Tumor treatment system based on sonoluminescence fatigue effect

Abstract

The invention discloses a tumor treatment system based on an ultrasonic fatigue effect, which directly damages a cancer cell membrane structure and destroys the integrity of the cancer cell membrane structure through the continuous action of low-intensity ultrasonic energy, specifically targets a disordered tumor extracellular matrix by utilizing the ultrasonic fatigue effect, and simultaneously causes mechanical damage to tumor blood vessels. The tumor core hypoxia caused by nutrition delivery is blocked, so that cell apoptosis is induced. And the multi-layer mechanical-biological effect jointly inhibits tumor growth. The tumor treatment system is combined with temozolomide for use, can synergistically amplify the anti-tumor effect, and is suitable for hypoxic tumors tolerated by a traditional therapy. According to the treatment system, a non-invasive and controllable tumor treatment mode is achieved through accurate regulation and control of energy, and a new thought is provided for tumor treatment.

Description

Technical Field

[0001] This invention relates to the field of medical device technology, and in particular to a tumor treatment system based on the sonoluminescence fatigue effect. Background Technology

[0002] Ultrasound, as a non-invasive and safe physical therapy method, has been widely used in the field of medical imaging diagnosis. Currently developing ultrasound therapy techniques, with their safe and reliable treatment process, significant clinical effects, precise targeting, and excellent tissue penetration capabilities, are receiving increasing attention from research and clinical fields. Existing sonodynamic therapy (SDT) relies on exogenous sonosensitive agents and an aerobic environment, while traditional ultrasound thermal ablation therapy carries the risk of tissue damage.

[0003] Ultrasonic fatigue effect can damage tumor tissue through cumulative mechanical stress, and can be used for efficient tumor ablation without relying on ultrasound sensitizers. Sonoluminescence refers to the phenomenon where liquids or bubbles emit visible or near-ultraviolet light during rapid collapse under the influence of ultrasound, generating instantaneous high temperature and pressure. The sonoluminescence effect (photoacoustic coupling phenomenon) allows for real-time monitoring of tissue state through the feedback of ultrasound-excited light signals. As an important physical phenomenon of ultrasound-matter interaction, it reveals the intrinsic mechanism by which ultrasound modulates the optical properties of a medium. When ultrasound propagates in a medium, bubbles in the liquid collapse inward and emit light under the excitation of sound waves. The bubbles are rapidly compressed under sound pressure, and the internal gas generates high temperature and emits light due to adiabatic heating. The duration of this light emission is extremely short (picoseconds), and the spectrum contains short-wavelength components. This effect not only changes the light propagation path but also affects the material transport and energy distribution of biological tissues through the dynamic modulation of the medium's microenvironment, providing theoretical support for the innovative application of ultrasound in tumor treatment. However, sonoluminescence has not yet been used to optimize ultrasound fatigue therapy.

[0004] Ultrasound, as a safe and controllable physical energy carrier, has demonstrated multi-dimensional application value in the medical field. In medical imaging, its advantages such as being radiation-free and providing real-time dynamic imaging have made it the preferred clinical detection method. In the treatment field, ultrasound's tissue penetration and energy controllability have given rise to innovative technologies such as sonodynamic therapy (SDT). However, traditional SDT destroys tumor cells by generating reactive oxygen species (ROS) through sound sensitizers. This technology relies on systemic administration of exogenous sensitizers, which has drawbacks such as insufficient targeting, allergy risks, and dependence on aerobic environments, especially limiting its effectiveness in the treatment of hypoxic solid tumors.

[0005] To address the aforementioned issues, this invention provides a device based on the sonoluminescence fatigue effect. This device differs significantly from traditional high-intensity focused ultrasound (HIFU) and sonodynamic therapy (SDT). It treats tumors through a mechanical fatigue damage mechanism, achieving a treatment mode that does not require exogenous sonosensitive agents, thus providing a new approach for safe tumor treatment. Summary of the Invention

[0006] The present invention aims to at least solve one of the aforementioned technical problems existing in the prior art. Therefore, the object of the present invention is to provide a tumor treatment system based on the sonoluminescence fatigue effect.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a tumor treatment system, the treatment system comprising an acoustic emission module and a monitoring module; The acoustic emission module includes an ultrasonic generator, an ultrasonic probe, and a power dynamic control system. The ultrasonic generator has an ultrasonic intensity of 0.1-2 W / cm². 2 Continuously adjustable, with the frequency adjustable from 0.5 to 3 MHz; The monitoring module includes a multispectral optical sensor; The multispectral optical sensor is used to detect changes in the refractive index of tumor tissue during treatment.

[0008] In some embodiments of the present invention, the multispectral optical sensor is also used to detect the blood oxygen saturation of tumor tissue.

[0009] In some embodiments of the present invention, the ultrasonic probe includes a transducer and a coupling layer.

[0010] In some embodiments of the present invention, the transducer includes a piezoelectric ceramic material for efficiently converting electrical energy into ultrasonic waves.

[0011] In some embodiments of the present invention, the coupling layer is made of a medical coupling agent or an elastic material.

[0012] In some embodiments of the present invention, the elastic material includes, but is not limited to, polyether block amide, silicone rubber and nitrile rubber.

[0013] In some embodiments of the present invention, the coupling layer is used to fill the air gap between the ultrasound probe and the human skin, reduce the energy loss of ultrasound waves during transmission, and ensure that ultrasound waves are efficiently transmitted to the treatment site.

[0014] In some embodiments of the present invention, the tumor treatment system further includes a navigation system; the navigation system includes a shear wave excitation unit, a multi-channel ultrasound receiving unit, and a three-dimensional positioning and navigation unit; the navigation system is used to measure the shear wave velocity of the tumor and guide the ultrasound beam to be precisely focused on the hypoxic core area.

[0015] In some embodiments of the present invention, the multi-channel ultrasound receiving unit includes an ultrasound signal receiver with 8-16 channels.

[0016] In some embodiments of the present invention, the ultrasonic signal receiver is used to receive the propagation signal of shear waves in tissues in real time, record the time difference of shear waves arriving at different spatial points, and generate a shear wave propagation velocity distribution map.

[0017] In some embodiments of the present invention, the three-dimensional positioning and navigation unit includes at least one of an optical positioning sensor and a tumor three-dimensional modeling module.

[0018] In some embodiments of the present invention, the monitoring module further includes a high-speed CCD camera and an acoustic-optical signal processing unit.

[0019] In some embodiments of the present invention, the acousto-optic signal processing unit is a cavitation effect analysis unit used to capture the dynamic behavior of microbubbles.

[0020] In some embodiments of the present invention, the tumor shear wave velocity >5m / s is determined to be a high disorder region.

[0021] In some embodiments of the present invention, measuring tumor shear wave velocity includes measuring the tumor extracellular matrix.

[0022] In some embodiments of the present invention, the power dynamic control system provides real-time feedback adjustment of the ultrasonic intensity to ≤2W / cm².

[0023] In some embodiments of the present invention, the adjustment of the ultrasonic intensity is 0.1-1.5 W / cm. 2 .

[0024] In some embodiments of the present invention, the adjustment of the ultrasound intensity is based on the tumor volume; the tumor volume is <200 mm². 3 Ultrasonic intensity 0.4-0.6 W / cm 2 The tumor volume is 200-400 mm. 3 The ultrasonic intensity is 0.6-0.8 W / cm. 2 The tumor volume is >400 mm. 3 The ultrasonic intensity is 0.8-1.2 W / cm. 2 .

[0025] In some embodiments of the present invention, the tumor treatment system further includes a safety protection device; the safety protection device includes a temperature sensor; the temperature sensor includes features for monitoring the treatment area to ensure that the temperature rise is less than 5°C.

[0026] In some embodiments of the present invention, the safety protection device has an over-temperature protection function.

[0027] In some embodiments of the present invention, the safety protection device further includes a current sensor and an automatic power-off protection device with overcurrent protection and overtime protection functions.

[0028] In some embodiments of the invention, the temperature sensor includes features for monitoring the temperature of the ultrasound probe, the ultrasound generator, and the treatment area.

[0029] In some embodiments of the present invention, the temperature sensor is used to monitor the treatment area to ensure that the temperature rise is less than 5°C.

[0030] In some embodiments of the present invention, the temperature sensor is used to monitor the treatment area to ensure that the temperature rise is less than or equal to 4°C.

[0031] In some embodiments of the present invention, the safe surface temperature of the ultrasonic probe is 43°C.

[0032] In some embodiments of the present invention, the safe temperature value for the internal components of the ultrasonic generator is 65°C.

[0033] In some embodiments of the present invention, the tumor treatment system automatically cuts off the power supply when the temperature exceeds a safe value.

[0034] In some embodiments of the present invention, the tumor treatment system further includes an ultrasound contrast imaging device; the ultrasound contrast imaging device includes a contrast agent injection control unit and a contrast signal acquisition unit; the ultrasound contrast imaging device is used to detect changes in tumor vascular density, and stops the operation of the tumor treatment system when the microvessels decrease by more than 30%.

[0035] In some embodiments of the present invention, the ultrasound imaging device further includes a blood vessel density analysis algorithm module.

[0036] In some embodiments of the present invention, the contrast agent injection control unit includes at least one of a dose adjustment module, a rate control module, and a timing synchronization module.

[0037] In some embodiments of the present invention, the dosage adjustment module precisely controls the total injection volume according to the ratio of contrast agent dosage to patient weight of 0.1-0.2 mL / kg.

[0038] In some embodiments of the present invention, the rate control module is used to adjust the injection rate; the injection rate is 1-3 mL / s.

[0039] In some embodiments of the present invention, the contrast signal acquisition unit includes at least one of a high-frequency ultrasound acquisition component, a grayscale signal conversion module, and a signal preprocessing component.

[0040] In some embodiments of the present invention, the high-frequency ultrasound acquisition component is used to acquire contrast agent perfusion signals.

[0041] In some embodiments of the present invention, the grayscale signal conversion module is used to generate grayscale change curves.

[0042] In some embodiments of the present invention, the signal preprocessing component is used to acquire dynamic perfusion signals of contrast agent in tumor blood vessels (frame rate ≥30fps) and capture the grayscale change curve of microvascular blood flow perfusion.

[0043] In some embodiments of the present invention, the tumor treatment system further includes an acoustic-optical signal processing unit.

[0044] In some embodiments of the present invention, the signal processing unit includes an integrated signal processing chip.

[0045] In some embodiments of the present invention, the signal processing unit is responsible for receiving, processing and analyzing data from the subunit, and is linked with the control device in real time.

[0046] In some embodiments of the present invention, the subunit includes a cavitation effect analysis subunit and a multispectral signal filtering subunit.

[0047] A second aspect of the present invention provides the use of the tumor treatment system described in the first aspect in the preparation of tumor treatment products.

[0048] In some embodiments of the present invention, the tumor includes at least one of glioma, meningioma, and neuroblastoma.

[0049] A third aspect of the present invention provides the use of the tumor treatment system and tumor treatment drug described in the first aspect in the preparation of tumor treatment products.

[0050] In some embodiments of the present invention, the tumor treatment drug includes a chemotherapy drug or an immunotherapy drug.

[0051] In some embodiments of the present invention, the chemotherapy drug includes temozolomide, lomustine, cisplatin, or gemcitabine.

[0052] In some embodiments of the invention, the application includes using the tumor treatment system 1 to 4 hours after chemotherapy or immunotherapy.

[0053] The beneficial effects of this invention are: This invention provides a tumor treatment system based on the sonoluminescence fatigue effect. This system directly damages the cell membrane structure of cancer cells through the continuous application of low-intensity ultrasound energy, disrupting their integrity. Simultaneously, the sonoluminescence fatigue effect specifically targets the disordered tumor extracellular matrix (ECM), promoting ECM degradation by activating signaling pathways such as TGF-β / PI3K-AKT. It can also cause mechanical damage to tumor blood vessels, blocking nutrient delivery and leading to hypoxia in the tumor core. Furthermore, it exacerbates ECM remodeling and induces apoptosis through the HIF-1α pathway. This multi-layered mechano-biological effect collectively inhibits tumor growth, significantly differentiating it from traditional high-intensity focused ultrasound (HIFU) and sonodynamic therapy (SDT).

[0054] The tumor treatment system of this invention can also be used in combination with temozolomide (TMZ) to synergistically amplify the anti-tumor effect, and is particularly suitable for hypoxic tumors resistant to traditional therapies. The treatment system of this invention achieves a non-invasive and controllable tumor treatment mode through precise energy regulation, which is of great significance to tumor treatment and provides new ideas for tumor treatment. Attached Figure Description

[0055] Figure 1 This describes the effect of the therapeutic device of the present invention on CT-2A cells in vitro. A is 1.75 W / cm². 2 Image showing the survival of CT-2A cells after treatment; B represents 1 W / cm². 2 Image showing the survival of CT-2A cells after treatment; C is 0.75 W / cm². 2 Image showing the survival of CT-2A cells after treatment; D = 1.75 W / cm². 2 Bar chart of CT-2A cell proliferation after treatment; E = 1 W / cm² 2 Bar graph of CT-2A cell proliferation after treatment; F = 0.75 W / cm² 2 Bar graph of CT-2A cell proliferation after treatment; G = 1.75 W / cm² 2 Image of the proliferation activity of CT-2A cells after treatment; H represents 1 W / cm² 2 Image showing the proliferation activity of CT-2A cells after treatment; I = 0.75 W / cm². 2 The proliferation activity of CT-2A cells after treatment; the statistical analysis between the two groups was performed using a two-tailed Student's t-test. P<0.05, P<0.01, P<0.001, n=3.

[0056] Figure 2 This illustrates the effect of the treatment system of the present invention on tumors in a mouse model. A represents ultrasound intensity (1 W / cm²). 2 Tumor growth curve; B represents ultrasound intensity at 1 W / cm.2 A bar chart showing changes in tumor volume; C represents ultrasound intensity at 1 W / cm². 2 Tumor resection body mass diagram; D represents ultrasound intensity at 0.5 W / cm. 2 Tumor growth curve; E represents ultrasound intensity of 0.5 W / cm. 2 A bar chart showing changes in tumor volume; F represents ultrasound intensity at 0.5 W / cm². 2 Tumor resection body mass chart; G represents ultrasound intensity (1 W / cm). 2 Time-dependent weight changes in CT-2A tumor-transplanted mice; H represents ultrasound intensity at 0.5 W / cm². 2 Time-dependent weight changes in CT-2A tumor-transplanted mice; I represents ultrasound intensity at 1 W / cm². 2 Mouse survival rate plot; J represents ultrasound intensity of 0.5 W / cm. 2 The survival rate of mice is shown in the figure; ns indicates no significant difference, n=6, and the statistical analysis between the two groups was performed using a two-tailed Student's t-test. P<0.05, P<0.01, P<0.001, n=4.

[0057] Figure 3 This is a bar chart showing the relative tumor volume and tumor inhibition rate of the treatment system of the present invention in a mouse model; A represents the ultrasound intensity at 1 W / cm². 2 A bar chart comparing relative tumor volume (experimental group / control group) on day 11 of tumor treatment; B represents ultrasound intensity at 1 W / cm². 2 A bar chart comparing tumor inhibition rates in groups G2 and G5; C represents ultrasound intensity at 0.5 W / cm². 2 A bar chart comparing relative tumor volume (experimental group / control group) on day 11 of tumor treatment; D represents ultrasound intensity at 0.5 W / cm². 2 At that time, a bar chart comparing the tumor inhibition rates of groups G7 and G9.

[0058] Figure 4 This is a bar chart showing the relative tumor volume and tumor inhibition rate of the treatment system of the present invention in a mouse model; A represents the ultrasound intensity at 1 W / cm². 2 A bar chart comparing the relative tumor volume of groups G1, G3, and G10 on day 11 of tumor treatment; B represents ultrasound intensity at 1 W / cm². 2 A bar chart comparing tumor inhibition rates in groups G3 and G10; C represents ultrasound intensity at 0.5 W / cm². 2 A bar chart comparing the relative tumor volume of groups G6, G11, G12, and G9 on day 11 of tumor treatment; D represents ultrasound intensity at 0.5 W / cm². 2 A bar chart comparing the tumor inhibition rates of groups G11, G12, and G9.

[0059] Figure 5 The therapeutic effect of the treatment system of this invention combined with tumor drugs in vivo is shown in Figure A. Schematic diagram of the treatment process; Figure B. Images of tumors removed after treatment in different groups; Figure C. Bar chart comparing the weight of tumors removed in mice after different treatments; Figure D. Curve of weight change in mice after different treatments; Figure E. Bar chart comparing tumor volume after different treatments; Figure F. Tumor growth curve in mice; Statistical analysis between two groups was performed using a two-tailed Student's t-test, and multiple comparisons between groups were performed using one-way ANOVA. P<0.05, P<0.01, P<0.001, n=5.

[0060] Figure 6 This is a graph showing the individual tumor growth of mice carrying tumors in vivo, where the treatment system of this invention and the tumor drug synergistically treat the tumor.

[0061] Figure 7 The images show the histochemical and immunofluorescence staining results of tumor sections on day 10 after treatment in this example. A shows the histochemical staining results of the tumor sections; B shows the immunofluorescence staining results of the tumor sections; C shows a bar chart comparing the necrosis rate (necrotic area relative to the total tumor area) under different treatments; D shows a bar chart comparing the number of tumor cells per square millimeter under different treatments; E shows a bar chart comparing the density of green-positive cells in the non-necrotic area outside the tumor sections; F shows a bar chart comparing the density of green-positive cells in the entire tumor section; G shows CD31 antibody staining in tumor sections after different treatment groups, with yellow arrows indicating ruptured blood vessels. Scale bar = 100 micrometers. Statistical analysis between the two groups was performed using a two-tailed Student's t-test. P<0.05, P<0.01, P<0.001.

[0062] Figure 8 Immunohistochemical staining images of HIF-1α, Ki67 and MMP9 in tumor sections after day 10 of treatment in Example 4, scale bar = 100 micrometers. Detailed Implementation

[0063] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments and comparative examples are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the test or experimental methods are conventional methods in the art.

[0064] Example 1 This embodiment provides the preparation of a tumor treatment system.

[0065] The tumor treatment system consists of the following components: The tumor treatment system consists of an acoustic emission device, a control device, a monitoring device, an elastography navigation system, an ultrasound imaging device, a safety protection device, a power supply device, and a treatment interface.

[0066] 1. The acoustic emission device includes an ultrasonic generator, an ultrasonic probe, and a power dynamic control system.

[0067] The ultrasound generator can produce ultrasound waves with a frequency of 0.8-3MHz and an adjustable power of 0.1-2W / cm². Its core component is a high-frequency oscillation circuit, which can precisely adjust the frequency and power of the ultrasound waves by controlling the circuit parameters to meet different treatment needs.

[0068] An ultrasound probe consists of a transducer and a coupling layer. The transducer uses piezoelectric ceramic material, which can efficiently convert electrical energy into ultrasound waves. The coupling layer is made of medical coupling agent or a specific elastic material (such as silicone rubber), and its function is to fill the air gap between the ultrasound probe and the human skin, reduce the energy loss of ultrasound waves during transmission, and ensure that ultrasound waves are efficiently transmitted to the treatment site. The power dynamic control system adjusts the ultrasonic intensity ≤2 W / cm based on the real-time power displayed on the screen. 2 .

[0069] 2. The safety protection devices include temperature sensors, current sensors, and automatic power-off protection devices with over-temperature protection, over-current protection, and over-time protection functions.

[0070] Over-temperature protection monitors the temperature of the ultrasonic probe and ultrasonic generator using a temperature sensor (integrated infrared sensor). Specific safe temperature settings are as follows: (1) Safe temperature value of ultrasonic probe surface: ≤43℃, to avoid burns at the point of contact between the probe and the skin. This temperature is the upper limit of human skin's tolerance to long-term contact. (2) The safe temperature value of the internal components of the ultrasonic generator is ≤65℃ to prevent the high-frequency oscillation circuit from being damaged by overheating, and to avoid heat conduction to the probe and affecting the treatment. (3) The safe temperature rise of the treatment area tissue is ≤4℃. This ensures that the temperature rise of the treatment area tissue is less than 4℃. The integrated infrared temperature sensor monitors the temperature in real time to ensure that the treatment effect is achieved only through the mechanical effect of ultrasound, thus avoiding thermal damage that could interfere with the treatment mechanism.

[0071] The power supply will be automatically cut off when the temperature exceeds the preset safety value. Overcurrent protection monitors the current in the circuit using a current sensor. The safe current value is set as follows: (1) Safe operating current range of ultrasonic generator: ≤5A, for frequencies of 0.8-3MHz and 0.1-2W / cm². 2 Matching current for power output to prevent circuit overload; (2) The safe operating current range of the control device and auxiliary modules, including the elastic imaging navigation system, is ≤2A; When the current of any module exceeds 1.2 times the safe value (e.g., ultrasonic generator current ≥ 6A, control device current ≥ 2.4A), the current sensor immediately sends a signal to the safety protection device, disconnects the corresponding circuit within 10ms, and displays the module prompt "overcurrent protection activated".

[0072] The automatic power-off protection device has a timer control unit. When the treatment time exceeds the set value, it will send a shutdown command to the ultrasound generator to automatically stop the equipment and prevent unnecessary harm to the human body.

[0073] 3. Ultrasound contrast imaging device The ultrasound contrast imaging device includes a contrast agent injection control unit, a contrast signal acquisition unit, and a vessel density analysis algorithm module. The device is used to assess changes in tumor vessel density; operation is stopped when microvessels decrease by more than 30%. This module is linked with monitoring and control devices, and its specific structure and workflow are as follows: Contrast agent injection control unit: Core components: Dosage adjustment module: Precisely controls the total injection volume according to the standard of 0.1-0.2mL / kg; Rate control module: Supports injection rate adjustment of 1-3mL / s; Timing synchronization module: Linked with the ultrasound probe to match the arrival time of contrast agent and signal acquisition.

[0074] It can precisely control the injection dose (0.1-0.2 mL / kg) and injection rate (1-3 mL / s) of ultrasound contrast agents (such as SonoVue), and synchronize with the ultrasound probe triggering sequence to ensure that ultrasound signal acquisition is initiated when the contrast agent reaches the tumor area; The contrast signal acquisition unit is integrated with the ultrasound probe and includes a high-frequency ultrasound acquisition component with a frame rate ≥30fps to acquire contrast agent perfusion signals; a grayscale signal conversion module to generate grayscale change curves; and a signal preprocessing component to reduce noise, amplify the original signal, acquire dynamic perfusion signals of contrast agent in tumor vessels (frame rate ≥30fps), and capture the grayscale change curves of microvascular blood flow perfusion.

[0075] The vascular density analysis algorithm module, embedded in the central processing unit of the control device, quantifies the microvascular density (MVD) of the tumor area by calculating parameters such as the "peak intensity" and "area under the curve" of the contrast agent perfusion, and compares it with the baseline value before treatment. When it is determined that the reduction ratio of microvessels is >30%, it automatically sends a "stop irradiation" command to the control device, and the central processing unit cuts off the power output of the ultrasound generator to terminate the treatment.

[0076] During treatment, a short-duration ultrasound angiography is initiated every 5-10 minutes (lasting 1-2 minutes, low power mode to avoid interfering with treatment). The angiography data is initially filtered by the signal processing unit of the monitoring device and then transmitted to the vascular density analysis module. If the threshold of "microvascular reduction >30%" is triggered, the control device immediately performs a shutdown operation and a warning message pops up on the display module.

[0077] 4. The elastography navigation system consists of four parts: a shear wave excitation unit, a multi-channel ultrasound receiving unit, a tissue elasticity parameter calculation module, and a three-dimensional positioning and navigation unit. It is used to measure the shear wave velocity of the tumor ECM (>5m / s is considered a high-disorder area) and guide the ultrasound beam to be precisely focused on the hypoxic core area to avoid excessive damage to normal tissues.

[0078] (1) Shear wave excitation unit: a low-frequency pulse transmitter (frequency 50-100kHz) integrated with the ultrasound probe, used to emit low-energy shear waves (power <0.1W / cm², to avoid tissue damage) to the tumor area. The shear wave propagates in the tumor and the surrounding normal tissue, and the difference in its velocity reflects the elastic stiffness of the tissue (the stiffness of the tumor ECM is higher than that of normal tissue).

[0079] (2) Multi-channel ultrasound receiving unit: containing 8-16 channel ultrasound signal receivers, connected to the transducer array of the ultrasound probe, used to receive the propagation signal of shear waves in the tissue in real time, record the time difference of shear waves arriving at different spatial points, and generate a shear wave propagation velocity distribution map (spatial resolution ≤1mm).

[0080] (3) Tissue elasticity parameter calculation module: A dedicated algorithm chip embedded in the control device calculates the shear wave velocity (SWV) at each point through time difference data and sets a judgment threshold—the area with SWV>5m / s is judged as the tumor ECM high disorder area (usually the tumor hypoxic core area, which is more sensitive to the ultrasonic mechanical effect), and the area with SWV<3m / s is judged as the normal tissue or the low stiffness area of ​​the tumor edge, and a "high disorder area heat map" is generated and transmitted to the display module.

[0081] (4) Three-dimensional positioning and navigation unit: including optical positioning sensor (fixed to the treatment bed, accuracy ±0.5mm) and tumor three-dimensional modeling module. The optical positioning sensor tracks the spatial position of the ultrasound probe in real time. Combined with the tumor three-dimensional model constructed by preoperative CT / MRI images, the "high disorder area" obtained by shear wave velocity analysis is matched with the tumor anatomical position, guiding the ultrasound probe to adjust the angle so that the ultrasound beam is accurately focused on the hypoxic core area (focusing error ≤1mm). At the same time, the display module displays the positional deviation of "ultrasound beam focus - tumor high disorder area" in real time to assist the operator in fine adjustment.

[0082] 5. Power supply: A switching power supply is adopted, which can convert the mains power into a stable DC voltage required by each component, providing stable power to the ultrasonic generator, control device, safety protection device, etc., and ensuring the normal operation of the equipment.

[0083] 6. The monitoring device includes a multispectral optical sensor (400-900nm), a high-speed CCD camera (frame rate ≥1000fps), and an acousto-optic signal processing unit; the multispectral optical sensor is used to detect the refractive index change Δn, and the acousto-optic signal processing unit is a cavitation effect analysis unit used to capture the dynamic behavior of microbubbles.

[0084] 7. Multispectral optical sensors: Key parameters: Detection wavelength range 400-900nm (covering the visible to near-infrared band), spectral resolution ≤5nm, spatial resolution ≤10μm, sampling rate ≥100Hz; Multispectral optical sensors primarily detect changes in the refractive index (Δn) of tumor tissue during treatment. When ultrasound waves act on the tissue, the tissue density changes periodically with mechanical vibration, resulting in a change in refractive index Δn (range 10). -5 -10 -4 Multispectral optical sensors collect changes in the intensity of transmitted / reflected light at different wavelengths, invert the Δn value, and indirectly reflect the mechanical vibration amplitude of the tissue (the larger the Δn, the stronger the vibration). Assisted detection of tissue oxygen saturation (SpO2): The oxygen level in the tumor area is calculated by the ratio of light intensity of 660nm (red light) and 940nm (near-infrared light). When SpO2 < 60% (indicating worsening hypoxia, which may affect the treatment effect), the display module will issue a "low blood oxygen" prompt.

[0085] 8. Signal Processing Unit The signal processing unit contains an integrated signal processing chip, a data bus interface, and a linkage control module. It is responsible for receiving and analyzing data from the sub-units and linking with the control device in real time. It includes a cavitation effect analysis sub-unit and a multispectral signal filtering sub-unit, both of which link with the control device in real time. Cavitation effect analysis sub-unit: Signal receiving module: Adapted to ultrasonic probes, receiving microbubble scattered signals with 1 / 2 or 2 times frequency offset; Parameter quantization module: calculates broadband noise intensity and subharmonic signal amplitude; High-speed sampling module (≥10MHz): records microbubble expansion-contraction cycle (10-50μs) data; The output module compares the threshold values ​​(noise 0.1V, subharmonic 20% incident amplitude, bubble diameter 1-5μm) and sends a "reduce power" command to the power control system.

[0086] The specific functions are as follows: The system receives microbubble scattering signals (with a frequency offset of 1 / 2 or 2 times the incident ultrasound frequency) from an ultrasound probe. The cavitation effect intensity is quantified using two parameters: "broadband noise intensity" and "subharmonic signal amplitude." When the broadband noise intensity > 0.1V (corresponding to violent microbubble oscillation) or the subharmonic amplitude > 20% of the incident wave amplitude (indicating a risk of cavitation bubble rupture), a "power reduction" command is sent to the power dynamic control system to reduce the ultrasound intensity to ≤ 2W / cm². The system also captures microbubble dynamics: the "expansion-contraction cycle" of microbubbles (typically 10-50μs) is recorded through high-speed signal sampling (sampling rate ≥ 10MHz), and the range of bubble diameter variation is analyzed (controlled within 1-5μm during normal treatment to avoid excessively large bubbles clogging microvessels). Multispectral signal filtering subunit: Signal input module: Receives raw signals from multispectral optical sensors; Filtering and correction modules: Gaussian filtering (filters ambient light), motion artifact correction (eliminates bed sway deviation); Data standardization module: processes Δn and SpO2 data; Transmission interface: split-path docking control device display module and elastic imaging navigation system. The specific functions are as follows: The raw signals acquired by the multispectral optical sensor are subjected to "Gaussian filtering" (to remove ambient light interference) and "motion artifact correction" (to eliminate signal deviations caused by slight shaking of the treatment bed). After standardizing the filtered Δn and SpO2 data, they are transmitted to the display module of the control device (updated in real time) and the elastic imaging navigation system (to assist in verifying the correlation between tissue stiffness changes and ultrasound effects).

[0087] 9. The combined treatment interface is used to connect chemotherapy or phototherapy equipment to enable linkage between different treatment devices. 10. Control device The control unit includes a central processing unit, an input module, and a display module. The input module uses buttons or a touch screen for operators to set and adjust parameters such as the frequency, power, and treatment time of the ultrasound. The central processing unit processes the input parameters and controls the ultrasound generator to operate according to the set parameters. The display module uses a high-definition LCD screen to display the current frequency, power, treatment time, and other parameters in real time, facilitating operator observation and monitoring.

[0088] Example 2 This embodiment provides the effect of the tumor treatment system prepared in Example 1 on the survival and proliferation of mouse CT-2A glioblastoma cells under different ultrasound intensities and durations. The specific experimental steps are as follows.

[0089] A culture dish containing mouse CT-2A glioblastoma cells was placed on the ultrasound probe of the tumor treatment system prepared in Example 1, and the ultrasound intensity was set to 0.75, 1, and 1.75 W / cm². 2 The cells were sonicated for 0, 3, 9, 18, and 36 minutes. After sonication, live / dead cell staining was used to assess cell viability, and the CCK-8 assay was used to assess cell proliferation. The specific experimental steps are shown below.

[0090] CT-2A cells were prepared at 1 × 10⁶ cells per dish 5 Cells were seeded at a density of 1,000 cells per 35 mm² culture dish. All samples were randomly divided into five groups: control group (0 minutes of sonication), 3 minutes of sonication, 9 minutes of sonication, 18 minutes of sonication, and 36 minutes of sonication. Subsequently, different intensities of sonication (1.75 W / cm²) were used. 2 1 W / cm 2 and 0.75 W / cm 2 The cells were then treated with a power setting of 1 MHz and a 50% duty cycle. The cells were then cultured for an additional 8 hours.

[0091] After culture, cell proliferation was assessed using the standard CCK-8 assay. After culturing for another 8 hours, the old culture medium was discarded, and the cells were gently washed twice with PBS. Then, fresh culture medium containing CCK-8 solution (10 µL of CCK-8 solution per 100 µL of medium) was added, and the cells were incubated for another 1 hour. The OD value at 450 nm was then measured using a microplate reader.

[0092] Simultaneously, using the Live&Dead™ Animal Cell Viability / Toxicity Assay Kit (UElandy), Calcein-AM / PI co-staining experiments were performed according to the manufacturer's instructions to observe cell death. After culture, cells were treated with calcein-AM (4 µg / mL) and propidium iodide (PI, 10 µg / mL) for 30 minutes, followed by washing three times with PBS at 37°C. Finally, the Calcein-AM / PI co-stained cells were observed under a fluorescence microscope (Axio Vert A1, Leica). The ratio of dead to live cells was quantitatively analyzed using ImageJ software.

[0093] Experimental results are as follows Figure 1 As shown in the AI, Figure 1 The results of the AC assay for Calcein-AM / PI co-staining show that at an ultrasound intensity of 1.75 W / cm², the results indicate that the AC signal is within the range of 1.75 W / cm². 2 ( Figure 1 (A), 1W / cm 2 ( Figure 1 (B), 0.75W / cm 2 ( Figure 1 Under the conditions of C), the proportion of dead cells increased with time.

[0094] Figure 1 The DI (Discrete Induction Difference) indicator shows that when the ultrasonic power density is 1.75 W / cm², 2 When the treatment time was 3 minutes, the viability of glioma cells decreased to 32%. Figure 1 (Middle D), further extending the treatment time at this power density, the inhibitory effect on cell proliferation can reach IC25 ( Figure 1 (G); when the ultrasonic power density is 1W / cm² 2 When the treatment time was 9 minutes, cell viability decreased significantly. Figure 1 The inhibitory effect on cell proliferation reached IC25 (E), which is effective against cell proliferation. Figure 1 (H); when the ultrasonic power density is 0.75 W / cm² 2 When the action time is 3 minutes, it has almost no inhibitory effect on cell viability. Figure 1 (F), extending the action time to 18 minutes, the inhibitory effect on cell proliferation reached IC25 ( Figure 1 Middle I).

[0095] The experimental results of the CCK-8 assay and live / dead cell staining show that the lower the ultrasound intensity, the longer the treatment time required to achieve the same killing effect, indicating that the inhibitory effect on tumor cells can be enhanced by prolonging the ultrasound treatment time based on the ultrasound fatigue effect.

[0096] Example 3 This embodiment demonstrates the therapeutic effect of the tumor treatment system prepared in Example 1 on a BALBc / nu mouse model carrying Ct-2A gliomas. The BALBc / nu mouse model carrying Ct-2A gliomas was divided into groups (4-week-old female BALB / c nude mice were provided by Beijing Huafu Biotechnology Co., Ltd. (China). All animal experiments obtained ethical approval from Shenzhen Topbio Biotechnology Co., Ltd. (Approval No.: TOP-IACUC-2023-0114)). The specific experimental details are as follows.

[0097] Mouse model construction: CT-2A cells were subcutaneously injected into the axilla of the right forelimb to establish a xenograft model of brain tumor.

[0098] The experimental groups are as follows: 1W / cm 2 Intensity groups: control group (0 minutes of ultrasound, G1), 3 minutes of ultrasound (G2), 9 minutes of ultrasound (G3), 18 minutes of ultrasound (G4), and 36 minutes of ultrasound (G5). 0.5W / cm 2 Intensity groups: control group (0 minutes ultrasound, G6), 3 minutes ultrasound group (G7), 15 minutes ultrasound group (G8), 30 minutes ultrasound group (G9).

[0099] Medical coupling gel was applied to the probe of the ultrasound generator and the exterior of the mouse tumor. The ultrasound probe coated with medical coupling gel was placed on the mouse tumor at a 0.5 cm interval, and the above-mentioned groups were administered ultrasound at a power of 0.5 W / cm. 2 and 1W / cm 2 Ultrasound treatment at different intensities for varying durations. Ultrasound was administered once daily for 11 days. The change in tumor volume was calculated as: tumor volume on day 11 / tumor volume on day 1 (before treatment).

[0100] Experimental results are as follows Figure 2 As shown in AJ, 1W / cm 2 In the ultrasound treatment groups, groups G3 (9 minutes / day) and G4 (18 minutes / day) showed significant inhibitory effects on tumor growth after 11 days. The tumor volume in group G3 was 61% of that in the control group, and the tumor growth ratio (V11 / V1, where V1 is the tumor volume before treatment and V11 is the tumor volume after treatment) (20.8 times) was significantly lower than that in the control group (32.2 times). The tumor weight was also significantly lower in group G3 than in group G1. Figure 2 (AC). In the G5 group (36 minutes / day), extending the treatment time to over 18 minutes did not enhance the therapeutic effect, and due to excessive energy, the survival rate of mice was only 33.3%. Figure 2 Middle I).

[0101] 0.5W / cm 2In the low-power ultrasound treatment groups, the tumor growth rate in group G8 (15 minutes / day) was significantly lower than that in the control group (31.6 times). Group G9 (30 minutes / day) showed even better results, with the tumor growth rate decreasing to 19.1 times. The tumor weight in both groups G8 and G9 was significantly lower than that in the control group. Figure 2 In terms of safety, the survival rate of mice in the G9 group (30 minutes / day) was 100%. Figure 2 (J), and the mice's weight remained stable during the treatment period ( Figure 2 (Glucose-containing compound) has good biocompatibility.

[0102] Figure 3 Display 1W / cm 2 In the ultrasound treatment group, the G3 group had the smallest relative tumor volume. Figure 3 (Among them, A), and the tumor growth inhibition rate is the highest ( Figure 3 (B) 0.5W / cm 2 In the ultrasound treatment group, the G9 group had the smallest relative tumor volume. Figure 3 (C), and the highest tumor growth inhibition rate ( Figure 3 (D).

[0103] Based on the above experiments, further tests were conducted at different time points: 1W / cm 2 Intensity groups: control group (G1), ultrasound for 9 minutes (G3), ultrasound for 20 minutes (G10), n=3 in each group. 0.5W / cm 2 Intensity groups: control group (G6), ultrasound for 9 minutes (G11), ultrasound for 18 minutes (G12), ultrasound for 30 minutes (G9).

[0104] Experimental results are as follows Figure 4 As shown, 1W / cm 2 In the ultrasound treatment group, the G3 group had the smallest relative tumor volume. Figure 4 (Among them, A), and the tumor growth inhibition rate is the highest ( Figure 4 (B) 0.5W / cm 2 In the ultrasound treatment group, the G9 group had the smallest relative tumor volume. Figure 4 (C), and the highest tumor growth inhibition rate ( Figure 4 (D).

[0105] In summary, 1W / cm 2 A 9-18 minute / day ultrasound regimen can effectively inhibit tumors, but excessively prolonged treatment time or increased power can damage surrounding normal tissues, limiting the treatment effect and reducing efficacy. 0.5W / cm² 2 Low-power ultrasound showed stronger tumor suppression effects and significantly better safety than high-power ultrasound when the treatment time was extended to 30 minutes / day.

[0106] Example 4 Based on the results of in vivo tumor treatment in the mouse model of Example 3, 1W / cm 2 Ultrasound intensity was excluded due to its increased mortality rate in mice, while 0.5 W / cm² was excluded. 2 Although the ultrasound intensity was safe, the TGI value did not reach 50% in any of the treatment duration groups (TGI = (1 - tumor volume in the treatment group / tumor volume in the control group) × 100%), therefore 0.75 W / cm² was used. 2 Ultrasound, combined with different treatment durations, was used to investigate the efficacy of ultrasound fatigue therapy in combination with temozolomide (a first-line chemotherapy drug for gliomas) in C57BL / 6 mice carrying CT-2A gliomas. The C57BL / 6J black mice (female) were provided by Beijing Huafu Biotechnology Co., Ltd. (China). The establishment of the Ct-2A glioma mouse model was the same as in Example 3. Figure 5 A diagram in the center illustrates the treatment process, as shown below: The experimental groups were as follows: Mice were randomly divided into 8 groups, namely: G1: PBS control group; G2: PBS + 5 minutes of ultrasound therapy daily; G3: PBS + 15 minutes of ultrasound therapy daily; G4: PBS + 30 minutes of ultrasound therapy daily; G5: Temozolomide monotherapy; G6: Temozolomide + 5 minutes of ultrasound therapy daily; G7: Temozolomide + 15 minutes of ultrasound therapy daily; G8: Temozolomide + 30 minutes of ultrasound therapy daily.

[0107] Two hours prior to ultrasound therapy, mice were injected with temozolomide or PBS at a dose of 10 mg per kilogram of body weight, with an ultrasound intensity of 0.75 W / cm². 2 Mice underwent ultrasound treatment according to the set time intervals for each group, daily for 10 days. Body weight and tumor volume (length × width × width / 2) were measured every other day. After treatment, mice were euthanized, and tumor tissue was collected for photographing, weighing, and subsequent research. The change in volume was calculated as: tumor volume on day 10 / day 1 (before treatment).

[0108] Tumor sections obtained on day 10 post-treatment were stained with H&E, TUNEL, Ki67, HIF-1α, and CD31 for systematic pathological analysis. Tumor samples from different treatment groups were collected, then formalin-fixed, paraffin-embedded, and sectioned for staining. Major organs (heart, liver, lung, spleen, and kidney) were also collected for H&E staining. The specific experimental procedure is as follows: 1. Sample collection and fixation (1) After the mice were euthanized, the tumor tissue (weighed and photographed) and major organs (heart, liver, lung, spleen and kidney) were immediately separated and rinsed three times with pre-cooled PBS (pH 7.4) to remove residual blood and connective tissue; (2) Place the tissue in 4% neutral formalin solution (5-10 times the volume of the tissue) and fix at room temperature for 24 hours (avoid over-fixation to prevent antigen destruction); if delayed treatment is required, the fixed tissue can be transferred to 70% ethanol and stored at 4°C for no more than 7 days.

[0109] (3) Dehydration and paraffin embedding: Dehydrate the tissue according to the following gradient: 70% ethanol (1 hour) → 80% ethanol (1 hour) → 90% ethanol (1 hour) → 95% ethanol (1 hour) → anhydrous ethanol I (30 minutes) → anhydrous ethanol II (30 minutes) → xylene I (15 minutes) → xylene II (15 minutes) (each step is performed at room temperature to ensure that the tissue is completely dehydrated and transparent); After gradient dehydration, the tissue is paraffin-embedded: The transparent tissue is placed in 60°C melted paraffin I (1 hour) → paraffin II (1 hour) → paraffin III (1 hour), and the temperature is kept constant at 60°C at each step to avoid paraffin crystallization; After paraffin embedding, the tissue is embedded: The paraffin-embedded tissue is placed in a paraffin embedding box, the tissue orientation is adjusted (tumor tissue must include the boundary between the lesion area and the normal tissue at the edge, and organ tissue must maintain the anatomical orientation), melted paraffin is poured in, and after the paraffin solidifies, it is stored at 4°C for later use.

[0110] (4) Use a rotary microtome to cut the paraffin block prepared in step (3) into continuous slices with a thickness of 4-5 μm. Float the slices in 45℃ warm water to flatten them and avoid wrinkles. Then, transfer them to a glass slide coated with poly-L-lysine (to prevent detachment). Place the glass slide in a 60℃ oven to dry for 2 hours to ensure that the slices are tightly attached to the glass slide. Then, transfer them to a 4℃ drying oven for storage until staining (storage time not exceeding 1 month).

[0111] 2. H&E staining H&E staining was performed on tumor tissue and major organs to assess tissue morphology and damage, as detailed below.

[0112] (1) Place the obtained tissue sections in xylene I (10 minutes) → xylene II (10 minutes) → anhydrous ethanol I (5 minutes) → anhydrous ethanol II (5 minutes) → 95% ethanol (3 minutes) → 90% ethanol (3 minutes) → 80% ethanol (3 minutes) → 70% ethanol (3 minutes) → distilled water (5 minutes) at room temperature; (2) Place the dewaxed sections from step (1) into Harris hematoxylin stain (Sigma-Aldrich) and stain at 37°C for 5-8 minutes, then rinse with distilled water for 2 minutes. (3) Place the cells in a 1% hydrochloric acid-ethanol solution (prepared by mixing hydrochloric acid and 70% ethanol at a volume ratio of 1:99) for 3-5 seconds to differentiate (observe under a microscope until the nucleus is dark blue and the cytoplasm is colorless), then immediately transfer them to 0.5% ammonia water (volume ratio) to turn blue again for 30 seconds, and rinse with distilled water for 5 minutes. (4) Stain in 0.5% eosin Y staining solution (water-soluble, Solarbio) for 3-5 minutes, then rinse quickly with distilled water for 10 seconds; (5) 70% ethanol (1 minute) → 80% ethanol (1 minute) → 90% ethanol (1 minute) → 95% ethanol (1 minute) → anhydrous ethanol I (3 minutes) → anhydrous ethanol II (3 minutes) → xylene I (5 minutes) → xylene II (5 minutes); Mounting: Add 1-2 drops of neutral resin per section (Servicebio), cover with a coverslip (avoid air bubbles), and allow to air dry at room temperature for 24 hours before microscopic examination.

[0113] 3. TUNEL staining The Roche In Situ Cell Death Detection Kit (Fluorescein, Roche) was used to detect apoptosis in tumor tissues according to the instructions, as detailed below.

[0114] (1) Follow the staining steps according to the kit; (2) Dewaxing to water: Same as step 1 of H&E staining; (3) Place the tissue sections in 0.01M citrate buffer (pH 6.0), heat on high in a microwave oven until boiling, then maintain on medium for 10 minutes, allow to cool naturally to below 30°C at room temperature, and rinse with PBS 3 times for 5 minutes each time; (4) Add 0.1% Triton X-100 (dissolved in PBS), incubate at room temperature for 15 minutes, and rinse with PBS 3 times, 5 minutes each time; (5) Add PBS solution containing 5% BSA by volume, block at 37°C for 30 minutes, and remove the blocking solution (without rinsing). (6) Prepare TUNEL reaction solution (enzyme solution: label solution volume ratio = 1:9) according to the kit instructions. Add 50 μL to each slice and incubate at 37°C in the dark for 60 minutes. Rinse three times with PBS for 5 minutes each time. (7) Add DAPI staining solution (1 μg / mL, Beyotime), incubate at room temperature in the dark for 5 minutes, and wash with PBS 3 times for 5 minutes each time; (8) Add anti-fluorescence quenching mounting solution (Servicebio, catalog number G1401), cover with a coverslip, and observe under a fluorescence microscope (excitation wavelength 488nm, emission wavelength 520nm; DAPI excitation wavelength 350nm, emission wavelength 460nm).

[0115] TUNEL staining should be performed in the dark throughout the entire process. After staining, the sections should be stored at 4°C in the dark and examined under a microscope within 24 hours to prevent fluorescence quenching.

[0116] 4. Ki67 immunohistochemical staining Cell proliferation was detected using the Ki67 immunohistochemical staining kit (Saiwell Biotechnology Co., Ltd.), as detailed below.

[0117] (1) Use the reagent kit's fluorescent labeling detection system for staining (immunofluorescence two-step method); (2) Dewaxing to water and antigen retrieval: Same as TUNEL staining steps (1) and (2); (3) Add 3% H2O2 (Solarbio), incubate at room temperature for 15 minutes, and wash with PBS 3 times, 5 minutes each time; (4) Add 5% BSA (dissolved in PBS), block at 37°C for 30 minutes, and remove the blocking solution (without rinsing); (5) Add rabbit anti-mouse Ki67 primary antibody (dilution ratio 1:200, dissolved in 5% BSA, Abcam), incubate overnight at 4℃, and wash 3 times with PBS for 5 minutes each time; (6) Add fluorescein-labeled goat anti-rabbit IgG secondary antibody (fluorescein selected as FITC, dissolved in PBS, dilution ratio 1:500), incubate at 37°C in the dark for 30 minutes, and wash with PBS 3 times, 5 minutes each time; (7) Add DAPI staining solution (final concentration 0.5 μg / mL, dissolved in PBS), incubate at room temperature in the dark for 5 minutes, rinse once with distilled water to stop staining; (8) Add anti-fluorescence quenching mounting medium (Solarbio), cover with coverslip (avoid air bubbles), and complete the mounting; (9) After mounting, store at 4°C protected from light. Observe under a fluorescence microscope (equipped with the corresponding fluorescence filter) within 24 hours. Ki67 positive signal is specific fluorescence, and DAPI counterstaining of cell nuclei will turn blue. (Ki67: excitation wavelength 488nm, emission wavelength 525nm; DAPI excitation wavelength 350nm, emission wavelength 460nm) 5. HIF-1α immunohistochemical staining The tumor tissue was subjected to HIF-1α immunohistochemical staining kit (Saiwell Biotechnology Co., Ltd.) to detect the hypoxic state, as detailed below.

[0118] (1) Staining steps (using the two-step immunofluorescence method) (2) Same as step 1 for H&E staining; (3) Place the slices in 0.01M EDTA buffer (pH 8.0, Solarbio), heat to 121°C in an autoclave, maintain for 2 minutes, cool naturally at room temperature, and rinse 3 times with PBS for 5 minutes each time (HIF-1α is a nucleoprotein, and EDTA buffer has a better repair effect than citrate buffer). (4) Same as steps 2-3 for Ki67 staining; (5) Add rabbit anti-mouse HIF-1α primary antibody (dilution ratio 1:150, dissolved in 5% BSA, Cell Signaling Technology), incubate overnight at 4°C, and wash 3 times with PBS for 5 minutes each time; Secondary antibody incubation, nuclear counterstaining, and mounting: Same as steps 6-8 of Ki67 immunofluorescence staining (where the secondary antibody is fluorescein-labeled goat anti-rabbit IgG, FITC is selected as the fluorescein, and the dilution ratio is 1:500; the nuclear counterstaining uses DAPI staining solution; the excitation wavelength is 488nm and the emission wavelength is 525nm; the DAPI excitation wavelength is 350nm and the emission wavelength is 460nm).

[0119] 6. CD31 immunohistochemical staining For tumor tissue, microvessel density was detected using a CD31 immunohistochemical staining kit (Saiwell Biotechnology Co., Ltd.), as detailed below.

[0120] (1) Staining steps (using the two-step immunofluorescence method) (2) Same as HIF-1α staining steps 1-2 (CD31 is a membrane protein, and EDTA buffer retrieval can better expose the antigen epitope). (3) Endogenous peroxidase blocking and inhibition, same as steps 2-3 of Ki67 staining; (4) Add rat anti-mouse CD31 primary antibody (dilution ratio 1:100, dissolved in 5% BSA, BD Biosciences), incubate overnight at 4°C, and wash 3 times with PBS for 5 minutes each time; (5) Add fluorescein-labeled goat anti-rat IgG secondary antibody (fluorescein selected as FITC, dissolved in PBS, dilution ratio 1:500), incubate at 37°C in the dark for 30 minutes, and wash with PBS 3 times, 5 minutes each time; Nuclear counterstaining and mounting: Same as steps 7-8 of Ki67 immunofluorescence staining (nuclear counterstaining uses DAPI staining solution; excitation wavelength 488nm, emission wavelength 525nm; DAPI excitation wavelength 350nm, emission wavelength 460nm). Microvessel counting: Five tumor hotspot areas were selected under a microscope (200x field of view) and CD31-positive vascular endothelial cells or luminal structures were counted. The microvessel density (MVD) was expressed as "average number of microvessels per field of view".

[0121] Statistical analysis was performed using GraphPad Prism 8, and a two-tailed Student's t-test was used to analyze the data from both groups. Multiple comparisons between groups were conducted using one-way ANOVA.

[0122] Results of tumor treatment efficacy experiments, such as Figure 5 As shown, after 10 days of treatment, the tumor size in each treatment group was significantly smaller than that in the control group. Figure 5 (B), and the longer the ultrasound treatment time, the smaller the tumor volume (B). Figure 5 (E and F) and weight ( Figure 5 The more significant the decrease in C compared to the control group ( Figure 5 B, Figure 6 The tumor volumes in groups G2, G3, and G4 were 60.9%, 31.7%, and 22.0% of the control group, respectively. The relative changes in tumor volume (V10 / V1, where V1 is the tumor volume before treatment and V10 is the tumor volume after treatment) were 15.0 times, 7.8 times, and 5.4 times, respectively, significantly lower than the 24.6 times in the control group. Figure 5 In the middle group (E), the TGI values ​​of groups G3 and G4 reached 68.3% and 80.0%, respectively.

[0123] Within a 10-day treatment cycle, the combined treatment group (ultrasound fatigue + temozolomide (TMZ)) exhibited a faster tumor suppression effect: the TMZ combined with 15 minutes / day ultrasound treatment group (G7) showed a significant reduction in tumor volume as early as day 5 (compared to the control group, p). <0.05); the mean tumor volume and relative tumor volume in the combined treatment group on days 5, 7, and 10 were all lower than those in the ultrasound-only group (G3) with the same parameters. The body weight of mice in all treatment groups remained stable, confirming that the combined regimen had no systemic toxicity. Figure 5 (D). By combining 0.75W / cm² ultrasound with TMZ chemotherapy, the combined group showed tumor-suppressing effects in the early stage of treatment (≤5 days), which was superior to ultrasound therapy alone, and the synergistic effect was maintained even after the treatment was extended to 10 days.

[0124] The tumor treatment trial results showed 0.75 W / cm². 2 Ultrasound therapy is superior to 1.0 W / cm² in both efficacy and safety. 2 and 0.5W / cm 2 0.75W / cm 2 The ultrasound combined with TMZ (temozolomide) regimen achieved a tumor inhibition rate of 68.7% with good safety.

[0125] Experimental results are as follows Figure 7 As shown, Figure 7 In Figure A, the results of H&E staining of tumor tissue are shown. It can be seen that the tumors in each experimental group have clear tissue boundaries and their areas are smaller than those in the control group. Figure 7 BF and G represent the TUNEL and CD31 immunofluorescence staining results of tumor sections, respectively. Immunohistochemical analysis revealed the mechanism of action and safety of acoustic fatigue therapy and its combination with temozolomide (TMZ): acoustic fatigue therapy alone significantly induced tumor cell death. With prolonged treatment time (5 minutes → 15 minutes → 30 minutes), the tumor necrosis rate increased from 7% in the control group to 16%, 36%, and 37%, respectively. Furthermore, the tumor cell density in the treatment group decreased and the gaps between cells became more pronounced, indicating that acoustic fatigue can disrupt the extracellular matrix (ECM). Figure 7 In the group treated solely with acoustic fatigue (G2-G4), the density of apoptotic cells (positive for green fluorescence) in the whole tumor sections was significantly higher than that in the control group (C). Figure 7 In the D and E groups, apoptotic cells in the temozolomide monotherapy and combination therapy groups (G5-G8) were mainly enriched in the non-necrotic area at the outer edge of the tumor, and the overall apoptosis rate in the combination therapy group (G8) was lower than that in the acoustic fatigue group alone (G4). Figure 7 (E); Acoustic fatigue therapy can directly damage tumor blood vessels, but when combined with temozolomide, it can inhibit the formation of new blood vessels and simultaneously counteract the damage to tumor blood vessels caused by ultrasound. Figure 7 (G).

[0126] Figure 8 The results of immunofluorescence staining for HIF-1α, Ki67, and MMP9 showed that HIF-1α expression, expressed as green fluorescence, was significantly increased after ultrasound fatigue treatment and combination therapy with temozolomide. Figure 8 This indicates that ultrasound fatigue-based treatment enhanced intratumoral hypoxia. Following ultrasound fatigue treatment and its combination therapy, Ki67 protein expression was significantly lower than in the control group. This confirms that ultrasound fatigue-based therapy inhibits tumor growth. Figure 8 Immunofluorescence assays of MMP9 showed a significant increase in expression (displayed as red fluorescence) after ultrasound fatigue treatment, particularly in groups receiving 15-minute and 30-minute ultrasound fatigue treatments (G3 and G4). Figure 8 ).

[0127] Optimized combination therapy regimen: Optimal ultrasound intensity: 0.75 W / cm² 2 Optimal timing: 15-30 minutes per dose; optimal dosing interval: 2 hours.

[0128] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A tumor treatment system, characterized in that, The treatment system includes an acoustic emission module and a monitoring module; The acoustic emission module includes an ultrasonic generator, an ultrasonic probe, and a power dynamic control system. The ultrasonic generator has an ultrasonic intensity of 0.1-2 W / cm². 2 Continuously adjustable, with the frequency adjustable from 0.5 to 3 MHz; The monitoring module includes a multispectral optical sensor; The multispectral optical sensor is used to detect changes in the refractive index of tumor tissue during treatment.

2. The tumor treatment system according to claim 1, characterized in that, The multispectral optical sensor is also used to detect blood oxygen saturation in tumor tissue.

3. The tumor treatment system according to claim 1, characterized in that, The tumor treatment system also includes a navigation system; the navigation system includes a shear wave excitation unit, a multi-channel ultrasound receiving unit, and a three-dimensional positioning and navigation unit; the navigation system is used to measure the shear wave velocity of the tumor and guide the ultrasound beam to be precisely focused on the hypoxic core area.

4. The tumor treatment system according to claim 1, characterized in that, The tumor treatment system also includes a safety protection device; the safety protection device includes a temperature sensor; the temperature sensor includes features for monitoring the treatment area to ensure that the temperature rise is less than 5°C.

5. The tumor treatment system according to claim 1, characterized in that, The tumor treatment system also includes an ultrasound contrast imaging device; the ultrasound contrast imaging device includes a contrast agent injection control unit and a contrast signal acquisition unit; the ultrasound contrast imaging device is used to detect changes in tumor blood vessel density, and stops the operation of the tumor treatment system when the microvessels decrease by more than 30%.

6. The use of the tumor treatment system according to any one of claims 1-5 in the preparation of tumor treatment products.

7. The application according to claim 6, characterized in that, The tumor includes at least one of glioma, meningioma, and neuroblastoma.

8. The use of the tumor treatment system and tumor treatment drug according to any one of claims 1-5 in the preparation of tumor treatment products.

9. The application according to claim 8, characterized in that, The tumor treatment drugs include chemotherapy drugs or immunotherapy drugs.

10. The application according to claim 9, characterized in that, The chemotherapy drugs include at least one of temozolomide, lomustine, cisplatin, and gemcitabine.

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