Systems and devices for treating brain glioma

Abstract

The invention relates to the technical field of tumor treatment, in particular to a system and device for treating brain glioma. The system for treating brain glioma comprises: a brain glioma diagnosis module; an engineered bacterium providing module; an ultrasonic signal control module; an ultrasonic stimulation intervention module; and an intervention effect evaluation module. Based on the functions of all the modules, the engineered bacteria marked by the near-infrared two-region fluorescent probe are injected into a subject, ultrasonic stimulation parameters are regulated and controlled, the engineered bacteria are stimulated to secrete interferon, and the brain glioma is intervened in combination with the ultrasonic effect and the immune effect of the engineered bacteria; and the effect of intervening the brain glioma is further evaluated, so that the intervening treatment of the brain glioma by the noninvasive transcranial ultrasonic regulation engineering bacteria can be realized, and the brain glioma can be well treated.

Description

Technical Field

[0001] The present application belongs to the technical field of tumor treatment, and in particular, relates to a system and device for treating brain glioma. Background Art

[0002] Gliomas are intracranial tumors that originate from glial cells. They are one of the most common primary tumors in the brain. They are highly malignant, grow rapidly, have a short course, and are prone to recurrence after surgery. They are considered to be one of the most difficult and refractory tumors in neurosurgery. Surgery and postoperative chemotherapy, radiotherapy, and electric field therapy are the main treatment options for gliomas. In addition, immunotherapy is an emerging technology for the treatment of gliomas. However, due to the existence of the blood-brain barrier and the complex microenvironment of gliomas (high interstitial pressure, hypoxia, acidification, etc.), current treatment methods are ineffective.

[0003] In the brain, the blood-brain barrier includes: (1) a barrier between plasma and brain cells formed by the walls of brain capillaries and glial cells; (2) a barrier between plasma and cerebrospinal fluid formed by the choroid plexus. The blood-brain barrier is a barrier structure that protects the brain from damage by foreign harmful substances. It can limit most substances in the blood from entering the brain and is of great significance for maintaining the stability of the internal environment of brain tissue. Due to the existence of the blood-brain barrier, large molecule drugs cannot or are difficult to be delivered into the brain, which greatly reduces the efficacy of the drugs. This has become a key bottleneck in the treatment of gliomas. Summary of the invention

[0004] The purpose of this application is to provide a system and device for treating brain glioma, aiming to solve the technical problem of how to better treat brain glioma.

[0005] In order to achieve the above application purpose, the technical solution adopted in this application is as follows:

[0006] In a first aspect, the present application provides a system for treating brain glioma, comprising:

[0007] Brain glioma diagnosis module: used to determine the pathological information of brain glioma of the subject;

[0008] An engineered bacteria providing module: used to determine the dosage of the engineered bacteria according to the pathological information of the brain glioma, and inject the engineered bacteria into the subject; wherein the engineered bacteria are labeled with a near-infrared zone II fluorescent probe to track the process of the engineered bacteria crossing the blood-brain barrier and the colonization and growth in the brain glioma tissue;

[0009] Ultrasonic signal control module: used to confirm ultrasonic stimulation parameters through an electronic control system according to the pathological information of the brain glioma;

[0010] Ultrasonic stimulation intervention module: used to stimulate the engineered bacteria to secrete interferon by using the ultrasonic stimulation parameters, and to intervene in brain glioma by combining the ultrasonic effect and the immune effect of the engineered bacteria;

[0011] Intervention effect evaluation module: Evaluate the effect of intervention for brain glioma by examining the health status of the subjects.

[0012] In a second aspect, the present application provides a device for treating brain glioma, comprising:

[0013] Medical imaging diagnostic equipment and electrophysiological monitors;

[0014] Engineered bacteria labeled with near-infrared region II fluorescent probe;

[0015] An electronic control system controls multiple ultrasonic transducers.

[0016] The system for treating glioma provided in the first aspect of the present application includes: a glioma diagnosis module, an engineered bacteria providing module, an ultrasonic signal control module, an ultrasonic stimulation intervention module, and an intervention effect evaluation module. Based on the functional effects of the above modules, the present application injects engineered bacteria labeled with near-infrared zone II fluorescent probes into subjects with gliomas, stimulates the engineered bacteria to secrete interferon by regulating ultrasonic stimulation parameters, and intervenes in the treatment of gliomas by combining the ultrasonic effect and the immune effect of the engineered bacteria, and further evaluates the effect of intervention in gliomas, thereby realizing non-invasive transcranial ultrasonic regulation of engineered bacteria for intervention treatment of gliomas; therefore, the system of the present application has the advantages of non-invasive transcranial ultrasound, focusability, and high tissue penetration, and can treat gliomas well.

[0017] The second aspect of the present application provides a device for treating gliomas, including: medical imaging diagnostic equipment and electrophysiological monitors; engineered bacteria labeled with near-infrared zone II fluorescent probes; and multiple ultrasonic transducers controlled by an electronic control system. In the device of the present application, the medical imaging diagnostic equipment and the electrophysiological monitor can diagnose gliomas and conduct treatment evaluations, and the combination of engineered bacteria labeled with near-infrared zone II fluorescent probes and ultrasonic transducers can achieve a combination of ultrasonic effects and the immune effects of engineered bacteria, and perform combined intervention treatment on gliomas, thereby achieving non-invasive transcranial ultrasonic regulation of engineered bacteria for intervention treatment of gliomas. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0019] Figure 1 is a schematic diagram of a system for treating brain glioma provided in an embodiment of the present application;

[0020] Figure 2 This is a comparison diagram of the effect of ultrasound-assisted near-infrared zone II fluorescent probe-labeled engineered Escherichia coli on the production of reactive oxygen species (ROS) in brain glioma C6 cells provided in the examples of the present application;

[0021] Figure 3 This is a comparison diagram of the effect of ultrasound-assisted near-infrared zone II fluorescent probe-labeled engineered Escherichia coli on the cell activity of brain glioma C6 cells provided in the examples of the present application;

[0022] Figure 4 This is a flow cytometry analysis comparison diagram of the intervention of engineered Escherichia coli labeled with ultrasound-assisted near-infrared zone II fluorescent probe on brain glioma C6 cells provided in the examples of the present application;

[0023] Figure 5 This is a comparison diagram of DNA fluorescence microscopic images of engineered Escherichia coli labeled with ultrasound-synergistic near-infrared zone II fluorescent probe intervening in brain glioma C6 cells provided in the examples of the present application. DETAILED DESCRIPTION

[0024] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present application more clearly understood, the present application is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0025] In this application, the term "and / or" describes the association relationship of associated objects, indicating that there may be three relationships. For example, A and / or B can mean: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural. The character " / " generally indicates that the associated objects are in an "or" relationship.

[0026] In the present application, "at least one" means one or more, "more than one" means two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single or plural items.

[0027] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution, some or all of the steps can be executed in parallel or sequentially, and the execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0028] The terms used in the embodiments of the present application are only for the purpose of describing specific embodiments, and are not intended to limit the present application. The singular forms "a", "said" and "the" used in the embodiments of the present application and the appended claims are also intended to include plural forms, unless the context clearly indicates other meanings.

[0029] The weight of the relevant components mentioned in the embodiment description of the present application can not only refer to the specific content of each component, but also represent the proportional relationship between the weights of the components. Therefore, as long as the content of the relevant components is proportionally enlarged or reduced according to the embodiment description of the present application, it is within the scope disclosed in the embodiment description of the present application. Specifically, the mass described in the embodiment description of the present application can be a mass unit known in the chemical industry such as μg, mg, g, kg, etc.

[0030] The terms "first" and "second" are used only for descriptive purposes to distinguish objects such as substances from each other, and should not be understood as indicating or implying relative importance or implicitly indicating the number of indicated technical features. For example, without departing from the scope of the embodiments of the present application, the first XX may also be referred to as the second XX, and similarly, the second XX may also be referred to as the first XX. Thus, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features.

[0031] Ultrasound is a mechanical wave with a frequency higher than 20,000 Hz. Ultrasound has three basic acoustic effects: mechanical effect, thermal effect, and cavitation effect, and can be widely used in the biomedical field. Ultrasound can penetrate the skull non-invasively to radiate deep brain tissues, and can become a non-invasive means of intervening in brain diseases.

[0032] Bacteria have the ability to break through the blood-brain barrier and adapt to the complex microenvironment of glioma, and activate the immune system through surface proteins and metabolites to exert anti-cancer effects. The immunotherapy performance of engineered bacteria can be enhanced through the transformation strategy of engineered anaerobic or facultative anaerobic bacteria (referred to as engineered bacteria or engineered bacteria).

[0033] Based on the fact that it is difficult for general drugs to enter the blood-brain barrier, the treatment effect on glioma is not ideal. In the treatment process using light stimulation combined with engineered bacteria, the depth of light stimulation penetrating brain tissue is insufficient, and the immune effect of lysing bacteria is limited. To this end, the application embodiment proposes a system and device for intervening in glioma based on non-invasive transcranial ultrasound regulation of engineered bacteria, which utilizes the advantages of ultrasound non-invasively crossing the skull, focusing, and high tissue penetration, and is used to regulate the immunotherapy effect of engineered bacteria. The engineered bacteria labeled with near-infrared zone II fluorescent probes can be used to observe the distribution of engineered bacteria in the process of crossing the blood-brain barrier and colonizing in glioma tissues. Fluorescence imaging technology can be used to visualize the process of engineered bacteria crossing the blood-brain barrier and the colonization and growth in glioma tissues. Therefore, the engineered bacteria are stimulated by ultrasound, combined with the ultrasound effect and the immune effect of the engineered bacteria themselves, to achieve efficient treatment of gliomas. The specific scheme is as follows.

[0034] In a first aspect, the present application provides a system for treating brain glioma. Figure 1 As shown, the system for treating brain glioma in the embodiment of the present application includes the following modules:

[0035] (1) Brain glioma diagnosis module; (2) Engineered bacteria provision module; (3) Ultrasonic signal control module; (4) Ultrasonic stimulation intervention module; (5) Intervention effect evaluation module.

[0036] Among them, the glioma diagnosis module can be used to determine the glioma pathological information of the subject; the engineered bacteria providing module can determine the dosage of the engineered bacteria based on the glioma pathological information obtained by the glioma diagnosis module, and inject the engineered bacteria into the subject, and the engineered bacteria used are labeled with a near-infrared zone II fluorescent probe, so that the process of the engineered bacteria crossing the blood-brain barrier and the colonization and growth in the glioma tissue can be tracked; the ultrasonic signal control module has an electronic control system, which can confirm and regulate the ultrasonic stimulation parameters through the electronic control system based on the previously determined glioma pathological information; the ultrasonic stimulation intervention module can use the previous ultrasonic stimulation parameters to stimulate the injected engineered bacteria to secrete interferon, so that the ultrasonic effect and the immune effect of the engineered bacteria can be combined to intervene in the glioma; finally, the health status of the subject is checked through the intervention effect evaluation module to evaluate the effect of the intervention in glioma.

[0037] Therefore, based on the functional effects of the above-mentioned modules, the embodiments of the present application can realize non-invasive transcranial ultrasound-regulated engineered bacteria intervention treatment for glioma. Such a system has the advantages of non-invasive ultrasound across the skull, focusability, and high tissue penetration, and can effectively treat glioma.

[0038] For the glioma diagnosis module: used to determine the glioma pathological information of the subject.

[0039] The subject may be a human or a mammal or other animal with a brain structure, for example, including but not limited to: mice, rats, monkeys, rabbits, pigs, etc. The glioma diagnosis module is used to diagnose and grade the glioma condition in the subject's brain.

[0040] In some embodiments, the subject's glioma pathological information includes the location, size and morphology of the glioma measured by medical imaging diagnostic equipment, and the glioma is diagnosed and graded based on the glioma pathological information and brain anatomical structure characteristics. For example, gliomas can be examined and identified by medical magnetic resonance imaging equipment (MR), positron emission tomography-X-ray computed tomography (PET / CT) and other imaging technologies, and the brain anatomical structure characteristics and brain tumor morphological characteristics, such as the location, size, peripheral edema state, tissue uniformity in the glioma lesion area, space-occupying effect, degree of blood-brain barrier damage and other combined signs caused by glioma lesions, can be clearly and accurately displayed to achieve the diagnosis and grading of gliomas.

[0041] A module is provided for engineered bacteria: it is used to determine the dosage of engineered bacteria based on the glioma pathological information of the subject obtained by the glioma diagnosis module, and inject the engineered bacteria into the subject.

[0042] The engineered bacteria are labeled with near-infrared II fluorescent probes. The wavelength range of near-infrared II (NIR-II) is 900 to 1700 nm. Near-infrared II fluorescent probe labeled imaging has lower tissue autofluorescence, deeper tissue penetration depth and higher imaging resolution.

[0043] In some embodiments, the engineered bacteria include at least one of Lactococcus lactis, Listeria, Salmonella typhimurium, Escherichia coli and Bifidobacterium longum labeled with near-infrared region II fluorescent probe. For example, Escherichia coli labeled with albumin-indocyanine green-glucose nanoprobe or indocyanine green-albumin-glucose nanoprobe can be selected.

[0044] Specifically, the preparation steps of Escherichia coli labeled with near-infrared zone II fluorescent probe include: (a) the first step is to prepare near-infrared zone II fluorescent probe: one method is to prepare albumin-indocyanine green-glucose nanoprobe: carboxyindocyanine green (ICG-COOH), 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) are mixed in a molar ratio of 1:1.2:1.5 to obtain activated indocyanine green; then the activated indocyanine green and glucosamine are mixed in a molar ratio of 1:1.5 to obtain glucose-indocyanine green of small size; then the albumin (BSA, 50 mg / mL) solution and the glucose-indocyanine green (4.5 mg / mL) aqueous solution are mixed at 4°C for 24 hours, and then dialyzed (MWCO 500Da). 10kDa) was purified to remove free glucose-indocyanine green, and BSA and glucose-indocyanine green formed small-size albumin-indocyanine green-glucose nanoprobes through molecular interaction. Another method is to prepare indocyanine green-albumin-glucose nanoprobes: ICG dimethyl sulfoxide solution (concentration 1.5mg / mL, 8mL) was slowly added to BSA aqueous solution (BSA weight 67mg, solution volume 20mL) at a speed of 1mL / min. Stirring for 12 hours in dark and dialyzing in distilled water for 24 hours to obtain BSA-ICG nanoparticles, then activating the protein surface carboxyl group by EDC / NHS, and further adding glucosamine to obtain indocyanine green-albumin-glucose nanoprobe particles by chemical coupling. (b) The second step is to label the engineered bacteria with a near-infrared zone II fluorescent probe: flow cytometry analysis, culture the engineered bacteria (e.g., Escherichia coli) in a shaker until the OD value is about 0.1-0.5, add the above-mentioned near-infrared zone II fluorescent probe solution (ICG concentration: 10 μg / mL) and incubate with the bacteria for 8 hours, remove the unlabeled probe by centrifugation, and use flow cytometry to measure the probe-labeled engineered bacteria signal.

[0045] In the examples of this application, the correlation between the size of the near-infrared zone II fluorescent probe and the labeling efficiency of engineered bacteria is also studied. Confocal imaging: The bacteria are cultured in a shaker until the OD value is about 0.1-0.5, and near-infrared zone II fluorescent probe solutions of different sizes (ICG concentration: 10 μg / mL) are added and incubated with the bacteria for 2 hours, 4 hours, 8 hours, and 16 hours. The unlabeled probes are removed by centrifugation, and then the zone II fluorescent probes are collected to label the engineered bacteria, and samples are prepared for confocal imaging; the results show that the near-infrared zone II fluorescent probes with a particle size of about 5 to 20 nm have better labeling efficiency for engineered bacteria.

[0046] Specifically, in the engineered bacteria providing module, the dosage of the engineered bacteria is further calculated based on the subject's glioma pathological information and the subject's personal information, such as measurement information: age and weight, etc.; after determining the dosage of the engineered bacteria labeled with the near-infrared zone II fluorescent probe, it is injected into the subject.

[0047] In some embodiments, engineered bacteria labeled with near-infrared zone II fluorescent probes are injected into the subject by intravenous injection. Fluorescence imaging technology is then used to observe the distribution of engineered bacteria, visualize the process of engineered bacteria crossing the blood-brain barrier and colonization and growth in brain glioma tissue.

[0048] For the ultrasonic signal control module: it is used to confirm the ultrasonic stimulation parameters through the electronic control system according to the pathological information of the glioma in the glioma diagnosis module. For example, the ultrasonic stimulation parameters are determined according to the location, size and depth of the glioma; at the same time, the corresponding ultrasonic stimulation parameters can also be adjusted according to the feedback results of the ultrasonic stimulation intervention module and the intervention effect evaluation module.

[0049] In some embodiments, the ultrasound signal control module further includes: a reference delay determination submodule for calculating the excitation pulse signal sent by the ultrasound stimulation intervention module, and an electronic modulation submodule for adjusting ultrasound stimulation parameters in real time.

[0050] The ultrasonic signal control module mainly includes the electronic control system that controls the array elements in each ultrasonic transducer of the ultrasonic stimulation intervention module. Each array element has a separate lead, and different ultrasonic transducers are controlled by independent electronic systems. The reference delay determination submodule included in the ultrasonic signal control module is used to calculate the reference delay of the excitation pulse signal emitted by each array element. According to the size and depth of the stimulation target, different types of electronic modulation submodules are selected to achieve accurate and dynamically adjustable stimulation of the target area of ​​glioma.

[0051] Specifically, different ultrasound parameters can be confirmed according to the type of glioma disease, the severity of the disease, the selection of stimulation targets and the stimulation effect: including basic frequency, pulse amplitude, pulse repetition frequency, pulse length, etc. for independent ultrasound stimulation of multiple targets. And according to the feedback, the stimulation parameters are adjusted in real time. According to the degree of glioma, the age of the subject, the type of engineered bacteria used and other information, the ultrasound stimulation parameters that the subject needs to receive, such as the ultrasound stimulation dose, are determined. Ultrasound stimulation parameters also include: transducer center frequency, pulse repetition frequency, pulse duration, fundamental frequency, number of fundamental waves and number of pulses, etc.

[0052] For the ultrasound stimulation intervention module: it is used to stimulate the secretion of interferon by engineered bacteria using ultrasound stimulation parameters, and to intervene in brain glioma by combining the ultrasound effect and the immune effect of engineered bacteria. The optimal transcranial ultrasound transducer irradiation time window can be selected, and ultrasound can be used to stimulate engineered bacteria such as Escherichia coli to secrete interferon-γ, combining the ultrasound effect and the immune effect of the bacteria themselves to achieve efficient treatment of brain glioma.

[0053] In some embodiments, the ultrasound stimulation intervention module includes a three-dimensionally adjustable fixation module that prevents the subject from walking or a wearable fixation module that allows the subject to walk freely.

[0054] Specifically, the stimulation methods can be: 1. Three-dimensional adjustable fixed module: the subject adopts a lying or reclining position, and uses a three-dimensional adjustable fixed module for ultrasound stimulation; 2. Wearable fixed module: the subject has a certain degree of freedom of movement. The fixed module has a fine-tuning function. According to the needs of research and treatment, ultrasonic transducers with different frequencies, different focusing effects, different output energies, and different focal spot sizes and focal lengths can be selected.

[0055] In some embodiments, the ultrasonic stimulation intervention module uses an ultrasonic transducer with a base frequency of 200kHz-4MHz to ultrasonically stimulate the engineered bacteria; the ultrasonic transducer contacts the subject's brain and begins stimulation under the guidance of magnetic resonance. The ultrasonic transducer material can be a magnetically compatible material, but is not limited to other materials. Depending on the size and location of the tumor, a scanning stimulation method can be used for intervention.

[0056] In some embodiments, the ultrasound stimulation parameters in the ultrasound stimulation intervention module include ultrasound with a frequency of 0.5M-5MHz. Due to the presence of the skull, the transcranial ultrasound frequency becomes lower, and excessive ultrasound will be reflected and absorbed by the skull. 0.5M-5MHz can better process stimulation. Furthermore, the stimulation time can be 10-15min, and the input voltage during ultrasound stimulation is 55-65mVpp. These ultrasound stimulation parameters can be confirmed and modulated by the ultrasound signal control module.

[0057] In some embodiments, an ultrasound stimulation intervention module for transcranial ultrasound precision stimulation can also be constructed through an acoustic artificial structure constructed by 3D printing technology, and by adjusting the material and size of the acoustic artificial structure layer, focused sound field modulation stimulation in different areas and depths can be achieved. Due to the scattering and diffraction effects of its structure on sound waves, the propagation of sound waves can be flexibly controlled.

[0058] By reducing the ultrasonic energy loss through the acoustic artificial structure, the mechanical energy of ultrasound is efficiently converted into thermal effect, and the interferon secretion of engineered bacteria is enhanced, which can reduce economic costs. The easy-to-process, flexible-design, and low-cost acoustic artificial structure is combined with a single-element ultrasonic transducer to achieve deep acoustic wave stimulation, making ultrasonic stimulation more convenient. The 3D acoustic artificial structure is coupled to the ultrasonic transducer, which has the characteristics of small size and low weight, and can realize ultrasonic stimulation in the awake state, with little impact on the daily activities of the subjects.

[0059] By modulating a special, high-precision focused sound field through 3D-printed acoustic artificial structures, the engineered bacteria in the glioma site can be enhanced to efficiently secrete interferon, thereby better immunotherapy for glioma. For example, multi-focal sound field regulation can be designed; acoustic holographic acoustic artificial structures can be designed to flexibly modulate any sound field through amplitude and phase; the modulated sound field can be used for 3D printing of organ scaffolds, bone repair, and other applications through acoustic thermal effects.

[0060] For the intervention effect evaluation module: the effect of intervention on brain glioma can be evaluated by checking the health status of the subjects. The tumor volume can be observed to be reduced or increased by imaging techniques such as magnetic resonance imaging, and the efficacy of treating brain glioma can be judged by combining the detection of glioma molecular biological markers. After treatment, the tumor treatment effect can be evaluated by imaging and pathological tests.

[0061] Specifically, medical imaging diagnostic equipment and electrophysiological monitors can be used. For example, at least one of an ultrasound machine, a magnetoencephalogram (MEG), an X-ray computed tomography (CT), a functional magnetic resonance imaging (f-MRI), and a magnetic resonance spectrometer (MRS) can be used to evaluate the therapeutic effect. Electrophysiological monitoring: EEG, ECG, EMG, heart rate, body temperature, blood oxygen, blood oxygen saturation, etc. After a course of treatment, the therapeutic effect is evaluated through clinical biochemical index examinations, medical imaging diagnostic technology, etc., and the treatment plan is modified.

[0062] In summary, the system of the embodiment of the present application combines the advantages of ultrasound non-invasive, focusable, and high tissue penetration, and combines the engineered bacteria labeled with near-infrared zone II fluorescent probes to synergistically treat brain gliomas. First, by using imaging techniques such as magnetic resonance imaging in the brain glioma diagnosis module to observe brain gliomas, identify the location, size, and properties of gliomas, and diagnose. Then, the engineered bacteria provide a module to complete the dosage calculation of the engineered bacteria labeled with near-infrared zone II fluorescent probes, inject (such as by intravenous injection) into the subject, and use fluorescence imaging technology to observe the distribution of engineered bacteria, visualize the process of engineering bacteria crossing the blood-brain barrier and the colonization and growth in brain glioma tissue. The ultrasonic stimulation parameters are confirmed by the ultrasonic signal control module, and a better transcranial ultrasonic transducer irradiation time window is selected. The engineered bacteria are stimulated to secrete gamma interferon by ultrasonic stimulation intervention module, and the ultrasonic effect and the immune effect of the bacteria themselves are combined to achieve efficient treatment of brain gliomas. At the same time, the tumor volume can be observed to be reduced or increased by imaging techniques such as magnetic resonance imaging in the intervention effect evaluation module, and the efficacy of treating brain gliomas can be judged by combining glioma molecular biological marker detection.

[0063] In a second aspect, the present application provides a device for treating glioma. The device for treating glioma in the present application includes the following instruments and reagents:

[0064] (1) Medical imaging diagnostic equipment and electrophysiological monitors; (2) Engineered bacteria labeled with near-infrared zone II fluorescent probes; (3) Multiple ultrasonic transducers controlled by an electronic control system.

[0065] The second aspect of the present application provides a device for treating gliomas, including: medical imaging diagnostic equipment and electrophysiological monitors; engineered bacteria labeled with near-infrared zone II fluorescent probes; and multiple ultrasonic transducers controlled by an electronic control system. In the device of the present application, the medical imaging diagnostic equipment and the electrophysiological monitor can diagnose gliomas and conduct treatment evaluations, and the combination of engineered bacteria labeled with near-infrared zone II fluorescent probes and ultrasonic transducers can achieve a combination of ultrasonic effects and the immune effects of engineered bacteria, and perform combined intervention treatment on gliomas, thereby achieving non-invasive transcranial ultrasonic regulation of engineered bacteria for intervention treatment of gliomas.

[0066] In some embodiments, the medical imaging diagnostic equipment includes: medical magnetic resonance imaging equipment (MR) for diagnosing brain glioma, positron emission tomography-computed tomography (PET / CT), etc., and at least one of an ultrasound machine, magnetoencephalogram (MEG), computed tomography (CT), functional magnetic resonance imaging (f-MRI), and magnetic resonance spectrometer (MRS) for evaluating the effect of treatment. Electrophysiological monitors can monitor EEG, ECG, EMG, heart rate, body temperature, blood oxygen, blood oxygen saturation, etc.

[0067] In some embodiments, the engineered bacteria include: at least one of Lactococcus lactis, Listeria, Salmonella typhimurium, Escherichia coli and Bifidobacterium longum labeled with a near-infrared zone II fluorescent probe; specifically, the near-infrared zone II fluorescent probe is selected from albumin-indocyanine green-glucose nanoprobe or indocyanine green-albumin-glucose nanoprobe, and the particle size of the near-infrared zone II fluorescent probe can be 5 to 20 nm. For example, the engineered bacteria can use Escherichia coli labeled with an albumin-indocyanine green-glucose nanoprobe with a particle size of 10 nm. The specific preparation process is described above. In the embodiment of the present application, by constructing an albumin-indocyanine green-glucose nanoprobe that has both optical properties and ultrasonic response properties, it can be applied to zone II fluorescence imaging and can respond to ultrasonic thermal effects.

[0068] In some embodiments, the ultrasonic transducer includes a bulk wave transducer or a surface acoustic wave transducer with a fundamental frequency of 200kHz-4MHz. Specifically, the array elements in the ultrasonic transducer are individually wired and controlled by an independent electronic control system to calculate the reference delay of the excitation pulse signal and adjust the ultrasonic stimulation parameters. Furthermore, a 3D acoustic artificial structure coupled ultrasonic transducer can also be used.

[0069] In the examples of the present application, in order to verify the therapeutic effect of the system for treating brain glioma, an in vitro cell experiment on brain glioma was carried out using an ultrasonic transducer to provide ultrasonic stimulation combined with Escherichia coli labeled with albumin-indocyanine green-glucose nanoprobes. The albumin-indocyanine green-glucose nanoprobe (a near-infrared second-zone fluorescent molecule) was compounded with Escherichia coli to successfully prepare Escherichia coli labeled with albumin-indocyanine green-glucose nanoprobes. Ultrasound synergized Escherichia coli labeled with albumin-indocyanine green-glucose nanoprobes to intervene in brain glioma C6 cells, significantly increasing the production of reactive oxygen species by cells, causing DNA breakage in brain glioma C6 cells, and killing brain glioma C6 cells.

[0070] The following describes the invention in conjunction with specific embodiments.

[0071] Example

[0072] 1. Preparation of Escherichia coli labeled with albumin-indocyanine green-glucose nanoprobe

[0073] 1.1 Carboxy indocyanine green (ICG-COOH), 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were mixed in a molar ratio of 1:1.2:1.5 to obtain activated indocyanine green; then the activated indocyanine green and glucosamine were mixed in a molar ratio of 1:1.5 and dialyzed with a dialysis bag (MWCO 500Da) to obtain small-sized glucose-indocyanine green; then the albumin solution (BSA concentration: 50 mg / mL) and the glucose-indocyanine green aqueous solution (glucose-indocyanine green concentration: 4.5 mg / mL) were mixed at 4°C for 24 hours, and then the free glucose-indocyanine green was purified by dialysis (MWCO 10kDa). The albumin and glucose-indocyanine green formed albumin-indocyanine green-glucose nanoprobes with a particle size of about 10 nm through molecular interaction.

[0074] 1.2 Shake E. coli MG1655 in LB liquid medium overnight (220 rpm, 37°C); centrifuge (4500 rpm, 5 min) to obtain bacterial precipitate, wash twice with PBS and resuspend in PBS; use a microplate reader to measure the optical density (OD) at 600 nm to determine the number of bacteria in the solution; dilute the bacterial solution with PBS to make its OD 600 The value was 0.1, 10 mL of Escherichia coli PBS solution was taken, and 10 μL of the above-mentioned albumin-indocyanine green-glucose nanoprobe solution (probe concentration: 10 mg / mL, solvent: dimethyl sulfoxide) was added; incubated at 220 rpm, 37°C for 2 hours, and centrifuged to remove the free probe to obtain the albumin-indocyanine green-glucose nanoprobe-modified Escherichia coli for subsequent experiments.

[0075] In subsequent experiments, the probe mentioned is the albumin-indocyanine green-glucose nanoprobe, the Escherichia coli mentioned is Escherichia coli MG1655, and the probe-labeled Escherichia coli is the prepared albumin-indocyanine green-glucose nanoprobe-modified Escherichia coli MG1655.

[0076] 2. Active oxygen detection

[0077] DCFH-DA fluorescent probe was used to detect reactive oxygen species (ROS) in brain glioma C6 cells. DCFH-DA (2',7'-dichlorofluorescein diacetic acid) itself has no fluorescence, but can freely pass through the cell membrane and enter the cell. In the cell, DCFH-DA is hydrolyzed by esterase into DCFH (2',7'-dichlorofluorescein diphenol), and DCFH cannot penetrate the cell membrane, so it is effectively loaded into the cell. When there is reactive oxygen in the cell, it will oxidize DCFH to generate highly fluorescent 2',7'-dichlorofluorescein (DCF) and emit green fluorescence.

[0078] The experimental steps include: inoculating 30,000 to 50,000 glioma C6 cells in a 24-well plate and culturing for 24 hours to allow the cells to fully adhere to the wall and grow; dividing the cells into six groups, and each group is treated with different drugs; Figure 2 As shown in the figure, the six groups were named as follows: PBS is the first group, Bac is the second group, Bac-ICG is the third group, PBS+ultrasound is the fourth group, Bac+ultrasound is the fifth group, and Bac-ICG+ultrasound is the sixth group. The first group was added with PBS, and the second group was added with E. coli (the number of bacteria was 1*10 7 CFU / mL, 300 μL), and the third group added albumin-indocyanine green-glucose nanoprobe labeled Escherichia coli (bacterial number was 1*10 7 CFU / mL, probe concentration was 8 μg / mL, 300 μL), the fourth group was added with PBS, and the fifth group was added with Escherichia coli (the number of bacteria was 1*10 7 CFU / mL, 300 μL), and the sixth group added albumin-indocyanine green-glucose nanoprobe labeled Escherichia coli (bacterial count was 1*10 7 CFU / mL, probe concentration was 8μg / mL, 300μL); after incubation in the incubator for 4h, the fourth, fifth and sixth groups were treated with ultrasound (ultrasonic transducer frequency: 1MHZ, input voltage: 60mVpp, stimulation time: 15min), and then continued to be cultured for 1h; the culture medium was removed, the cells were washed 1-2 times with PBS, 300μL of serum-free culture medium containing DCFH-DA (10μM) was added, incubated in the incubator for 20min, and washed 1-2 times with serum-free culture medium to remove DCFH-DA that did not enter the cells; the production of reactive oxygen species by cells in each group was observed using a cell imaging instrument.

[0079] When used for flow cytometry analysis, the cells are digested with trypsin and collected by centrifugation for flow cytometry analysis. 200 μL of trypsin digestion is added to the wells, and 400 μL of complete culture medium is added to terminate the digestion. The cells are then centrifuged and resuspended in PBS.

[0080] The results are as follows Figure 2 As shown: In a, green represents the reactive oxygen species produced in the cells, and b is the flow cytometry analysis of the production of reactive oxygen species. The results showed that the sixth group can significantly increase the production of reactive oxygen species (ROS) in brain glioma cells.

[0081] 3. Cell activity detection

[0082] 3.1 Calcein-AM / PI cell double staining kit (produced by Beyotime) was used to simultaneously observe live and dead cells in the same cell culture dish under a fluorescence microscope. Calcein-AM stains live cells with green fluorescence, while propidium iodide (PI) stains dead cells with red fluorescence. The maximum excitation wavelength of Calcein, the hydrolysis product of Calcein-AM, is 494nm, and the maximum emission wavelength is 517nm; the maximum excitation wavelength of the PI-DNA complex is 535nm, and the maximum emission wavelength is 617nm.

[0083] The experimental steps include: inoculating 30,000 to 50,000 glioma C6 cells in a 24-well plate and culturing for 24 hours to allow the cells to fully adhere to the wall and grow; dividing the cells into six groups, each group receiving a different drug treatment; Figure 3 As shown in a, the six groups were named as Control, Bac, Bac-ICG, PBS+ultrasound, Bac+ultrasound, and Bac-ICG+ultrasound. PBS was added to the first group, and E. coli was added to the second group (the number of bacteria was 1*10 7 CFU / mL, 300 μL), and the third group added albumin-indocyanine green-glucose nanoprobe labeled Escherichia coli (bacterial number was 1*10 7 CFU / mL, probe concentration was 8 μg / mL, 300 μL), the fourth group was added with PBS, and the fifth group was added with Escherichia coli (bacterial count was 1*10 7 CFU / mL, 300 μL), and the sixth group added albumin-indocyanine green-glucose nanoprobe labeled Escherichia coli (bacterial count was 1*10 7CFU / mL, probe concentration 8μg / mL, 300μL); after incubation in the incubator for 4h, the fourth, fifth and sixth groups were subjected to ultrasonic treatment (transducer frequency: 1MHZ, input voltage: 60mVpp, stimulation time: 15min); after continued cultivation for 1h after illumination, the culture medium was removed, and the cells were washed 1-2 times with PBS; a sufficient amount of staining working solution was added to each well to ensure that the monolayer of cells was covered; after incubation at 37℃ for 15 minutes, the staining solution was washed off, and the cells were washed twice with PBS, and finally 100μL of PBS was added; the live and dead cells were observed under a fluorescence microscope. The steps for preparing the staining working solution are as follows: take out Calcein AM Solution and PI Solution, and equilibrate at room temperature for 30 minutes; add 5 μl of PI Solution and 5 μl of Calcein AM Solution to 10 ml of PBS, and vortex to mix to prepare the working solution. At this time, the concentration of Calcein AM is 2 μM, and the concentration of PI is 8 μM. The obtained working solution (2 μM Calcein AM and 8 μM PI) can be directly used for staining cells.

[0084] 3.2 Detect cell activity using CCK-8. CCK-8 kit (produced by Beyotime) is a kit based on WST-8 and widely used for cell proliferation and cell activity. WST-8 can be reduced by some tolhydrogenases in mitochondria to generate orange-yellow color. The more and faster the cell proliferation, the darker the color; the greater the cytotoxicity, the lighter the color.

[0085] The experimental steps include: 30,000 to 50,000 glioma C6 cells are seeded in a 24-well plate and cultured for 24 hours to allow the cells to fully adhere to the wall and grow; the cells are divided into six groups, each group is treated with different drugs, such as Figure 3 As shown in b, brain glioma C6 cells were incubated with different concentrations of albumin-indocyanine green-glucose nanoprobes labeled with Escherichia coli: the concentrations represent the number of bacteria, which are Control (the first group), 1x10 6 That is the second group, 1x10 7 That is the third group, 1x10 8 That is the fourth group, 1x10 9 That is the fifth group, 5x10 9 That is, the sixth group; after incubating in the incubator for 4 hours; remove the culture medium and wash the cells 1-2 times with PBS; add 100 μL of fresh culture medium containing 10% CCK-8 reagent to each well; continue to incubate in a 37°C, 5% CO2 incubator for half an hour; measure the absorbance value at OD450 with an enzyme marker.

[0086] 3.3 The cell activity was also detected using the above CCK-8 kit.

[0087] The experimental steps include: inoculating 30,000 to 50,000 glioma C6 cells in a 24-well plate and culturing for 24 hours to allow the cells to fully adhere to the wall and grow; dividing the cells into six groups, each group receiving different drug treatments, such as Figure 3 As shown in c, the six groups were named as follows: PBS is the first group, Bac is the second group, Bac-ICG is the third group, PBS+L is the fourth group, Bac+L is the fifth group, and Bac-ICG+L is the sixth group. Among them, PBS was added to the first group, and the number of E. coli added to the second group was 1*10 7 CFU / mL, 300 μL), and the third group added albumin-indocyanine green-glucose nanoprobe labeled Escherichia coli (bacterial number was 1*10 7 CFU / mL, probe concentration was 8 μg / mL, 300 μL), the fourth group was added with PBS, and the fifth group was added with Escherichia coli (the number of bacteria was 1*10 7 CFU / mL, 300 μL), and the sixth group added albumin-indocyanine green-glucose nanoprobe labeled Escherichia coli (bacterial count was 1*10 7 CFU / mL, probe concentration is 8μg / mL, 300μL); after incubation in the incubator for 4h, the fourth, fifth and sixth groups were subjected to ultrasonic treatment (transducer frequency: 1MHZ, input voltage: 60mVpp, stimulation time: 15min); culture was continued for 1h; the culture medium was removed and the cells were washed 1-2 times with PBS; 100μL of fresh culture medium containing 10% CCK-8 reagent was added to each well; the cells were continued to be incubated in a 37°C, 5% CO2 incubator for half an hour; the absorbance value at OD450 was measured with an enzyme marker.

[0088] The results are as follows Figure 3 Shown: a is a confocal fluorescence image of C6 cells stained with calcein AM (CAM) and PI, b is the effect of different concentrations of fluorescent probe-labeled engineered Escherichia coli (Bac-ICG) on cell activity of brain glioma C6 cells, and c is a quantitative characterization diagram of brain glioma C6 cells. The results showed that the sixth group could better kill brain glioma C6 cells.

[0089] 4. Quantitative detection of live and dead cells by flow cytometry

[0090] Annexin-FITC and PI double staining kit (produced by Beyotime) is a kit for detecting cell apoptosis. It combines two dyes, Annexin-FITC and PI, and can distinguish living cells (Annexin V- / PI-), early apoptotic cells (Annexin V+ / PI-), and late apoptotic or necrotic cells (Annexin V+ / PI+).

[0091] The experimental steps include: inoculating 30,000 to 50,000 glioma C6 cells per well in a 24-well plate and culturing for 24 hours to allow the cells to fully adhere to the wall and grow; dividing the cells into six groups, each group undergoing different intervention treatments; Figure 4 As shown in the figure, the six groups were named as follows: control group (group 1), E. coli (group 2), engineered E. coli (group 3), ultrasound group (group 4), E. coli + ultrasound (group 5), and engineered E. coli + ultrasound (group 6). PBS was added to group 1, and E. coli (bacterial count was 1*10 7 CFU / mL, 300 μL), and the third group added albumin-indocyanine green-glucose nanoprobe labeled Escherichia coli (bacterial number was 1*10 7 CFU / mL, probe concentration was 8 μg / mL, 300 μL), the fourth group was added with PBS, and the fifth group was added with Escherichia coli (the number of bacteria was 1*10 7 CFU / mL, 300 μL), and the sixth group added albumin-indocyanine green-glucose nanoprobe labeled Escherichia coli (bacterial count was 1*10 7 CFU / mL, probe concentration is 8μg / mL, 300μL); after incubation in the incubator for 4h, the fourth, fifth and sixth groups were subjected to ultrasonic treatment (transducer frequency: 1MHZ, input voltage: 60mVpp, stimulation time: 15min); culture was continued for 1h, the culture medium was removed, and the cells were washed 1-2 times with PBS; 100μL of trypsin was added for digestion for 1 minute, and the well plate was tapped to confirm that all cells were digested under a microscope, and 200μL of complete culture medium was added to terminate the digestion, and the cells were collected by centrifugation at 1000rpm for 3min; the supernatant was removed, and the cells were resuspended and washed once with PBS to remove the active esterase contained in the culture medium, and the cells were resuspended with 200μl of staining working solution after centrifugation; incubated at 37℃ for 15 minutes, and directly tested on the machine.

[0092] The results are as follows Figure 4 As shown, the sixth group, namely ultrasound synergistically fluorescent probe-labeled Escherichia coli intervened in brain glioma C6 cells and significantly killed brain glioma C6 cells.

[0093] 5. TUNEL (TdT-mediated dUTP Nick-End Labeling) staining to detect cell apoptosis

[0094] Experimental steps: 30,000 to 50,000 glioma C6 cells were seeded in a 24-well plate and cultured for 24 hours to allow the cells to fully adhere to the wall and grow; the cells were divided into six groups, and each group was treated with different drugs; Figure 5As shown in the figure, the six groups were named as Control (Group 1), Bac (Group 2), Bac-ICG (Group 3), Control+ultrasound (Group 4), Bac+ultrasound (Group 5), and Bac-ICG+ultrasound (Group 6). PBS was added to Group 1, and Escherichia coli (the number of bacteria was 1*10 7 CFU / mL, 300 μL), and the third group added albumin-indocyanine green-glucose nanoprobe labeled Escherichia coli (bacterial number was 1*10 7 CFU / mL, probe concentration was 8 μg / mL, 300 μL), the fourth group was added with PBS, and the fifth group was added with Escherichia coli (the number of bacteria was 1*10 7 CFU / mL, 300 μL), and the sixth group added albumin-indocyanine green-glucose nanoprobe labeled Escherichia coli (bacterial count was 1*10 7 CFU / mL, probe concentration is 8μg / mL, 300μL); after incubation in the incubator for 4h, the fourth, fifth and sixth groups were subjected to ultrasonic treatment (transducer frequency: 1MHZ, input voltage: 60mVpp, stimulation time: 15min); culture was continued for 1h, the culture medium was removed, and the cells were washed 1-2 times with PBS; 300μL 4% paraformaldehyde was added to each well for fixation at room temperature for 20min, and PBS was washed 1-2 times; 300μL permeabilization solution was added to each well for permeabilization for 10min, and PBS was washed 1-2 times; 300μL diluted staining solution was added, incubated at room temperature in the dark for 30 minutes, and washed 1-2 times with PBS; 300μL DAPI nuclear staining solution was added, incubated at room temperature for 15min, and washed 1-2 times with PBS; the conditions of cells in each group were observed with a cell imager.

[0095] The results are as follows Figure 5 As shown, blue represents the cell nucleus and red represents the cell nuclear DNA breakage, that is, the sixth group can better cause DNA breakage in brain glioma C6 cells.

[0096] It can be seen from this that the present application embodiment can combine ultrasound stimulation with engineered bacteria (such as E. coli) labeled with near-infrared zone II fluorescent probes, and combine the ultrasound effect and the immune effect of engineered bacteria to synergistically perform non-invasive, cross-skull, focusable, and highly tissue-penetrating intervention on brain gliomas, which can achieve a good therapeutic effect. Therefore, the combination of the two can be used to prepare a system and device for treating brain gliomas. For example, a system for treating glioma may include the following: (1) a glioma diagnosis module, used to determine the glioma pathology information of a subject; (2) an engineered bacteria provision module, used to determine the dosage of the engineered bacteria based on the glioma pathology information, and to inject the engineered bacteria into the subject; wherein the engineered bacteria are labeled with a near-infrared zone II fluorescent probe to track the process of the engineered bacteria crossing the blood-brain barrier and the colonization and growth in the glioma tissue; (3) an ultrasonic signal control module, used to confirm the ultrasonic stimulation parameters through an electronic control system based on the glioma pathology information; (4) an ultrasonic stimulation intervention module, used to stimulate the engineered bacteria to secrete interferon using ultrasonic stimulation parameters, and to intervene in glioma by combining the ultrasonic effect and the immune effect of the engineered bacteria; and (5) an intervention effect evaluation module, which evaluates the effect of intervention in glioma by checking the health status of the subject.

[0097] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims

1. A system for treating brain glioma, characterized in that: include: Brain glioma diagnosis module: used to determine the pathological information of brain glioma of the subject; An engineered bacteria providing module: used to determine the dosage of the engineered bacteria according to the pathological information of the brain glioma, and inject the engineered bacteria into the subject; wherein the engineered bacteria are labeled with a near-infrared zone II fluorescent probe to track the process of the engineered bacteria crossing the blood-brain barrier and the colonization and growth in the brain glioma tissue; Ultrasonic signal control module: used to confirm ultrasonic stimulation parameters through an electronic control system according to the pathological information of the brain glioma; Ultrasonic stimulation intervention module: used to stimulate the engineered bacteria to secrete interferon by using the ultrasonic stimulation parameters, and to intervene in brain glioma by combining the ultrasonic effect and the immune effect of the engineered bacteria; Intervention effect evaluation module: Evaluate the effect of intervention for brain glioma by examining the health status of the subjects.

2. The system for treating brain glioma according to claim 1, characterized in that: The engineered bacteria include: at least one of Lactococcus lactis, Listeria, Salmonella typhimurium, Escherichia coli and Bifidobacterium longum labeled with near-infrared region II fluorescent probe; And / or, the engineered bacteria is injected into the subject by intravenous injection.

3. The system for treating brain glioma according to claim 2, characterized in that: The engineered bacteria are selected from Escherichia coli labeled with albumin-indocyanine green-glucose nanoprobe or indocyanine green-albumin-glucose nanoprobe; And / or, the particle size of the near-infrared second-region fluorescent probe is 5 to 20 nm.

4. The system for treating brain glioma according to claim 1, characterized in that: The ultrasonic signal control module further includes: a reference delay determination submodule for calculating the excitation pulse signal sent by the ultrasonic stimulation intervention module, and an electronic modulation submodule for adjusting the ultrasonic stimulation parameters in real time.

5. The system for treating brain glioma according to claim 1, characterized in that: The ultrasound stimulation intervention module includes a three-dimensionally adjustable fixation module that prevents the subject from walking or a wearable fixation module that enables the subject to walk freely; And / or, the ultrasonic stimulation intervention module utilizes an ultrasonic transducer with a fundamental frequency of 200kHz-4MHz or a 3D acoustic artificial structure coupled ultrasonic transducer to ultrasonically stimulate the engineered bacteria; And / or, the ultrasound stimulation parameters in the ultrasound stimulation intervention module include ultrasound with a frequency of 0.5M-5MHz.

6. The system for treating brain glioma according to any one of claims 1 to 5, characterized in that: The brain glioma pathological information includes the location, size and pathological morphology of the glioma measured by medical imaging diagnostic equipment. The brain glioma is diagnosed and graded based on the brain glioma pathological information and brain anatomical structure characteristics.

7. A device for treating brain glioma, characterized in that: include: Medical imaging diagnostic equipment and electrophysiological monitors; Engineered bacteria labeled with near-infrared region II fluorescent probe; An electronic control system controls multiple ultrasonic transducers.

8. The device for treating brain glioma according to claim 7, characterized in that: The medical imaging diagnostic equipment comprises: At least one of a medical magnetic resonance imaging device and a positron emission tomography-computed tomography device for diagnosing brain glioma; At least one of an ultrasound machine, a magnetoencephalogram, an X-ray computed tomography machine, a functional magnetic resonance imaging machine, and a magnetic resonance spectrometer for evaluating the therapeutic effect of brain glioma.

9. The device for treating brain glioma according to claim 7, characterized in that: The engineered bacteria include: at least one of Lactococcus lactis, Listeria, Salmonella typhimurium, Escherichia coli and Bifidobacterium longum labeled with the near-infrared second region fluorescent probe; And / or, the near-infrared second-region fluorescent probe is selected from albumin-indocyanine green-glucose nanoprobe or indocyanine green-albumin-glucose nanoprobe.

10. The device for treating brain glioma according to any one of claims 7 to 9, characterized in that: The ultrasonic transducer includes a 3D acoustic artificial structure coupled ultrasonic transducer, or a body wave transducer or a surface acoustic wave transducer with a fundamental frequency of 200kHz-4MHz; And / or, each array element in the ultrasonic transducer is individually wired and controlled by an independent electronic control system to calculate a reference delay for sending an excitation pulse signal and adjust ultrasonic stimulation parameters.

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