably unless otherwise stated.

[0103] As used herein, “gas vesicle (GV)” or “GV” are used interchangeably unless otherwise stated.

[0104] As used in this article, the unit vg / mL represents the number of viral genome copies contained in one milliliter of viral solution.

[0105] As used in this article, “exogenous mechanosensitive ion channels” refer to mechanosensitive ion channels introduced into or to be introduced into target cells by various means (such as transduction, transfection, etc.), even though the target cells themselves may already have the specific type of endogenous mechanosensitive ion channels introduced.

[0106] When references to the methods of this disclosure are made as "in vivo methods" or "performed in vivo" or similar expressions, it is understood that the "target neurons" do not need to be isolated from the subject, or that the target neurons express the exogenous mechanosensitive ion channels within the subject. Those skilled in the art will understand that devices for applying ultrasound, such as ultrasound generators, can be located outside the body, or ultrasound can be applied outside the subject's body.

[0107] As used herein, "nucleic acid vector" or "nucleic acid vector encoding an exogenous mechanosensitive ion channel" or similar expressions refer to a nucleic acid vector containing a polynucleotide encoding an exogenous mechanosensitive ion channel. Those skilled in the art will understand that the term "nucleic acid vector" as used herein does not exclude the possibility that it may contain other elements, such as promoters.

[0108] As used in this document, “particle size” refers to the average particle size unless otherwise specified. Similarly, “length”, “diameter (width)”, and “aspect ratio” as used herein refer to the average length, average diameter (width), and average aspect ratio, unless otherwise specified.

[0109] As used in this article, “relief” of a subject’s disease (e.g., a brain disease) or similar expressions include reducing or alleviating the symptoms of a disease (e.g., a brain disease), reducing, alleviating, or eliminating a portion of the multiple symptoms of a disease (e.g., a brain disease).

[0110] As used in this article, “treatment” includes reducing or alleviating the symptoms of a disease (such as a brain disease), or reducing, alleviating, or eliminating a portion of the many symptoms of a disease (such as a brain disease).

[0111] As used in this article, "Mscl (Mechanosensitive Channel of Large Conductance)" refers to a mechanosensitive, high-conductivity ion channel. This type of channel, commonly found in bacteria, is an ion channel that opens in response to mechanical forces.

[0112] In this disclosure, when it is mentioned that nanobubbles / nanocapsules do not contain molecules or portions connected to mechanosensitive ion channels, nucleic acid carriers / mechanosensitive ion channels do not contain molecules or portions capable of connecting to the nanobubbles / nanocapsules, or similar expressions, the "molecules or portions" include peptides, modified peptides, adhesive peptides, antibodies, antibody fragments, or groups, etc., which have a linking function.

[0113] In this disclosure, when references are made to administering the kit to a subject, such as "the composition or kit is administered to the subject" or similar expressions, it should be understood that the composition contained in the kit (i.e., the nucleic acid carrier and / or nanobubbles) is administered to the subject. As used herein, when references are made to "use in combination," "combination," such as "use of a combination of a nucleic acid carrier encoding an exogenous mechanosensitive ion channel and nanobubbles in the preparation of a composition," "use of a combination of an ultrasound generator, a nucleic acid carrier encoding an exogenous mechanosensitive ion channel, and nanobubbles in the preparation of a kit," or similar expressions, it indicates the combined use of component A (e.g., nanobubbles), component B (e.g., a nucleic acid carrier encoding an exogenous mechanosensitive ion channel), and / or component C (e.g., an ultrasound generator), but does not necessarily mean that the components used in the combined use must be administered simultaneously. The above description includes cases where the components are applied sequentially, sequentially, or simultaneously. For example, A (nanobubbles) can be applied first (e.g., delivered), followed by B (a nucleic acid carrier encoding an exogenous mechanosensitive ion channel), and finally C (ultrasound); or B can be applied first, followed by A, and finally C; or A and B can be applied simultaneously, followed by C; or A, B, and C can be applied simultaneously; and so on.

[0114] As used herein, the term "approximately," when used before a quantitative value, includes the quantitative value itself, unless otherwise expressly stated. As used herein, the term "approximately" means a variation of ±10%, ±7%, ±5%, ±3%, ±1%, or ±0% compared to the stated value or range, unless otherwise expressly stated.

[0115] Unless otherwise stated, the singular as used in this article includes the plural (and vice versa).

[0116] Unless otherwise stated or contrary to common general knowledge in the art, one or more features described in the above embodiments of this disclosure may be combined with each other in any way.

[0117] Example

[0118] Experimental Materials and Methods

[0119] 1. Preparation and characterization of nanobags

[0120] Anabaena flos-aquae (FACHB-1255, Freshwater Algae Bank, Institute of Hydrobiology, Chinese Academy of Sciences) was cultured under controlled conditions in sterile BO-II medium. Nanoparticles (GVs) were extracted with sucrose solution via hyperosmolar lysis according to Walsby's method (Buckland and Walsby, 1971). They were then separated by centrifugation and purification.

[0121] The purity of GV was confirmed by measuring the optical density at 500 nm using a UV-Vis spectrophotometer (2100Pro; GE Healthcare, Piscataway, New Jersey, USA), and the size and morphology of GV were determined by transmission electron microscopy (TEM) at an operating voltage of 200 kV.

[0122] The GV used in this study is as follows Figure 2 As shown, it is modified by surface grafting of PEG.

[0123] 2. Characterization of sound field characteristics

[0124] To achieve precise ultrasonic stimulation, the sound field characteristics were characterized. The following experimental setup was used: a planar ultrasonic transducer with a center frequency of 1.0 MHz, a function generator, and a power amplifier. To ensure accuracy, the transducer and hydrophone were immersed in degassed water. A computer-controlled three-axis translation stage was used to perform point-by-point scanning to acquire sound field parameters such as sound intensity distribution.

[0125] Figure 3 The spatial distribution of sound pressure generated by the ultrasonic device used in the experiments of this disclosure is shown.

[0126] 3. Statistical Analysis

[0127] Statistical analysis was performed using GraphPad Prism software to analyze all data and create charts. Two-tailed ANOVA was used to determine statistical significance, with a significance level set at p < 0.05. Data are expressed as mean ± standard deviation (SD) (where applicable).

[0128] Example 1: In vitro experimental evaluation

[0129] This experiment verified the in vitro effect of the combination of nanobubbles and exogenous mechanosensitive ion channels under ultrasound.

[0130] 1. Primary cortical neuron culture

[0131] Rat embryos at day 18 of embryonic development were selected, carefully dissected, and treated with trypsin to isolate neurons from the cerebral cortex. The isolated neurons were then seeded into PA gel-coated culture dishes. A suitable culture medium for neurons was used to ensure adequate conditions for neuronal development.

[0132] On day 7 of neuronal culture, the cultured neurons were transduced with rAAV virus containing MScL / control rAAV virus without MScL (also known as "empty vector"). The transduced neurons were used for subsequent experiments 3-4 days post-infection, i.e., 10-12 days of in vitro culture (DIV).

[0133] 2. In vitro neuronal calcium imaging

[0134] The culture medium for culturing neurons was replaced with Fluo-4AM solution in a calcium-containing buffer at pH 7.4. The cells were then incubated in the dark at 37°C for 30 minutes to allow for sufficient uptake of the fluorescent dye. Before initiating sonication, excess dye was carefully rinsed off the cell surface with fresh calcium-containing solution.

[0135] Imaging was performed using a custom-designed inverted epifluorescence microscope setup. This setup is equipped with a dual-color LED light source, whose excitation light is filtered before being applied to the sample for illumination of the calcium sensor. To prevent phototoxicity, the LED trigger frequency was set to 1 Hz and synchronized with sCMOS time-delay imaging. A confocal culture dish containing dye-loaded cells was placed above the objective lens for imaging.

[0136] Nanoparticles were introduced into the culture medium just before ultrasonic stimulation began. Intracellular Fluo-4AM images were captured at predetermined time intervals using an excitation wavelength of 494 nm. Seamless coordination between the microscope and monochromator was achieved via software to ensure efficient imaging operations.

[0137] Figure 4 This demonstrates the effect of ultrasound stimulation on MScL before and after treatment in the presence of nanobags. + Representative images of calcium response in MscL cells. After sonication, the arrows indicate the MscL cells. + The cells have more intracellular calcium. 2+ This indicates that the combination of nanobubbles and exogenous MScL can significantly improve the ultrasonic responsiveness of neurons.

[0138] Example 2: In vivo experimental evaluation in the LH brain region

[0139] This experiment verified the in vivo effect of the combination of nanobubbles and exogenous mechanosensitive ion channels under ultrasound.

[0140] 1. Stereotactic injection of viruses and nano-airbags

[0141] (1) Stereotactic injection of virus

[0142] Eight-week-old male C57BL / 6 mice were anesthetized via intraperitoneal injection of ketamine and methaqualone. The anesthetized mice were placed in a stereotaxic instrument (RWD, Shenzhen, China), and the viral vector / empty vector was injected into the designated brain region (LH region) at a controlled rate (0.05-0.1 μL / min) using a standard stereotaxic procedure. The injection volume was 0.5 μL (1 × 10⁻⁶ mcg). 12 (vg / mL). After injection, the incision site was sutured and disinfected. The mouse was then placed on a heating pad to recover and returned to its rearing environment after recovery.

[0143] (2) Stereotactic injection of nano-airbags

[0144] For the target brain region (LH region), a 1.0 μL nanoparticle balloon (4 nM / L) was injected stereotactically at a controlled rate, or an equal volume of saline was injected (as a control). After injection, the puncture site was disinfected and sutured.

[0145] The recovery process was monitored in the mouse rearing area. No abnormalities were observed in the mice after injection.

[0146] This experiment included four experimental groups, in which the target brain regions of mice were injected with: empty AAV vector + saline (empty vector + saline); empty AAV vector + nanobubbles (empty vector + NB); AAV viral vector containing MscL + saline (MscL + saline); and AAV viral vector containing MscL + nanobubbles (MscL + NB). All four viral vectors contained the glutamatergic neuron-specific promoter CaMKIIα, enabling them to specifically target glutamatergic neurons, and all contained the red fluorescent protein mCherry.

[0147] 2. In vivo ultrasound stimulation

[0148] A specialized miniature wearable ultrasonic transducer operating at 1.0 MHz was used. This transducer was encapsulated within a custom-designed 3D-printed cap, specifically tailored to fit a mounting plate on the mouse's head. The transducer was strategically positioned above the target brain region. Prior to behavioral assessment, the mounting plate was coated with degassed ultrasonic gel, and the capped ultrasonic transducer was carefully inserted into the plate.

[0149] Ultrasound waves were applied to the target brain region of mice at a frequency of 1.0 MHz and a sound pressure of 0.31 MPa.

[0150] Figure 5 The photo shows the miniature ultrasonic transducer worn by the mice in this experiment, with the ultrasonic adapter having a diameter of 9 mm.

[0151] 3. Mouse behavior recording

[0152] Mice were monitored before, during, and after ultrasound stimulation using a Logitech camera to track their movement.

[0153] To assess feeding behavior, mice were placed in an open box measuring 40cm x 40cm, with a food tray in the center containing highly sweet chocolate. This setup allowed for the quantification of food consumption and time spent in the designated food area. Mice were allowed free exploration for 5 minutes, followed by 5 minutes of ultrasonic stimulation when their heads approached the food source. During this time, the mice were free to move around within the box.

[0154] To assess the reversible activation of on-demand feeding, satiety-enriched mice were observed for 5 minutes before, during, and after ultrasound stimulation (a total of 15 minutes). Spatial location, time spent in the food zone, speed, and behavior of the mice were captured at 15 frames per second using a commercial digital camera. Data for each mouse were recorded in a semi-dark environment, and video clips were processed using ANY-maze software (ANYmaze Video Tracking System 7.20). Results (not shown) indicated that foraging behavior in mice was inhibited upon ultrasound application; when ultrasound was removed, mouse activity returned to baseline, demonstrating that ultrasound stimulation can reversibly activate mouse behavior.

[0155] Prior to the behavioral test, all animals underwent an acclimatization period of at least 1 hour in the behavior room (i.e., the box) and were familiar with the box environment and equipment operating sounds for 15 minutes without stimulation at least 3 days prior.

[0156] Figure 6 shows the experimental results of this embodiment, demonstrating that the method exemplified in this embodiment can effectively regulate the feeding behavior of mice. Each group had N = 6 mice, ns indicates no statistically significant difference, **p < 0.01. Two-tailed ANOVA analysis was performed on Figures b) and c), and the error bars represent the mean ± SD.

[0157] Figure 6a The figures show the movement paths of mice before (top) and during (bottom) ultrasound stimulation. Dashed circles indicate specific observation areas (food zones); only mice in the MScL+NB treatment group completely left the food zone during ultrasound treatment.

[0158] Figure 6b The data shows the amount of food consumed by mice in different treatment groups before and after ultrasound stimulation. Figure 6cThe results showed that the time mice in different treatment groups spent in the food zone before and after ultrasound stimulation was significantly different in terms of food intake and food exploration time under ultrasound stimulation compared to MScL or NB alone, indicating that the combination of MScL and NB produced a synergistic effect in vivo under ultrasound stimulation.

[0159] 4. In vivo biosafety assessment

[0160] To assess the in vivo biosafety of the method described in this embodiment, mice were carefully returned to their rearing area for observation after undergoing the four treatment groups described above. Mouse weight was monitored daily to assess the overall health status of mice receiving the viral vector / NB, as weight changes generally reflect overall health conditions.

[0161] Ten days later, the mice were euthanized, and their brains were sectioned. The brain sections were then histologically stained using hematoxylin and eosin (H&E) staining for a comprehensive tissue safety assessment.

[0162] Figure 7 The results showed that viral vector / NB injection did not have a significant adverse effect on the health of mice.

[0163] The above description of the embodiments is provided to enable those skilled in the art to understand and apply this disclosure. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, this disclosure is not limited to the specific embodiments disclosed herein, and any improvements and modifications made by those skilled in the art based on the principles of this disclosure without departing from its scope should be within the protection scope of this disclosure.

[0164] This disclosure also includes the following implementation schemes

[0165] Implementation Scheme A1: A method for enhancing the ultrasound responsiveness of target neurons in a target brain region of a subject, comprising:

[0166] This induces target neurons in the target brain region of the subject to express exogenous mechanosensitive ion channels; and

[0167] Ultrasound is applied to the target brain region in the presence of nanobubbles.

[0168] Implementation Scheme A2: A method for treating, preventing, or alleviating a subject's brain disease or a behavior-related disease of the subject, or controlling the subject's behavior, by enhancing the ultrasound responsiveness of target neurons in a target brain region of the subject, comprising:

[0169] This induces target neurons in the target brain region of the subject to express exogenous mechanosensitive ion channels; and

[0170] Ultrasound is applied to the target brain region in the presence of nanobubbles.

[0171] Implementation Scheme A3: The method according to Implementation Scheme A1 or A2, wherein the exogenous mechanosensitive ion channel includes MScL, TRAAK, TRPA such as TRPA1 and hsTRPA1, TRPC such as TRPC6, Piezo such as Piezo1 and Piezo2, or any combination thereof.

[0172] Implementation Scheme A4: The method according to any one of Implementation Schemes A1-A3, wherein the exogenous mechanosensitive ion channel is specifically targeted and expressed in the target neuron, for example, by means of a promoter connection specific to the target neuron.

[0173] Implementation Scheme A5: The method according to any one of Implementation Schemes A1-A4, wherein the method includes delivering to a subject a nucleic acid vector encoding the exogenous mechanosensitive ion channel, thereby causing the exogenous mechanosensitive ion channel to be expressed in the target neuron.

[0174] Implementation Scheme A6: According to the method of Implementation Scheme A5, wherein the nucleic acid vector comprises a polynucleotide encoding the exogenous mechanosensitive ion channel; optionally, the nucleic acid vector further comprises a target neuron-specific promoter to specifically target the neuron, thereby specifically expressing the exogenous mechanosensitive ion channel in the target neuron.

[0175] Implementation Scheme A7: According to the method of Implementation Scheme A5 or A6, the nucleic acid vector is a viral vector, such as including but not limited to adeno-associated virus vectors, adenovirus vectors, lentivirus vectors, or any combination thereof.

[0176] Implementation scheme A8: The method according to any one of implementation schemes A1-A7, wherein the nanobubbles have a particle size of 30-600 nm, and / or the nanobubbles are modified, for example, by surface grafting PEG.

[0177] Implementation Scheme A9: The method according to any one of Implementation Schemes A1-A8, wherein the nanobubbles are not connected to molecules or portions of the mechanosensitive ion channel or nucleic acid carrier, or the nucleic acid carrier does not contain molecules or portions connected to the nanobubbles.

[0178] Implementation Scheme A10: The method according to any one of Implementation Schemes A1-A9, wherein the nanobubbles are nanobladders, the nanobladders having a width of about 60-90 nm or about 65-85 nm; a length of about 300-600 nm or about 350-550 nm; and / or an aspect ratio of about 4.5-10 or 5-8.

[0179] Implementation Scheme A11: The method according to any one of Implementation Schemes A1-A10, wherein the method includes applying the ultrasonic wave at a frequency of 0.5-5 MHz, and / or, the method includes applying the ultrasonic wave at a sound pressure of 0.1-0.6 MPa.

[0180] Implementation Scheme A12: The method according to any one of Implementation Schemes A1-A11, wherein the ultrasound is applied by an ultrasound generator, the ultrasound generator including a benchtop ultrasound generator, a portable ultrasound generator, a wearable ultrasound generator, an implantable ultrasound generator, or any combination thereof.

[0181] Implementation Plan A13: According to the method of any one of Implementation Plans A1-A12, including delivering approximately 2 × 10⁻⁶ mmol / L to the target brain region of the subject. 10 -2.5×10 10 vg / kg or approximately 2.2×10 10 -2.4×10 10 Vg / kg of nucleic acid or viral vector, and / or, approximately 1.6 × 10⁻⁶. -4 -2.0×10 -4 nM / kg or approximately 1.8 × 10 -4 nanobubbles or nanobags with a density of nM / kg.

[0182] Implementation Scheme A14: The method according to any one of Implementation Schemes A1-A13, wherein the target neuron includes, but is not limited to, glutamatergic neurons, GABAergic neurons, dopaminergic neurons, cholinergic neurons, serotonergic neurons, adrenergic neurons, or any combination thereof, or

[0183] The target neurons include, but are not limited to, excitatory neurons, inhibitory neurons, interneurons, or any combination thereof.

[0184] Implementation scheme A15: The method according to implementation schemes A1-A14, wherein the method is performed in vivo.

[0185] Implementation Scheme A16: The subject is an animal, such as a mammal, such as a rodent like a mouse or rat, or a primate like a rabbit, bovine, equine, sheep, canine, feline, human, monkey, chimpanzee, or gorilla, according to any one of the implementation schemes A1-A15.

[0186] Implementation Scheme A17: The method according to any one of Implementation Schemes A2-A16, wherein the brain disease includes, but is not limited to, Alzheimer's disease, Parkinson's disease, multiple sclerosis, epilepsy, stroke, traumatic brain injury, sleep disorder, depression, schizophrenia, pain, migraine, obsessive-compulsive eating disorder, anorexia, or any combination thereof; the disease related to the behavior of the subject includes, but is not limited to, obesity, diabetes, cardiovascular disease, or any combination thereof; or, the behavior of the subject includes, but is not limited to, foraging, escape, reward, punishment, reproduction, socialization, aggression, memory, learning, or any combination thereof.

[0187] Implementation Scheme A18: The method according to any one of Implementation Schemes A5-A17, the method comprising delivering nanobubbles and a nucleic acid carrier encoding an exogenous mechanosensitive ion channel separately or simultaneously to the subject by injection.

[0188] Implementation Scheme A19: According to the method of any one of Implementation Schemes A5-A18, the nucleic acid carrier and nanobubbles are respectively at 5×10 11 -5×10 12 concentrations of vg / mL and 4-6 nM / L exist in different solutions or in the same solution.

Claims

1. Use of a combination of a nucleic acid carrier encoding an exogenous mechanosensitive ion channel and nanobubbles in the preparation of a composition for enhancing the ultrasound responsiveness of target neurons in a target brain region of a subject, wherein the nucleic acid carrier targets the target neurons in the target brain region and expresses the exogenous mechanosensitive ion channel therein.

2. Use of a combination of a nucleic acid carrier encoding an exogenous mechanosensitive ion channel and nanobubbles in the preparation of a composition for treating, preventing or alleviating a brain disease or a disease related to the behavior of a subject, or for controlling the behavior of the subject, wherein the nucleic acid carrier targets target neurons in the target brain region and expresses the exogenous mechanosensitive ion channel therein.

3. Use of a combination of an ultrasound generator, a nucleic acid carrier encoding an exogenous mechanosensitive ion channel, and nanobubbles in the preparation of a kit for enhancing the ultrasound responsiveness of target neurons in a target brain region of a subject, wherein the nucleic acid carrier targets the target neurons in the target brain region and expresses the exogenous mechanosensitive ion channel therein.

4. Use of a combination of an ultrasound generator, a nucleic acid carrier encoding an exogenous mechanosensitive ion channel, and nanobubbles in the preparation of a kit for treating, preventing, or alleviating a brain disease or a disease related to the behavior of a subject, or for controlling the behavior of the subject, wherein the nucleic acid carrier targets target neurons in the target brain region and expresses the exogenous mechanosensitive ion channel therein.

5. Use of a combination of a nucleic acid carrier encoding an exogenous mechanosensitive ion channel and nanobubbles in the preparation of a composition, wherein the composition is used in a method for enhancing the ultrasound responsiveness of target neurons in a target brain region of a subject, the method comprising: The target neurons in the target brain region of the subject express the exogenous mechanosensitive ion channel; as well as Ultrasound waves are applied to the target brain region in the presence of the nanobubbles.

6. Use of a combination of an ultrasound generator, a nucleic acid carrier encoding an exogenous mechanosensitive ion channel, and nanobubbles in the preparation of a kit, wherein the kit is used in a method for enhancing the ultrasound responsiveness of target neurons in a target brain region of a subject, the method comprising: The target neurons in the target brain region of the subject express the exogenous mechanosensitive ion channel; as well as Ultrasound waves are applied to the target brain region in the presence of nanobubbles.

7. The use according to any one of claims 5-6, wherein the method is used to treat, prevent or alleviate a brain disease of the subject or a disease related to the subject's behavior or to control the subject's behavior by enhancing the ultrasound responsiveness of target neurons in a target brain region of the subject.

8. The use according to any one of claims 1-7, wherein the composition or kit comprises about 5 × 10 11 -5×10 12 vg / mL or approximately 9×10 11 -2×10 12 vg / mL, 1×10 12 Nucleic acid carriers at a concentration of vg / mL, and / or nanobubbles at a concentration of approximately 4-6 nM / L or approximately 4-5.5 nM / L; Optionally, the nucleic acid carrier and nanobubbles are present in the same or different solutions.

9. The use according to any one of claims 1-8, wherein the nucleic acid vector comprises a polynucleotide encoding the exogenous mechanosensitive ion channel; and Optionally, the nucleic acid vector further comprises a target neuron-specific promoter to specifically target the neuron, thereby specifically expressing the exogenous mechanosensitive ion channel in the target neuron.

10. The use according to any one of claims 1-9, wherein the nucleic acid vector is a viral vector, optionally including, but not limited to, adeno-associated virus vectors, adenovirus vectors, lentiviral vectors, or any combination thereof.

11. The use according to any one of claims 1-10, wherein said method is performed in vivo; and Optionally, the ultrasonic wave is applied at a frequency of 0.5-5 MHz, and / or the ultrasonic wave is applied at a sound pressure of 0.1-0.6 MPa.

12. The use according to any one of claims 1-11, wherein the exogenous mechanosensitive ion channel comprises MScL, TRAAK, TRPA such as TRPA1 and hsTRPA1, TRPC such as TRPC6, Piezo such as Piezol and Piezo2, or any combination thereof; and Optionally, the nanobubbles have a particle size of 30-600 nm, and / or the nanobubbles are modified, for example, by grafting PEG onto the surface to increase biocompatibility.

13. The use according to any one of claims 1-12, wherein the nanobubbles do not contain molecules or portions connected to the mechanosensitive ion channels, or the nucleic acid carrier does not contain molecules or portions connected to the nanobubbles.

14. The use according to any one of claims 1-13, wherein the ultrasound is applied by an ultrasound generator, such as a benchtop ultrasound generator, a portable ultrasound generator, a wearable ultrasound generator, an implantable ultrasound generator, or any combination thereof.

15. The use according to any one of claims 1-14, wherein said composition or kit is configured to deliver approximately 2 × 10⁻⁶ cells / day to a target brain region of a subject. 10 -2.5×10 10 vg / kg or approximately 2.2×10 10 -2.4×10 10 Vg / kg of nucleic acid or viral vector, and / or, approximately 1.6 × 10⁻⁶. -4 -2.0×10 -4 nM / kg or approximately 1.8 × 10 -4 nM / kg nanobubbles or nanobags.

16. The use according to any one of claims 1-15, wherein the target neuron includes, but is not limited to, glutamatergic neurons, GABAergic neurons, dopaminergic neurons, cholinergic neurons, serotonergic neurons, adrenergic neurons, or any combination thereof, or The target neurons include, but are not limited to, excitatory neurons, inhibitory neurons, interneurons, or any combination thereof.

17. The use according to any one of claims 1-16, wherein the subject is an animal, such as a mammal, such as a rodent like a mouse or rat, or a primate like a rabbit, bovine, equine, sheep, canine, feline, human, monkey, chimpanzee, or gorilla.

18. The use according to any one of claims 2, 4-5 and 8-17, wherein: The brain diseases mentioned include, but are not limited to, Alzheimer's disease, Parkinson's disease, multiple sclerosis, epilepsy, stroke, traumatic brain injury, sleep disorders, depression, schizophrenia, pain, migraine, obsessive-compulsive eating disorder, anorexia, or any combination thereof; or Diseases related to the subject's behavior include, but are not limited to, obesity, diabetes, cardiovascular disease, or any combination thereof; or The subjects' behaviors include, but are not limited to, foraging, escaping, rewarding, punishing, reproducing, socializing, aggression, memory, learning, or any combination thereof.

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